ß-lactamases
■ Are enzymes that chemically inactive ß-lactam
molecules by disrupting the ß-lactam ring
componentof the molecule.
■ Production of these enzymes by some bacteria
such as Staphylococci, H. influenza, N.
gonorrhea, Eneterobacteriacease,
Pseudomonas, M. catarrhalis, and Bacteroides
spp, is a significant mechanism contributing to
resistance to some ß-lactam drugs
■ Production of ß-lactamases in staphylococci is
inducible, and exposure to an inducing agent
(another ß- lactam agent, like oxacillin) is often
required to obtain enough concentrations of
the enzyme for detection with conventional ß-
lactamases tests.
■ As with other ß-lactamase-producing bacteria,
production of these enzymes is constitutive,
meaning the same amount regardless of
exposure to an inducing agent.
■ Most clinically important ß- lactamases: Class A
and C ß-lactamases
 Class A enzymes are found on plasmids,
 Class C are chromosomally located and
inducible by exposure to ß-lactamase.
■ In Gram-positive bacteria, they are secreted as
exoenzymes and offer less protection the
organisms while in Gram- negative bacteria,
the ß- lactamases are localized to the
periplasmic space.
Extended Spectrum ß-lactamases (ESBLs)
■ These are ß- lactamases that can inactivate
extended- spectrum cephalosporins, penicillin
and aztreonam.
■ Are derivatives of TEM, SHV and OXA type B ß-
lactamases that differ by one or more amino
acid substitutions near the reactive site of the
enzymes.
■ Most K. pneumoniae, K. oxytoca and many
E.coli are resistance to ampicillin because of
production of a plasmid- mediated ß-
lactamases known as TEM-1 or SHV-1.
■ These enzymes are characterized and
numbered based on their relationship to the
parent enzymes.
■ The carbapenems (imipenem, meropenem,
ertapenem) are active against ESBL-producing
strains.
■ Indicator drugs for detection of ESBLs:
aztreonam, ceftriazxone and cefotaxime –these
drugs are selected based on the likelihood of
their being readily hydrolyzed by one of the
many types o ESBLs.
ß-lactamases hydrolyze ß-lactam antibiotics using 2
mechanisms:
1. Metallo-based mechanism
 Expressed by class B ß-lactamases
(cephalosporinases)
 S.maltophilia, K.pneumonia and P.aeruginosa
encode class B enzymes.
2. Serine-based mechanism
 Expressed by classes A,C,D ß-lactamases –
evolved from PBPs.
 TEM-1 and SHV-1 = are Class A ß-lactamases.
a. Acquired Resistance
 It is present only on certain isolates that are
different from the parental strain.
 It arises as a result of chromosomal mutations
(transformation and recombination), acquisition
of extrachromosomal DNA or by horizontal
transfer of preexisting resistance genes.
 It is usually expressed phenotypically as efflux,
modification or acquisition of targer sites,
enzymatic modification or degradation of
antibiotics.
NOTES TO REMEMBER:
 The substrate of ß-lactamases is the ß-lactam
ring structure, which the enzyme selectively
hydrolyze to form the inactive penicilloic acid.
 PBPs and ß-lactamases can serve as receptors
for ß- lactam antibiotics entering the cell.
 It is the binding equilibrium between PBP, ß-
lactamases, and ß-lactam antibiotics that
determines that fate and ultimate survival of
the microorganism.
 ß-lactamase inhibitors: clavulanic acid,
sulbactam, and tazobactam – structural
analogues of the ß-lactam antibiotics and
function as substrates for ß-lactamase, thus
reducing their detrimental effects on the ß-
lactam antibiotic.
 Penicillin resistance is due to the presence of
altered penicillin-binding proteins (drug targets
in the cell wall).
 It is important to test penicillin-resistant
isolates with other clinically relevant
antimicrobial agents such as cefotaxime or
ceftriaxone (MIC method not disk diffusion),
erythromycin, floroquinolone, vancomycin,
clindamycin or tetracycline.
  Leptospirosis – 1st to be positive in blood
and CSF Typhoid fever – 1sts to be positive in
blood and bone marrow
4. Quantify sufficient, prompt delivery, proper
sterile containers
Collection
1. Swab (aerobic)s
 Cotton - toxic to Neisseria, good for virus
 Calcium alginate – toxic to virus, good for
Neisseria
2. Needle aspiration: aerobic and anaerobic
3. Catheterization: aspirate from catheter tube
 Foley Catheter – urine
 IV catheter – blood
Storage
 1-2 hours delay Refrigerated: stool, sputum,
VACCINES
 urine, swab except genital (because Neisseria is
 Killed- Polio ( IM- Salk), HepaA; Hepa B
cold sensitive)
 Live attenuated ( weakened) – Rabies, MMR,
 Room temperature (not ref): CSF, blood, body
varicella Rotavirus
fluids, genital swab of N.gonorrhea
 Toxoid ( modified to be non toxic) – Tetauns,
Diphtheria
Clinical specimen
 Subunits ( Specific protein)– recombinant
Hepatitis B
 Conjugate( Polysaccharide with carrier) –
Hemophilus influenzae b

Killed Live
# of doses Multiple Single
# of adjuvant Yes No
Duration of immunity Shorter Longer
Effectiveness of protection Lower Greater
Immunoglobulin produced IgG IgA and IgG
Mucosal immunity produced Poor Yes
Cell mediated immunity Poor Yes
produced
Residualvirulent virus Possible No
vaccine 2. Urine
Reversion to virulence No Possible ■ Random, catheterized, midstream, suprapubic
Excretion of vaccine virus No Possible (anaerobic)
and transmission to non ■ Centrifuge: collect sediment for culture and GS
immune contacts ■ Quantitative: use BAP and MAC
Interference by other virus No Possible
– For suspected infection like UTI
Stability High Low
– (E.coli – gram negative; Enterococcus
and S.saprophyticus – gram positive)
SPECIMEN COLLECTION, PROCESSING AND TRANSPORT
– Colony count in CFU/ml = number of
Type of Specimen
colony x 1000 (if 0.001mL loop)
1. Sterile – without normal flora
– >100,000CFU/ml = ID and AST of UTI
2. Non-sterile – use selective medium
– <10,000 CFU/ml – ID only
Rules
3. CSF
1. Collect before antibiotic theraphy
■ placed on bottle 2, not refrigerated only
2. Aseptic collection
incubator
 Betadine/iodophor – blood culture
■ Routine test: India ink and gram stain
 Bottle #2 - CSF
■ Isolation of: Neisseria and Haemophilus
3. Acute stage
■ Centrifuge: sediment for culture and GS
 – BAP, CAP (5-10% CO2)
– BHIB, MAC (incubator – No CO2)
– India ink method (capsule): CSF Latex
agglutionation (capsular Ag)
4. Wound
■ BAP, MAC, THIO, GS
■ Swab collected at edge after NSS – aerobic
culture
■ Needle aspiration - anaerobic culture

5. Stool
■ do not GS
■ 1-2grams stool on sterile vial
■ Rectal swab on Cary Blair
■ MAC, BAP, CAP, SSA, TCBS, HEA, XLD
■ SELENITE F (SSA), APW (TCBS), CBAP (42C for
48 hours)
■ Presumptive: oxidase test, biochemical test
■ Confirmatory: Serotyping
6. Respiratory (Sputum, NPS)
■ Processing done on BSC
■ Gram stain (>25PMN, <10EC)
■ Gentamicin BAP – S.pneumoniae
■ Bacitracin CAP – H.influenza (5-10% CO2
incubator, MAC 35C incubator)
■ Do gram stain and AFS
■ Bartlett’s Classification
■ Assess the quality of sputum
■ Enumerate the number of neutrophils and
epithelial cells/LPF
■ 0 score or less – saliva (no inflammation)
■ >1 score – inflammation/infection