Kenneth S. Thomson

Extended-Spectrum-β-Lactamase, AmpC, and Carbapenemase Issues

Optimal use of microbiology laboratories is essential to combat the spread of multiply antibiotic-resistant pathogens. This is vital for patient care, as advocated by Jarvis in stressing the importance of active detection and isolation to control methicillin-resistant Staphylococcus aureus and other resistant hospital-acquired pathogens (17). It is also vital for hospital accreditation in the United States, where the Joint Commission has set requirements for the control of acquisition and transmission of multidrug-resistant organisms (http: //www.jointcommission.org/NR/rdonlyres/31666E86-E7F4-423E -9BE8-F05BD1CB0AA8/0/HAP_NPSG.pdf-NPSG.07.03.01). These requirements cannot be met without excellence in diagnostic microbiology. The absence of new, effective anti-gram-negative antibiotics makes infection control the most important countermeasure against multidrug-resistant gram-negative pathogens. Infection control can prevent additional infections and the spread of resistant pathogens and thereby reduce the need to use antibiotics. Infection control is most effective when directed by rapid, accurate laboratory results. In short, excellence in diagnostic microbiology is critical to quality initiatives in hospitals. Some resistant pathogens may not be recognized because they are falsely susceptible in routine tests. This can lead to patients receiving ineffective antibiotics and contribute to the spread of the pathogens. Because the detection of such “hidden” resistance is so critical, this Commentary focuses on its detection in gram-negative pathogens. Because susceptibility tests may be unreliable, special tests are required to detect the resistance mechanisms involved. The mechanisms include extended-spectrum -lactamases (ESBLs), AmpC -lactamases, and carbapenemases of molecular classes A and B. ESBLs ESBLs are typically inhibitor-susceptible -lactamases that hydrolyze penicillins, cephalosporins, and aztreonam and are encoded by mobile genes. The most frequently encountered ESBLs belong to the CTX-M, SHV, and TEM families. ESBL producers are usually multiply drug resistant (5, 30), but their cephalosporin and aztreonam resistance is not reliably detected by susceptibility tests (33). Many labs have adopted CLSI recommendations and only attempted to detect ESBLs in Escherichia coli, Klebsiella pneumoniae, K. oxytoca, and Proteus mirabilis (8). Since ESBL genes are transmissible, it is important that ESBLs be tested for in other organisms in hospital and long-term care facility patient populations where ESBLs are encountered. This may be unnecessary for community isolates, which at this time appear to be predominantly CTX-M-producing E. coli. The need for ESBL detection is under challenge on the supposition that it is possible to set breakpoints for injectable cephalosporins and aztreonam that accurately discriminate which ESBL-producing isolates can and cannot be reliably treated with these drugs. This approach is controversial (19) but has been adopted in slightly different forms by the European Committee on Antimicrobial Susceptibility Testing and the CLSI. It is based on limited therapeutic outcome data (3, 31), pharmacokinetic/pharmacodynamic data (3), and the concept that the lower the cephalosporin MIC the greater the

Cefepime, piperacillin-tazobactam, and the inoculum effect in tests with extended-spectrum beta-lactamase-producing Enterobacteriaceae

ABSTRACT There is little information about the clinical effectiveness of cefepime and piperacillin-tazobactam in the treatment of infections caused by extended-spectrum β-lactamase (ESBL)-producing pathogens. Some inferences have been drawn from laboratory studies, which have usually involved only one or a few strains of ESBL-producingKlebsiella pneumoniae or Escherichia colithat produced only a limited range of ESBLs. Such studies are indirect, sometimes conflicting, indicators of efficacy. To extend previous laboratory findings, a study was designed to investigate organism-drug interactions by determining the in vitro activities of eight parenteral β-lactam agents against 82 clinical and laboratory strains ofKlebsiella, Escherichia,Enterobacter, Citrobacter,Serratia, Morganella, andProteus species that produced 22 different ESBLs, alone or in combination with other β-lactamases. Activities were determined in broth microdilution MIC tests using standard and 100-fold-higher inocula. An inoculum effect, defined as an eightfold or greater MIC increase on testing with the higher inoculum, was most consistently detected with cefepime, cefotaxime, and ceftriaxone and least frequently detected with meropenem and cefoteten. Piperacillin-tazobactam was intermediate between these two groups of agents. Although the inoculum effect is an in vitro laboratory phenomenon, if it has any predictive value in identifying increased risk of therapeutic failure in serious infections, these results support suggestions that cefepime may be a less-than-reliable agent for therapy of infections caused by ESBL-producing strains.

Detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests.

The three-dimensional and clavulanate double-disk potentiation tests were compared as procedures for the detection of extended-spectrum beta-lactamase production in 32 strains of Escherichia coli and Klebsiella pneumoniae, 31 of which produced TEM-1, TEM-2, TEM-3, TEM-4, TEM-5, TEM-7, TEM-8, TEM-9, TEM-10, TEM-12, TEM-101, SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, CAZ-2, MIR-1, or an unidentified extended-spectrum beta-lactamase with a pI of 5.95, with some strains producing multiple beta-lactamases. The three-dimensional test, which was performed in conjunction with a routine disk diffusion test, detected extended-spectrum beta-lactamase production in 26 of 28 (93%) of the strains that produced extended-spectrum beta-lactamases. The clavulanate double-disk potentiation test detected extended-spectrum beta-lactamases in only 22 of the 28 strains (79%) when it was performed as currently recommended. The three-dimensional test, when performed in conjunction with the disk diffusion test, offered the advantages of providing simultaneous information about both antibiotic susceptibility and extended-spectrum beta-lactamase production, coupled with a greater sensitivity and earlier detection of extended-spectrum beta-lactamases. Images

AmpC Disk Test for Detection of Plasmid-Mediated AmpC β-Lactamases in Enterobacteriaceae Lacking Chromosomal AmpC β-Lactamases

ABSTRACT Although plasmid-mediated AmpC β-lactamases were first reported in the late 1980s, many infectious disease personnel remain unaware of their clinical importance. These enzymes are typically produced by isolates of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. and are associated with multiple antibiotic resistance that leaves few therapeutic options. Plasmid-mediated AmpC β-lactamases have been associated with false in vitro susceptibility to cephalosporins. Many laboratories do not test for this resistance mechanism because current tests are inconvenient, subjective, lack sensitivity and/or specificity, or require reagents that are not readily available. In this study a new test, the AmpC disk test, based on filter paper disks impregnated with EDTA, was found to be a highly sensitive, specific, and convenient means of detection of plasmid-mediated AmpC β-lactamases in organisms lacking a chromosomally mediated AmpC β-lactamase. Using cefoxitin insusceptibility as a screen, the test accurately distinguished AmpC and extended-spectrum β-lactamase production and differentiated AmpCs from non-β-lactamase mechanisms of cefoxitin insusceptibility, such as reduced outer membrane permeability. The test is a potentially useful diagnostic tool. It can provide important infection control information and help to ensure that infected patients receive appropriate antibiotic therapy.

Prevalence of Newer β-Lactamases in Gram-Negative Clinical Isolates Collected in the United States from 2001 to 2002

ABSTRACT Newer β-lactamases such as extended-spectrum β-lactamases (ESBLs), transferable AmpC β-lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC β-lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, β-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC β-lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC β-lactamases were widespread. Clinical laboratories must be able to detect important β-lactamases to ensure optimal patient care and infection control.

Occurrence of Newer β-Lactamases in Klebsiella pneumoniae Isolates from 24 U.S. Hospitals

ABSTRACT Despite the discovery of novel β-lactamases such as extended-spectrum β-lactamases (ESBLs), imported AmpC, and carbapenem-hydrolyzing β-lactamases at least a decade ago, there remains a low level of awareness of their importance and how to detect them. There is a need to increase the levels of awareness of clinical laboratories about the detection of newer β-lactamases. Therefore, a study was conducted in 2000 to investigate the occurrence of these β-lactamases in Klebsiella pneumoniae isolates at 24 U.S. medical centers. To enhance the likelihood of detecting imported AmpC and carbapenem-hydrolyzing β-lactamases, participating laboratories were permitted to include archived strains (1996 to 2000) that were intermediate or resistant to either cefoxitin or imipenem. The β-lactamase production of 408 isolates positive by screening of 1,123 isolates was investigated by ESBL phenotypic confirmation tests; and for AmpC and carbapenem-hydrolyzing β-lactamases, three-dimensional tests, isoelectric focusing, β-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular tests were used. ESBL-producing isolates were detected at 18 of the 24 sites (75%), imported AmpC-producing isolates were detected at 10 sites (42%), inducible imported AmpC-producing isolates were detected at 3 sites (12.5%), and a molecular class A carbapenem-hydrolyzing enzyme was detected at 1 site (4%). No class B or D carbapenem-hydrolyzing enzymes were detected. ESBLs and imported AmpC β-lactamases were detected at a significant number of sites, indicating widespread penetration of these enzymes into U.S. medical institutions. Because these enzymes may significantly affect therapeutic outcomes, it is vital that clinical laboratories be aware of them and be able to detect their occurrence.

Version 2000 : the new β-lactamases of Gram-negative bacteria at the dawn of the new millennium

beta-lactamases of Gram-negative bacteria are evolving dynamically. New developments include the production of enzymes with novel substrate profiles, reduced susceptibility to beta-lactamase inhibitors, and the simultaneous production of multiple types of beta-lactamases. The changes represent evolutionary upgrades which provide modern pathogens with a greater potential to resist beta-lactam antibiotics and cause formidable therapeutic, infection control, and diagnostic challenges. This review is a clinically oriented outline of recent developments in the beta-lactamase production of Gram-negative bacteria.

Plasmid-Mediated Carbapenem-Hydrolyzing Enzyme KPC-2 in an Enterobacter sp.

ABSTRACT A strain of an Enterobacter sp. with reduced susceptibility to imipenem, which produced a plasmid-mediated class A carbapenem-hydrolyzing enzyme, KPC-2 β-lactamase, was isolated from a patient with sepsis at a Boston hospital. This is the first report of the production of a plasmid-encoded KPC-2 β-lactamase by an Enterobacter sp.

PLASMID-MEDIATED RESISTANCE TO EXPANDED- SPECTRUM CEPHALOSPORINS AMONG ENTEROBACTER AEROGENES STRAINS

ABSTRACT Resistance to expanded-spectrum cephalosporins commonly develops inEnterobacter aerogenes during therapy due to selection of mutants producing high levels of the chromosomal Bush group 1 β-lactamase. Recently, resistant strains producing plasmid-mediated extended-spectrum β-lactamases (ESBLs) have been isolated as well. A study was designed to investigate ESBL production among 31 clinical isolates of E. aerogenes from Richmond, Va., with decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk potentiation test. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. β-Lactamases were investigated by an isoelectric focusing overlay technique which simultaneously determined isoelectric points (pIs) and substrate or inhibitor profiles. Decreased susceptibility to cefotaxime, ceftazidime, and aztreonam (MIC range, 1 to 64 μg/ml) was detected and associated with resistance to gentamicin and trimethoprim-sulfamethoxazole. All strains produced an inducible Bush group 1 β-lactamase (pI 8.3). Twenty-nine of the 31 isolates also produced an enzyme similar to SHV-4 (pI 7.8), while 1 isolate each produced an enzyme similar to SHV-3 (pI 6.9) and to SHV-5 (pI 8.2). The three different SHV-derived ESBLs were transferred by transconjugation to Escherichia coli C600N and amplified by PCR. Plasmid profiles of the clinical isolates showed a variety of different large plasmids. Because of the linkage of resistance to aminoglycosides and trimethoprim-sulfamethoxazole with ESBL production, it is possible that the usage of these drugs was responsible for selecting plasmid-mediated resistance to extended-spectrum cephalosporins in E. aerogenes. Furthermore, it is important that strains such as these be recognized, because they can be responsible for institutional spread of resistance genes.

Escherichia coli: Development of Carbapenem Resistance During Therapy

A 76-year-old woman had recurrent urosepsis due to extended-spectrum beta -lactamase-positive Escherichia coli. Imipenem resistance was detected after long-term imipenem-meropenem therapy. The carbapenem-hydrolyzing enzyme gene was identified as blaKPC-3. To our knowledge, this is the first documented case in which carbapenem-resistant E. coli emerged during therapy with imipenem and meropenem, and the first identification of the carbapenem-hydrolyzing enzyme in E. coli isolates.