Cumulative Techniques

and Procedures in
Clinical Microbiology
I
New Developments in
Antimicrobial Agent
Susceptibility Testing:
a Practical Guide I

American Society
for Microbiology
Washington, DC
Cumitech IA Blood Cultures II June 1982 l l

Cumitech 2A Laboratory Diagnosis of Urinary Tract Infections March 1987

l l

Cumitech 3A Quality Control and Quality Assurance Practices in Clinical Microbiology

l l

May 1990
Cumitech 4 Laboratory Diagnosis of Gonorrhea October 1976
l l

Cumitech 5 Practical Anaerobic Bacteriology April 1977

l l

Cumitech 6 New Developments in Antimicrobial Agent Susceptibility Testing September

l l

1977
Cumitech 7A Laboratory Diagnosis of Lower Respiratory Tract Infections September 1987
l l

Cumitech 8 Detection of Microbial Antigens by Counterimmunoelectrophoresis

l December l

1978
Cumitech 9 Collection and Processing of Bacteriological Specimens August 1979
l l

Cumitech IO Laboratory Diagnosis of Upper Respiratory Tract Infections December 1979

l l

Cumitech 1I Practical Methods for Culture and Identification of Fungi in the Clinical
l

Microbiology Laboratory August 1980 l

Cumitech 12 Laboratory Diagnosis of Bacterial Diarrhea October 1980

l l

Cumitech 13 Laboratory Diagnosis of Ocular Infections May 1981

l l

Cumitech 14 Laboratory Diagnosis of Central Nervous System Infections January 1982

l l

Cumitech 15 Laboratory Diagnosis of Viral Infections March 1982

l l

Cumitech 16 Laboratory Diagnosis of the Mycobacterioses

l March 1983 l

Cumitech 17 Laboratory Diagnosis of Female Genital Tract Infections August 1983

l l

Cumitech 18 Laboratory Diagnosis of Hepatitis Viruses January 1984

l l

Cumitech 19 Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections August 1984

l l

Cumitech 20 Therapeutic Drug Monitoring: Antimicrobial Agents October 1984

l l

Cumitech 21 Laboratory Diagnosis of Viral Respiratory Disease March 1986

l l

Cumitech 22 Immunoserology of Staphylococcal Disease August 1987

l l

Cumitech 23 Infections of the Skin and Subcutaneous Tissues June 1988

l l

Cumitech 24 Rapid Detection of Viruses by Immunofluorescence

l August 1988 l

Cumitech 25 Current Concepts and Approaches to Antimicrobial Agent Susceptibility

l

Testing December 1988 l

Cumitech 26 Laboratory Diagnosis of Viral Infections Producing Enteritis September 1989

l l

Cumitechs should be cited OS follows, e,g.: Neumann, M, A., D. F. Sahm, C. Thornsberry, and J. E.
McGowan, Jr. 1991. Cumitech 6A New developments in antimicrobial agent susceptibility testing: a practical
guide. Coordinating ed., J. E. McGowan, Jr. American Society for Microbiology, Washington, DC

Editorial Board for MM Cumitechs: Steven C. Specter, Chairman, Carl Abramson, Ellen Jo Baron,
Mary J. R. Gilchrist, Willram J, Martone, John E. McGowan, Jr., Frederrck S Nolte Arne C. Rodloff. James
W. Smrth, John A. Smith, Thomas J. Trnghrtella, and Alice S. Weissfeld.

The purpose of the Cumitech series is to provide consensus recommendations by the authors CIS to appropriate
state-of-the4 operating procedures for clinical microbiology laboratories which may lack the facilities for fully
evaluating routine or new methods.
The procedures given are not proposed as “standard” methods.

Copyright 0 1991 Amerlcon Society for Mlcroblolom

1325 Massachusetts Ave , NW
Washington. DC 20005
 NEW DEVELOPMENTS IN ANTIMICROBIAL AGENT
SUSCEPTIBILITY TESTING: A PRACTICAL GUIDE
MARK A. NEUMANN, Clinical Microbiology, Immunology and Virology Laboratories and Clinical
Pharmacology Investigations Division, Diagnostic Services, Inc., and Department of Pathology and
Laboratory Medicine, Naples Community Hospital, Naples, Florida 33940

DANIEL F. SAHM, Clinical Microbiology Laboratories and Department of Pathology, University of Chicago
Medical Center, Chicago, Illinois 60637

CLYDE THORNSBERRY, Institutes for Microbiology Research, 357 Riverside Drive, Franklin,
Tennessee 37064

JOHN E. MCGOWAN, JR., Department of Pathology and Laboratory Medicine, Emory University School of
Medicine, and Clinical Laboratory, Grady Memorial Hospital, Atlanta, Georgia 30335

COORDINATING EDITOR

JOHN E. MCGOWAN, JR., Department of Pathology and Laboratory Medicine, Emory University School of
Medicine, and Clinical Laboratory, Grady Memorial Hospital, Atlanta, Georgia 30335

Since the first printing of Cumitech 6 (182) in smallteaching and nonteachinghospitals(1, 21,
1977,a number of developmentshave occurred 32). Community-acquiredORSA outbreaks, par-
with regard to our understandingand recogni- ticularly in intravenous drug abusers and pa-
tion of antimicrobial resistancefactors, trendsin tients with serious underlying disease, have
occurrence for certain bacterial pathogens,and been reported as well (153).
recognition of the importance of certain fastidi- Coagulase-negative staphylococci are cur-
ous or unusual bacteria in infectious diseases. rently recognized as significant nosocomial
These factors have led to several changesand pathogens found in association with surgical
developments in antimicrobial susceptibility wounds, intravenous catheters, shunts, joint
testing of microorganisms. In certain cases, prostheses,prosthetic valves, septicemia, and
modificationsof the current standardagar disk catheter-relatedurinary tract infections (4, 167).
diffusion (124) and broth and agar dilution Staphylococcus epidermidis is a leadingcauseof
(121-123, 125) susceptibility tests have been nosocomialbacteremia in someU.S. hospitals,
necessaryto produce reliable and reproducible resulting in a crude-mortality rate of 35% (138,
test results. This Cumitech reviews theserecent 167). Immunocompromisedpatients, particu-
changes, developments, and observations. A larly those in critical care units, are at greatest
companionCumitech (no. 25)provides a general risk of infection with this organism(167). One-
guide to help laboratories decide what testing third to one-half of these isolates have been
methodswill be usedand how the methodswill reported to be resistantto penicillinase-resistant
be implemented. Both Cumitechs supplement penicillins (e.g., oxacillin, nafcillin, and methi-
publications such as the American Society for cillin). Several types of resistance have been
Microbiology’s Manual of Clinical Microbiol- noted.
ogy (180) and the documents of the National
Committee for Clinical Laboratory Standards Intrinsically Resistant(Heteroresistant)
(NCCLS) (121-125), to which readers are re- Staphylococci
ferred for detailed descriptions of the most The most important intrinsic mechanismof
recently recommendedtest methods. staphylococcalresistanceto oxacillin is a chro-
mosomallymediatedalteration in the penicillin-
OXACILLIN-RESISTANT STAPHYLOCOCCI binding protein (PBP) called 2’ or 2a (56,66-68).
Uxacillin-resistant Staphylococcus aureus Several reports have shown that this PBP of
(ORSA), described by some as methicillin-re- ORSA has a low affinity for penicillinase-resis-
sistantS. aureus, hasemergedinternationally as tant penicillins, and probably for all beta-lactam
a major nosocomialpathogen (179, 197, 198). antimicrobial agents,at physiologic pH (66-68).
The incidence of ORSA in U.S. hospitals has Becauseof this low affinity, the concentration of
increasedsteadily through the 198Os,not only in oxacillin necessaryto inhibit growth of the or-
large tertiary care teaching centers but also in ganismsfar exceedsthat which can be achieved

1
2 NEUMANN ET AL.
therapeutically. The presence of beta-lactams
also may induce increased production of low-
affinity PBPs (143, 187).
The term “heteroresistant” denotesthe phe-
notypic heterogeneity of the staphylococci that
are genotypically (intrinsically) oxacillin resist-
ant. Even though all of the cells within this
population have the genetic potential to express
oxacillin resistance, it has been estimated that
only 1 in 104to IO6 organismsdoes so in the
presenceof a penicillinase-resistantpenicillin at
37°C (3, 146, 172). Heterogeneity occurs not
only with the penicillinase-resistantpenicillins
but also with most of the cephalosporinsand
imipenem.The proportion of this resistantpop-
ulation can be influenced by factors such as
temperature, osmolarity, length of incubation,
presenceof beta-lactams,and other factors (1,
18, 146). For example, lowering the incubation
temperature to between 30 and 35°C or using
hypertonic growth mediumcan considerably en-
hancethe size and growth rate of the population
manifestingresistance.
The difficulty in accurately detecting intrinsi-
cally oxacillin-resistant’staphylococci has been
the topic of several studies(2,6, 11, 14, 29,95).
It was clearly shown that the standard disk
diffusion and broth microdilution tests, as well
ascommercially available susceptibility test sys-
tems, did not reliably detect staphylococcal re-
sistanceto penicillinase-resistantpenicillins and
cephalosporins(2, 12, 13, 64, 103, 183). A sig-
nificant number of truly oxacillin-resistant
staphylococci therefore may have beenreported
to be falsely susceptible to the penicillinase-
resistant penicillins and cephalosporins,poten-
tially compromising patient managementand
very likely underestimating the incidence of
oxacillin-resistant staphylococci in certain hos-
pital settings(55, 134).
In response,changesin antimicrobial suscep-
tibility test methodologieshave focused on en-
hancingthe growth rate, and thus proportion, of
the resistant population of cells. Lowering the
incubation temperature, increasingthe inoculum
size, and increasing the osmolarity of the me-
dium have beenusedto modify standardsuscep-
tibility test methodsto increaseoxacillin-resist-
ant staphylococcal detection rates (103, 124,
125157,183). Currently NCCLS is recommend-
ing the following techniquesfor the increased
detection of resistanceto penicillinase-resistant
penicillins and cephalosporinsamong staphylo-
coccal isolates(124, 125).
Agar disk diffusion test. For the agar disk
diffusion test use the procedure outlined in
NCCLS M2-A4 (124), but be certain to care-
fully perform the following essential steps to
optimize detection of oxacillin-resistant staph-
ylococci: (i) use the direct inoculum prepara-
CUMITECH 6A
tion method (124), (ii) use a 1-pg oxacillin disk
since it is most likely to detect cross resistance
to other penicillinase-resistantpenicillins, (iii)
incubate inoculated Mueller-Hinton agar plates
for a full 24 h (not less,not more) at 35°C(avoid
37”C), and (iv) examine and interpret suscepti-
bility zone diameters in a manner that will
maximize detection of significant inner zone
colonies or a film of growth within the zone.
Interpret zone diameter breakpoints by using
NCCLS criteria (124); zone diameters of 2 13
mm are interpreted as susceptible,resistanceis
equated with zone diameters of ~10 mm, and
zone diameters of 11 to 12 mm reflect “inter-
mediate” susceptibility. Staphylococcal iso-
lates demonstratingintermediate susceptibility
by the disk test shouldbe further tested using a
broth or agar dilution method.
Some studiessuggestthat agar disk diffusion
testing with a 4-pg oxacillin disk might better
correlate with oxacillin susceptibility and resis-
tance MIC breakpoints (103). However, 4-pg
oxacillin disks are not commercially available,
andthe needto start testingwith a 4-Fg oxacillin
disk has not been resolved by NCCLS.
Broth microdilution test. For the broth micro-
dilution test use the procedure outlined in
NCCLS M7-A2 (125), but be certain to care-
fully perform the following essential steps to
optimize detection of oxacillin-resistant staph-
ylococci: (i) use the direct inoculum prepara-
tion method, (ii) oxacillin is preferred for test-
ing sinceit is the most reliable for testing and is
the most likely to detect cross resistance to
other penicillinase-resistantpenicillins, (iii) use
cation-supplemented Mueller-Hinton broth
containing 2% NaCl, and (iv) incubate the
inoculated microdilution trays for a full 24 h at
35OC. Interpret results by using the current
NCCLS MIC breakpoints (52 kg/ml, suscep-
tible; ~4 pg/ml, resistant) (125). Some (103)
suggestusingoxacillin susceptibility breakpoints
of ~2 pg/ml as susceptible,2 pg/ml as border-
line, 4 kg/ml as intermediate, and 28 pg/ml as
resistant; to date there are few data to suggest
that thesebreakpointsare more clinically useful
than current NCCLS recommendations.
A major clue that a staphylococcal isolate is
oxacillin resistant is the concurrent manifesta-
tion of multiple in vitro resistancesto antibiot-
ics, including other beta-lactams,erythromycin,
clindamycin, aminoglycosides,tetracycline, and
chloramphenicol in various combinations. Al-
though ORSA strainswill always be resistantto
beta-lactamantimicrobial agents, in vitro resis-
tance to other antimicrobial agentsvaries. Until
1985,essentiallyall oxacillin-resistant staphylo-
coccal strains were susceptibleto vancomycin,
teichoplanin, and ciprofloxacin. However, resis-
tance of oxacillin-resistant staphylococci to van-
CUMITECH 6A ANTIMICROBIAL
comycin (in coagulase-negative staphylococci)
(52, 158) and ciprofloxacin (71, 126, 156) has
been reported, thus necessitating routine sus-
ceptibility testing of ORSA against these antimi-
crobial agents.
Screening tests. It may be advisable for labo-
ratories to screen S. aureus isolates;either meth-
icillin ( 10pg /ml), oxacillin (6 pg/ml), or nafcillin
(6 Fg/ml) incorporated into Mueller-Hinton agar
supplementedwith 4% NaCl can be used for
testing. The mediumis “spot” inoculated using
a swabsaturatedwith a freshly standardized(0.5
McFarland standard equivalent [approximately
10’ CFU/ml]) cell suspension,incubatedfor 24 h
at 35”C, and observed for growth (resistant) or
no growth (susceptible)(157). The test is reli-
able, economical,and simpleto perform. These
screening media are available in plated form
through several commercialsuppliers.
Intrinsically oxacillin-resistant (heteroresis-
tant) staphylococci (both ORSA and coagulase-
negative staphylococci) should be reported as
resistantto all beta-lactamantimicrobial agents,
including all cephalosporins and imipenem,
regardlessof in vitro test results, becausemost
patients with documentedinfection with intrin-
sically resistant strains respondpoorly to these
agents.
Although the currently recommendedsuscep-
tibility test modifications have significantly im-
proved the detection of oxacillin-resistant staph-
ylococci, further modifications for certain test
systemsmay becomenecessary(12, 13, 64, 65,
103).
Acquired Oxacillin-ResistantS. aureus
(AORSA)
In 1986, McDougal and Thomsberry (104)
offered convincing evidence that borderline (2
pg/ml) or intermediate (4 kg/ml) susceptibility
to oxacillin in strains of S. aureus is causedby
beta-lactamaseactivity. These strains produce
largequantities (are “hyperproducers”) of beta-
lactamasewhich is capableof slowly inactivat-
ing penicillinase-resistantpenicillins. Resistance
via hyperproduction of beta-lactamaseis an ac-
quired (plasmid- or transposon-mediated)trait
and appearsto be unrelated to the “classical”
chromosomally mediated intrinsic resistance
(i.e., PBP2amediated).The beta-lactamasepro-
duced by these strains is inhibited by beta-
lactamaseinhibitors such asclavulanic acid and
sulbactam.One convenient methodfor differen-
tiating plasmid-mediatedhyper-beta-lactamase
oxacillin resistancefrom intrinsic resistancein
S. aureus is to test these isolatesagainstamox- StaphylococcalToleranceto Beta-Lactam
icillin-clavulanic acid and oxacillin. Generally,
plasmid-mediatedhyper-beta-lactamase-produc- Another form of resistance described for
ing strains are susceptibleto amoxicillin-clavu- staphylococci is the phenomenonof tolerance in
AGENT SUSCEPTIBILITY TESTING 3
lanic acid, while intrinsically resistantstrainsare
resistantto both antimicrobial agents.
The clinical, epidemiologic, and therapeutic
significance of AORSA is largely unknown.
There is some debate over whether to report
suchstrainsassusceptibleor resistant(104, 163,
200). Two retrospective casestudiesreported a
combinedtotal of 22 patients with AORSA in-
fections who demonstrateda favorable clinical
outcome when patients were treated with a
penicillinase-resistantpenicillin or beta-lacta-
maseinhibitor-penicillin combination. This sug-
geststhat infection with such strainsmay possi-
bly be reported as susceptible(65, 101). How-
ever, these limited data do not permit a
definitive recommendationof this approach at
present. In clinical situations (e.g., bacteremia,
endocarditis, meningitis, or osteomyelitis) in
which bactericidal levels of an antimicrobial
agent are needed,penicillinase-resistantpenicil-
lins and beta-lactamaseinhibitor-penicillin com-
binations will likely be less effective against
these strains than are other antistaphylococcal
antimicrobial agents(e.g., vancomycin, teicho-
planin, or daptomycin) (96). Resolution of the
clinical, epidemiologic, and therapeutic signifi-
canceof AORSA through larger, controlled pro-
spective studies,however, is greatly needed.
Other Oxacillin-ResistantStrains
Two additional subgroupsof S. aureus strains
with intermediate-rangeoxacillin MICs (2 to 4
pg/ml) were reported in 1988by Sierra-Madero
and co-workers (163). One subgroupof isolates
produced beta-lactamase, but clavulanic acid
did not lower the oxacillin MIC; a second sub-
group of isolatesdid not produce beta-lactamase
and were susceptibleto penicillin. These inves-
tigators observed that some of their strains
would be called susceptibleif incubated at 35°C
but resistantif incubatedat 3O*C,suggestingthat
they are heteroresistant(163). These subgroups
of S. aureus isolates, therefore, appear to be
distinct from the classicacquired borderline ox-
acillin-resistantstrainspreviously reportedby Mc-
Dougal and Thomsberry (104). The mechanism
responsiblefor this phenomenonis currently un-
known; however, there is somesuspicionthat the
“regular” PBPs (not PBP2a)with low &nity to
oxacillin may be important (163). As with
AORSA, the clinical, epidemiologic,and thera-
peutic significanceof these strainsis largely un-
known but deservesfurther evaluation. Unless
they can be proven susceptibleby clinical re-
sponse,the strainsshouldbe reportedasresistant.
Antimicrobial Agents
4 NEUMANN ET AL. CUMITECH 6A

which the growth of somestrains of staphylo- Resistanceto High Levelsof Aminoglycosides

cocci is inhibited at low concentrations of beta- Until the mid-198Os,enterococcal isolatesal-
lactam antimicrobial agents, but much higher most universally demonstratedsome degree of
concentrations of drug are required for tidal susceptibility to penicillin, ampicillin, and van-
(killing) activity. The mechanismof tolerance is comycin. Although the enterococci are often
thought to involve antagonismof autolytic en- refractory to the bactericidal activity of such
zyme activity by cell-wall-associatedsubstances agentswhen tested or usedalone, the combina-
(e.g., lipoteichoic acid) or substancesin the
bacterial milieu. tion of a penicillin or vancomycin with an ami-
noglycoside has been shown to act synergisti-
Tolerance is determined in vitro by perform-
ing an MIC and MBC test. Tolerance is defined cally, achieving bactericidal activity in vitro
as an MBC-to-MIC ratio of ~32: 1. The repro- (105, 112-114, 155). The bactericidal action of
ducibility is low due to a large extent to meth- this synergistic combination is believed to be
odologic errors such as the use of a stationary- causedby the disruptionof the bacterial cell wall
growth-phaseinoculum, organismadherenceto by penicillin or vancomycin, which facilitates
the walls of the tube or well above the broth the penetration and uptake of the aminoglyco-
meniscus,andwhen a 24-h incubation is allowed side (81). Therefore, it has become standard
before MBC subculture. practice to treat seriousenterococcal infections
Pelletier and Baker (135)reported a 48-h mod- (e.g., bacteremia, endocarditis, meningitis, and
ification of the MBC method of Taylor et al. osteomyelitis) with combination antimicrobial
(177)that avoided thesepitfalls. Theseworkers therapy (69, 82, 108, 110, 113, 155). The recog-
determinedthat antimicrobial toleranceis in fact nition of the phenomenonof high-level amino-
quite rare even among preselected, allegedly glycosideresistance,however, now requiresthe
oxacillin-tolerant staphylococcal isolates (135). performance of additional tests to support this
The experience of these authors supports the practice.
view that tolerance among staphylococcal iso- Synergy between penicillin or vancomycin
lates is more a laboratory artifact than a true and an aminoglycosidecan be predicted reliably
biological phenomenon. As a result, routine by in vitro susceptibility testing. Clinical isolates
testing for tolerance in clinical microbiology of enterococci may be tested using a singlehigh
laboratories should not be performed (149). concentration of aminoglycosidein a modifica-
Standardization of methods for MBC testing tion of the agardilution, broth microdilution, or
(122) is in its infancy. Thus, such testing, if agar disk diffusion test. Generally, for the agar
clinically justifiable, should be performed by and broth dilution methods, clinical isolatesare
experiencedreference laboratories only. tested against2,000 pg of streptomycin per ml
and either 500or 2,000 p,gof gentamicinper ml.
If the agar dilution screeningmethod is used, an
ENTEROCOCCI inoculum of lo6 CFU provides reliable test re-
During the last 10to 15years we have recog- sults (150). Although the type of agar medium
nized an apparent increasein the frequency and used does not affect the test outcome, use of
mortality of serious infections associatedwith Mueller-Hinton agar supplemented with 5%
the enterococci (58, 83, 114). In the, 1980sen- sheepblood provides reliable and easily inter-
terococci emerged as significant nosocomial pretable test results (150). The broth microdilu-
pathogens, accounting for over 10% of noso- tion screening method is included in several
comial infections, and were associatedwith an commercially prepared antimicrobial suscepti-
overall mortality rate of 19.6to 71.4%in casesof bility test systems. However, as yet, certain
bacteremia(178). It hasbeen suggestedthat the commercial test systems do not perform con-
increase in nosocomialenterococcal infections sistently (164); therefore, commercial test sys-
has been correlated with an increased use of tems must be carefully evaluated for reliability
broad-spectrum antimicrobial agents, particu- of test results before formal implementation
larly the cephalosporins(108). and test reporting are initiated. Growth in the
Approximately 90% of clinical isolatesof en- presence of a high level of aminoglycoside
terococci are Enterococcus faecalis, with E. indicates that synergy between the amino-
faecium accounting for most of the balance of glycoside tested and penicillin or vancomycin
clinical isolates and E. durans and E. avium is unlikely. Conversely, absenceof growth in
being rarely encountered. The enterococcal in- the presenceof a high level of aminoglycoside
fections recognized most frequently involve the predicts probable bactericidal synergism with
urinary tract or heart valves; however, soft combination therapy.
tissue abscesses,osteomyelitis, meningitis, and Sahmand Torres (151) evaluated a simpleand
peritonitis due to enterococci also occur, albeit reliable modified agar disk diffusion test for
infrequently. screeningisolates of enterococci for high-level
CUMITECH 6A ANTIMICROBIAL
aminoglycoside resistance (142). Because of its
ease of performance, this method is outlined in
brief below.
Modified agar disk diffusion test (151). A4e-
dium. Use Mueller-Hinton agar supplemented
with 5% sheep blood.
Inoculum. Use pure fresh growth from an
overnight blood agar plate to prepare a suspen-
sion in 5 ml of Mueller-Hinton broth to a turbid-
ity equivalent to a 0.5 McFarland standard.
Test procedure. (i) Susceptibility disks may be
prepared in-house, using sterile blank &mm
disks (Difco Laboratories, Detroit, Mich.).
Disk-drug combinations may be prepared by
applying 25 ~1 of aminoglycoside stock solution
containing 40x the final desired drug potency.
The final concentration of aminoglycoside per
disk is 120 pg of gentamicin or 300 pg of
streptomycin. A disk containing 120 pg of kan-
amycin should be prepared since amikacin disks
do not predict amikacin synergy. Disks may be
stored at 4°C for up to 4 months or longer at
-2OOC.
(ii) Perform the agar disk diffusion test accord-
ing to the procedure outlined in NCCLS M2-A4
(124). Incubate susceptibility test plates at 35°C
for 20 h.
(iii) Perform quality control tests each time
that the test is performed, using isolates of
enterococci with known aminoglycoside suscep-
tibility (e.g., E. faecalis ATCC 29212) and resis-
tance (strain acquired from a reliable reference
laboratory or ATCC).
Test interpretation. Zone diameters of 210
mm predict synergy between the aminoglyco-
side tested and penicillin or vancomycin. Con-
versely, zone diameters of 59 mm predict lack
of synergy.
The recognition of high-level aminoglycoside-
resistant enterococci has clearly increased dur-
ing this past decade. Medical centers in the
United States and elsewhere are reporting resis-
tance rates ranging from 4.5 to 55% (83, 119,
203, 204). The prevalence of high-level amino-
glycoside resistance shows considerable geo-
graphic variation. Approximately 40 to 60% of
clinical isolates of enterococci from the United
States presently exhibit high-level resistance to
streptomycin, and approximately 25 to 40% are
resistant to synergism with penicillin (or vanco-
mycin) in combination with kanamycin or ami-
kacin (109). Ten to 50% of enterococcal isolates
are resistant to high-level gentamicin; however,
25 to 33% of these strains are susceptible to
streptomycin (109). Therefore, as a minimum
routine, high-level aminoglycoside screening
should include gentamicin, which is the amino-
glycoside of choice. Preferably, both gentamicin
and streptomycin should be tested. High-level
aminoglycoside resistance in enterococci is re-
AGENT SUSCEPTIBILITY TESTING 5
portedly conferred by resistance genes or trans-
ferable plasmids which code for inactivating
enzymes, similar to the plasmid-mediated resis-
tance found in gram-negative bacilli (88, 111,
155). Enterococci isolated from blood, cerebro-
spinal fluid (CSF), and other normally sterile
body fluids (except urine), bone, or single-patho-
gen soft-tissue abscesses should routinely be
screened for high-level aminoglycoside suscep-
tibility. If all aminoglycosides are inactive by
high-level aminoglycoside screening, bacteri-
cidal therapy probably will not be achieved
using these agents and should not be expected.
Therapeutic options beyond this point may in-
clude high-dose penicillin, ampicillin, or vanco-
mycin, depending on the site of infection.
Enterococci Resistant to Beta-Lactams
In addition to the therapeutic problems posed
by the emergence of high-level aminoglycoside-
resistant enterococci, treatment of enterococcal
infections has been complicated further by the
discovery of strains exhibiting higher-than-nor-
mal resistance (MIC, >lOO Fg/ml) to penicillin
and in some cases resistance to vancomycin.
These observations suggest that the choice of
antimicrobial agents for treating enterococcal
infections may become extremely limited.
Penicillin-resistant enterococci were first dis-
covered in the early 1980s (116). Resistance to
E. faecalis occurs as a result of a plasmid-
mediated beta-lactamase. In the case of E. fue-
cium, resistance (penicillin MIC, ?lOO pg/ml)
appears to be due to altered PBPs with dimin-
ished binding affinity for penicillins (109, 115,
116, 133, 155, 161). Resistance due to alteration
of PBP constitutes the more common of the two
enterococcal penicillin resistance mechanisms,
but currently the prevalence of such strains is
low (152). Beta-lactamase-producing strains ap-
pear to make an enzyme similar to the plasmid-
mediated enzyme found in S. aureus; since this
plasmid is self-transferable, the potential exists
for spread (109, 116, 133, 155, 161). The beta-
lactamase is a constitutively produced cell-
bound enzyme and is readily detected by the
nitrocefin test (109, 133). Beta-lactamase can be
inhibited by clavulanic acid and sulbactam, im-
plying a favorable therapeutic response with
combinations of beta-lactamase inhibitors and
penicillins (161). All beta-lactamase-producing
strains reported to date also have demonstrated
high-level resistance to gentamicin, and no syn-
ergy seems to occur when a beta-lactam and an
aminoglycoside are used concurrently (109,
155). Even though the prevalence of beta-
lactamase-producing enterococci appears to be
low, it is important to note that these strains
exhibit a striking inoculum effect; at a low
inoculum, the MICs for these strains are simi-
6 NEUMANNETAL. CUMITECH 6A

lar to those for beta-lactamase-negative ampi- penicillin, vancomycin, and cephalothin (154).
cillin-susceptible strains and therefore may go Therefore, if a nonenterococcal group D strep-
undetected by routine broth microdilution or agar tococcus is isolated from blood, particularly in
disk di#!usion testing (109). Thus, routine screen- cases of endocarditis, an MIC should be deter-
ing using the nitrocefin disk test is the optimal mined for penicillin, using the broth microdilu-
method for detection of beta-lactamase-producing tion method (125). An isolate requiring a peni-
enterococci. As a minimum, enterococcal isolates cillin MIC of 20.5 Fg/ml should not be consid-
which exhibit high-level gentamicin resistance and ered for therapy with either penicillin or
isolates from treatment failures should be ampicillin alone. Penicillin or ampicillin plus an
screened for beta-lactamase (109, 115, 133). aminoglycoside, or vancomycin with or without
an aminoglycoside, is a reasonable therapeutic
Enterococci Resistant to Vancomycin and alternative.
Teichoplanin
Enterococci exhibiting resistance to the gly- NUTRITIONALLY DEFICIENT
copeptide antimicrobial agents (e.g., vancomy- STREPTOCOCCI
tin and teichoplanin) were first reported in 1988 The term “nutritionally deficient” strepto-
(92, 189). In some strains, resistance appears to cocci (NDS) refers to viridans streptococci that
be plasmid mediated, but the precise genetic or are unable to grow on media lacking thiol or the
biochemical mechanism of resistance to vanco- active forms of vitamin B,, pyridoxal, or pyri-
mycin and teichoplanin is largely unknown (92, doxamine (19). The major clinical significance of
189). Some plasmids encoding vancomycin re- these organisms is their role in causing en-
sistance have been shown to be transferable to docarditis. Viridans streptococci have been
E. fuecalis, various streptococcal species, and cited as the cause of approximately 50% of the
Listeria species, sparking concern about the cases of endocarditis, and the NDS comprise
potential for dissemination of resistance among about 5 to 6% of the cases of viridans strepto-
these organisms (93). By the standard agar disk coccal endocarditis. A corollary to this is that
diffusion method, current NCCLS interpretive approximately 17 to 33% of isolates of NDS
criteria for vancomycin may not accurately iden- require MICs to penicillin G of >O.l Fg/ml,
tify all vancomycin-resistant enterococci (147, suggesting possible therapeutic failure if penicil-
174). Swenson and colleagues (174) have deter- lin G is used alone to treat systemic infection
mined that enterococcal susceptibility to vanco- (3 1, 70). As a result of these findings, combina-
mycin is more accurately determined using in- tion therapy with penicillin or ampicillin and an
terpretive zone sizes of 514 mm as resistant and aminoglycoside has been prescribed frequently.
215 mm as susceptible. In addition, there is Stein and Nelson (166) reported a clinical failure
some concern that commercial susceptibility rate of approximately 40% with this combina-
test systems may not accurately predict vanco- tion. Alternative combination therapy with van-
mycin resistance in enterococci (147). comycin and rifampin offers more promise for
To date, all of the enterococci resistant to therapy of endocarditis due to NDS (165).
beta-lactams, high-level aminoglycosides, and Because of the- fastidious nature of NDS,
vancomycin have demonstrated susceptibility to attempts to perform antimicrobial susceptibility
the lipopeptide antimicrobial agent daptomycin tests with inappropriately supplemented media
(171, 194), and many of the strains exhibit in may lead to false susceptibility patterns (19).
vitro susceptibility to ciprofloxacin (148, 171). Generally, these organisms can be grown quite
However, the clinical importance of this suscep- easily on agar medium containing 0.001% pyri-
tibility is unclear, as daptomycin is still in the doxal or thiol, or satellite growth can be pro-
early stages of clinical investigation and cipro- duced around an S. aureus streak.
floxacin therapy of enterococci with high-level A standard method for susceptibility testing of
gentamicin resistance has been unsuccessful NDS does not, as yet, exist. Two simple and
(155). reproducible methods will be described below.
Medium. Use cation-supplemented Mueller-
NONENTEROCOCCALGROUPD Hinton broth or agar plus 5% lysed horse blood
STREPTOCOCCI and 0.001% pyridoxal, or use double-strength
In contrast to the enterococcal species of Schaedler broth with 10% Fildes enrichment,
group D streptococci, in vitro data indicate that 10% horse serum, and 0.001% pyridoxal.
Streptococcus bovis and other nonenterococcal Inoculum. Prepare the inoculum by inoculat-
group D streptococci are generally highly sus- ing tryptic (Trypticase) soy broth containing
ceptible to penicillin, ampicillin, cephalothin, 10% (vol/vol) of a 1-mgldl pyridoxal solution.
clindamycin, and erythromycin (18 1). However, Incubate overnight; then adjust the suspension
some strains of S. bovis associated with en- to equal the optical density of a 0.5 McFarland
docarditis have been reported to be tolerant to standard.
CUMITECH 6A ANTIMICROBIAL
Test procedure. Perform susceptibility testing
as describedby NCCLS (125). Incubate at 35°C
for 24 h. An enriched CO, atmospherewill be
required for strainsthat will not grow well in its
absence.
Test interpretation. Interpret MIC endpoints
according to NCCLS criteria (125).
How well data from thesein vitro susceptibil-
ity methodscorrelate with therapeutic responses
of patients infected with NDS has not been
firmly established. Thus, combination therapy
with penicillin and an aminoglycosideor therapy
with vancomycin alone or in combination with
an aminoglycosideor rifampin is recommended
in casesof systemic disease(e.g., endocarditis)
becauseof the risk of relapsefrom these strains
of NDS.
PENICILLIN- AND CHLORAMPHENICOL-
RESISTANT STREPTOCOCCUS
PNEUMONIAE
Penicillin-resistantS. pneumoniae strains are
defin(edas those for which the MIC of penicillin
is 21.0 pg/ml. In addition, strains that require
MICs of 0.12 to 1.0 pg/ml are defined as rela-
tively resistant. Infections due to these bacteria
have been detected over the past 20 years in
many countries (130, 195).In the United States,
the reported incidence of S. pneumoniue with
decreasedsusceptibility to penicillin (resistant
or relatively resistant) is rather low (3.7%) (24).
However, these strains of S, pneumoniae are
endemic in certain areas of the United States,
with reported rates of relative resistanceranging
from 6.9 to 15.5%(72, 145).
The resistanceof S. pneumoniae to penicillin,
as well as to other beta-lactam antibiotics, ap-
pearsto be due to alterations in PBPs (62, 63).
Not infrequently, penicillin-resistantS. pneumo-
niae strainsare alsoresistantto other antimicro-
bial agents, including chloramphenicol,tetracy-
cline, and trimethoprim-sulfamethoxazole (73,
130).Also, these strains show a decreasedsus-
ceptibility to other beta-lactam antimicrobial
agents, including extended-spectrum cephalo-
sporins,althoughthe decreasemay be muchless
than with penicillin, dependingon the specific
cephalosporinbeing tested.
S. pneumoniae isolatedfrom CSF, blood, and
other closed body sites should be tested rou-
tinely for susceptibility to penicillin, as should
strainsfrom treatment failures.
Agar disk diffusion test. Screeningof S. pneu-
moniae for resistanceto penicillin by agar diffu-
sion shouldbe performed using a I-lg oxacillin
disk (124, 173).Use of a methicillin disk results
in an unacceptableincidence of false suscepti-
bility readings and should not be used (173).
Since nafcillin cannot be reliably tested on
blood-containing media, this drug also should
AGENT SUSCEPTIBILITY TESTING 7
not be used. When 10-U penicillin disks are
used, penicillin-resistantS. pneumoniae strains
frequently produce zone diameters of >30 mm
and occasionally even >35 mm; therefore, a
penicillin disk should not be used to screen for
decreasedsusceptibility to penicillin. Indiscrim-
inate use of the penicillin disk results in report-
ing false susceptibility to this drug and has been
cited as a continuing problem in somelaborato-
ries participating in Collegeof American Pathol-
ogists Proficiency Surveys (74, 75). Thus, only
oxacillin disks shouldbe usedto predict suscep-
tibility to penicillin therapy for S. pneumoniae.
Medium. Use Mueller-Hinton agar supple-
mented with 5% sheep,rabbit, or horse blood.
Znoculum. Remove several colonies from a
blood agar plate that has been incubated over-
night, and prepare a suspensionin Mueller-
Hinton broth. Adjust the turbidity to equal that
of a 0.5 McFarland standard.
Test procedure. Inoculate the surface of the
agar plate with the adjusted inoculum, using a
sterile cotton swab,and apply in three directions
asdescribedfor the standardagar disk diffusion
test (124).Allow the surfaceto dry. Place a 1-p,g
oxacillin disk on the agar surface, and press
firmly with sterile forceps. (A 30,pg chloram-
phenicol disk may also be placed on the agar
surface to screen for chloramphenicol resis-
tance.) Incubate the platesat 35°Cfor 18to 24 h.
Incubation in CO* is unnecessary, except for
rare strainsthat grow poorly or not at all without
it.
Test interpretation. Penicillin-susceptible
(MIC,
SO.06pg/ml) strainswill produce oxacillin zones
of ~20 mm. Penicillin-resistant(MIC, >1 pg/ml)
or relatively resistant(MIC, 0.12 to 1.Okg/ml) S.
pneumoniue will produce oxacillin zones of 519
mm (124, 173).The ‘diskdiffusion test shouldnot
be usedto separatestrainsthat are resistantfrom
those that are relatively resistant.To accomplish
this, follow-up testingof all strainswith zone sizes
of 519 mm shouldbe done usinga broth or agar
dilution test (131).Usethe NCCLS zone diameter
breakpointsfor interpretingchloramphenicolsus-
ceptibility (124).
Determinationof MICs. For the determination
of MICs, usethe generalmethodsrecommended
by NCCLS (125), except that Mueller-Hinton
agar or broth is supplementedwith blood (33).
For the agar dilution method, supplementwith
5% defibrinated blood, but for the broth micro-
dilution method 5% lysed horse blood is pre-
ferred. Jorgensenet al. (78) have demonstrated
consistently accurate susceptibility test results
for S. pneumoniae, usinga clear highly supple-
mentedbroth, haemophilustest medium(HTM),
now recommendedfor testing Haemophihs in-
jluenzae. This protocol is currently being evalu-
ated by NCCLS. For MIC tests, incubation
8 NEUMANN ET AL.
shouldbe at 35°Cfor 18to 24 h. Incubation in a
CO,-enriched atmosphere is generally not re-
quired but may be used if necessaryto support
growth. Antimicrobial agents that should be
consideredfor testing include penicillin, chlor-
amphenicol, tetracycline, erythromycin, tri-
methoprim-sulfamethoxazole, cephalothin, and
vancomycin. In laboratories where penicillin-
resistant or relatively resistant strains are iso-
lated with frequency, testing with cefotaxime or
ceftriaxone shouldbe considered,especiallyfor
S. pneumoniae isolatesfrom casesof meningitis.
Penicillin MIC results should be interpreted as
follows: (0.06 Fg/ml, susceptible;0.12 to 1.0
pg/ml, relatively resistant; and >l.O pg/ml,
resistant. Susceptibility breakpoints for other
antimicrobial agents should be interpreted ac-
cording to NCCLS criteria (125).
When S. pneumoniae is being tested, com-
mercially prepared broth microdilution systems
have shown an unacceptably high rate of false-
susceptibleMIC test resultsfor penicillin and, to
some extent, chloramphenicol (159, 175). This
underscoresthe recommendationthat the 1-pg
oxacillin disk test should serve as the standard
methodfor screeningS. pneumoniae isolatesfor
resistanceto penicillin.
Disagreementexists concerning the useful-
nessof penicillin in the treatment of relatively
resistantpneumococci(130,195).Pneumococcal
meningitis due to relatively resistant strains
clearly shouldnot be treated with penicillin (131,
195). Infections at sites other than the central
nervous systemmay respondto higher dosesof
penicillin therapy (195). Pneumococcalpneumo-
nia due to strainsthat require MICs of ~0.1 and
52 pg/ml may respondfavorably to high doses
of penicillin therapy (130). Treatment of infec-
tions due to penicillin-resistantor relatively re-
sistant pneumococci should be carefully based
on results of MIC testing of alternative drugs,
suchas chloramphenicol,vancomycin, cefotax-
ime, or ceftriaxone (186, 190).
AMPICILLIN- AND CHLORAMPHENICOL-
RESISTANT H.ZNFLUENZAE
H. influenzae causesa variety of human dis-
eases,including epiglottitis, sinusitis,otitis me-
dia, meningitis, septicemia,cellulitis, and pneu-
monia. Until 1974,H. injluenzae wasconsidered
to be susceptible to ampicillin, the drug of
choice. In that year, however, a beta-lactamase-
producing strain of H. influenzae was isolated
from the CSF of a child who later died of
meningitis(59). Beta-lactamase-producing H. in-
fluenzae serotype b strainsare now recovered at
rates varying from 0 to 50% (meanrate of 21%)
in U.S. hospitals(41). No geographicresistance
trends have been observed, but higher beta-
lactamaseprevalence rates are more commonly
CUMITECH 6A
associatedwith (i) children’s hospitals or labo-
ratories serving active pediatric medicalgroups,
(ii) patients between 3 months and 5 years, and
(iii) isolates from invasive infections such as
meningitisand bacteremia.
The primary mechanismof resistance of H.
influenzae to ampicillin is plasmidmediated, by
the production of a transposon-encoded,consti-
tutively produced, extracellular, TEM-l-type
beta-lactamaseor a lesscommonone designated
ROB-l. In a multicenter national collaborative
study conducted in 1986,20% of 2,811 clinical
isolatesof H. injluenzae were found to be resist-
ant to ampicillin by virtue of TEM-1 beta-lacta-
mase production (42). Encapsulated type b
strains of H. influenzae were found twice as
commonly as non-type b strains to produce
TEM- 1 beta-lactamase. Other Haemophilus
spp. may also contain these plasmidsand pro-
duce beta-lactamase,but they are much less
frequently involved in systemic infections. The
problem of ampicillin resistancewith H. in&-
enzae is further complicated by the subsequent
description of ampicillin-resistant isolates that
lacked the typical TEM-1 beta-lactamase(8,99).
Resistanceto ampicillin among these isolates
appears to be the result of a chromosomally
mediated alteration in PBPs (8, 132, 144). The
true incidence of non-beta-lactamase-producing
ampicillin-resistantstrains is believed to be rel-
atively low. A nationwide surveillance study
conducted in 1986 revealed that 2 of 2,250
(CO.1%) isolatesof beta-lactamase-negativeH,
intuenzae demonstratedclear resistanceto am-
picillin (42). A more recent report by Gutmann
and colleagues(61)revealed chromosomallyme-
diated ampicillin resistance rates of 2 to 4%.
Thesestrainsappearto be more commonamong
nontypable respiratory tract isolates. Non-
typable H. influenzae has been increasingly im-
plicated in community-acquired and nosocomial
pneumoniain those adultswith chronic obstruc-
tive pulmonary disease.In addition, nontypable
strains of H. influenzae play a major role in
certain pediatric diseases,such as otitis media
and pneumonia.Of clinical concern are data that
suggestthat these ampicillin-resistant strains
exhibit decreasedsusceptibility (four- to eight-
fold-greater MICs) to extended- andbroad-spec-
trum cephalosporins(137).
Somestrainsof H. injluenzae are resistantto
chloramphenicol(15,84, 140, 188).Although the
exact prevalence of chloramphenicolresistance
is largely unknown, two publishednational col-
laborative studies conducted in 1984and 1988
reveal chloramphenicolresistancerates approxi-
mating 0.5% (41, 42). In a few strainsthe mech-
anismof resistanceto chloramphenicolmay be
chromosomally mediated, but in most strains
chloramphenicolresistance is due to plasmid-
CUMITECH 6A ANTIMICROBIAL
mediated reduction of the enzyme chloramphen-
icol acetyltransferase (15, 140). Nearly all chlor-
amphenicol-resistant strains are also resistant to
tetracycline, and most produce TEM-l-type beta-
lactamase. “Since most chloramphenicol resis-
tance is plasmid-mediated and may be linked to
the R-plasmid encoding for beta-lactamase pro-
duction, a plasmid that is readily exchanged
among bacteria by conjugation, genetic transfer
of chloramphenicol resistance is almost a certain-
ty” (37).
Because invasive hl. inJEuenzaeinfections are
quite serious, the early and accurate recognition
of ampicillin, chloramphenicol, and other drug
resistance is crucial to guide the selection of
effective antimicrobial chemotherapy. II. infEu-
enzae isolated from CSF, blood, and other body
fluids or associated with otitis media, ocular
infections, and epiglottitis should be tested for
beta-lactamase and, if resistant, tested for sus-
ceptibility to other antimicrobial agents.
Beta-lactamase of Ii. influenzae may be
readily detected using the rapid acidometric
method (49), the rapid iodometric technique
(20), or the rapid chromogenic cephalosporin
method (129), using either nitrocefin or pyridine-
2-azo-dimethylaniline cephalosporin as a sub-
strate. Commercially available filter paper disk
tests based on either the acidometric or the
chromogenic cephalosporin method may be
used. In performing beta-lactamase tests, it is
extremely important to test more than 1 colony
(about IO is preferred) from a culture plate
because both beta-lactamase-positive and -neg-
ative strains have been simultaneously recov-
ered from the same patient specimen (87). Beta-
lactamase-producing strains of hl. infEuenzae
should be considered resistant to ampicillin. It is
equally important to note that beta-lactamase-
negative strains are not necessarily ampicillin
susceptible since at least a small percentage of
strains of H. influenzae are resistant to ampicil-
lin via mechanisms other than the production of
TEM-1 beta-lactamase. Because ampicillin re-
sistance may occur in the absence of beta-
lactamase production, susceptibility testing of
all isolates from systemic infections, including
epiglottitis, cellulitis, CSF, and blood isolates as
well as isolates from pulmonary infections in
which treatment has failed, should be performed
using the agar disk diffusion method or broth or
agar dilution test. All beta-lactamase-negative,
ampicillin-resistant H. influenzcre isolates should
be referred to a public health laboratory for
confirmatory testing.
If screening for resistance to chloramphenicol
is clinically or epidemiologically indicated, the
agar disk diffusion test or a broth or agar dilution
test is preferred. A rapid (ca, 70-min) tube test
(5) to quickly screen for chloramphenicol acetyl-
AGENT SUSCEPTIBILITY TESTING 9
transferase-producing II. iltfluenzue is a sensi-
tive assay that is simple to perform and useful if
positive; however, isolates yielding negative re-
sults should be tested using the disk diffusion or
a dilution test. Commercially available chloram-
phenicol acetyltransferase disk tests may also be
used for screening H. infl~enzue isolates for
chloramphenicol resistance (37, 39).
There is little need or justification to test H.
influenzae for susceptibility to antimicrobial
agents other than ampicillin and chloramphen-
icol. Routine susceptibility testing is of mini-
mal value with antimicrobial agents that are
uniformly active (e.g., newer cephalosporins,
imipenem, aztreonam, fluoroquinolones, and
beta-lactam inhibitor combinations), that are
relatively inactive (e.g., erythromycin and sul-
fonamides), that lack a clinical indication for
treatment (e.g., tetracycline and penicillins
other than penicillin G and ampicillin), or for
which interpretive criteria and standards do not
exist (42). Examples of drugs for which suscep-
tibility tests may be of some potential usefulness
in illnesses that are not life threatening include
trimethoprim-sulfamethoxazole, rifampin, and
selected cephalosporins (e.g., cefaclor, cefa-
mandole, or cefuroxime). When susceptibility is
being tested to other antimicrobial agents, dis-
cretion must be used to restrict testing to organ-
ism-drug combinations for patients with an in-
fectious process for which that particular anti-
microbial agent is of potential therapeutic
benefit (example: test isolates from CSF to cef-
triaxone, not cefaclor, cephalothin, or cefaman-
dole) (42).
Because susceptibility testing of hl. injhenzue
requires modification of the standard agar disk
diffusion and dilution procedures, these methods
will be briefly detailed. The most significant
modification recommended for susceptibility
testing of H. injluenzue is the employment of
HTM in agar or broth formulation (79, 81, 124,
125). HTM theoretically offers advantages over
previously used media formulations due to its
optical clarity that results in easier test interpre-
tation, better reproducibility of test results,
greater stability, and utility for testing sulfon-
amide-containing antimicrobial agents (40).
While HTM is commercially available, quality
control problems have occurred. As a result,
great care must be exercised in its use. Revised
interpretive breakpoints are reported in the
NCCLS M2-A4 (124) and M7-A2 (125) docu-
ments (Tables 1 and 2). Quality control testing
for HTM, using appropriate test ranges for H.
illfluenzae ATCC 49247, is summarized in Ta-
bles 3 and 4.
Agar disk diffusion test. The modified disk
diffusion test continues to most accurately as-
sess H. influenzue resistance to ampicillin and
10 NEUMANN ET AL. CUMITECH 6A

TABLE 1. Zone diameter interpretive standards and equivalent MIC breakpoints

for Haemophilus species (124)”
Equivalent MIC
Zone diam (nearest whole mm)
Antimicrobial agent Disk content (p,g) breakpoint (pg/ml)
Resistant Intermediateb Susceptible Resistant Susceptible
Amoxicillin-clavulanic acid 20110 519 220 r8/4
Ampicillin 10 521 22-24 225 24
Ampicillin-sulbactam lo/lo 519 220 2412
Aztreonamb 30 226
Cefaclor 30 ~18 19-23 224 232
Cefamandole 30 520 2 l-23 224 116
Cefiximebpc 5 230
Cefonicid 30 520 21-23 224 216
Cefotaxime’ 30 ~26
Ceftazidimeb 30 226
Ceftriaxoneb 30 226
Ceftizoximeb 30 126
Cefuroxime 30 520 2 l-23 224 116
Chloramphenicol 30 525 26-28 229 28
Ciprofloxacii? 5 221
Imipenem 10 216
OfloxacirP 5 216
Rifampin 5 ~16 17-19 220 24 51
Tetracycline 30 525 26-28 229 28 52
Trimethoprim-sulfamethoxazole 1.25i23.75 510 11-15 216 24176 50.519.5
aThese zone diameters and MIC breakpoints apply only with Haemophilus species, using HTM.
‘The category “Intermediate” should be reported. It generally indicates that the result should be considered
equivocal or indeterminate.
“Information for cefixime and ofloxacin is considered tentative for 1 year after publication of reference 124.

other clinically applicable antimicrobial agents
(124).
Medium. UseHTM (Mueller-Hinton agarsup-
plementedwith 15 Fg of purified bovine hematin
per ml, 15pg of NAD per ml, and 5 mg of yeast
extract per ml) (81).
Inoculum. Usepure fresh growth (10 colonies)
from an overnight chocolate agar plate to inoc-
ulate 5 ml of Mueller-Hinton broth to a turbidity
equivalent to a 0.5 McFarland standard, prefer-
ably usinga photometric device. (Note: A major
source of error in performing and interpreting
agar disk diffusion susceptibility test results for
H. h..uenzae is due to improper inoculum prep-
aration. It is therefore critical that the inoculum
be prepared carefully.)
Testprocedure. Perform the test accordingto
the procedure outlined in NCCLS M2-A4 (124).
Incubate HTM at 35°Cin 5 to 7% CO, for 16to
18h.
Test interpretation. Interpret zone diameters
according to the current NCCLS M2-A4 docu-
ment, usingbreakpointsfor testingH. injluenzae
on HTM (Table 1). The 2-pg ampicillin disk may
be better than the lo-pg ampicillin disk for
predicting in vitro resistance for all plasmid-
mediatedand particularly chromosomallymedi-
ated ampicillin-resistant H. injhenzae isolates
(59, 106, 107). Chromosomally mediated non-
beta-lactamase-producing isolates express a
lower level of resistance compared with plas-
mid-mediatedbeta-lactamase-producingstrains.
Vis-&vis, asreported, there may be a 40 to 50%
failure rate for detecting chromosomally medi-
ated ampicillin-resistantH. infiuenzae when the
lO+g ampicillin disk is used (61). This impor-
tant observation deserves further evaluation.
Occasionally, double zones of inhibition will be
observed when testing the susceptibility of H.
injluenzae against certain drugs on HTM. This
most notably occurs when testing cefaclor, cef-
uroxime, cefonicid, and some newer cephalo-
sporins, particularly when performing quality
control testing with H. influenzae ATCC 49247
(76a). The inner zones generally contain nonvi-
able organisms;thus, the outer zone should be
usedfor determining susceptibility.
Broth microdilution susceptibility test (125).
Medium. UseHTM (cation-supplementedMuel-
ler-Hinton broth with 15 pg of purified bovine
hematin per ml, 15 pg of NAD per ml, 5 mg of
yeast extract per ml, and 0.2 IU of thymidine
phosphorylaseper ml). Usea final volume of 100
~1of broth per well.
Inoculum. Use pure fresh growth (10colonies)
from an overnight chocolate agar plate to inoc-
ulate HTM broth or plain Mueller-Hinton broth
to a turbidity equivalent to a 0.5 McFarland
standard,preferably usinga photometric device.
CUMITECH 6A ANTIMICROBIAL AGENT SUSCEPTIBILITY TESTING 11

TABLE 2. MIC interpretive standards for Haemophilus species (125)”

MIC breakpoint (pg/ml)
Antimicrobial agent
Susceptible Intermediateb Resistant
Amoxicillin-clavulanic acid 5412 2814
Ampicillin 51 2 24
Ampicillin-sulbactam 52/l ~412
Aztreonamc 52
Cefaclor 58 16 232
Cefamandole 54 8 216
Cefiximecpd 51
Cefonicid 54 8 ~16
Cefotaxime” 52
Ceftazidime” 52
Ceftriaxone” 52
Ceftizoxime” 52
Cefuroxime 54 8 216
Chloramphenicol 52 4 18
Ciprofloxacin” 51
ImipenemC 54
Ofloxacincpd 52
Rifampin 51 2 r4
Tetracycline 52 4 28
Trimethoprim-sulfamethoxazole (l/19) 50.5l9.5 l/19-2/38 ~4/76
aThese interpretive standards are applicable only to broth microdilution susceptibility tests with Haemophilus
species, using HTM.
?I’he intermediate category should be construed as indicating an equivocal or indeterminant result.
=For these antimicrobial agents, the absence of resistant strains precludes defining any result categories other
than “susceptible.” Strains yielding results suggestive of a nonsusceptible category should be submitted to a
reference laboratory for further testing.
“See Table 1, footnote c.

Dilute to producea final concentration of 5 x lo5 ing strains.To date, no isolatesof H. injluenzae
CFWlml in the broth in each well. have been reported to be resistant to ciproflox-
Test procedure. Perform the test according to acin, albeit this drug is contraindicated for the
the procedure outlined in NCCLS M7-A2 (125). treatment of meningitisor for infected patients
Incubate MIC trays at 35°Cin ambientair for 20 who are lessthan 18years of age.
to 24 h. There is growing concern about the emer-
Test interpretation. Interpret MIC endpoints gence of rifampin-resistantH. influenzae since
according to the current NCCLS M7-A2 docu- this antimicrobial ‘agent has been the drug of
ment, usingbreakpointsfor testingH. inJIuenzae choice for chemoprophylaxis when sporadic
in HTM (Table 2). outbreaks or epidemics occur (91, 127). Ri-
Multiply resistantisolatesof hl. ,inJluenzaeare fampin MIC and disk diffusion susceptibility
not an infrequent occurrence (17, 77). Such breakpoints for H. influenzae have been estab-
strainsoften showresistanceto ampicillin, chlor- lished by NCCLS (124, 125) (Tables 1 and 2).
amphenicol,tetracycline, or trimethoprim-sulfa- However, in outbreak situationswhere rifampin
methoxazole in various combinations. Often, susceptibility is not known, chemoprophylaxis
multiply antibiotic-resistant, beta-lactamase- with rifampin plus minocycline for adults, and
producingstrainsof H. injluenzaeare susceptible rifampin plus trimethoprim for children, has
to beta-lactamaseinhibitors in combinationwith been reported to demonstrateepidemiologicef-
beta-lactams,as well asto other newer cephalo- ficacy (10, 102).
sporins (e.g., ceftriaxone and cefonicid) (91,
170). Conversely, non-beta-lactamase-producing PENICILLIN-RESISTANT MORAXELLA
ampicillin-resistantstrainsfrequently demonstrate (BRANHAMELLA) CATARRHALIS
reducedsusceptibility(higherMICs) tobeta-lactam- M. catarrhalis hasbecomeincreasingly recog-
aseinhibitor--beta-la&amcombinationsand ex- nized as an important human pathogen capable
tended-spectrumcephalosporins(170). Suscep- of causing a diverse spectrum of human infec-
tibility of suchstrainsto newer cephalosporinsis tions. These include acute and chronic otitis
conserved although the MICs for these strains media, acute and chronic maxillary sinusitis,
are up to lo-fold higher than those observed for andbronchopulmonaryinfections particularly in
ampicillin-susceptibleor beta-lactamase-produc- patients with obstructive pulmonary disease
12 NEUMANN ET AL. CUMITECH 6A

TABLE 3. Control limits for monitoring antimicrobial disk susceptibility tests with Huemophilus zone
diameter limits for individual tests (124)”
Antimicrobial agent Disk content (p,g) Zone diam (mm)
Amoxicillin-clavulanic acid 20/10 15-23
Ampicillin 10 13-21
Ampicillin-sulbactam lo/lo 14-22
Aztreonam 30 30-38
Cefaclor 30 14-22
Cefamandole 30 17-25
Cefixime 5 25-33
Cefonicid 30 19-27
Cefotaxime 30 31-39
Ceftazidime 30 27-35
Ceftizoxime 30 29-39
Ceftriaxone 30 31-39
Cefuroxime 30 17-25
Chloramphenicol 30 3 l-40
Ciprofloxacin 5 34-42
Imipenem 10 21-29
Rifampin 5 22-30
Tetracycline 30 14-22
Trimethoprim-sulfamethoxazole 1.25123.75 24-32
uThese quality control limits apply only to tests conducted with H. injfuenzae ATCC 49247, using HTM.

(36). This organism is rarely recovered from
blood, CSF, and other “sterile sites.”
Beta-lactamase-producingstrains of 1M. ca-
tarrhalis were first reported in 1977(98). Cur-
rently, most clinically significant isolatesof 1M.
ca tarrhalis produce beta-lactamase and are
therefore resistant to penicillin and ampicillin
(36, 38, 44, 86, 128). The beta-lactamases(see
below) of M. catarrhalis are cell-associated,
constitutively produced enzymes that are chro-
mosomallymediatedand are more active against
penicillins than cephalosporin antimicrobial
agents(38, 44, 50).
M. catarrhalis strainsmay produceone of two
types of beta-lactamase,each with a distinct
isoelectric profile, designatedRavasio and 1908
(97, 120). Most strains in the United States
produce the Ravasio-type beta-lactamase, al-
though a smaller but significant number of
strains produce the 1908-type beta-lactamase
(38, 120), The hydrolytic activity of both types
of beta-lactamaseis inhibited by clavulanic acid
and sulbactam. The Ravasio-type strains are
beta-lactamasepositive by the nitrocefin test,
and for these strainsthe MICs are generally ~2
PgIml by the broth microdilution test, thus indi-
cating frank resistanceto ampicillin and penicil-
lin. In contrast, the 1908-type strains produce
lo- to lOO-fold-lower quantities of beta-lactam-
ase, and although these strains demonstrate a
positive nitrocefin test, the penicillin and ampi-
cillin MICs for the strains could generally be
interpreted as susceptible (40, 97, 168). This
observation may explain why some infections
due to beta-lactamase-producing M. catarrhalis
have responded favorably to treatment with
penicillin, ampicillin, or amoxicillin (43). There-
fore, detection of beta-lactamasefrom clinical
isolatesof M. catarrhalis doesnot always imply
in vitro or in vivo resistance to penicillin or
ampicillin (40).
All clinically relevant isolates of M. catarrh-
alis should be routinely tested for beta-lactam-
ase production. The procedure of choice for
testing M. catarrhalis isolatesfor susceptibility
to penicillin and ampicillin is to determine
beta-lactamaseactivity (46, 139, 199). Of the
beta-lactamase-screeningtests, the nitrocefin
chromogenic cephalosporin method is report-
edly superior to the acidometric and iodometric
tests (36, 44, 46, 50, 90). The superior perfor-
mance and increased sensitivity of the nitro-
cefin chromogenic cephalosporin reagent are
most likely due to the fact that M. catarrhalis
beta-lactamaseis produced in small amounts
and is strongly cell associated(46, 50, 76). A
survey conducted by the College of American
Pathologistsfor a single strain of M. catarrha-
lis indicated that the nitrocefin substrate was
96% accurate in predicting the beta-lactamase
production whereas other approved methods
were less than 80% accurate (76). Since the
nitrocefin test cannot differentiate Ravasio-
type M. catarrhalis strains from 1908-type
strains, all beta-lactamase-producing strains
shouldbe interpreted and reported as resistant
to penicillins.
SinceM. catarrhalis is predictably susceptible
to cephalosporins,extended-spectrum penicil-
lins (e.g., mezlocillin and piperacillin), imi-
penem, trimethoprim-sulfamethoxazole,aztreo-
nam, ticarcillin-clavulanate, amoxicillin-clavu-
CUMITECH 6A ANTIMICROBIAL AGENT SUSCEPTIBILITY TESTING 13

TABLE 4. Acceptable quality control ranges of MICs resistance has not been reported. Because of
for dilution tests with Haemophilus species (125)a this, it is not possibleto know whether zone size
Antimicrobial agent MIC range @g/ml) interpretive criteria, establishedwith other bac-
teria, alsoconsistently apply to detecting poten-
Amoxicillin-clavulanic acid 2/l-16/8 tially resistant M. catarrhalis.
Ampicillin 2-8
Ampicillin-sulbactam 211-814 Broth microdilution susceptibilitytest (40). Me-
Aztreonam 0.12-O-5 dium. Use Mueller-Hinton broth.
Cefaclor 4-16 Inoculum. Inoculum suspensions are prepared
Cefamandole 2-8 as per NCCLS (125), using a final inoculum
Cefixime 0.12-l concentration of 5 x lo5 CFU/ml. Incubate the
Cefonicid 0.5-2 MIC trays at 35°Cin ambient air for 20 to 24 h.
Cefotaxime 0.12-0.5 With the exception of penicillin and ampicil-
Ceftazidime 0.12-l lin, the MIC interpretive criteria published by
Ceftizoxime 0.0&0.5 NCCLS for usewith nonfastidiousaerobicbacte-
Ceftriaxone 0.0&0.25
Cefuroxime 2-8 ria may alsobe appliedto M. catarrhalis. Ampi-
Chloramphenicol 0.25-l cillin andpenicillinMICs shouldbe interpretedas
Ciprofloxacin 0.004-0.03 follows: ~1 pg/ml, resistant;0.125to 0.5 Fg/ml,
Imipenem 0.12-l moderatelysusceptible;and (0.06 Fg/ml, suscep-
Rifampin 0.25-l tible (40). Thesediffer from NCCLS interpretive
Tetracycline 4-32 criteria (125).
Trimethoprim- 0.03/0.57-0.2514.75
sulfamethoxazole
“These quality control ranges are only applicable to PENICILLIN- AND TETRACYCLINE-
H. injluenzae ATCC 49247 tested by a broth microdi- RESISTANT NEZSSERZAGONORRHOEAE
lution procedure using HTM. In 1976,strains of N. gonorrhoeae that pro-
duced plasmid-mediatedbeta-lactamase(peni-
cillinase) were reported in the United States
lanate, ampicillin-sulbactam,ciprofloxacin, and (136). Currently, penicillinase-producing N.
erythromycin, routine susceptibility testing is gonorrhoeae (PPNG) annually accounts for
generally unnecessary(40,45,47). However, in 1.6%(ca. 17,000)of the casesof gonorrheain the
vitro susceptibility testsperformed by useof the United States that are reported to the Centers
disk diffusion or the broth microdilution or agar for DiseaseControl (CDC). PPNG strains are
dilution test may be relevant in selected cases now endemicin certain regions of the country,
(e.g., meningitisor refractory or recurrent bac- with resistancerates of as high as 35% reported
teremia). The disk difIusion and broth microdi- in Dade County, Florida (22, 27).
lution tests may be performed as follows. A secondmechanismof penicillin resistancein
Agar disk diffusiontest (40, 45). Medium. Use N. gonorrhoeae wasreportedby Dougherty et al.
unsupplementedMueller-Hinton agar (chocola- (48) in 1980. They observed penicillin-resistant
tized Mueller-Hinton agar may be needed to strainsof N. gonorrhoeae that did not produce
support growth of somestrains). plasmid-mediatedpenicillinasebut in fact were
Inoculum. Inoculum suspensions are prepared resistant to penicillin due to a chromosomally
as per NCCLS (124). mediatedalterationof PBPs. In 1983,a localized
Test procedure. Inoculate the surface of the outbreakof gonorrheadueto chromosomallyme-
Mueller-Hinton agar plate as described for the diated resistantN. gonorrhoeae (CMRNG) was
standarddisk diffusion procedure (124). Allow reportedin North Carolina(51). By October 1984,
the surfaceto dry. Placea lO+g ampicillin disk 446casesof CMRNG infection hadbeenreported
(or a 10-U penicillin disk or both) and other to CDC from 23 statesthat had been screening
additional drug disks to be tested on the surface for this type of resistance (25). Unlike PPNG,
of the agar plate, and gently pressonto the agar CMRNG cannot be detected by screening for
surfacewith sterile forceps. Incubate the plate at beta-lactamase. However, by direct suscepti-
35°Cin ambient air for 20 to 24 h. bility test methods, CMRNG strains produce a
Test interpretation. An ampicillin zone diam- zone diameter of 525 mm with a 10-U penicil-
eter of 519 mm reflects resistanceto ampicillin lin disk and for these strains the MICs are > 1.O
and penicillin, a zone of ~38 mm indicates pg/ml; for 75% of these strains, the MICs are
susceptibility, and a zone of 20 to 37 mm indi- >2.0 pg/ml by the agar dilution susceptibility
catesmoderatesusceptibility to thesedrugs. For test method (23, 28). Most of these strains
other drugs that are tested, follow interpretive show moderate resistanceto tetracycline (e.g.,
criteria outlined by NCCLS (124); however, MIC, 22.0 kg/ml) and decreasedsusceptibility
definitive susceptibility rangesfor most of these to erythromycin, cefoxitin, and trimethoprim-
agents are uncertain at this time since frank sulfamethoxazole (25, 51).
14 NEUMANN ET AL.
In 1985, CDC reported the isolation of strains
of N. gonorrhoeae from Georgia, New Hamp-
shire, and Pennsylvania which were penicillin
susceptible but demonstrated plasmid-mediated
high-level resistance to tetracycline (MIC, 2 16
pg/ml) (26). Most of the tetracycline-resistant
N. gonorrhoeae isolates were recovered from
cases of treatment failure with oral tetracycline.
All clinical isolates of Iv. gonorrhoeae in areas
where therapy other than ceftriaxone is being
used should be screened for resistance to peni-
cillin. PPNG may be easily and accurately de-
tected using one of the rapid beta-lactamase
tests previously described (20, 49, 124, 129).
Due to the relatively low prevalence of CMRNG
strains, there is little justification or need to
routinely examine primary isolates which are
beta-lactamase negative. However, antimicro-
bial susceptibility testing may need to be per-
formed on isolates from cases of treatment fail-
ure or isolates recovered in laboratories that
serve an active CMRNG-endemic area. Tests
that detect both CMRNG and tetracycline-resis-
tant N. gonorrhoeae should be used.
Although the agar dilution test is the most
accurate and reproducible method for determin-
ing antimicrobial susceptibility of N. gonor-
rhoeae isolates, it is not practical for most
clinical laboratories, and results vary greatly
according to the method used for inoculum
preparation (35). Those laboratories that wish to
use the agar dilution test, due to high-volume
test demands, should follow currently recom-
mended test standards (40).
The conventional broth microdilution test has
been generally unsuccessful for determining
MICs for gonococcal isolates due to organism
autolysis in Mueller-Hinton broth. However,
newer broth-based formulations that avoid orga-
nism autolysis have been developed and proven
reliable for manual and automated broth micro-
dilution tests (40, 161). Briefly, the broth formu-
lation now recommended for testing gonococcal
isolates consists of peptone broth no. 3 supple-
mented with 1% IsoVitaleX. Susceptibility test-
ing is performed according to the NCCLS
M7-A2 document guidelines (125). This method
provides reliable and reproducible results com-
parable to those obtained with the NCCLS agar
dilution procedure for the penicillins, doxycy-
cline, trimethoprim-sulfamethoxazole, erythro-
mycin, spectinomycin, and the fluoroquinolones
(40, 160). Additional work and experience with
the broth microdilution method for testing gono-
coccal isolates will be necessary before testing
in routine clinical microbiology laboratories can
be recommended.
Because the modified agar disk diffusion test
is the most practical and widely used procedure
bv routine clinical microbiology laboratories for
CUMITECH 6A
antimicrobial susceptibility testing of gonococ-
cal isolates, it has been outlined briefly below.
However, there are a few important limitations
that must be carefully considered. With the
exception of penicillin and spectinomycin, test-
ing and interpretive criteria for alternative anti-
microbial agents (e.g., tetracycline, ceftriaxone,
and cefoxitin) have not as yet been thoroughly
standardized. Use of the penicillin interpretive
criteria given in the NCCLS M7-A2 document
effectively categorizes beta-lactamase-produc-
ing strains as being resistant since PPNG strains
typically produce zone sizes of 519 mm. How-
ever, these criteria may fail to detect CMRNG
strains. In 1987, the CDC Sexually Transmissi-
ble Diseases Section addressed this issue, rec-
ommending that a disk zone breakpoint of (25
mm be used to identify CMRNG strains (28).
Susceptibility testing guidelines and interpretive
breakpoints for testing N. gonorrhoeae against
penicillin, tetracycline, spectinomycin, and cef-
triaxone are available (Table 5), along with qual-
ity control performance standards for N. gonor-
rhoeae ATCC 49226 (Table 6) (124, 125).
Agar disk diffusion test. Medium. Use GC agar
base supplemented with 1% IsoVitaleX.
Inoculum. Use pure fresh growth from an
overnight chocolate agar plate to inoculate 5 ml
of Mueller-Hinton broth (or sterile physiologic
nonbacteriostatic saline) to a turbidity equiva-
lent to a 0.5 McFarland standard. Do not delay
inoculation once the inoculum is prepared as
autolysis will decrease viability of the inoculum.
Test procedure. Perform the test according to
the procedure outlined in NCCLS M2-A4 (124).
Incubate plates at 35°C in 5 to 7% CO, for 24 h.
Test interpretation. Results for penicillin
(10-U disk) and spectinomycin (lOO-pg disk)
may be interpreted using the NCCLS M2-A4
document. Gonococcal strains with penicillin
zone sizes of ~47 or 526 mm are interpreted as
susceptible and resistant, respectively. Gono-
coccal strains with spectinomycin zone sizes of
~18 or 514 mm are interpreted as susceptible
and resistant, respectively. Interpretive catego-
ries established by CDC for penicillin, spectino-
mycin, and other alternative antimicrobial
agents are provided in Table 5. However, at this
time, these interpretive criteria should be re-
garded as tentative until validated by more ex-
tensive corroborative studies.
NEZSSERZA MENZNGZTZDZS RESISTANT TO
PENICILLIN OR RIFAMPIN
Until 1983, N. meningitidis was considered to
be universally susceptible to penicillin and
chloramphenicol when meningitis due to this
organism was being treated. However, Dillon et
al. (34) in 1983 reported isolating a strain of N.
meningitidis resistant to penicillin. Extensive
CUMITECH 6A ANTIMICROBIAL AGENT SUSCEPTIBILITY TESTING 15

TABLE 5. Zone diameter interpretive standards and equivalent MIC breakpoints for N. gonorrhoeae (124)”
Equivalent MIC
Zone diam (nearest whole mm)
breakpoint (pg/ml)
Antimicrobial agent Disk content
Moderately
Resistant Intermediateb Susceptible Resistant Susceptible
susceptibleC
Ceftriaxoned 30 w 235 SO.25
Penicillin” 10 u 526 27-46 247 22 SO.06
Spectinomycin 100 l43 514 15-17 218 1128 532
Tetrac yclinef 30 ha 530 31-37 238 22 ~0.25
aAll information in this table is considered tentative for 1 year after publication of reference 124.
‘An intermediate or indeterminate result for an antimicrobial agent indicates either a technical problem that
should be resolved by repeated testing or lack of clinical experience in treating organisms with these zones or
MICs. The latter seems to be the case for ceftriaxone and spectinomycin.
“Moderately susceptible organisms have a documented lower clinical cure rate (85 to 95%) compared with
~95% for susceptible strains.
‘For ceftriaxone, the current absence of resistant strains precludes defining any result categories other than
“susceptible.” Strains yielding results suggestive of a nonsusceptible category should be submitted to a
reference laboratory for further testing.
eGonococci with 10-U penicillin disk zone diameters of 519 mm are likely to be beta-lactamase-producing
strains. However, the beta-lactamase test remains preferred to other susceptibility methods for rapid, accurate
recognition of this plasmid-mediated penicillin resistance.
qetracycline 30.pg disk zone diameters of (: 19 mm usually indicate a plasmid-mediated tetracycline-resistant
N. gonorrhoeae isolate. These strains should be confirmed by the dilution test (MIC, 16 pg/ml) or referred to a
public health laboratory for epidemiologic investigation or both.

investigation revealed that penicillinaseproduc-
tion was plasmidmediatedand that the plasmid
wasidentical to the 45MDa “Asian-type” plas-
mid found in PPNG isolates. The samemenin-
gococcal isolate also carried a conjugative plas-
mid of the samesize (24.5 MDa) asthat found in
somegonococcal isolates. The presenceof the
transfer plasmid in this isolate “signals” the
possibletransfer of antibiotic resistancegenes
from other bacterial pathogenssuch asN. gon-
orrhoeae.
Only one beta-lactamase(penicillinase)-pro-
ducingN. meningitidisisolatehasbeenreported
to date. Therefore, routine susceptibility testing
of these organisms is not necessary (149).
Should beta-lactamaseproduction or relative
resistanceto penicillin becomea characteristic
of N. meningitidis, routine beta-lactamase
screeningor susceptibility testing of N. menin-
gitidis isolates in clinical laboratories would
becomenecessaryat somefuture time. Screen-
ing for beta-lactamase,and subsequentMIC
testing if a screeningtest is positive, should be
performed on all isolatesfrom suspectedtreat-
ment failures of casesof bacteremicmeningitis.
Penicillin-resistant isolates of N. meningitidis
shouldbe referred to a state public health labo-
ratory or CDC or both for further characteriza-
tion.
Although routine susceptibility testing of N.
meningitidisagainstrifampin is not necessaryto
direct therapy at this time, it may be used at
times for epidemiologicdata, for selection of a
drug for prophylaxis, or in casesof suspected
treatment failure. A simple, rapid disk diffusion
method is briefly describedbelow.
Agar disk diffusion test (9). Medium. Use
Mueller-Hinton agar.
Inoculum. Suspendgrowth from an overnight
agar plate into Mueller-Hinton broth and adjust
the turbidity to that of a 0.5 McFarland stan-
dard.
Test procedure. Inoculate a Mueller-Hinton
agarplate with the adjustedinoculum as done in
the standarddisk diffusion method (124).Place a
5-pg rifampin disk on the inoculated surface of
the plate. Incubate at 35°C in a 5% CO, atmo-
spherefor 18 to 24 h.
Test interpretation. A zone diameter of 220
mm indicates susceptibility, a zone diameter of
17to 19mm is interpreted asintermediate, and a
zone diameter of 516 mm indicates resistance
(124).

TABLE 6. Acceptable zone diameter quality control limits for N. gonorrhoeae (124)
Zone range (mm)
Organism
Penicillin Tetracycline Spectinomycin Ceftriaxone
N. gonorrhoeae ATCC 49226 26-34 3&42 23-29 39-51
S. aureus ATCC 25923 33-39 27-33 9-15 23-29
16 NEUMANN ET AL.
ANAEROBES
The indications for antimicrobial susceptibil-
ity testing of anaerobeshas been the subject of
considerabledebate among clinical microbiolo-
gistsfor many years (53, 54, 100, 117, 141).The
problem of increasingresistanceto certain con-
ventional antimicrobial agents (e.g., penicillin,
carbenicillin, clindamycin, and cefoxitin) among
anaerobic bacterial pathogensmight be usedas
an argumentin support of routine susceptibility
testing. However, the current availability of
several newer antimicrobial agents (e.g., imi-
penem, ampicillin-sulbactam,amoxicillin-clavu-
lanate, ticarcillin-clavulanate, piperacillin, and
metronidazole) that are essentially universally
active against clinically significant anaerobes
strongly suggeststhat testing of individual pa-
tient isolates need not be performed routinely
(149). Finegold (Wadsworth VA Medical Center
and UCLA) and the NCCLS Working Group on
Anaerobic Susceptibility Testing recommend
performing susceptibility testing of anaerobesin
the following four settings(53): (i) to determine
antibiogram patterns of anaerobesfor newer
antimicrobial agents,(ii) to periodically monitor
susceptibility patterns in local hospitals, (iii) to
periodically monitor susceptibility patterns and
trends in various geographicregions,and (iv) to
provide specific antimicrobial susceptibility test
resultsto guide physician managementof infec-
tions in selected individual patients. Examples
of infections where susceptibility testing of
anaerobesmay be usefulfor specificpatientsare
brain abscess,meningitis, endocarditis, refrac-
tory or recurrent bacteremia,osteomyelitis, sep-
tic arthritis, and prosthetic valve and vascular
graft infection (53). When two or more anaer-
obesare cultured in theseclinical settings,con-
sideration of which “organism-drug” combina-
tions to test remains a dilemma. A practical
approachin this setting is to assesswhich anaer-
obesare more virulent or are likely to be resis-
tant to conventional antimicrobial agents. An-
aerobic pathogenswhich should be considered
for susceptibility testing include subspeciesof
the Bacteroides fragilis group, pigmentedanaer-
obic gram-negativebacilli, Bacteroides gracilis,
Fusobacterium spp., Clostridium perfringens,
and Clostridium ramosum (53). In casesof poly-
microbial infection including two or more anaer-
obes (e.g., intra-abdominal sepsis),one of us
(M.A.N.) notes on the microbiology patient re-
port that “anaerobic pathogensare almost uni-
versally susceptibleto metronidazole, piperacil-
lin, ampicillin-sulbactam, ticarcillin-clavulanic
acid, amoxicillin-clavulanic acid, and imipenem
and generally susceptibleto cefoxitin and clin-
damycin.’ ’ This approach has worked well in
practice.
CUMITECH 6A
The selection of antimicrobial agents to test
against anaerobesmust be carefully based on
hospital formulary policy, the pathogen iso-
lated, the type and site of infection, and the
method of the susceptibility test used. The
accuracy of certain anaerobic susceptibility
tests is method dependentwith regard to deter-
mining susceptibility endpoints. In particular,
there is a lack of agreement regarding which
test method provides accurate susceptibility
test results for newer cephalosporins(53). This
problem is further compounded by general
disagreementwith regard to the levels of clin-
ical effectiveness, antimicrobial activity, and
predictability of in vivo correlation of suscep-
tibility results when anaerobesare being tested
against newer cephalosporins.
A variety of susceptibility test methods are
available for anaerobes.The standardagar disk
diffusion method, however, should not be used
to perform susceptibility tests on anaerobesbe-
cause this technique is intended for rapidly
growing aerobic or facultatively anaerobicbac-
teria. The interpretive standardsfor this method
are not meant to be used with anaerobes.The
agardilution method is the reference procedure
described by the NCCLS Working Group on
Anaerobic Susceptibility Testing. The agar di-
lution test has been questioned as a suitable
reference method since the medium, Wilkins-
Chalgrenagar, doesnot support the growth of all
anaerobesand certain organism-drugcombina-
tions do not producereproducibleresults(202).
NCCLS has published a proposed guideline
for alternative methodsfor antimicrobial suscep-
tibility testing of anaerobic bacteria (121, 123).
Both the broth disk elution (Kurzynski) (89) and
broth microdilution methodsare described. De-
spite its flexibility, ease of performance, and
recommendation as an alternative anaerobic
susceptibility test method, the disk elution test is
fraught with technical and interpretive prob-
lems. The major problems associatedwith the
disk elution methodinclude difficult-to-read end-
points (80, 201, 202) and false-negative and
false-positive results (7, 162, 201), particularly
with newer cephalosporinsand imipenem.As a
result, the NCCLS Working Group on Anaero-
bic Susceptibility Testing has reconsideredthe
suitability of the disk elution method for routine
use and hasabandonedit altogether (201).
“In the opinion of many experiencedworkers,
the best approach for anaerobic susceptibility
testing is clearly the broth microdilution tech-
nique” (202). However, further in-depth collab-
orative studiesare neededto achieve more stan-
dardization with this methodology. Key issues
that need to be evaluated or agreed upon by
consensusinclude the choice of medium, the
method of inoculumpreparation, and the inocu-
CUMITECH 6A ANTIMICROBIAL
lum size. At this time, microdilution tray well
volumes of 100 ~1 and incubation at 35°C for a
full 48 h in an anaerobic atmosphere have been
generally agreed upon.
Beta-lactamase testing of anaerobes (e.g.,
Bacteroides spp., Fusobacterium spp., and C.
perfringens) may offer useful information per-
taining to the susceptibility of these organisms
to penicillins; however, little, if any, informa-
tion pertaining to cephalosporin susceptibility
is gained (149).
In addition to, or in lieu of, routine antimicro-
bial susceptibility testing of anaerobicisolates,a
numberof rapid test methodsfor detecting beta-
lactamase production and clindamycin resis-
tance have been used successfullyto screenfor
and/or predict antimicrobial resistance,particu-
larly with clindamycin-resistant Bacteroides
spp. (16, 54, 118). With regard to performing
beta-lactamase(e.g., nitrocefin) testing, micro-
biologistsmust remain aware that resistanceto
beta-la&am drugs is not always mediated by
beta-lactamase(e.g., B. gracilis and Bacteroides
distasonis) (53).
MYCOBACTERZUM FORTUZTUM-
MYCOBACTERZUM CHELONAE COMPLEX
Since the early 197Os,the M. fortuitum-M.
chelonae complex has becomeincreasingly as-
sociatedwith a wide rangeof infectious compli-
cations. These include diseasessuch as post-
traumatic cellulitis, infections of prosthetic de-
vices, prosthetic valve endocarditis, ulcerative
keratitis, peritonitis, meningitis, osteomyelitis,
and a variety of postsurgical wound infections
(193).Frequently, infections with the M. fortui-
turn-M. chelonae complex are progressiveand
can become serious or life threatening; hence,
someform of therapy is essential. Successful
treatment of M. fortuitum or M. chelonae infec-
tion involves surgicaldebridementand appropri-
ate antimicrobial therapy. M. fortuitum and M.
chelvnae are resistant to the usual antitubercu-
losis drugs. This group of mycobacteria, how-
ever, demonstratesvariable susceptibility to the
tetracycline congeners, erythromycin, sulfon-
amides,trimethoprim-sulfamethoxazole,amika-
tin, kanamycin, gentamicin, and cefoxitin. Be-
cause of the unpredictable susceptibility to
many drugs among the six subgroups of M.
fortuitum-M. chelonae, susceptibility testing of
clinically significantisolatesis essentialto deter-
mine drugs likely to be effective in treatment.
Pendingresultsof susceptibility testing, empiric
therapy for patients with serious infections
shouldinclude the combination of cefoxitin and
amikacin.
Essentiallyall isolatesfrom wounds, bone, or
normally sterile sites should be identified and
tested for drug susceptibility. Since sputumiso-
AGENT SUSCEPTIBILITY TESTING 17
lates are lesslikely to be clinically significant,
only multiple positive specimens with large
numbers of organismspresent in each of the
cultures would support the need to perform
susceptibility testing.
Four different susceptibility test methods
have been used for testing M. fortuitum-M.
chelonae. They are agar dilution (196), disk
diffusion (191), broth microdilution (176), and
agardisk elution (169). A comparative review of
the advantagesand disadvantagesof these test
methodshasbeenpublished(193). Each method
has proven to be useful in selecting drugs for
treatment, but none have yet been well stan-
dardized for all drugs and each has certain
limitations and pitfalls that must be considered
(193).
NCCLS (125) currently recommends the
broth microdilution method, using cation-sup-
plemented Mueller-Hinton broth. Cultures are
incubated at 35°C (or 30°C for isolates from
cutaneoussites)for 72 h. Inoculum is prepared
from overnight growth in Mueller-Hinton broth
supplemented with 0.02% Tween 80 and
adjustedto a turbidity equivalent to a 0.5 Mc-
Farland standard.Antimicrobial agentsthat may
be tested are listed in the antimicrobial menu
outlined in Table 7. The NCCLS M7-A2 (125)
document may be used for interpretive break-
point guidelines.
Those laboratories using commercially pre-
pared broth microdilution susceptibility test
panelsmay be limited with regard to the choice
and concentrations of antimicrobial agentsthat
they can test M. fortuitum-M. chelonae isolates
against. Since the agar disk elution method has
the advantage of being simple to perform, has
flexibility in antimicrobial agents that can be
tested, is practical -for routine microbiology lab-
oratories, has good correlation with other test
methods(161), and is amenableto standardiza-
tion, it may serve as a useful alternative to the
broth microdilution test. Because of this, the
agar disk elution test is briefly describedbelow.
Agar disk elution susceptibilitytest. Medium.
Use OADC (oleic acid, albumin, dextrose, and
catalase) and molten (SOOC)Mueller-Hinton
agar.
Inoculum. Pick several colonies from fresh
growth on blood agaror Middlebrook 7HlO agar
and transfer them to cation-supplementedMuel-
ler-Hinton broth plus 0.02% Tween 80 (this
helps to produce a smooth suspension).Incu-
bate overnight at 35°C. Adjust the inoculum to
equal a 0.5 McFarland standard. Dilute the
suspensionto 1:100and 1:1,000.
Test procedure. (Prepare duplicate sets of
trays.) (i) Place the appropriate number of se-
lected antimicrobial disks (Table 7) into each
well of a 24-well tissue culture plate (Linbro,
18 NEUMANN ET AL.
TABLE 7. Preparation of antimicrobial
M. fortuitum-M.
Antimicrobial agent Disk content (pg)
Doxyc y cline
Amikacin
Kanamycin
Cefoxitin
Erythromycin
Sulfisoxazole
Trimethoprim-sulfamethoxazole
Imipenem
Ciprofloxacin
Tobramycin
Flow Laboratories, Inc.). Placethe disk(s)in the
center of the well. (Allow one well without
antimicrobial disksto serve as a growth control
well.)
(ii) Pipette 0.5 ml of OADC to each well,
being certain to immersethe disks. Allow 15min
for the drugs to elute.
(iii) Pipette 4.5 ml of melted Mueller-Hinton
agar into each well with a swirling motion to
adequately mix the drug eluent and OADC. Be
certain to center the disks with a sterile wooden
stick. Allow the agar to harden.
(iv) Pipette 10 lull of the adjusted inoculum
(1:100, 1:1,000) onto the agar surface in each
well of the duplicate trays, Spreadthe inoculum
evenly acrossthe agar.Incubate at 35°Cfor 72 h.
Somestrainsof A4. chelonaemay need a longer
incubation period or incubation at 30°C rather
than WC. Mycobacterium mar&m can alsobe
testedwith this methodbut shouldbe incubated
at 30°Cfor 7 to 14 days.
Test interpretation. Colony counts of the in-
oculum which grows 100 to 500 CFU in the
control well are reported. Susceptibility is de-
fined as no growth (for other than sulfonamide
antimicrobial agents)or greater than 80%inhibi-
tion of colony size (for sulfonamides)compared
with the control well.
Quality control. Quality control of the agar
and susceptibility disks can be done using stan-
dard NCCLS quality control bacterial strainsfor
both Mueller-Hinton agar and antimicrobial
disks. Possible mycobacterium control strains
are also available (169).
The only major problemencounteredwith this
method is that reading an endpoint for erythro-
mycin is difficult becauseof the trailing end-
point, and many strains that are erythromycin
susceptiblein the broth microdilution procedure
produce fine dysgonic colonies when tested by
the agar disk elution method. Therefore, some
erythromycin strains may be interpreted as re-
sistantby this method; clinical data to determine
CUMITECH 6A
agent concentrations for the agar disk elution method for testing
chelonae complex ( 169)
Final concn (pg) of
No. of disks per well antimicrobial agent per
ml of agar
30 1 6
30 2 12
30 2 12
30 5 30
15 1 3
300 1 60
1.25i23.75 5 l/19
10 5 10
5 2 2
10 3 6
which method provides the more appropriate
result are lacking.
NOCARDZASPECIES
Nocardia speciesare associatedwith a broad
spectrum of clinical disease,including wound
infections, mycetomas,lymphocutaneoussporo-
trichoid syndrome, posttraumatic keratitis, pul-
monary infection, and disseminateddisease(94,
185). Pulmonary and generalizedinfections are
particularly prominent in patients with debilitat-
ing diseasessuch as lymphoreticular neoplasms,
chronic pulmonary disorders including alveolar
proteinosis, collagen vascular disease, chronic
ileitis and colitis, cirrhosis, and immunosuppres-
sion(185).Pulmonary and disseminatednocardi-
osis has also been described in alcoholics. In
addition, renal and cardiac transplant recipients
have been reported to be at high risk for nocar-
diosis. Pulmonary diseasemimicking tuberculo-
sis is the most frequent presentationof nocardi-
osis; it can remain confined to the lungs or may
disseminateto various organs,especiallythe cen-
tral nervous systemwith involvment of the brain
and meninges(94).
Nocardia asteroides is the most frequent
causeof nocardiosis,although Nocardia brasil-
iensisand Nocardia caviae can also cause hu-
man disease.
Since mortality rates range from 40 to 80% in
untreated cases,effective medical management
of pulmonary or disseminatednocardiosis re-
quires prompt treatment with an appropriate
antimicrobial agent (185). Although sulfon-
amideshave been widely used as the drugs of
choice for treating nocardiosis, not all patients
show a favorable response and some are not
able to tolerate drug therapy. Sulfonamide
therapy results in treatment failure in 20% of
cases of pulmonary nocardiosis and 50% of
casesof central nervous system disease(185).
Becauseof this, alternative antimicrobial regi-
mens can be critically important in effective
CUMITECH 6A ANTIMICROBIAL
medical management of nocardiosis. Alterna-
tive antimicrobial agents, including trimetho-
prim-sulfamethoxazole, amikacin, ampicillin,
erythromycin, amoxicillin-clavulanate, mino-
cycline, newer cephalosporins, and imipenem,
have been used with promising results (30, 57,
60, 85).
Justification of and methods for the perfor-
mance of antimicrobial susceptibility testing of
clinical isolates of Nocardia were publishedin
1988by Wallace and Steele (192). In addition to
variable patient tolerance and treatment failure
with sulfonamides,these investigators note an-
other phenomenonthat calls for susceptibility
testing. When tested in vitro, N. asteroides in
particular demonstrates variable susceptibility
to a numberof antimicrobial agents(192). Since
a broad range of antimicrobial agents are now
available, susceptibility testing of clinical iso-
lates of Nocardia provides clinicians with a
pertinent guide in the selectionof antimicrobial
therapy for treating nocardiosis.
Although susceptibility testing of Nocardia
isolateshasnot beenconsideredor evaluatedby
NCCLS and is best performed by a competent
reference laboratory, experienced clinical mi-
crobiology laboratories may choose to provide
clinicians with rapid presumptive antimicrobial
susceptibility information by performing the
modified disk diffusion test described by Wal-
lace and Steele (192).
Agar disk diffusion test, At present, the mod-
ified disk diffusion test is the most reproducible
and practical methodfor susceptibility testing of
Nocardiu species(192).
Medium. Use a 1500mmMueller-Hinton agar
plate. Ten percent of Nocardia strainsmay not
grow on unsupplementedMueller-Hinton agar.
Therefore, if no growth is observed, retest the
isolate, using Mueller-Hinton agar supple-
mented with 5% sheep blood or chocolatized
Mueller-Hinton agar; this, however, may result
in an unreliable interpretation of sulfonamide
antimicrobial agents.
Inoculum. Usepure fresh growth from an agar
plate to inoculate a 5-ml tube of Mueller-Hinton
broth or other suitablenutrient broth. Incubate
the broth tube at 35°C to match the optical
density of a 0.5 McFarland standard. (Frequent
shakingof the broth tubesand/or vortexing with
a few sterile glassbeadsadded will provide a
smoother suspension.)Be careful to avoid too
heavy an inoculum (i.e., so confluent that no
spacesbetween the colonies are visible on the
plate) asthis could result in an interpretation of
false resistanceto sulfonamides.
Quality control shouldbe monitored eachtime
that Nocardia susceptibility testing is per-
formed, using N. asteroides ATCC 19247as
reported by Wallace and Steele (192).
AGENT SUSCEPTIBILITY TESTING 19
Test procedure. Inoculate the surface of the
agar plate with the adjusted inoculum, using a
sterilecotton swab,and apply in three directions
as describedfor the standardagar disk diffusion
test (124). Allow the surface to dry. Place the
disks to be tested on the surface of the agar
plate, allowing for a spatial distance of approx-
imately 70 mm; zone sizes of 30 to 60 mm may
be produced with some antimicrobial agents.
Antimicrobial agents tested should include
erythromycin, ampicillin, amoxicillin-clavu-
lanate, ampicillin-sulbactam,cefuroxime, cefti-
zoxime or cefotaxime, ceftriaxone, imipenem,
amikacin, sulfisoxazole or sulfamethoxazole or
trimethoprim-sulfamethoxazole, and minocy-
cline. Other antimicrobial agents, including flu-
oroquinolones, gentamicin, tobramycin, doxy-
cycline, and ticarcillin-clavulanate, may alsobe
tested. Incubate the inoculated plate(s) at 35°C
in ambient air and humidity for 48 to 72 h.
Test interpretation. Measure zone diameters
with a caliper or millimeter ruler. Assessand
record clear zones of inhibition except for
sulfisoxazole or trimethoprim-sulfamethoxazole,
for which an 80%inhibition of colony sizeis used.
Confirmatory readingson theseantibioticsat 72 h
shouldbe done. Colonieswithin the zone should
be regardedas significantgrowth. Carefully ob-
serve for smallercoloniesinside the zone with
aminoglycosidesand beta-lactam antimicrobial
agents. Breakpoint zone size interpretations
should be assessedand recorded as shown in
Table 8. Caution must be used in interpreting
zones due to antimicrobial agents adjacent to
amoxicillin-clavulanate and/or imipenem since
drug synergymay occur andresultin falsesuscep-
tibility readings.
UNUSUAL OR FASTIDIOUS BACTERIA
There is an ever-expanding list of unusualor
fastidiousbacteria associatedwith opportunistic
infections in compromised patients that chal-
lenge both clinicians and clinical microbiolo-
gists.Many of theseclinically important bacteria
have characteristics that preclude their being
testedby the standardizedagar disk diffusion, or
broth or agardilution susceptibility tests recom-
mendedby NCCLS. They may grow too slowly,
require special nutrients, require special atmo-
spheresor specialincubation temperatures, or
simply not have been tested adequately to show
that current standardized susceptibility test
methodsare valid. Although certain unusual or
fastidious pathogensmay not satisfy the neces-
sary criteria or perform well with currently
established susceptibility tests, clinicians de-
mand and deserve someform of antimicrobial
susceptibility information to guide them in dis-
tinguishingwhich antimicrobial agentswill likely
20 NEUMANN ET AL. CUMITECH 6A

TABLE 8. Preliminary interpretive disk diffusion susceptibility breakpoints for Nocardia species (192)
MIC breakpoint
Antimicrobial agent Zone diam (mm) Susceptibility categorya
(w/ml)
Ampicillin 235 51 S
16-34 2-16 I
515 ~16 R
Amikacin 130 S
*b
520 >16 R
Cefotaxime 125 4 S
20-24 16-32 I
519 >32 R

Ciprofloxacin 230 S
25-29 I
524 R

Doxycycline 235 51 S
20-34 2-4 I
519 >4 R

Erythromycin 229 so.5 S

20-28 l-4 I
519 >4 R
Gentamicin 225 54 S
16-24 8 I
515 >8 R
Minoc ycline 235 51 S
20-34 2-4 I
(-19 >4 R

Sulfisoxazole 235 532 S

*
515 ~32 R

Trimethoprim-sulfamethoxazoie 220 50.5-9.5 S

*
115 >2/38 R
Tobramycin 225 54 S
16-24 8 I
515 >8 R
“S, Susceptible; I, intermediate; R, resistant.
b*, Inadequate strains for evaluation of “intermediate” for disk diffusion testing. By broth microdilution
testing, isolates could be either susceptible or resistant.

not be effective versus those that at least dem-
onstrate potential antimicrobial activity.
Clinical microbiologists sometimeshave an
obligation to implement “impromptu” methods
in order to provide the physician with “best
guess” antimicrobial susceptibility test results.
In thesesituationswhere an isolatedoesnot test
appropriately with current standardizedsuscep-
tibility methods,one shouldevaluate the neces-
sary growth factors of the organismin order to
test it for susceptibility to a battery of antimicro-
bial agents. Factors to be considered are (i)
atmosphere(e.g., ambient, CO, enrichment,and
anaerobic versus microaerophilic), (ii) optimal
temperature to support growth (e.g., 30°C ver-
sus 35”C), (iii) nutrients or supplements(e.g.,
amino acids, blood, serum, carbohydrates, and
yeast extract), (iv) growth medium consistency
(e.g., agar and broth versus semisolid),and (v)
pH. Once these factors are determined, an at-
tempt to test antimicrobial agents by using a
modifieddisk diffusion, agaror broth dilution, or
disk elution can be made. Modified or unique
susceptibility test methodsand interpretive pa-
rameters are available for a wide range of
specialproblem pathogens(184). Susceptibility
CUMITECH 6A ANTIMICROBIAL AGENT SUSCEPTIBILITY TESTING 21

test results can be carefully interpreted by going cardiac surgery. Antimicrob. Agents Chemother.
using general NCCLS breakpoints for the type 17~269-272.
5. Azemum, P., T. Stull, M. Roberts, and A. L. Smith. 1981.
of test performed (124, 125), but it is necessary Rapid detection of chloramphenicol resistance in Hue-
for laboratories and physicians to exercise cau- mophilus ifluenzue. Antimicrob. Agents Chemother.
tion in interpreting results. Susceptibility test 20:168-170.
results reflecting total resistance to an antimi- Sa.Baker, C. N. CDC, personal communication.
crobial agent versus potential susceptibility 6. Barry, A. L., and R. N. Jones. 1987. Reliability of high-
content disks and modified broth dilution tests for detect-
should be cautiously reported and communi- ing staphylococcal resistance to the penicillinase-resis-
cated to physicians as a “best estimate” sus- tant penicillins. J. Clin. Microbial. 25:1897-1901.
ceptibility profile, informing them of the poten- 7. Barry, A. L., and R. R. Packer. 1984. Determination of
tial limitations of the method used to perform susceptibility of anaerobic bacteria to cefotetan and
cefoxitin by the thioglycolate disk elution method. J.
the test (149). In most cases the isolate should Clin. Microbial. 20:912-916.
also be referred to a reputable reference labo- 8. Bell, S. M., and D. Plowman. 1980. Mechanisms of
ratory for further testing. ampicillin resistance in Huemophilus irfluenzue from the
If the identity of the isolate is known, fre- respiratory tract. Lancet i:279-280.
quently the microbiologist and physician may 9. Bennett, J. V., H. M. Camp, and T. C. Eickhoff. 1968.
Rapid sulfonamide disc sensitivity test for meningococci.
refer to a published review article that provides Appl. Microbial. 16: 1056-1060.
an antimicrobial profile of that organism. Profes- 10. Berti, M., R. Scott, F. Ripamonti, and V. Arioli. 1979.
sional journals including Antimicrobial Agents Activity of rifampin plus trimethoprim against Huemo-
and Chemotherapy, Journal of Clinical Micro- philus influenzue. Curr. Microbial. 2:223-225.
biology, Antimicrobic Newsletter, Diagnostic 11. Blair, H. C., and T. J. Cleary, 1983. Susceptibility test-
ing of multidrug-resistant Staphylococcus uureus with
Microbiology and Infectious Diseases, Journal the Scepter microdilution system. J. Clin. Microbial.
of Infectious Diseases, Reviews of Infectious 18: 194-l%.
Diseases, and European Journal of Clinical Mi- 12. Boyce, J. M. 1984. Reevaluation of the ability of the
crobiology and Infectious Diseases commonly standardized disk diffusion test to detect methicillin-
print antibiogram studiesof specialorganisms. resistant strains of Stuphylococcus uureus. J. Clin. Mi-
crobiol. 19:813-817.
The Index Medicus also shouldserve asa useful 13. Boyce, J. M., L. S. Lytte, and D. A. Walsh. 1984. Detec-
informational resource. tion of methicillin-resistant Staphylococcus uureus by
When performing standardized susceptibility microdilution and disk elution susceptibility systems. J.
testing, microbiologistsmust alsoremain cogni- Clin. Microbial, 20: 1068-1075.
14. Boyce, J. M., R. L. White, M. C. Banner, and W. R.
zant that even commonly tested bacterial patho- Lockwood. 1982. Reliability of the MS-2 system in de-
gens may fail to grow adequately or may not tecting methicillin-resistant Staphylococcus uureus. J.
grow at all becauseof a mutation or alteration in Clin. Microbial. l&220-225.
15. Burns, J. L., P. M. Mendehnan, J. Levy, T. L. Stall, and
growth factor requirements.One exampleof this A. L. Smith. 1985. A permeability barrier as a mecha-
wasan isolate of Morganella morganii, cultured nism of chloramphenicol resistance in Huemophilus in-
from the blood of a patient with endocarditis, fluenzue. Antimicrob. Agents Chemother. 27:45-54.
that would not grow on plain Mueller-Hinton 16. Callihan, D. R., and F. S. Nolte. 1985. Disk diffusion
agar. When Mueller-Hinton agar supplemented method to screen for high-level resistance to clindamycin
and erythromycin in the Bucteroides fiugilis group. Di-
with 5% sheepblood wasused, the isolate grew agn. Microbial. Infect. Dis. 3:131-138.
luxuriantly and appropriate antimicrobial sus- 17. Campos, J., S. Garcia-Tornel, and I. Sanfeliu. 1984.
ceptibility test results were obtained (5a). Susceptibility studies of multiply resistant Huemophilus
. influenzae isolated from pediatric patients and contacts.
Antimicrob. Agents Chemother. 25:706-709.
ACKNOWLEDGMENTS. We are greatly indebted to Mary 18. Canawati, H. N., J. L. Witte, and F. L. Sapico. 1982.
Nelson-Jones and Agnes Suarez for their helpful suggestions Temperature effect on the susceptibility of methicillin-
and technical assistance in the preparation of this manuscript. resistant Staphylococcus uureus to four different cepha-
losporins. Antimicrob. Agents Chemother. 21:173-175.
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