Resistance in gram-negative bacteria: Enterobacteriaceae

David L. Paterson, MD, PhD Pittsburgh, Pennsylvania

The emergence and spread of resistance in Enterobacteriaceae are complicating the treatment of serious nosocomial infections and threatening to create species resistant to all currently available agents. Approximately 20% of Klebsiella pneumoniae infections and 31% of Enterobacter spp infections in intensive care units in the United States now involve strains not susceptible to thirdgeneration cephalosporins. Such resistance in K pneumoniae to third-generation cephalosporins is typically caused by the acquisition of plasmids containing genes that encode for extended-spectrum b-lactamases (ESBLs), and these plasmids often carry other resistance genes as well. ESBL-producing K pneumoniae and Escherichia coli are now relatively common in healthcare settings and often exhibit multidrug resistance. ESBL-producing Enterobacteriaceae have now emerged in the community as well. Salmonella and other Enterobacteriaceae that cause gastroenteritis may also be ESBL producers, which is of relevance when children require treatment for invasive infections. Resistance of Enterobacter spp to third-generation cephalosporins is most typically caused by overproduction of AmpC b-lactamases, and treatment with third-generation cephalosporins may select for AmpC-overproducing mutants. Some Enterobacter cloacae strains are now ESBL and AmpC producers, conferring resistance to both third- and fourthgeneration cephalosporins. Quinolone resistance in Enterobacteriaceae is usually the result of chromosomal mutations leading to alterations in target enzymes or drug accumulation. More recently, however, plasmid-mediated quinolone resistance has been reported in K pneumoniae and E coli, associated with acquisition of the qnr gene. The vast majority of Enterobacteriaceae, including ESBL producers, remain susceptible to carbapenems, and these agents are considered preferred empiric therapy for serious Enterobacteriaceae infections. Carbapenem resistance, although rare, appears to be increasing. Particularly troublesome is the emergence of KPC-type carbapenemases in New York City. Better antibiotic stewardship and infection control are needed to prevent further spread of ESBLs and other forms of resistance in Enterobacteriaceae throughout the world. (Am J Infect Control 2006;34:S20-8.)

Gram-negative bacteria of the Enterobacteriaceae family are important causes of urinary tract infections (UTIs), bloodstream infections, hospital- and healthcare-associated pneumonias, and various intra-abdominal infections. Within this family, Escherichia coli is a frequent cause of UTIs, Klebsiella spp and Enterobacter spp are important causes of pneumonia, and all of the Enterobacteriaceae have been implicated in bloodstream infections and in peritonitis, cholangitis, and other intra-abdominal infections. Additionally, organisms such as Salmonella produce gastroenteritis and subsequently, in some patients, invasive infection. Emerging resistance in Enterobacteriaceae is a signicant problem that requires immediate attention. Resistance related to production of extended-spectrum b-lactamases (ESBLs) is a particular problem in the handling of Enterobacteriaceae infections, but other mechanisms of resistance are also emerging, leading
From the Antibiotic Management Program, University of Pittsburgh Medical Center, Pittsburgh, PA. Reprint requests: David L. Paterson, MD, PhD, Suite 3A, Falk Medical Building, 3601 5th Avenue, Pittsburgh, Pennsylvania 15213. E-mail: patersond@dom.pitt.edu. 0196-6553/$32.00 Copyright 2006 by the Association for Professionals in Infection Control and Epidemiology, Inc. and Elsevier, Inc. doi:10.1016/j.ajic.2006.05.238

to multidrug resistance and threatening to create panresistant species.

OVERVIEW OF RESISTANCE TRENDS AND OUTCOMES

Third-generation cephalosporins were originally developed as b-lactams able to overcome resistance caused by common b-lactamases. When rst introduced, third-generation agents like ceftriaxone, cefotaxime, and ceftazidime were stable in the presence of common b-lactamases. However, within a few years, hospital-acquired gram-negative bacilli like Klebsiella pneumoniae and others began producing mutated versions of these b-lactamases that made them resistant to third-generation cephalosporins and to the monobactam aztreonam. According to the National Nosocomial Infections Surveillance (NNIS) System report, in 2003, 20.6% of all K pneumoniae isolates from patients in intensive care units (ICUs) in the United States were nonsusceptible to third-generation cephalosporins.1 This represented a 47% increase compared with resistance rates for 1998 to 2002. Nonsusceptibility to thirdgeneration cephalosporins was also observed in 31.1% of Enterobacter spp and 5.8% of E coli isolated from patients in ICUs in 2003. Those rates were approximately the same as for 1998 to 2002. International studies report even higher rates of nonsusceptible K pneumoniae in hospitals and particularly in ICU settings.2

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Enterobacteriaceae resistance to third-generation cephalosporins is typically caused by production of b-lactamases. An example are the ESBLs that hydrolyze broad- and extended-spectrum cephalosporins, monobactams, and penicillins.3,4 The genes that encode ESBLs are frequently found on the same plasmids as genes that encode resistance to aminoglycosides and sulfonamides, and many Enterobacteriaceae species possess changes that confer high-level resistance to quinolones. This means that ESBL-producing Enterobacteriaceae in hospitals and ICU settings are commonly multidrug resistant, which poses a particular challenge for the treatment of nosocomial infections, especially in critically ill patients. Inappropriate empiric antimicrobial treatment for nosocomial- or community-acquired infections has been reported to contribute to signicantly greater mortality rates in the ICU, and inadequate antimicrobial treatment of infection was the most important independent determinant of hospital mortality.5 Other studies have reported that inappropriate initial antibiotic treatment for nosocomial bacteremia caused by ESBL-producing K pneumoniae or E coli is associated with a signicantly higher mortality rate than is initial therapy involving an agent with activity against these ESBL-producing bacteria.6,7

aeruginosa and only rarely in Enterobacteriaceae.8 Another important fact about ESBLs is that they are typically plasmid mediated rather than chromosomally mediated b-lactamases. ESBLs should be distinguished from other b-lactamases capable of hydrolyzing extended-spectrum cephalosporins. Examples include AmpC and carbapenemases. Carbapenemases may be further grouped as either metallo-b-lactamases (class B) or serine carbapenemases (classes A and D). Like ESBLs, AmpC b-lactamases hydrolyze third-generation or expandedspectrum cephalosporins, but unlike ESBLs, they are also active against cephamycins and are resistant to inhibition by clavulanate or other b-lactamase inhibitors.3,10 Carbapenemases have broader-range activity, covering carbapenems as well as expanded-spectrum cephalosporins.10,11 Carbapenemase-producing Enterobacteriaceae are currently relatively rare, but there are concerns about the emergence and spread of these strains. Table 1 presents a brief summary of different b-lactamases produced by Enterobacteriaceae or other gram-negative bacteria.

In vitro susceptibility proles and clinical outcomes

In vitro susceptibility proles for ESBL-producing Enterobacteriaceae for cephalosporins can be misleading because they suggest that an isolated strain is susceptible to a given cephalosporin when in fact the drug may not be effective when used to treat a serious infection caused by such an organism. This phenomenon is sometimes known as hidden resistance. Susceptibility break points from the National Committee for Clinical Laboratory Standards (NCCLS), now known as the Clinical and Laboratory Standards Institute (CLSI), for third-generation cephalosporins in Enterobacteriaceae were created in the early 1980s, at a time when ESBL production was not a common occurrence. Thus, there is a problem in that ESBL-producing strains sometimes may be incorrectly recorded by the microbiology laboratory as susceptible to third-generation cephalosporins. In vitro studies show that the minimum inhibitory concentration (MIC) of third- and fourth-generation cephalosporins for ESBL-producing Enterobacteriaceae is elevated severalfold when high versus standard inocula are used in susceptibility testing.12,13 This can lead to a situation in which in vitro testing indicates the isolate is susceptible to late-generation cephalosporins, but clinical failure occurs when these agents are used. In a study of patients with ESBL-producing K pneumoniae bacteremia, 54% of patients receiving treatment with a susceptible cephalosporin, as determined by in vitro methods, experienced clinical failure.14 The

EXTENDED-SPECTRUM b-LACTAMASE PRODUCING ENTEROBACTERIACEAE General issues and nomenclature

Infections caused by ESBL-producing Enterobacteriaceae are serious concerns in the current environment. Many ESBLs represent enzymes that have evolved from class A b-lactamasesnamely, TEM-1, TEM-2, and SHV-1, which are frequently expressed in gramnegative bacteria and which confer resistance to ampicillin, amoxicillin, and other penicillins, as well as to early- but not later-generation cephalosporins. ESBLs arose when mutations of the genes encoding TEM-1, TEM-2, or SHV-1 gave rise to new b-lactamases that became able to hydrolyze third-generation cephalosporins and aztreonam.3,8 TEM- or SHV-type ESBLs are typically not active against cephamycins (eg, cefotetan, cefoxitin, or cefmetazole) or carbapenems (imipenem, ertapenem, and meropenem), and can generally be inhibited by b-lactamase inhibitors such as clavulanate, sulbactam, or tazobactam. Enterobacteriaceae may also express ESBLs that are not closely related to TEMor SHV-related species, including CTX-M- and OXAtype ESBLs, among others. CTX-M-type ESBLs typically hydrolyze cefotaxime more efciently than ceftazidime.9 Unlike most ESBLs that have been found in E coli, K pneumoniae, and other Enterobacteriaceae, OXAtype ESBLs have been found mainly in Pseudomonas

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Table 1. Selected b-lactamases of gram-negative bacteria

b-Lactamase Broad-spectrum Examples TEM-1, TEM-2, SHV-1 OXA family Extended-spectrum TEM family, SHV family CTX-M family OXA family Others (PER-1, PER-2, BES-1, GES/IBC family, SFO-1, TLA-1, VEB-1, VEB-2) ACC-1, ACT-1, CFE-1, CMY family, DHA-2, FOX family, LAT family, MIR-1, MOX-1, MOX-2 IMP family, VIM family, GIM-1, SPM-1 (metallo-b-enzymes) KPC-1, KPC-2, KPC-3 OXA-23, OXA-24, OXA-25, OXA-26, OXA-27, OXA-40, OXA-48 Substrates Penicillin G, aminopenicillins, carboxypenicillins, piperacillin, narrow-spectrum cephalosporins Substrates of the broad-spectrum group plus cloxacillin, methicillin, and oxacillin Substrates of the broad-spectrum group plus oxyimino-cephalosporins, and monobactam (aztreonam) Substrates of the expanded-spectrum group plus, for some enzymes, cefepime Same as for CTX-M family Same as for TEM family and SHV family Inhibition by clavulanate* 111 1 1111 1111 1 1111 Molecular class A D A A D A

AmpC

Substrates of expanded-spectrum group plus cephamycins

Carbapenemase

Substrates of the expanded-spectrum group plus cephamycins and carbapenems Same as for IMP family, VIM family, GIM-1, and SPM-1 Same as for IMP family, VIM family, GIM-1, and SPM-1

0 111 1

B A D

Adapted from N Engl J Mea.10 *1, 111, and 1111 denote relative sensitivity to inhibition.

data suggested that an antibiotic MIC of 8 mg/mL was universally associated with clinical failure and that high rates of failure were also observed with a MIC of 4 mg/mL (Table 2).14 These results are consistent with those from a variety of observational studies that show rates of clinical failure .90%, approximately 67%, and ,30% with MICs of 8, 4, and #2 mg/mL, respectively.7,15 Whether the susceptibility break points of Enterobacteriaceae for cephalosporins should be changed is currently under consideration. The 2005 CLSI guidelines recommend that laboratories report ESBL-producing isolates as resistant to all penicillins, cephalosporins, and aztreonam irrespective of in vitro tests results.16 The presence of inadequately identied ESBL producers has important implications for both antibiotic therapy and infection control.

Table 2. Outcome of cephalosporin treatment of serious infections due to extended-spectrum b-lactamase producing organisms
Patients % (n) MIC (mg/mL) 8 4 2 #1 Total* Experienced failure of cephalosporin therapy 100 (6/6) 67 (2/3) 33 (1/3) 27 (3/11) 54 (15/28) Died of bacteremia within 14 days 33 (2/6) 0 (0/3) 0 (0/3) 18 (2/11)

MIC, minimum inhibitory concentration. Adapted from J Clin Microbio.14 *Includes 5 patients with isolates for which MICs were recorded simply as 0.5 to 4 mg/L.

Treatment of ESBL producers

The presence of ESBL-producing Enterobacteriaceae complicates therapy, especially because these organisms are often multidrug resistant. When isolates from a patient indicate an ESBL-producing organism, the rst thing to consider is whether the patient has a true infection. Patients with positive isolates from urine or perhaps the respiratory tract may be only colonized, and clearly, there is no indication for treatment in those situations. Assuming the patient has a serious infection due to ESBL-producing Enterobacteriaceae, the choice of empiric therapy is made difcult by the likelihood of multidrug resistance and the fact that there are no

data from large, randomized, controlled trials designed to compare one antibiotic therapy with another for infections caused by ESBL-producing organisms. Moreover, for a variety of reasons, it is unlikely that such a study will ever be performed. Nonetheless, data from a number of studies strongly point to carbapenems as the drugs of choice for empiric treatment of serious infections involving ESBL-producing Enterobacteriaceae. Subgroup analysis from a randomized, evaluatorblind trial comparing cefepime with imipenem in patients with nosocomial pneumonia showed that 100% of patients (10 of 10) receiving imipenem for pneumonia caused by an ESBL producer experienced a positive clinical response compared with only 69% of patients (9 of 13) treated with cefepime.17 Similarly,

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a prospective, observational, international study of patients with K pneumoniae bacteremia reported an all-cause 14-day mortality rate of 3.7% (1 of 27) with carbapenem alone, compared with rates of 36.3% and 44.4% with quinolone and noncarbapenem b-lactam monotherapy, respectively.7 For patients infected with ESBL-producing K pneumoniae, the corresponding 14-day mortality rates were 4.8% (2 of 42) among patients receiving carbapenem monotherapy or combination therapy and 27.6% (8 of 29) among those receiving treatment with a noncarbapenem antibiotic. Although TEM- and SHV-type ESBLs do not effectively hydrolyze cephamycins (such as cefoxitin or cefotetan), Enterobacteriaceae may exhibit resistance to those agents due to plasmid-mediated expression18 or overexpression19 of AmpC b-lactamases. Additionally, resistance to b-lactamb-lactamase inhibitor combinations (such as piperacillin-tazobactam) may occur due to the coexistence of AmpC-type b-lactamases and ESBLs. The development of porin-decient mutants may also contribute to resistance to cephamycins and b-lactamb-lactamase inhibitor combinations.20 Such occurrences are not infrequent and argue against the use of cephamycins or b-lactamb-lactamase inhibitor combinations in patients with serious infections due to ESBL-producing Enterobacteriaceae. As mentioned earlier, the CLSI recommends that isolates found to be ESBL producing be considered resistant to all penicillins, all cephalosporins, and aztreonam. Similarly, quinolones, aminoglycosides, and trimethoprim-sulfamethoxazole (TMP-SMX) are generally not appropriate initial therapeutic choices for serious infections caused by ESBL-producing Enterobacteriaceae because ESBL producers are often resistant to these drugs as well.21-23 Moreover, multidrug resistance among ESBL-producing K pneumoniae and E coli species appears to be increasing.23 With quinolones, even in the presence of apparent susceptibility, there may be a substantial failure rate. In our international study discussed earlier, 36.4% of patients who received treatment with a quinolone for bacteria caused by ESBL-producing K pneumoniae died within 14 days.7 Quinolone resistance in Enterobacteriaceae is described in greater detail below.

Community-acquired ESBLs
ESBL-producing Enterobacteriaceae are prevalent in the hospital setting, and there is now evidence that they are emerging and spreading in the community as well.24 Most cases of ESBL-producing organisms in the community have been reported internationally; the situation in the United States is not yet known. Most commonly, the cases of community-acquired ESBLs (CA-ESBLs) involve urinary tract infections (UTIs),

although gastrointestinal infections in the community may also be important. A population-based laboratory surveillance study of ESBL-producing E coli infections in the Calgary Health Region of Canada reported that 71% of patients had community-onset disease.25 The study did not address whether the ESBL-producing E coli were necessarily acquired in the community, but the data do speak to the high prevalence of infections associated with ESBL-producing species in the community, and cautions that many clinical laboratories may not be aware of the importance of screening for ESBL-producing organisms when dealing with infections originating in the community.24 Failure to do so may lead to inappropriate treatment and adverse outcomes. The surveillance study also reported that 70% of the ESBL in E coli isolated from patients with communityonset infections were of the CTX-M-type.25 Other studies, too, have reported a high prevalence of CTX-M-type ESBLs in community-onset infections.24 This contrasts with the general preponderance of TEM- and SHV-type ESBLs in isolates from hospitalized patients with K pneumoniae or E coli infections both in the United States and worldwide. In fact, CTX-M-type ESBLs have only very recently been identied in patients with nosocomial E coli infections in the United States.26 In our international study of nosocomial bloodstream infections, CTX-M-type ESBLs were identied in isolates from patients in all countries except the United States.27 Outside the United States, among nonhospitalized patients, ESBL-producing E coli have been identied in various countriesincluding Spain, Israel, Canada, and the United Kingdomand frequently, the ESBLs have been of the CTX-M type.25,28-31 As with TEM- or SHV-type ESBLs, CTX-M-type ESBLs are often multidrug resistant.24 This underscores the importance of screening for ESBL-producing pathogens in certain groups in the community. The typical clinical picture for community-associated infection involving ESBLs is UTI (sometimes associated with bacteremia) due to CTX-M-producing E coli, with elderly women being most commonly affected. Isolates are resistant to typical rst-line agents for UTI, such as ciprooxacin, TMP-SMX, gentamicin, and ceftriaxone. So there is now the very real risk that treatment of community-acquired infections with E coli may be compromised because of multidrug resistance. Data for January 1998 through June 2004 from the US-based Intensive Care Antimicrobial Resistance Epidemiology (ICARE) project indicated that only 0.6% of E coli isolates and 1.8% of K pneumoniae isolates from so-called outpatient areas were nonsusceptible to third-generation cephalosporins.1 At present, the existence of CA-ESBL-producing Enterobacteriaceae appears to be very limited in the United States.

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Nonetheless, the healthcare community needs to be aware of the potential problem that CA-ESBL producers may present in the United States in the future, especially given what has been observed in the United Kingdom and Canada. It suggests that attention be focused on potential risk factors for community development of infection with ESBL-producing bacteria. In a Spanish study, those risk factors included diabetes mellitus, previous quinolone use, recurrent UTIs, a previous hospital admission, and older age in male patients.30 An Israeli study identied risk factors as previous hospitalization within the past 3 months, antibiotic treatment within the past 3 months, age .60 years, male sex, K pneumoniae infection, previous use of third-generation cephalosporins, previous use of second-generation cephalosporins, previous use of quinolones, and previous use of penicillin.28 Although both studies identied previous hospitalization as a risk factor, it should be noted that cases of infection with an ESBLproducing organism in the community have been reported in patients without recent hospitalization. So, cases of true CA-ESBL producers have been documented. As mentioned previously, ESBL-producing pathogens may also be involved in gastrointestinal infections acquired in the community. Bacterial species that have been reported to produce ESBLs leading to drug-resistant gastroenteritis include Salmonella spp, Shigella, Vibrio cholerae, and Shiga toxinproducing E coli.29,32-37 The possible emergence and spread of Salmonella strains resistant to antibiotics commonly used as treatment are concerns, because these infections can be invasive. Outside the United States, TEM-, SHV-, and CTX-M-type ESBLs, as well as AmpC b-lactamases, have been identied in infection-causing Salmonella.29,32,38 Within the United States, the mechanism of Salmonella resistance to third-generation cephalosporins has been linked to production of AmpC b-lactamases.39-41 In particular, resistance has been associated with the plasmid-mediated AmpC b-lactamase known as CMY-2. Salmonella strains resistant to thirdgeneration cephalosporins are of concern because (1) ceftriaxone and, secondarily, quinolones are the drugs of choice for invasive salmonella disease, and (2) quinolones are not indicated for use in children. Fortunately, ceftriaxone-resistant Salmonella are currently rare in the United States, but they represent an area that bears further watching.

Moreover, b-lactam exposure is capable of inducing expression of AmpC b-lactamases in Enterobacter spp with consequent resistance to third-generation cephalosporinsand mutations may result in permanent hyperproduction and persistent resistance. Treatment of Enterobacter infections with third-generation cephalosporins may select for mutant strains associated with hyperproduction of AmpC b-lactamase. The prevalence of Enterobacter spp resistant to third-generation cephalosporins has increased since the introduction and common use of these antibiotics. For example, in 1 study, resistance to third-generation cephalosporins emerged in approximately 20% of patients during treatment for Enterobacter bacteremia.43 Multidrugresistant Enterobacter spp in initial positive blood cultures were signicantly more prevalent (P , .001) among patients who had previously received thirdgeneration cephalosporins than among patients who had previously received other antibiotic treatments, and they were associated with higher mortality rates.43 In summary, third-generation cephalosporins should be avoided as treatment for serious infection with Enterobacter spp because their use in such situations results in selection of the small number of AmpC-overproducing mutants present in any collection of Enterobacter spp isolates and because the typical clinical scenario is characterized by initial response followed by recurrence of infection. In contrast, cefepime is comparatively stable to AmpC b-lactamases, and therefore has been regarded as a suitable option for treatment of Enterobacter infections.42 However, ESBL-producing Enterobacter spp, particularly Enterobacter cloacae, have been identied in the United States (Table 1),44-46 Europe,47 and Asia.48-50 At our medical center in Pittsburgh, approximately 33% of bloodstream isolates have been shown to contain E cloacae that produce ESBL as well as AmpC b-lactamases.51 The MIC of cefepime may be within that danger zone of 48 mg/mL, where cephalosporin activity may be compromised. Hence, not all resistance to later-generation cephalosporins in E cloacae may be the result of hyperproduction of AmpC b-lactamases, and ESBL-producing strains of E cloacae may be resistant to fourth- as well as third-generation cephalosporins. The advent of ESBLs in AmpC-producing E cloacae is certainly something to be aware of and is potentially clinically important.

ANTIBIOTIC RESISTANCE IN ENTEROBACTER SPECIES

Enterobacter spp are signicant causes of nosocomial infection and are intrinsically resistant to aminopenicillins, cefazolin, and cefoxitin due to production of constitutive chromosomal AmpC b-lactamases.42

EMERGING QUINOLONE AND CARBAPENEM RESISTANCE Quinolone resistance

Quinolones are used widely for the treatment of serious E coli UTIs and may also be used to treat other

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infections caused by other members of the Enterobacteriaceae family.52,53 Hence, quinolone resistance in Enterobacteriaceae may lead to treatment failures and is a signicant concern, as is the recent emergence of plasmid-mediated resistance to quinolones. According to the 2004 NNIS report, means of 7.3% and 8.2% of E coli isolates from US patients in both ICUs and nonICU areas of hospitals, respectively, and a mean of 3.6% from US outpatients exhibited quinolone resistance.1 Higher rates of quinolone resistance may be found in ESBL-producing strains of E coli and K pneumoniae. For example, 55.8% of infections in the University of Pennsylvania Health System caused by ESBL-producing E coli or K pneumoniae were uoroquinolone resistant.21 In Shanghai, China, 86.1% of ESBLproducing E coli and 45.6% of ESBL-producing K pneumoniae were reported to be resistant to levooxacin.54 We studied K pneumoniae bacteremia from 12 hospitals in 7 countries and found that overall, 18% of ESBL-producing isolates were ciprooxacin resistant and 60% of ciprooxacin-resistant isolates produced ESBLs.55 Prior receipt of a quinolone has been shown to be an independent risk factor for quinolone resistance.21,55 Quinolone resistance in Enterobacteriaceae is usually due to alterations in target enzymes (DNA gyrase and/or topoisomerase IV) or to impaired access to the target enzymes, occurring either because of changes in porin expression or because of efux mechanisms.56 Both of these principal means of resistance are caused by chromosomal mutations. More recently, plasmid-mediated quinolone resistance has emerged in K pneumoniae and E coli. The rst case of plasmidmediated resistance to quinolones in K pneumoniae was reported in the United States in 1998 and was from a strain isolated at the University of Alabama in 1994.57 The plasmid, pMG252, confers multidrug resistance and was shown to greatly increase quinolone resistance when transferred to strains of K pneumoniae decient in outer-membrane porins. The gene associated with that resistance has been designated qnr. Quinolone resistance associated with qnr-containing plasmids has now emerged in E coli and K pneumoniae strains.58-60 A recent study in the United States reported that 11.1% of K pneumoniae strains from 6 states exhibited plasmid-mediated quinolone resistance associated with the qnr gene, although none of the E coli strains examined contained qnr.61 Some of the strains contained the original pMG252 plasmid, but qnr was carried on different plasmids for others. The mechanism of quinolone resistance associated with qnrcontaining plasmids appears to involve inhibition of quinolone binding with DNA gyrase.62 The emergence of this new plasmid-mediated mechanism of quinolone resistance is particularly worrisome because it provides a mechanism for the rapid

development and spread of quinolone and multidrug resistance to important members of the Enterobacteriaceae family.

Carbapenem resistance
As mentioned earlier, carbapenems are currently considered to be the preferred agents for treatment of serious infections caused by ESBL-producing Enterobacteriaceae. Carbapenems are highly stable to b-lactamase hydrolysis, and porin penetration is facilitated by their general size and structure. Their susceptibility to most strains of Enterobacteriaceae makes them generally useful as treatment for multidrug-resistant organisms. Carbapenem resistance is currently rare among Enterobacteriaceae, but some worrisome signs have appeared in recent years. The expression of AmpC or class A (TEM- or SHVtype) ESBLs plus loss of outer-membrane proteins have been associated with carbapenem resistance in K pneumoniae.63-68 Resistance to carbapenems has also been reported in K pneumoniaeproducing class B b-lactamases (metallo-b-lactamases) in various countries outside the United States, including Brazil,69 Greece,70 China,71 and Singapore.72 A metallo-b-lactamaseproducing strain of E cloacae with reduced susceptibility to carbapenems has also recently been observed in Greece.73 Standard susceptibility testing may categorize metallo-b-lactamaseproducing Enterobacteriaceae as susceptible to carbapenems, but an inoculum effect has been observed, suggesting that the susceptibility testing may falsely predict the susceptibility of particular Enterobacteriaceae to carbapenems in the clinical environment.73,74 In the United States, carbapenem resistance has been observed in strains of K pneumoniaeproducing class A carbapenemases, namely, KPC-1, KPC-2, and KPC-3.75-82 The genes encoding these enzymes are apparently obtained via plasmid conjugation; the enzymes are capable of hydrolyzing and inactivating the carbapenems. KPC-producing strains have generally been shown to exhibit multidrug resistance that includes piperacillin/ tazobactam, third- and fourth-generation cephalosporins, uoroquinolones, and aminoglycosides, as well as carbapenems.82 KPC-1 expression itself was apparently associated with moderate- to high-level carbapenem resistance,75 while loss of outer-membrane proteins appeared to be a required cofactor for highlevel resistance in KPC-2 and KPC-3producing strains.76,78 In 96 isolates obtained from 10 New York hospitals, .80% of the KPC-producing organisms belonged to a single ribotype.82 This is worrisome because it suggests that these isolates are not just being selected by antibiotic use, but rather are being passed from person to person as a result of breakdown in infection

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control. As with metallo-b-lactamaseproducing Enterobacteriaceae, susceptibility testing may falsely indicate the clinical susceptibility of KPC-producing K pneumoniae due to an inoculum effect.79-81 In vitro testing suggests that tigecycline and polymyxins may exhibit the most consistent activity against KPCproducing strains of K pneumoniae, but this has yet to be demonstrated clinically.82 KPC-producing strains of Enterobacter81 and Salmonella spp83 have also been identied in the United States. Strains of clinically important Enterobacteriaceae have now emerged with broader multidrug resistance than has ever before been observed. As the list of antibiotics with potential activity against these strains continues to shrink, measures that prevent and slow the spread of multidrug-resistant Enterobacteriaceae strains throughout the world must be put into action.

K pneumoniae. Both improvement in antibiotic stewardship and infection control strategies are needed before it is too latebefore we have to deal with infections caused by multidrug-resistant Enterobacteriaceae for which there are no clinically effective drugs.

References
1. National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004. Am J Infect Control 2004;32:470-85. 2. Paterson DL, Ko WC, Von Gottberg A, et al. International prospective study of Klebsiella pneumoniae bacteremia: implications of extended-spectrum beta-lactamase production in nosocomial Infections. Ann Intern Med 2004;140:26-32. 3. Rupp ME, Fey PD. Extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae: considerations for diagnosis, prevention, and drug treatment. Drugs 2003;63:353-65. 4. Shah AA, Hasan F, Ahmed S, Hameed A. Extended-spectrum betalactamases (ESBLs): characterization, epidemiology, and detection. Crit Rev Microbiol 2004;30:25-32. 5. Kollef MH, Sherman G, Ward S, Fraser VJ. Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients. Chest 1999;115:462-74. 6. Du B, Long Y, Liu H, et al. Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae bloodstream infection: risk factors and clinical outcome. Intensive Care Med 2002;28: 1718-23. 7. Paterson DL, Ko WC, Von Gottberg A, et al. Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum beta-lactamases. Clin Infect Dis 2004;39:31-7. 8. Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933-51, table of contents. 9. Walther-Rasmussen J, Hoiby N. Cefotaximases (CTX-M-ases), an expanding family of extended-spectrum beta-lactamases. Can J Microbiol 2004;50:137-65. 10. Jacoby GA, Munoz-Price LS. The new beta-lactamases. N Engl J Med 2005;352:380-91. 11. Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev 2005;18: 306-25. 12. Thomson KS, Moland ES. Cefepime, piperacillin-tazobactam, and the inoculum effect in tests with extended-spectrum beta-lactamase-producing Enterobacteriaceae. Antimicrob Agents Chemother 2001;45: 3548-54. 13. Jett BD, Ritchie DJ, Reichley R, Bailey TC, Sahm DF. In vitro activities of various beta-lactam antimicrobial agents against clinical isolates of Escherichia coli and Klebsiella spp. resistant to oxyimino cephalosporins. Antimicrob Agents Chemother 1995;39:1187-90. 14. Paterson DL, Ko WC, Von Gottberg A, et al. Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum beta-lactamases: implications for the clinical microbiology laboratory. J Clin Microbiol 2001;39: 2206-12. 15. Wong-Beringer A, Hindler J, Loeloff M, et al. Molecular correlation for the treatment outcomes in bloodstream infections caused by Escherichia coli and Klebsiella pneumoniae with reduced susceptibility to ceftazidime. Clin Infect Dis 2002;34:135-46. 16. Data on le. Performance standards for antimicrobial susceptibility testing; fteenth informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2005. 17. Zanetti G, Bally F, Greub G, et al. Cefepime versus imipenem-cilastatin for treatment of nosocomial pneumonia in intensive care unit patients:

SUMMARY
Enterobacteriaceae are signicant causes of serious infections, and many of the most important members of this family are becoming increasingly resistant to currently available antibiotics. This is a troubling trend, and one that requires vigilance and intensied measures to control the further spread of resistance by these important gram-negative pathogens. Although improvements in antibiotic stewardship and infection control are discussed in greater detail by others in this supplement, it should be emphasized that such improvements are necessary if the steady rise in ESBLproducing Enterobacteriaceae and in other forms of resistance in these species is to be slowed or stopped. The widespread use of third-generation cephalosporins as the driving force behind the emergence of ESBL-producing organisms has been shown in many studies. Because of that risk, third-generation cephalosporins are no longer appropriate as workhorse antibiotics in our hospitals, and the restriction of their use in hospitals and other healthcare facilities has been shown to reduce the incidence of ESBL-producing Enterobacteriaceae like K pneumoniae. Quinolones seem to have replaced third-generation cephalosporins in hospitals, yet their overuse, particularly in light of plasmid-mediated quinolone resistance in Enterobacteriaceae, may be of concern. Infection control is the second aspect in restriction of the emergence and spread of resistant Enterobacteriaceae. With regard to ESBL producers, there is ample evidence of person-to-person spread. Combining reductions in third-generation cephalosporin use with traditional infection control measuressuch as the use of gloves, gowns, and hand hygiene in the care of colonized or infected patientshas been reported to control the hospital spread of multidrug-resistant

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a multicenter, evaluator-blind, prospective, randomized study. Antimicrob Agents Chemother 2003;47:3442-7. Alvarez M, Tran JH, Chow N, Jacoby GA. Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States. Antimicrob Agents Chemother 2004;48:533-7. Tracz DM, Boyd DA, Bryden L, et al. Increase in AmpC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT-PCR. J Antimicrob Chemother 2005;55:768-72. Martinez-Martinez L, Hernandez-Alles S, Alberti S, Tomas JM, Benedi VJ, Jacoby GA. In vivo selection of porin-decient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum-cephalosporins. Antimicrob Agents Chemother 1996;40:342-8. Lautenbach E, Strom BL, Bilker WB, Patel JB, Edelstein PH, Fishman NO. Epidemiological investigation of uoroquinolone resistance in infections due to extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae. Clin Infect Dis 2001;33:1288-94. DiPersio JR, Deshpande LM, Biedenbach DJ, Toleman MA, Walsh TR, Jones RN. Evolution and dissemination of extended-spectrum betalactamase-producing Klebsiella pneumoniae: epidemiology and molecular report from the SENTRY Antimicrobial Surveillance Program (19972003). Diagn Microbiol Infect Dis 2005;51:1-7. Hyle EP, Lipworth AD, Zaoutis TE, et al. Risk factors for increasing multidrug resistance among extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella species. Clin Infect Dis 2005;40:1317-24. Pitout JD, Nordmann P, Laupland KB, Poirel L. Emergence of Enterobacteriaceae producing extended-spectrum b-lactamases (ESBLs) in the community. J Antimicrob Chemother 2005;56:52-9. Pitout JD, Hanson ND, Church DL, Laupland KB. Population-based laboratory surveillance for Escherichia coli-producing extended-spectrum beta-lactamases: importance of community isolates with blaCTX-M genes. Clin Infect Dis 2004;38:1736-41. Moland ES, Black JA, Hossain A, Hanson ND, Thomson KS, Pottumarthy S. Discovery of CTX-M-like extended-spectrum beta-lactamases in Escherichia coli isolates from 5 US States. Antimicrob Agents Chemother 2003;47:2382-3. Paterson DL, Hujer KM, Hujer AM, et al. Extended-spectrum betalactamases in Klebsiella pneumoniae bloodstream isolates from 7 countries: dominance and widespread prevalence of SHV- and CTXM-type beta-lactamases. Antimicrob Agents Chemother 2003;47: 3554-60. Colodner R, Rock W, Chazan B, et al. Risk factors for the development of extended-spectrum beta-lactamase-producing bacteria in nonhospitalized patients. Eur J Clin Microbiol Infect Dis 2004;23: 163-7. Munday CJ, Whitehead GM, Todd NJ, Campbell M, Hawkey PM. Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum beta-lactamases in York, UK. J Antimicrob Chemother 2004;54:628-33. Rodriguez-Bano J, Navarro MD, Romero L, et al. Epidemiology and clinical features of infections caused by extended-spectrum beta-lactamase-producing Escherichia coli in nonhospitalized patients. J Clin Microbiol 2004;42:1089-94. Woodford N, Ward ME, Kaufmann ME, et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum betalactamases in the UK. J Antimicrob Chemother 2004;54:735-43. Kruger T, Szabo D, Keddy KH, et al. Infections with nontyphoidal Salmonella species producing TEM-63 or a novel TEM enzyme, TEM-131, in South Africa. Antimicrob Agents Chemother 2004;48:4263-70. Ishii Y, Kimura S, Alba J, et al. Extended-spectrum beta-lactamaseproducing Shiga toxin gene (Stx1)-positive Escherichia coli O26:H11: a new concern. J Clin Microbiol 2005;43:1072-5. Kim S, Kim J, Kang Y, Park Y, Lee B. Occurrence of extended-spectrum beta-lactamases in members of the genus Shigella in the Republic of Korea. J Clin Microbiol 2004;42:5264-9.

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