Characterization and Sequence Analysis of Extended-Spectrum-β-Lactamase-Encoding Genes from Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis Isolates Collected during Tigecycline Phase 3 Clinical Trials

ABSTRACT In concert with the development of novel β-lactams and broad-spectrum cephalosporins, bacterially encoded β-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-β-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the bla TEM, bla SHV, bla OXA, and bla CTX genes from clinical strains. Isolates were also screened for AmpC genes of the bla CMY, bla ACT, bla FOX, and bla DHA families as well as the bla KPC genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of ≥2 μg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, bla CTX-M-type β-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas bla SHV genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC90 = 2 μg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.

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The Occurrence of blaCTX-M, blaSHV, and blaTEM Genes in Extended-Spectrum β-Lactamase-Positive Strains of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis in Poland

International Journal of Antibiotics ◽

10.1155/2014/935842 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 18

Author(s):

Dominika Ojdana ◽

Paweł Sacha ◽

Piotr Wieczorek ◽

Sławomir Czaban ◽

Anna Michalska ◽

...

Keyword(s):

Escherichia Coli ◽

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Proteus Mirabilis ◽

Bacterial Strains ◽

E Coli ◽

Extended Spectrum ◽

Genes Encoding

Bacteria belonging to the Enterobacteriaceae family that produce extended-spectrum β-lactamase (ESBL) enzymes are important pathogens of infections. Increasing numbers of ESBL-producing bacterial strains exhibiting multidrug resistance have been observed. The aim of the study was to evaluate the prevalence of blaCTX-M, blaSHV, and blaTEM genes among ESBL-producing Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis strains and to examine susceptibility to antibiotics of tested strains. In our study, thirty-six of the tested strains exhibited blaCTX-M genes (blaCTX-M-15, blaCTX-M-3, blaCTX-M-91, and blaCTX-M-89). Moreover, twelve ESBL-positive strains harbored blaSHV genes (blaSHV-18, blaSHV-7, blaSHV-2, and blaSHV-5), and the presence of a blaTEM gene (blaTEM-1) in twenty-five ESBL-positive strains was revealed. Among K. pneumoniae the multiple ESBL genotype composed of blaCTX-M-15, blaCTX-M-3, blaSHV-18, blaSHV-7, blaSHV-2, and blaSHV-5 genes encoding particular ESBL variants was observed. Analysis of bacterial susceptibility to antibiotics revealed that, among β-lactam antibiotics, the most effective against E. coli strains was meropenem (100%), whereas K. pneumoniae were completely susceptible to ertapenem and meropenem (100%), and P. mirabilis strains were susceptible to ertapenem (91.7%). Moreover, among non-β-lactam antibiotics, gentamicin showed the highest activity to E. coli (91.7%) and ciprofloxacin the highest to K. pneumoniae (83.3%). P. mirabilis revealed the highest susceptibility to amikacin (66.7%).

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Evaluation of the Clinical and Laboratory Standards Institute phenotypic confirmatory test to detect the presence of extended-spectrum β-lactamases from 4005 Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates

Journal of Medical Microbiology ◽

10.1099/jmm.0.068981-0 ◽

2014 ◽

Vol 63 (4) ◽

pp. 556-561 ◽

Cited By ~ 9

Author(s):

Ian Morrissey ◽

Samuel K. Bouchillon ◽

Meredith Hackel ◽

Douglas J. Biedenbach ◽

Stephen Hawser ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Proteus Mirabilis ◽

Susceptibility Testing ◽

Klebsiella Oxytoca ◽

Confirmatory Test ◽

Predictive Values ◽

E Coli ◽

Extended Spectrum ◽

Phenotype Analysis

A subset of Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates collected for the Study for Monitoring Antimicrobial Resistance Trends that were positive for the Clinical and Laboratory Standards Institute (CLSI) extended-spectrum β-lactamase (ESBL) phenotypic confirmatory test (n = 3245) or had an ertapenem MIC of ≥0.5 µg ml−1 (n = 293), or both (n = 467), were analysed for ESBL genes. Most ESBL phenotype E. coli or K. pneumoniae possessed an ESBL gene (95.8 and 88.4 %, respectively), and this was 93.1 % if carbapenem-non-susceptible K. pneumoniae were removed. This rate was lower for P. mirabilis (73.4 %) and K. oxytoca (62.5 %). Virtually all ESBL-positive isolates (99.5 %) were cefotaxime non-susceptible [CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints)]. Fewer isolates (82 %) were ceftazidime non-susceptible (CLSI breakpoints). In addition, 21.1 % of E. coli, 25 % of K. oxytoca and 78.7 % of P. mirabilis isolates were ceftazidime susceptible but ESBL positive. This suggests that CLSI breakpoints for ceftazidime are too high to detect ESBLs. The lower EUCAST breakpoints detected ESBLs in E. coli and K. oxytoca better, but 59.6 % of ESBL-positive isolates of P. mirabilis were ceftazidime susceptible. For isolates with ertapenem MICs ≥0.5 µg ml−1, more accurate ESBL phenotype analysis was observed for E. coli and K. pneumoniae (sensitivity >95 % for both, specificity 94.4 and 54.1 %, respectively). If carbapenemase-positive K. pneumoniae were excluded, the specificity increased to 78 %. The positive predictive values for the ESBL phenotypic test with E. coli and K. pneumoniae were 97.6 and 81.8 %, respectively, and negative predictive values were 75.9 and 95.2 %, respectively. We therefore suggest that it would be prudent to confirm phenotypic ESBL-positive P. mirabilis, K. pneumoniae and K. oxytoca with molecular analysis.

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Community spread of extended-spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis: a long-term study in Japan

Journal of Medical Microbiology ◽

10.1099/jmm.0.059279-0 ◽

2013 ◽

Vol 62 (7) ◽

pp. 1038-1043 ◽

Cited By ~ 45

Author(s):

Yong Chong ◽

Shinji Shimoda ◽

Hiroko Yakushiji ◽

Yoshikiyo Ito ◽

Toshihiro Miyamoto ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Proteus Mirabilis ◽

Epidemiological Data ◽

Detection Rates ◽

E Coli ◽

Long Term Study ◽

Extended Spectrum ◽

Few Data

Community-acquired infections caused by extended-spectrum β-lactamase (ESBL)-producing bacteria, particularly CTX-M-producing Escherichia coli, are a rising concern worldwide. There are few data from Japan on the acquisition of ESBLs in the community or the influx of these bacteria into hospitals. Therefore, we examined the prevalence of ESBL carriage in outpatients, in order to estimate the spread of ESBLs in community settings. We analysed bacterial isolates from outpatient samples at our institution over a 9-year period from 2003 to 2011, with respect to epidemiological data on ESBL-producing bacteria and their genotypic features. Out of 5137 isolates, 321 (6.3 %) were ESBL producers, including E. coli, Klebsiella pneumoniae and Proteus mirabilis. The detection rates of the ESBL-producing isolates gradually increased and reached 14.3, 8.7 and 19.6 % for E. coli, K. pneumoniae and P. mirabilis strains, respectively, in 2011. Genotyping analysis showed that many of the strains produced multiple β-lactamases, including TEM, SHV and CTX-M, rather than just CTX-M. The CTX-M-9 group was dominant among the CTX-M genotypes; further, the CTX-M-1 and M-2 groups were also detected (~30 %). This is believed to be the first report from Japan showing a definite increase in ESBL detection in outpatients. In addition, our findings suggest the simultaneous community spread of diverse ESBL genotypes, not an expansion of particular ESBL genes.

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CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum β-lactamase in clinical isolates of Escherichia coli from Korea

Journal of Medical Microbiology ◽

10.1099/jmm.0.004507-0 ◽

2009 ◽

Vol 58 (2) ◽

pp. 261-266 ◽

Cited By ~ 53

Author(s):

Wonkeun Song ◽

Hyukmin Lee ◽

Kyungwon Lee ◽

Seok Hoon Jeong ◽

Il Kwon Bae ◽

...

Keyword(s):

Escherichia Coli ◽

Hydrolytic Activity ◽

Common Type ◽

Clinical Isolates ◽

Confirmatory Test ◽

Dominant Type ◽

E Coli ◽

Extended Spectrum ◽

Genes Encoding ◽

M 14

This study was performed to assess the prevalence and genotypes of plasmid-borne extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Escherichia coli in Korea. A total of 576 isolates of E. coli was collected from 12 Korean hospitals during May and July 2007. A phenotypic confirmatory test detected ESBLs in 82 (14.2 %) of the 576 E. coli isolates. The most common types of ESBLs identified were CTX-M-14 (n=32) and CTX-M-15 (n=27). The prevalence and diversity of the CTX-M mutants, including CTX-M-15, CTX-M-27 and CTX-M-57, with significant hydrolytic activity against ceftazidime were increased. PCR experiments detected genes encoding plasmid-borne AmpC β-lactamases in 15/56 cefoxitin-intermediate or cefoxitin-resistant isolates, and the most common type of AmpC β-lactamase identified was DHA-1 (n=10). These data suggest that the incidence of ESBLs in E. coli has increased as a result of the dissemination of CTX-M enzymes in Korea. In addition, CTX-M-22, CTX-M-27 and CTX-M-57 have appeared in Korea.

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Cefotaximases (CTX-M-ases), an expanding family of extended-spectrum β-lactamases

Canadian Journal of Microbiology ◽

10.1139/w03-111 ◽

2004 ◽

Vol 50 (3) ◽

pp. 137-165 ◽

Cited By ~ 57

Author(s):

Jan Walther-Rasmussen ◽

Niels Høiby

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Salmonella Typhimurium ◽

Vibrio Cholerae ◽

Proteus Mirabilis ◽

Aeromonas Hydrophila ◽

Insertion Sequences ◽

Class A ◽

Extended Spectrum ◽

Genes Encoding

Among the extended-spectrum β-lactamases, the cefotaximases (CTX-M-ases) constitute a rapidly growing cluster of enzymes that have disseminated geographically. The CTX-M-ases, which hydrolyze cefotaxime efficiently, are mostly encoded by transferable plasmids, and the enzymes have been found predominantly in Enterobacteriaceae, most prevalently in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis. Isolates of Vibrio cholerae, Acinetobacter baumannii, and Aeromonas hydrophila encoding CTX-M-ases have also been reported. The CTX-M-ases belong to the molecular class A β-lactamases, and the enzymes are functionally characterized as extended-spectrum β-lactamases. This group of β-lactamases confers resistance to penicillins, extended-spectrum cephalosporins, and monobactams, and the enzymes are inhibited by clavulanate, sulbactam, and tazobactam. Typically, the CTX-M-ases hydrolyze cefotaxime more efficiently than ceftazidime, which is reflected in substantially higher MICs to cefotaxime than to ceftazidime. Phylogenetically, the CTX-M-ases are divided into four subfamilies that seem to have descended from chromosomal β-lactamases of Kluyvera spp. Insertion sequences, especially ISEcp1, have been found adjacent to genes encoding enzymes of all four subfamilies. The class I integron-associated orf513 also seems to be involved in the mobilization of blaCTX-M genes. This review discusses the phylogeny and the hydrolytic properties of the CTX-M-ases, as well as their geographic occurrence and mode of spread.Key words: extended-spectrum β-lactamases, cefotaximases, phylogeny, dissemination, hydrolytic properties.

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Molecular Characterization and Epidemiology of Extended-Spectrum- β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolates Causing Health Care-Associated Infection in Thailand, Where the CTX-M Family Is Endemic

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00171-08 ◽

2008 ◽

Vol 52 (8) ◽

pp. 2818-2824 ◽

Cited By ~ 123

Author(s):

Pattarachai Kiratisin ◽

Anucha Apisarnthanarak ◽

Chaitat Laesripa ◽

Piyawan Saifon

Keyword(s):

Escherichia Coli ◽

Health Care ◽

Klebsiella Pneumoniae ◽

Antimicrobial Agents ◽

Upstream Region ◽

University Hospitals ◽

E Coli ◽

Extended Spectrum ◽

Genes Encoding ◽

Health Care Associated Infection

ABSTRACT Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae have rapidly spread worldwide and pose a serious threat for health care-associated (HA) infection. We conducted molecular detection and characterization of ESBL-related bla genes, including bla TEM, bla SHV, bla CTX-M, bla VEB, bla OXA, bla PER, and bla GES, among 362 isolates of ESBL-producing E. coli (n = 235) and ESBL-producing K. pneumoniae (n = 127) collected from patients who met the definition of HA infection at two major university hospitals in Thailand from December 2004 to May 2005. The prevalence of ESBL-producing E. coli and ESBL-producing K. pneumoniae, patient demographics and the susceptibilities of these bacteria to various antimicrobial agents were described. A total of 87.3% of isolates carried several bla genes. The prevalence of bla CTX-M was strikingly high: 99.6% for ESBL-producing E. coli (CTX-M-14, -15, -27, -40, and -55) and 99.2% for ESBL-producing K. pneumoniae (CTX-M-3, -14, -15, -27, and -55). ISEcp1 was found in the upstream region of bla CTX-M in most isolates. Up to 77.0% and 71.7% of ESBL-producing E. coli and ESBL-producing K. pneumoniae, respectively, carried bla TEM; all of them encoded TEM-1. ESBL-producing K. pneumoniae carried bla SHV at 87.4% (SHV-1, -2a, -11, -12, -27, -71, and -75) but only at 3.8% for ESBL-producing E. coli (SHV-11 and -12). bla genes encoding VEB-1 and OXA-10 were found in both ESBL-producing E. coli (8.5% and 8.1%, respectively) and ESBL-producing K. pneumoniae (10.2% and 11.8%, respectively). None of the isolates were positive for bla PER and bla GES. Pulsed-field gel electrophoresis analysis demonstrated that there was no major clonal relationship among these ESBL producers. This is the first study to report CTX-M-3, CTX-M-27, CTX-M-40, SHV-27, SHV-71, and SHV-75 in Thailand and to show that CTX-M ESBL is highly endemic in the country.

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Genotypic detection and molecular epidemiology of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a regional hospital in central Taiwan

Journal of Medical Microbiology ◽

10.1099/jmm.0.015818-0 ◽

2010 ◽

Vol 59 (6) ◽

pp. 665-671 ◽

Cited By ~ 20

Author(s):

Chin-Fu Lin ◽

Shih-Kuang Hsu ◽

Chao-Hsien Chen ◽

Jr-Rung Huang ◽

Hsueh-Hsia Lo

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Regional Hospital ◽

Specific Primers ◽

E Coli ◽

Extended Spectrum ◽

Genes Encoding ◽

Central Taiwan ◽

Esbl Producers ◽

M 14

This study was conducted to detect the genes encoding extended-spectrum β-lactamases (ESBLs) and determine the epidemiological relatedness of 69 Escherichia coli and 33 Klebsiella pneumoniae isolates collected from a regional hospital in central Taiwan, mostly from inpatients (E. coli 87.0 %; K. pneumoniae 88.0 %). The phenotypes of these isolates were examined according to the combination disc method recommended by the Clinical and Laboratory Standards Institute. Most of the ESBL-producing E. coli and K. pneumoniae isolates (98.6 % and 97 %, respectively) could be detected using cefotaxime discs with and without clavulanate. Genotyping was performed by PCR with type-specific primers. CTX-M-14 type (53.6 %) was the most prevalent ESBL among E. coli isolates while SHV type (57.6 %) was the most dominant among K. pneumoniae isolates. Six E. coli and three K. pneumoniae isolates did not carry genes encoding ESBLs of types TEM, SHV, CTX-M-3, CTX-M-14, CMY-2 and DHA-1. The co-existence of two or more kinds of ESBL in a single isolate was common, occurring in 40.6 % and 72.7 % of E. coli and K. pneumoniae isolates, respectively. PFGE analysis revealed that ESBL producers isolated in this setting were genetically divergent.

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Prevalence and Characterization of Extended-Spectrum β-Lactamase-Producing Antibiotic-Resistant Escherichia coli and Klebsiella pneumoniae in Ready-to-Eat Street Foods

Antibiotics ◽

10.3390/antibiotics10070850 ◽

2021 ◽

Vol 10 (7) ◽

pp. 850

Author(s):

Shobha Giri ◽

Vaishnavi Kudva ◽

Kalidas Shetty ◽

Veena Shetty

Keyword(s):

Escherichia Coli ◽

Food Safety ◽

Klebsiella Pneumoniae ◽

Rural Areas ◽

Significant Risk ◽

Microbiological Quality ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

E Coli ◽

Extended Spectrum

As the global urban populations increase with rapid migration from rural areas, ready-to-eat (RTE) street foods are posing food safety challenges where street foods are prepared with less structured food safety guidelines in small and roadside outlets. The increased presence of extended-spectrum-β-lactamase (ESBL) producing bacteria in street foods is a significant risk for human health because of its epidemiological significance. Escherichia coli and Klebsiella pneumoniae have become important and dangerous foodborne pathogens globally for their relevance to antibiotic resistance. The present study was undertaken to evaluate the potential burden of antibiotic-resistant E. coli and K. pneumoniae contaminating RTE street foods and to assess the microbiological quality of foods in a typical emerging and growing urban suburb of India where RTE street foods are rapidly establishing with public health implications. A total of 100 RTE food samples were collected of which, 22.88% were E. coli and 27.12% K. pneumoniae. The prevalence of ESBL-producing E. coli and K. pneumoniae was 25.42%, isolated mostly from chutneys, salads, paani puri, and chicken. Antimicrobial resistance was observed towards cefepime (72.9%), imipenem (55.9%), cefotaxime (52.5%), and meropenem (16.9%) with 86.44% of the isolates with MAR index above 0.22. Among β-lactamase encoding genes, blaTEM (40.68%) was the most prevalent followed by blaCTX (32.20%) and blaSHV (10.17%). blaNDM gene was detected in 20.34% of the isolates. This study indicated that contaminated RTE street foods present health risks to consumers and there is a high potential of transferring multi-drug-resistant bacteria from foods to humans and from person to person as pathogens or as commensal residents of the human gut leading to challenges for subsequent therapeutic treatments.

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Evaluation of extended spectrum beta lactamase enzymes prevalence in clinical isolates of Escherichia coli

Microbiology Research ◽

10.4081/mr.2011.e8 ◽

2011 ◽

Vol 2 (1) ◽

pp. 8

Author(s):

Ronak Bakhtiari ◽

Jalil Fallah Mehrabadi ◽

Hedroosha Molla Agamirzaei ◽

Ailar Sabbaghi ◽

Mohammad Mehdi Soltan Dallal

Keyword(s):

Escherichia Coli ◽

Disk Diffusion ◽

Confirmatory Test ◽

Diffusion Method ◽

Multi Drug Resistance ◽

Beta Lactamase ◽

Disk Diffusion Method ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Extended Spectrum

Resistance to b-lactam antibiotics by gramnegative bacteria, especially Escherichia coli (E. coli), is a major public health issue worldwide. The predominant resistance mechanism in gram negative bacteria particularly E. coli is via the production of extended spectrum beta lactamase (ESBLs) enzymes. In recent years, the prevalence of b-lactamase producing organisms is increased and identification of these isolates by using disk diffusion method and no-one else is not satisfactory. So, this investigation focused on evaluating the prevalence of ESBL enzymes by disk diffusion method and confirmatory test (Combined Disk). Five hundred clinical samples were collected and 200 E. coli isolates were detected by standard biochemical tests. To performing initial screening of ESBLs was used from Disk diffusion method on E. coli isolates. A confirmation test (Combined Disk method) was performed on isolates of resistant to cephalosporin's indicators. Up to 70% isolates exhibited the Multi Drug Resistance phenotype. In Disk diffusion method, 128(64%) E. coli isolates which resistant to ceftazidime and cefotaxime while in Combined Disk, among 128 screened isolates, 115 (89.8%) isolates were detected as ESBLs producers. This survey indicate beta lactamase enzymes are playing a significant role in antibiotic resistance and correct detection of them in phenotypic test by using disk diffusion and combined Disk is essential for accurate recognition of ESBLs.

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