Molecular epidemiology and resistance patterns of blaOXA-48 Klebsiella pneumoniae and Escherichia coli: A nationwide multicenter study in Taiwan

BackgroundWe describe the molecular epidemiology and resistance patterns of bla_OXA-48 Klebsiella pneumoniae and Escherichia coli in Taiwan.MethodsIn this multicenter surveillance study from January 2012 to August 2015, the identified bla_OXA-48 Enterobacteriaceae isolates were subjected to antibiotics susceptibility testing. PCR method was used for detecting concomitant other beta-lactamases. Outer membrane porins were analyzed. Genetic relatedness and molecular epidemiology of the isolates were determined through pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid incompatibility was determined using PCR-based replicon typing.ResultsForty-three bla_OXA-48 K. pneumoniae and two E. coli isolates were analyzed. The annual incidence of bla_OXA-48 K. pneumoniae isolates from 2012 to 2015 were 0%, 1.1%, 2.4%, and 7.6%, respectively. Forty-three (95.5%) of 45 isolates were non-susceptible to broad-spectrum beta-lactams (ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam), Forty-two (93.3%) of the 45 isolates showed resistance against all tested carbapenems (imipenem, meropenem, doripenem, and ertapenem). Molecular characterization revealed that they co-produced at least one extended-spectrum beta-lactamases or AmpC beta-lactamases, with at least one outer membrane porin loss. Thirty-eight (88.3%) of the 43 K. pneumoniae isolates belonged to ST11. PFGE analysis of 43 K. pneumoniae isolates revealed dissemination of multiple clones. Six of the 12 tested K. pneumoniae representatives of different pulso-types belonged to IncA/C.ConclusionConcomitant loss of porins and production of other beta-lactamases renders the bla_OXA-48-producing isolates in Taiwan a high-level carbapenem resistance and broad resistance against many beta-lactam antibiotics. Following dissemination of multiple clones of bla_OXA-48 K pneumoniae ST 11, a trend of increased bla_OXA-48 prevalence was noted.     

Detection of β-lactamase and urovirulence genes in Escherichia coli serogroups isolated from urinary tract infection in cats

Sixty-five Escherichia coli isolates were recovered from cats with urinary tract infection and their serogroups were determined. The isolates were screened for the presence of β-lactamase (bla _TEM) and urovirulence (cnf1, cdtB, afaIB-C, papE-F, sfa/focD-E and iutA) genes. Serogroup determination of 65 uropathogenic E. coli isolates showed that 23 (35.38%) were O-typeable and belonged to 11 different O serogroups including: O1, O2, O4, O6, O8, O11, O25, O49, O75, O115 and O125. Fourteen (21.54%) isolates were positive for the bla _TEM gene. The bla _TEM-positive isolates were distributed in both typeable (five) and non-typeable (nine) serogroups. Eleven bla _TEM-positive isolates showed combination patterns of virulence genes, whereas the combination of cnf1-hlyA-papE-F-sfa/focDE was the most prevalent pattern. Polymerase chain reaction tests revealed that 38 isolates (58.46%) contained at least two urovirulence genes. Among the examined isolates, 56.92% had cnf1, 52.30% hlyA, 50.76% sfa/focD-E, 47.69% papE-F, 13.84% iutA and 6.15% afaIB-C. None of the isolates contained the cdtB gene. Eight different combinations of virulence genes were detected. The combination pattern of cnf1-hlyA-papE-F-sfa/focD-E was the most prevalent (30.77%), whereas the positive isolates fell into O2 (three), O6 (two), and O49 (one) serogroup and 14 isolates were O-non-typeable. In conclusion, feline E. coli isolates from urinary tract infections belonged to several serogroups and may possess β-lactamase and urovirulence coding genes.     

Characterization of carbapenem-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> from a healthcare region in Hong Kong

Carbapenem-resistant Enterobacteriaceae represents a major public health issue. This study investigated the clonality and resistance mechanisms of 92 carbapenem-resistant E. coli (n?=?21) and K. pneumoniae (n?=?71) isolates collected consecutively from clinical specimens and patients at high risk of carriage between 2010 and 2012 in a healthcare region in Hong Kong. Combined disk tests (CDTs) and the Carba NP test were used for phenotypic detection of carbapenemases. PCR assays were used to detect carbapenemase genes. All isolates were intermediate or resistant to at least one carbapenem. Nine (9.8 %) isolates were genotypic carbapenemase producers and included six K. pneumoniae (one ST1306/bla _IMP-4, one ST889/bla _IMP-4, two ST11/bla _KPC-2, one ST258/bla _KPC-2, one ST483/bla _NDM-1) and three E. coli (one ST131/bla _IMP-4, two ST744/ bla _NDM-1) isolates. All nine isolates carrying carbapenemase genes could be detected by the CDTs and the Carba NP test. PCR identified bla _CTX-M and bla _AmpC alone or in combination in 77.8 % (7/9) and 96.4 % (80/83) of the carbapenemase-producers and non-producers, respectively. Porin loss was detected in 22.2 % (2/9) and 59.0 % (49/83) of the carbapenemase-producers and non-producers, respectively. Overall, the E. coli clones were diverse (14 different STs), but 36.6 % (26/71) of the K. pneumoniae isolates belonged to ST11. In conclusion, the prevalence of carbapenemases among carbapenem-nonsusceptible E. coli and K. pneumoniae remained low in Hong Kong. Porin loss combined with AmpC and/or CTX-M type ESBL was the major mechanism of carbapenem resistance in the study population.     

Klebsiella pneumoniae: development of a mixed population of carbapenem and tigecycline resistance during antimicrobial therapy in a kidney transplant patient

Nine isolates of Klebsiella pneumoniae were isolated from a renal transplant patient suffering from recurrent urosepsis over a period of 4 months. Imipenem resistance was detected after imipenem-ertapenem therapy. When treatment was switched to tigecycline the K. pneumoniae developed resistance to tigecycline (MIC = 8 mg/L). The nine isolates were tested by determination of agar dilution MICs, phenotypic carbapenemase, extended-spectrum beta-lactamases and metallo-beta-lactamase (MBL) testing and pulsed-field gel electrophoresis. Polymerase chain reaction and sequencing analysis were employed for identification of bla genes and mapping of the integron carrying the MBL gene. The nine isolates were clonally related and all produced the SHV-12 enzyme. Five MBL-producing isolates showed imipenem MICs ranging from 2 to 64 mg/L and all were detected by testing with imipenem and EDTA. The five isolates harboured the bla_VIM-1 gene. Three isolates showed increased tigecycline MICs (4–8 mg/L). Serial blood cultures obtained on the same day resulted in a VIM-positive/tigecycline-susceptible and a VIM-negative/tigecycline-resistant K. pneumoniae isolate. No isolate developed concurrent imipenem and tigecycline resistance. The patient had a persistent urinary tract infection and recurrent bacteraemia caused by a mixed population of Klebesiella pneumoniae isolates adapting to the selective pressure of antimicrobial therapy at the time. The present study is a worrisome example of what could happen when an immunocompromised host is subjected to the pressures of antimicrobial therapy. In addition, we report the first treatment-emergent MIC increase of tigecycline from 0.5 to 8 mg/L in K. pneumoniae.     

Differences in the antibiotic susceptibility of human Escherichia coli with poultry-associated and non-poultry-associated extended-spectrum beta-lactamases

The concurrent presence of bla _CTX-M-1 and bla _TEM-52 genes on similar plasmids of Escherichia coli isolated from poultry, chicken meat and humans supports the occurrence of food-borne transmission of extended-spectrum beta-lactamase (ESBL) genes. ESBL-producing E. coli (ESBL-E. coli) are most frequently detected in hospitalised patients and are known to spread in healthcare settings. We hypothesised that poultry-associated (PA) ESBL genes are predominant in the community, where acquisition is fuelled by food contamination, whereas non-PA ESBL genes are predominant in hospitals, with acquisition fuelled by cross-transmission. Then, differences in antimicrobial selective pressure in hospitals and poultry would create differences in co-resistance between PA and non-PA ESBL-E. coli. We, therefore, determined the prevalence and co-resistance of PA and non-PA ESBL-E. coli in community-acquired and nosocomial urinary tract infections in humans and bla _CTX-M-1 and bla _TEM-52 isolates from poultry. A total of 134 human ESBL-E. coli urine isolates were included in this study. Isolates containing bla _CTX-M-1 or bla _TEM-52 were considered to be PA, with the remainder being non-PA. Also, 72 poultry ESBL-E. coli were included. Minimum inhibitory concentration (MIC) values were determined by broth microdilution. The prevalence of PA ESBL genes in isolates obtained in general practice and hospitals was 28 % versus 30 % (n.s.). Human PA ESBL-E. coli were more frequently susceptible to ciprofloxacin (51 % vs. 25 %; p?=?0.0056), gentamicin (86 % vs. 63 %; p?=?.0.0082), tobramycin (91 % vs. 34 %; p?=?0.0001) and amikacin (98 % vs. 67 %; p?=?0.0001) compared to human non-PA ESBL-E. coli. PA ESBL-E. coli are not more prevalent in community acquired than nosocomial urine samples, but are more often susceptible to ciprofloxacin and aminoglycosides than non-PA ESBL-E. coli. This does not support the existence of different reservoirs of ESBL genes.     

Risk factors for extended-spectrum β-lactamase positivity in uropathogenic Escherichia coli isolated from community-acquired urinary tract infections

The aim of this prospective cohort study was to determine the risk factors for community-acquired urinary tract infections (UTIs) caused by extended-spectrum β-lactamase (ESBL)-positive Escherichia coli and the distribution of the ESBL enzyme types. Structured forms were filled in for patients diagnosed with community-acquired UTI in four different geographical locations in Turkey. The forms and the isolates were sent to the central laboratory at Baskent University Hospital, Ankara. Antimicrobial susceptibility was determined according to the CLSI criteria. PCR and DNA sequencing were used to characterize the bla_TEM, bla_CTX-M and bla_SHV genes. Multivariate analysis was performed using logistic regression. A total of 510 patients with UTI caused by Gram-negative bacteria were included in this study. ESBLs were detected in 17 of 269 (6.3%) uropathogenic E. coli isolates from uncomplicated UTIs and 34 of 195 (17.4%) E. coli isolates from complicated UTIs (p <0.001). According to multivariate analysis, more than three urinary tract infection episodes in the preceding year (OR 3.8, 95% CI 1.8–8.1, p <0.001), use of a β-lactam antibiotic in the preceding 3 months (OR 4.6, 95% CI 2.0–0.7, p <0.001) and prostatic disease (OR 9.6, 95% CI 2.1–44.8, p 0.004) were found to be associated with ESBL positivity. The percentages of isolates with simultaneous resistance to trimethoprim-sulphamethoxazole, ciprofloxacin and gentamicin were found to be 4.6% in the ESBL-negative group and 39.2% in the ESBL-positive group (p <0.001). Forty-six of 51 ESBL-positive isolates (90.2%) were found to harbour CTX-M-15. Therapeutic alternatives for UTI, particularly in outpatients, are limited. Further clinical studies are needed to guide the clinicians in the management of community-acquired UTIs.     

Metallo-β-lactamases among Enterobacteriaceae from routine samples in an Italian tertiary-care hospital and long-term care facilities during 2008

The emergence of metallo-β-lactamase (MBL)-producing Enterobacteriaceae is a serious public health concern. Producers have been repeatedly isolated from patients and long-term care facility (LTCF) residents around Bolzano, and we sought to assess their prevalence and clinical impact. All routine Enterobacteriaceae isolates from a Bolzano tertiary-care hospital and associated long-term care facilities in 2008 (n = 5500) were screened for MBLs, with case details reviewed for the source patients. In total, 36 producers were obtained from 29 patients, comprising 14 Escherichia coli, six Klebsiella pneumoniae, four Klebsiella oxytoca, four Citrobacter freundii, two Enterobacter cloacae and two Morganella morganii, as well as single Citrobacter amalonaticus, Enterobacter aerogenes, Providencia stuartii and Proteus mirabilis isolates. All were PCR-positive for bla_VIM and 25 were PCR-positive for qnrS; 19 non-K. pneumoniae had bla_SHV and one had bla_CTX-M-group1; 13 were from 12 LTCF residents and 23 were from 17 acute-care patients. All these patients had serious underlying diseases with prolonged hospitalization or LTCF stay; only seven had infections due to the MBL producers, comprising four urinary tract infections, two catheter-related bloodstream infections and one patient with both a surgical site infection and pneumonia. Five patients had more than one MBL-producing organism. Pulsed-field gel electrophoresis identified a cluster of six related E. coli, whereas pairs of K. pneumoniae and C. freundii isolates had >85% profile similarity. Transformants prepared from two isolates were shown to be PCR-positive for bla_VIM, qnrS and bla_SHV; their plasmids gave similar restriction fragment length polymorphism patterns, and bla_VIM-1, qnrSI and bla_SHV-12 were detected by sequencing.     

Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases

Objectives: The objective of this study is to determine the prevalence of antibiotic resistance factors, including the production of extended-spectrum beta-lactamases (ESBLs) and the presence of class 1 integrons among Escherichia coli isolated from clinical specimens. Materials and Methods: Bacterial species identification was performed using a VITEK-2 system (VITEK2 GN-card; bioMérieux, France). Antimicrobial susceptibility testing was determined using the disk diffusion method according to the 2010 Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was used to detect integrons and amplify variable regions of the bla_TEM, bla_SHV and blaCTX-M genes. Gene cassettes were detected by deoxyribonucleic acid sequencing. Results: In this study, 58% (100/172) of clinical E. coli isolates were identified as ESBL producers. We found that 90% of the ESBL-producing E. coli isolates harbored the bla_CTX-M gene, whereas only 59% and 32% possessed the bla_TEM and bla_SHV genes respectively. The presence of class 1 integrons was based on the detection of the integrase gene by PCR. A total of 69% of the ESBL-producing isolates were integron-positive. Resistance to 10 antibiotics, including quinolones, sulfonamides and β-lactam/enzyme inhibitors, was significantly higher in the class 1 integron-positive isolates (P < 0.05). The occurrence of class 1 integrons in bla_TEM, bla_SHV and bla_CTX-M gene carriers was 72.9%, 84.4% and 68.9%, respectively. Class 1 integrons were detected in 61.5% of the isolates with only one ESBL genotype, but in 69.0% and 92.3% of the isolates with two or three different ESBL genotypes, respectively. Conclusions: Our findings indicate that clinical strains of bacteria with multiple ESBL genotypes may have greater opportunities to carry class 1 integrons.     

Prevalence and antimicrobial susceptibility of extended-spectrum beta lactamases-producing Escherichia coli and Klebsiella pneumoniae isolated in selected hospitals of Anyigba,Nigeria

BackgroundEscherichia coli and Klebsiella pneumoniae are commonly implicated in urinary tract infections accounting for majority of the antimicrobial resistance encountered in hospitals.ObjectivesTo determine the prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamases (ESBLs) producing E. coli and K. pneumoniae among patients in Anyigba, Nigeria.MethodsThis hospital-based cross-sectional study was conducted using urine samples from 200 patients of Grimmard Catholic hospital and Maria Goretti hospital. Urine samples were processed to identify ESBL-producing E. coli and K. pneumoniae using standard microbiological techniques. Isolates were then tested against antimicrobial agents.ResultsA total of 156 bacterial isolates were recovered consisting 128 of E. coli and 28 of K. pneumoniae. Extended spectrum beta-lactamases production was observed in 69% of E. coli and 31% of K. pneumoniae. These pathogens were resistant to 3 or more antibiotics. Of the antimicrobials tested, cefotaxime demonstrated the highest rates of resistance (100%) for both ESBL-producing E. coli and K. pneumoniae. Fifty-four isolates of ESBL-producing E. coli showed a high level of resistance to amoxicillin clavulanic acid (83.3%), ciprofloxacin (83.3%), and ceftazidime (79.6%). ESBL-positive K. pneumoniae isolates were highly resistant to ciprofloxacin (75%), and amoxicillin clavulanic acid (83.3%). Cefoxitin (62.5%) and gentamicin (66.7%) showed substantially higher rates of resistance against these isolates while all 24 strains were resistant to imipenem.ConclusionThis study indicated the prevalence of ESBL-positive Gram-negative pathogens in these study sites and also demonstrated their resistance to a few antibiotics. This highlights the need for new antimicrobials that are potent and improved policy on use of antibiotics.     

Escherichia coli clonal group A causing bacteraemia of urinary tract origin

Escherichia coli clonal group A (CgA) causes disease in humans. This is the first study investigating the prevalence of CgA among E. coli from non-urine, extraintestinal infections in a northern European country. E. coli blood (n = 196) and paired urine (n = 195) isolates from the same patients with bacteraemia of urinary tract origin were analysed. The isolates were collected from January 2003 through May 2005 at four hospitals in Copenhagen, Denmark. Pulsed-field gel electrophoresis (PFGE) patterns, antimicrobial resistance and patient characteristics were determined for all CgA isolates; presence of virulence-associated genes (VAGs) and serotypes were determined for the blood CgA isolates. Thirty blood isolates (15%) belonged to CgA. CgA blood isolates were associated with female patients and sulfamethoxazole-trimethoprim resistance and they harboured a distinctive VAG profile. The blood and urine isolates from each pair were found to be related in 26 of 27 CgA blood/urine pairs, confirming a urinary tract origin of infection. Furthermore, a relationship between the PFGE patterns of CgA blood/urine isolates and CgA isolates from UTI patients in general practice and a CgA isolate from a community-dwelling human reported previously, was found, suggesting a community origin of CgA. The finding of CgA strains in 15% of the E. coli bloodstream infections with a urinary tract origin in Denmark suggests that CgA constitutes an important clonal lineage among extraintestinal pathogenic E. coli. A reservoir of this pathogenic E. coli group in the community causing not only UTI but also more severe infections such as bacteraemia has implications for public health.     

Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii

Purpose: The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla_NDM-1 gene using TaqMan probes among clinical isolates. Materials and Methods: Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. Results: TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla_NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12–17) cycles (mean Cp value 14), indicating number of bla_NDM-1 gene copies of 106–108. The wider range of Cp values (15–34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla_NDM-1 gene copies of 103–108. Conclusions: The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla_NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla_NDM-1 gene copies per specimen of DNA.     

Outbreak of KPC-3-producing,and colistin-resistant,Klebsiella pneumoniae infections in two Sicilian hospitals

We report the first outbreak caused by colistin-resistant Klebsiella pneumoniae producing KPC-3 carbapenamase in two Italian hospitals. This spread occurred in 1 month, and was caused by eight colistin-resistant and carbapenem-resistant Klebsiella pneumoniae isolates from eight patients. A further three isolates were obtained from the intestinal tract and pharyngeal colonization. All isolates were multidrug-resistant (MDR), including being resistant to colistin, but they were susceptible to gentamicin and tigecycline. PCR detection showed that all isolates harboured the bla_KPC-3 gene associated with bla_SHV-11, bla_TEM-1 and bla_OXA-9. All K. pneumoniae isolates, genotyped by pulsed-field gel electrophoresis and multilocus sequence typing, belonged to the same sequence type (ST)258 clone. From our data and a review of the international literature, K. pneumoniae ST258 seems to be the most widespread genetic background for KPC dissemination in Europe.     

Emergence of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae during the years 2000 and 2004 in Helsinki,Finland

The molecular epidemiology of 33 Escherichia coli and 81 Klebsiella pneumoniae extended-spectrum β-lactamase-producing health-care-associated and community-acquired isolates collected in the Helsinki district during 2000–2004 was investigated. Clonality studies, antimicrobial susceptibility and genotyping of the isolates were performed. Newly emerging CTX-M-producing E. coli and bla_SHV-12-producing K. pneumoniae isolates were detected. Clonal clusters of both species persisted throughout the study period.     

Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014–2018 national laboratory surveillance

ObjectivesCarbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system.MethodsMeropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS).ResultsIn total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with bla_OXA-48 being predominant (38%, 336/892), followed by bla_NDM-1 (16%, 145/892). For the first time in the Netherlands, bla_OXA-181, bla_OXA-232 and bla_VIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse.ConclusionsThe CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are bla_OXA-48 and bla_NDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem.     

Characterization of plasmid-mediated AmpC-producing E. coli from Swedish broilers and association with human clinical isolates

A selection of plasmid-mediated AmpC-producing Escherichia coli isolates carrying bla_CMY-2 from Swedish broilers were characterized to establish their relatedness to and a possible overlap with human clinical E. coli isolates. The results showed diversity among the E. coli isolated from broilers, indicating that the spread in the population was not due to one strain. However, only one type of plasmid belonging to replicon type incK was identified. Furthermore, there were no indications of spread of bla_CMY-2 E. coli isolates from broilers to human clinical settings, although Swedish broilers may be a source of bla_CMY-2 and/or the plasmid carrying bla_CMY-2.     

Prevalence and types of extended spectrum β-lactamases among urinary Escherichia coli isolates in Moroccan community

This study was designed to characterize extended-spectrum-β-lactamases (ESBL) produced by Escherichia coli isolates causing community urinary tract infections over a 2-year period (2010 and 2011) in a Moroccan large geographical region. Molecular characterization was done by using PCR and sequencing of the β-lactamases genes and plasmid-mediated quinolone resistance determinants. Among 1174 isolates, 49 (4.1%) were ESBL producers. The bla_CTx-M-15 (n = 31) was the most frequent ESBL gene detected, followed by bla_CTx-M-1 (n = 5), bla_SHV-12 (n = 6), bla_PER-2 (n = 3), then bla_TEM-3, bla_TEM-20, bla_TEM-158, bla_SHV-27, bla_SHV-28, bla_SHV-36, bla_SHV-125, bla_CTx-M-14 and bla_CTx-M-27 with one isolate for each. The non-ESBL genes detected were bla_TEM-70 (n = 1), bla_TEM-176 (n = 1), bla_TEM-104 (n = 6), bla_TEM-1 (n = 15) and bla_OxA-1 (n = 12). Plasmid mediated AmpC β-lactamases genes; bla_ACT-5 (n = 1), bla_DHA-1(n = 2) and bla_CMY-2 (n = 4) were detected in seven isolates (14.2%). The bla_OxA-48 (n = 1) and bla_IMP-1 (n = 1) carbapenemases genes were detected among five carbapenem-resistant E. coli. Five isolates (10.2%) harboured qnr genes, qnrB1 (n = 3), qnrB2 (n = 1) and qnrS1 (n = 1) type were detected. Thirty isolates (61.2%) were positive for aac(6′)-Ib-cr gene. The class 1 integron was detected in twenty two (44.8%) isolates. Phylogenetic grouping revealed that 22 (44.8%) isolates belonged to group A, while 15 (30.6%), 11 (22.4%) and 1 (2%) belonged to B2, D and B1. Results of conjugation experiments indicated that bla_CTx-M-15, bla_TEM-1, bla_OxA-1, aac(6′)-Ib-cr and qnrB1 genes were co-transferred and that these genes were carried by a conjugative plasmid of high molecular weight. The results of this work reports the genetic diversity of ESBL genes, with the CTX-M-15 enzyme being the most common among ESBL-producing E. coli in Moroccan community.     

Incidence of blaNDM-1 gene in Escherichia coli isolates at a tertiary care referral hospital in Northeast India

Purpose: Increasing reports on New Delhi metallo-β-lactamase-1 (NDM-1) producing Escherichia coli constitute a serious threat to global health since it is found to be highly resistant to most of the currently available antibiotics including carbapenems. This study has been performed to find out the incidence bla_NDM-1 in E. coli isolates recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. Materials and Methods: A total of 270 non-duplicated E. coli isolates were recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. All isolates with reduced susceptibility to meropenem or ertapenem (diameter of zones of inhibition, ≤21 mm) were further phenotypically confirmed for carbapenemase production by modified Hodge test. All screened isolates were also subjected to the polymerase chain reaction detection of bla_NDM-1 gene and additional bla genes coding for transmission electron microscopy, SHV, CTX-M, and AmpC. Results: Out of 270 E. coli isolates, 14 were screened for carbapenemase production on the basis of their reduced susceptibility to meropenem or ertapenem. All screened isolates were found to be positive for bla_NDM-1. Each of the bla_NDM-1 possessing isolate was also positive for two or more additional bla genes, such as bla_TEM, bla_CTX-M and bla_AmpC. Phylogenetic analysis showed very less variation in bla_NDM-1 gene with respect to bla_NDM-1 possessing E. coli isolates from other parts of India and abroad. Conclusions: Our findings highlight the incidence of bla_NDM-1 in E. coli isolates with a reduced susceptibility to meropenem or ertapenem.     

Multiclonal epidemic of Klebsiella pneumoniae isolates producing DHA-1 in a Spanish hospital

Between June 2007 and January 2008, 26 Klebsiella pneumoniae isolates carrying bla_DHA-1 on an IncL/M plasmid were obtained from clinical samples at Granollers Hospital, Barcelona, Spain. Three of the isolates also carried a bla_CTX-M-15 gene. The 26 isolates showed 11 pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing showed that PFGE patterns A, B and C belonged to sequence type (ST)17, D to ST13, E to ST427, F and G to ST416, H to ST37, I to ST440, J to ST326, and K to ST428. Results demonstrated the effectiveness of the infection control programme in place at the centre. This study reports the first characterization of STs for bla_DHA-1-producing K. pneumoniae isolates.     

Characterization of blaCTX-M-14 transposition from plasmid to chromosome in Escherichia coli experimental strain

Mostly, bla_CTX-M is found on transferable plasmids as a component of the bla_CTX-M transposition unit containing an insertion sequence, ISEcp1, which exists on the upstream region of bla_CTX-Ms. Several recent studies conducted in clinical and community settings have reported the presence of chromosomally located bla_CTX-M in extended spectrum β-lactamase (ESBL)-producing bacterial isolates. In this study, we observed the frequency and molecular nature of the ISEcp1-mediated transposition of bla_CTX-M-14 from a plasmid to a chromosome by using an experimental strain of Escherichia coli. We determined 102 different chromosomal transposition sites of bla_CTX-M-14 in 126 E. coli isolates following five independent screening procedures. The characterization of the 102 different chromosomal transposition sites of bla_CTX-M-14 observed in this study revealed the presence of 5-bp direct repeat (DR) sequences and identical left terminal inverted sequences in 80 E. coli isolates. However, 5′-flanking sequences of the right terminal DR sequences in the 80 E. coli isolates were highly diverse, and consensus sequences of the right terminal inverted repeat sequences were not observed. In case of our E. coli experimental strain, the frequency of the ISEcp1-mediated transposition of bla_CTX-M-14 from a plasmid to a chromosome was determined to be 0.51% (SD = 0.37). Collectively, the molecular nature of ISEcp1 could plausibly be a factor contributing to the high detection rates of E. coli possessing chromosomally located bla_CTX-M-14 in both clinical and community settings.     

Extensive Within-Host Diversity in Fecally Carried Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolates: Implications for Transmission Analyses

Studies of the transmission epidemiology of antimicrobial-resistant Escherichia coli, such as strains harboring extended-spectrum beta-lactamase (ESBL) genes, frequently use selective culture of rectal surveillance swabs to identify isolates for molecular epidemiological investigation. Typically, only single colonies are evaluated, which risks underestimating species diversity and transmission events. We sequenced the genomes of 16 E. coli colonies from each of eight fecal samples (n = 127 genomes; one failure), taken from different individuals in Cambodia, a region of high ESBL-producing E. coli prevalence. Sequence data were used to characterize both the core chromosomal diversity of E. coli isolates and their resistance/virulence gene content as a proxy measure of accessory genome diversity. The 127 E. coli genomes represented 31 distinct sequence types (STs). Seven (88%) of eight subjects carried ESBL-positive isolates, all containing bla_CTX-M variants. Diversity was substantial, with a median of four STs/individual (range, 1 to 10) and wide genetic divergence at the nucleotide level within some STs. In 2/8 (25%) individuals, the same bla_CTX-M variant occurred in different clones, and/or different bla_CTX-M variants occurred in the same clone. Patterns of other resistance genes and common virulence factors, representing differences in the accessory genome, were also diverse within and between clones. The substantial diversity among intestinally carried ESBL-positive E. coli bacteria suggests that fecal surveillance, particularly if based on single-colony subcultures, will likely underestimate transmission events, especially in high-prevalence settings.