Nancy D. Hanson

DETECTION OF PLASMID-MEDIATED AMPC BETA-LACTAMASE GENES IN CLINICAL ISOLATES BY USING MULTIPLEX PCR

ABSTRACT Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC β-lactamases are limited because these organisms are usually resistant to all the β-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC β-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC β-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC β-lactamase expression in organisms with or without a chromosomal AmpC β-lactamase gene.

Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms

SUMMARY Treatment of infectious diseases becomes more challenging with each passing year. This is especially true for infections caused by the opportunistic pathogen Pseudomonas aeruginosa, with its ability to rapidly develop resistance to multiple classes of antibiotics. Although the import of resistance mechanisms on mobile genetic elements is always a concern, the most difficult challenge we face with P. aeruginosa is its ability to rapidly develop resistance during the course of treating an infection. The chromosomally encoded AmpC cephalosporinase, the outer membrane porin OprD, and the multidrug efflux pumps are particularly relevant to this therapeutic challenge. The discussion presented in this review highlights the clinical significance of these chromosomally encoded resistance mechanisms, as well as the complex mechanisms/pathways by which P. aeruginosa regulates their expression. Although a great deal of knowledge has been gained toward understanding the regulation of AmpC, OprD, and efflux pumps in P. aeruginosa, it is clear that we have much to learn about how this resourceful pathogen coregulates different resistance mechanisms to overcome the antibacterial challenges it faces.

Phenotypic and Molecular Detection of CTX-M-β-Lactamases Produced by Escherichia coli and Klebsiella spp.

ABSTRACT Organisms producing CTX-M-β-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-β-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of Escherichia coli and Klebsiella species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum β-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-β-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for blaCTX-M-1-like genes, 95 (54%) were positive for blaCTX-M-14-like genes, and the remaining 56 (32%) were negative for blaCTX-M genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.

Population-Based Laboratory Surveillance for Escherichia coli–Producing Extended-Spectrum β-Lactamases: Importance of Community Isolates with blaCTX-M Genes

A prospective, population-based laboratory surveillance study was conducted to define the epidemiology of extended-spectrum beta -lactamase-producing Escherichia coli infections in the Calgary Health Region during the years 2000-2002. The incidence was 5.5 cases per 100,000 population per year. The annualized incidence of 3.9 cases per 100,000 population for January through March was significantly lower than the incidence for the other quarters of the year (6.0 per 100,000 population; P<.01). Seventy-one percent of subjects had community-onset disease. Patients aged > or =65 years (22.0 vs. 3.8 cases per 100,000 population per year; P<.0001) and women (9.2 vs. 1.7 cases per 100,000 population per year; P<.0001) had significantly higher rates of infection. Polymerase chain reaction identified 23 (15%) of 157 isolates as positive for blaCTX-M genes from the CTX-M-I subgroup and 87 (55%) from the CTX-M-III subgroup. Ciprofloxacin resistance was independently associated with CTX-M- beta -lactamases (odds ratio, 14.2; 95% confidence interval, 3.69-54.84). Strains of E. coli with blaCTX-M genes commonly cause community-onset infections, and women and older patients are at highest risk.

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.

Molecular epidemiological analysis of Escherichia coli sequence type ST131 (O25:H4) and bla CTX-M-15among extended-spectrum-β- lactamase-producing E. coli from the United States, 2000 to 2009

ABSTRACT Escherichia coli sequence type ST131 (from phylogenetic group B2), often carrying the extended-spectrum-β-lactamase (ESBL) gene blaCTX-M-15, is an emerging globally disseminated pathogen that has received comparatively little attention in the United States. Accordingly, a convenience sample of 351 ESBL-producing E. coli isolates from 15 U.S. centers (collected in 2000 to 2009) underwent PCR-based phylotyping and detection of ST131 and blaCTX-M-15. A total of 200 isolates, comprising 4 groups of 50 isolates each that were (i) blaCTX-M-15 negative non-ST131, (ii) blaCTX-M-15 positive non-ST131, (iii) blaCTX-M-15 negative ST131, or (iv) blaCTX-M-15 positive ST131, also underwent virulence genotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Overall, 201 (57%) isolates exhibited blaCTX-M-15, whereas 165 (47%) were ST131. ST131 accounted for 56% of blaCTX-M-15-positive- versus 35% of blaCTX-M-15-negative isolates (P < 0.001). Whereas ST131 accounted for 94% of the 175 total group B2 isolates, non-ST131 isolates were phylogenetically distributed by blaCTX-M-15 status, with groups A (blaCTX-M-15-positive isolates) and D (blaCTX-M-15-negative isolates) predominating. Both blaCTX-M-15 and ST131 occurred at all participating centers, were recovered from children and adults, increased significantly in prevalence post-2003, and were associated with molecularly inferred virulence. Compared with non-ST131 isolates, ST131 isolates had higher virulence scores, distinctive virulence profiles, and more-homogeneous PFGE profiles. blaCTX-M-15 was associated with extensive antimicrobial resistance and ST131 with fluoroquinolone resistance. Thus, E. coli ST131 and blaCTX-M-15 are emergent, widely distributed, and predominant among ESBL-positive E. coli strains in the United States, among children and adults alike. Enhanced virulence and antimicrobial resistance have likely promoted the epidemiological success of these emerging public health threats.

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.

Association between Handling of Pet Treats and Infection with Salmonella enterica Serotype Newport Expressing the AmpC β-Lactamase, CMY-2

ABSTRACT Resistance to the extended-spectrum cephalosporins can occur in Salmonella species via the production of extended-spectrum and AmpC β-lactamases. We describe human infections with Salmonella enterica serotype Newport phage type 14 strains resistant to ceftazidime (CAZ) and cefoxitin (FOX) related to the handling of pet treats containing dried beef. These strains were isolated from five patients in Calgary, Alberta, Canada, during 2002 and were compared to a strain cultured from a commercial pet treat present at the property of one of the patients. The strains were resistant to FOX, CAZ, cefpodoxime, ampicillin, and chloramphenicol; intermediate resistant to ceftriaxone and cefotaxime; and sensitive to the aminoglycosides, ciprofloxacin, cefepime, and imipenem. Isoelectric focusing, multiplex PCR, and sequencing of the amplicons showed that all strains produced the plasmid-encoded AmpC β-lactamase, CMY-2. Restriction analysis of plasmid DNA following transformation demonstrated that blaCMY-2 was encoded on an approximately 140-kb plasmid. Pulsed-field gel electrophoresis showed the human and pet treat Salmonella strains to be highly related. This study is the first to implicate the transfer of multidrug-resistant Salmonella species through the handling of commercial pet treats containing animal products. In addition to documenting the first cases of human infection caused by CMY-2-producing S. enterica serotype Newport strains in Canada, this study illustrates the necessity of rapid and accurate laboratory-based surveillance in the identification of novel types of antimicrobial resistance.