To investigate the β-lactamase background of ertapenem non-susceptible isolates for the presence of the most commonly detected carbapenemase genes, blaKPC, blaOXA-48 and blaVIM, and the newly described blaNDM-1.
Two hundred and thirty-five ertapenem-non-susceptible (MIC ≥0.5 mg/L) isolates of Enterobacteriaceae from the worldwide Study for Monitoring Antimicrobial Resistance Trends (SMART) 2009 programme were screened using a multiplex PCR for the presence of blaKPC, blaOXA-48, blaVIM and blaNDM-1 genes. Extended-spectrum β-lactamase (ESBL) and AmpC genes (blaESBL and blaAmpC) were identified using the Check-MDR CT101 microarray. DNA sequencing was performed to identify the blaESBL, blaKPC and blaNDM-1 genes. Molecular typing was also performed to genetically characterize these isolates.
Sixty-six isolates (28%) had a carbapenemase gene, with blaNDM-1 identified in 33 isolates including 2 isolates carrying both blaNDM-1 and blaOXA-48; other carbapenemase genes found included blaKPC (n = 23), blaVIM (n = 7) and blaOXA-48 (n = 3). All blaNDM-1-carrying isolates were from patients in India and comprised five different species. With the exception of one isolate carrying only blaNDM-1, all blaNDM-1 carbapenemase-possessing isolates carried additional β-lactamases in various combinations: blaESBL and blaAmpC (n = 18); blaESBL (n = 10); blaESBL, blaAmpC and blaOXA-48 (n = 2); and blaAmpC (n = 2). Except for blaOXA-48-carrying isolates, novel multilocus sequence types or enterobacterial repetitive intergenic consensus PCR patterns were observed along with clonal dissemination within and among sites.
A range of carbapenemase genes, associated with diverse ESBLs and/or AmpC backgrounds, were found among Enterobacteriaceae isolated during the study. Many of these ertapenem non-susceptible strains were clonally related and carried various combinations of β-lactamases.
carbapenemases; Enterobacteriaceae; microarray analysis
Although β-lactam antibiotics are heavily used in many developing countries, the diversity of β-lactamase genes (bla) is poorly understood. We screened for major β-lactamase phenotypes and diversity of bla genes among 912 E. coli strains isolated from clinical samples obtained between 1992 and 2010 from hospitalized and non-hospitalized patients.
None of the isolates was resistant to carbapenems but 30% of all isolates were susceptible to cefepime, cephamycins and piperacillin-tazobactam. Narrow spectrum β-lactamase (NSBL) phenotype was observed in 278 (30%) isolates that contained blaTEM-1 (54%) or blaSHV-1 (35%) or both (11%). Extended Spectrum β-lactamase (ESBL) phenotype was detected in 247 (27%) isolates which carried blaCTX-M-14 (29%), blaCTX-M-15 (24%), blaCTX-M-9 (2%), blaCTX-M-8 (4%), blaCTX-M-3 (11%), blaCTX-M-1 (6%), blaSHV-5 (3%), blaSHV-12 (5%), and blaTEM-52 (16%). Complex Mutant TEM-like (CMT) phenotype was detected in 220 (24%) isolates which carried blaTEM-125 (29%), while blaTEM-50, blaTEM-78, blaTEM-109, blaTEM −152 and blaTEM-158 were detected in lower frequencies of between 7% and 11%. Majority of isolates producing a combination of CTX-M-15 + OXA-1 + TEM-1 exhibited resistance phenotypes barely indistinguishable from those of CMT-producers. Although 73 (8%) isolates exhibited Inhibitor Resistant TEM-like (IRT) phenotype, blaTEM-103 was the only true IRT-encoding gene identified in 18 (25%) of strains with this phenotype while the rest produced a combination of TEM-1 + OXA-1. The pAmpCs-like phenotype was observed in 94 (10%) isolates of which 77 (82%) carried blaCMY-2 while 18% contained blaCMY-1.
Isolates from urine accounted for 53%, 53%, 74% and 72% of strains exhibiting complex phenotypes such as IRT, ESBL, CMT or pAmpC respectively. On the contrary, 55% isolates from stool exhibited the relatively more susceptible NSBL-like phenotype. All the phenotypes, and majority of the bla genes, were detected both in isolates from hospitalized and non-hospitalized patients but complex phenotypes were particularly common among strains obtained between 2000 and 2010 from urine of hospitalized patients.
The phenotypes and diversity of bla genes in E. coli strains implicated in clinical infections in non-hospitalized and hospitalized patients in Kenya is worryingly high. In order to preserve the efficacy of β-lactam antibiotics, culture and susceptibility data should guide therapy and surveillance studies for β-lactamase-producers in developing countries should be launched.
Objective: To detect genes encoding carbapenem resistance in urinary isolates of Escherichia coli recovered from hospitalized patients in tertiary care centre in Pune, India.
Methods: From Jan 2012 to Dec 2012, a total of 300 consecutive non-duplicate (one isolate per patient) clinical isolates of Escherichia coli were recovered from urine cultures of hospitalized patients including hospital acquired infection cases admitted to the medical and surgical intensive care units. Polymerase chain reaction (PCR) assays and sequencing was used to determine the presence of beta-lactamase encoding genes. Conjugation experiments were performed to determine the transferability of beta-lactamase.
Results: All the isolates were completely resistant to the second and third generation cephalosporins tested as well as carbapenems. All the isolates showed 100% susceptibility to tigecycline and colistin in vitro. Conjugation experiments demonstrated that blaNDM-1 was transferable via plasmid. All the isolates showed presence of blaNDM-1 and co-association of blaOXA-48 was 25/45(55%) of the isolates. Repetitive element based PCR (REP PCR), Enterobacterial Repetitive Intergenic Consensus (ERIC PCR) and Randomly Amplified Polymorphic DNA (RAPD) revealed a diversity of six clonal types among E.coli isolates.
Conclusion: Co-production of NDM-1with OXA-48 in urinary isolates of E. coli was detected for the first time in India. Transmission of plasmid carrying these resistant genes to other members of Enterobacteriaceae will increase incidence of multidrug resistance. Early detection of these genes will help in prevention and adequate infection control by limiting the spread of these organisms.
Multidrug resistant; Carbapenem resistance; Conjugation; Beta-lactamase; Uropathogenic E.coli
Escherichia coli sequence type 131 (O25b:H4), associated with the CTX-M-15 extended-spectrum beta-lactamases (ESBLs) and linked predominantly to the community-onset antimicrobial-resistant infections, has globally emerged as a public health concern. However, scant attention is given to the understanding of the molecular epidemiology of these strains in high-burden countries such as India. Of the 100 clinical E. coli isolates obtained by us from a setting where urinary tract infections are endemic, 16 ST131 E. coli isolates were identified by multilocus sequence typing (MLST). Further, genotyping and phenotyping methods were employed to characterize their virulence and drug resistance patterns. All the 16 ST131 isolates harbored the CTX-M-15 gene, and half of them also carried TEM-1; 11 of these were positive for blaOXA groups 1 and 12 for aac(6′)-Ib-cr. At least 12 isolates were refractory to four non-beta-lactam antibiotics: ciprofloxacin, gentamicin, sulfamethoxazole-trimethoprim, and tetracycline. Nine isolates carried the class 1 integron. Plasmid analysis indicated a large pool of up to six plasmids per strain with a mean of approximately three plasmids. Conjugation and PCR-based replicon typing (PBRT) revealed that the spread of resistance was associated with the FIA incompatibility group of plasmids. Pulsed-field gel electrophoresis (PFGE) and genotyping of the virulence genes showed a low level of diversity among these strains. The association of ESBL-encoding plasmid with virulence was demonstrated in transconjugants by serum assay. None of the 16 ST131 ESBL-producing E. coli strains were known to synthesize carbapenemase enzymes. In conclusion, our study reports a snapshot of the highly virulent/multiresistant clone ST131 of uropathogenic E. coli from India. This study suggests that the ST131 genotypes from this region are clonally evolved and are strongly associated with the CTX-M-15 enzyme, carry a high antibiotic resistance background, and have emerged as an important cause of community-acquired urinary tract infections.
The association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and blaCTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids.
The ESBL-encoding genes (blaCTX-M, blaTEM and blaSHV), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and blaCTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-blaCTX-M were also analyzed. The clonal relatedness was investigated by PFGE.
Of the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried blaCTX-M, with blaCTX-M-14 the most common. We observed a significant higher prevalence of blaCTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and blaCTX-M was found in 18 of the 127 isolates carrying oqxAB-blaCTX-M. These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination.
blaCTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-blaCTX-M genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, blaCTX-M, and floR on the same plasmids in E. coli.
Resistance to cephalosporins in Enterobacteriaceae is mainly due to the production of extended-spectrum beta-lactamase (ESBL). Little is known about ESBL-producing bacteria in Bangladesh. Therefore, the study presents results of phenotypic and molecular characterization of ESBL-producing Escherichia coli from hospitals in Bangladesh.
A total of 339 E. coli isolated from patients with urinary tract and wound infections attending three different medical hospitals in urban and rural areas of Bangladesh between 2003–2007 were screened for ESBL-production by the double disk diffusion test. Isolates with ESBL-phenotype were further characterized by antibiotic susceptibility testing, PCR and sequencing of different β-lactamase and virulence genes, serotyping, and XbaI-macrorestriction followed by pulsed-field gel electrophoresis (PFGE).
We identified 40 E. coli with ESBL phenotype. These isolates were resistant to ceftriaxone, ceftazidime, cefotaxime, aztreonam, cefepime, and nalidixic acid but remained susceptible to imipenem. All but one isolate were additionally resistant to ciprofloxacin, and 3 isolates were resistant to cefoxitin. ESBL genes of blaCTX-M-1-group were detected in all isolates; blaTEM-type and blaOXA-1-type genes were detected in 33 (82.5%) and 19 (47.5%) isolates, respectively. Virulence genes that are present in diarrhoeagenic E. coli were not found. Class-1 integron was present in 20 (50%) isolates. All the ESBL-producing E. coli isolates harbored plasmids ranging between 1.1 and 120 MDa. PFGE-typing revealed 26 different pulsotypes, but identical pulsotype showed 6 isolates of serotype O25:H4.
The prevalence of multidrug-resistant ESBL-producing E. coli isolates appears to be high and the majority of the isolates were positive for blaCTX-M. Although there was genetic heterogeneity among isolates, presence of a cluster of isolates belonging to serotype O25:H4 indicates dissemination of the pandemic uropathogenic E. coli clone in Bangladesh.
The trend of extended-spectrum beta-lactamases producing Escherichia coli (ESBL-EC) is increasing in Nepal. Limited studies have been reported investigating ESBL types and carbapenemases in E. coli.
A cross sectional study was conducted between June 2012 to January 2013 in Kathmandu Medical College and Teaching Hospital, Nepal. Non-repetitive clinical samples from out-patient department (OPD) and Intensive Care Units (ICU) were processed for bacteriological culture and identification of E. coli. Antibiotic susceptibility test, screening and phenotypic confirmation for ESBLs and carbapenemases and PCR (blaCTX-M, blaSHV and blaTEM-type ESBLs, blaVIM, blaIMP and blaNDM-1-type carbapenemases, and class 1 integron element integrase gene) were performed. Clones were resolved by PCR-Randomly Amplified Polymorphic DNA.
Out of 332 non-repetitive clinical specimens processed for culture and identification 160 (48.2%) were culture positive. Of which, 93 (58.1%) were E. coli. Of these, 24 (25.8%) were phenotypically confirmed as ESBL-EC and 3 (12.50%) of 24 ESBL-EC were carbapenemase producers. blaCTX-M-type ESBL was most common (23, 95.8%) followed by blaTEM (7, 29.2%) and blaSHV (3, 12.5%). blaVIM, blaIMP and blaNDM-1 were present in 3, 2 and 2 ESBL-EC, respectively. Class 1 integron element was present in 18 (75.0%) ESBL-EC. Nine isolates possessed more than one type of beta-lactamases. Interestingly, all carbapenemase producers were isolated form ICU and co-existence of blaCTX-M, blaSHV, blaTEM, blaIMP, blaVIM and blaNDM-1 beta-lactamases was documented in one ESBL-EC (EC104). All most all isolates had different RAPD patterns.
For the first time in Nepal, high prevalence of blaCTX-M-type ESBL and co-existence of ESBLs and carbapenemases has been described. Continuous monitoring and surveillance and proper infection control and prevention practices will limit the further spread of these super-bugs within this hospital and beyond.
ESBL producing Escherichia coli; Carbapenemases; Clinical isolates; Integron element
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 blaTEM, blaSHV, blaCTX-M, blaVEB, blaOXA, blaPER, and blaGES, 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 blaCTX-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 blaCTX-M in most isolates. Up to 77.0% and 71.7% of ESBL-producing E. coli and ESBL-producing K. pneumoniae, respectively, carried blaTEM; all of them encoded TEM-1. ESBL-producing K. pneumoniae carried blaSHV 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 blaPER and blaGES. 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.
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.
TEM-, SHV-, and OXA-type β-lactamases were studied by PCR with 124 ampicillin-resistant (AMPr) Escherichia coli isolates recovered from foods of animal origin (n = 20) and feces of humans (n = 49) and healthy animals (n = 55). PCR showed that 103 isolates were positive for TEM and negative for SHV and OXA. Three E. coli isolates showed a positive reaction for OXA, and one showed a positive reaction for SHV. The remaining 17 E. coli isolates were negative for the three enzymes by PCR. Fifty-seven of the 103 blaTEM amplicons were sequenced. Different molecular variants of blaTEM-1 were found in 52 isolates: blaTEM-1a (n = 9), blaTEM-1b (n = 36), blaTEM-1c (n = 6), and blaTEM-1f (n = 1). Four inhibitor-resistant TEM (IRT) β-lactamase-encoding genes were also detected: blaTEM-30c (IRT-2), blaTEM-34b (IRT-6), blaTEM-40b (IRT-11), and blaTEM-51a (IRT-15). A new blaTEM gene, named blaTEM-95b, which showed a mutation in amino acid 145 (P→A) was detected. It was found in a food isolate of chicken origin (AMPr, amoxicillin-clavulanic acid susceptible). The promoter region in 24 blaTEM amplicons was analyzed, and the weak P3 promoter was found in 23 of them (blaTEM-1 in 20 amplicons and blaTEM-51a, blaTEM-30c, and blaTEM-95b in 1 amplicon each). The strong Pa/Pb promoter was found only in the blaTEM-34b gene. No extended-spectrum β-lactamases were detected. Mutations at position −42 or −32 in the ampC gene promoter were demonstrated in 4 of 10 E. coli isolates for which the cefoxitin MIC was ≥16 μg/ml. Different variants of blaTEM-1 and IRT blaTEM genes were found among the AMPr E. coli isolates from foods and the feces of humans and healthy animals, and a new gene, blaTEM-95b (P3), was detected.
In this study, we focused on evaluating the occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified as E. coli were screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 had E. coli isolates with ceftiofur MICs higher than 8 μg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples). blaCTX-M, blaSHV-12, blaCMY-2, and blaDHA-1 were identified in 108, 5, 9, and 1 isolates, respectively. The most common blaCTX-M genes were blaCTX-M-27 (n = 34), blaCTX-M-55 (n = 27), blaCTX-M-24e (n = 22), and blaCTX-M-105 (n = 20), followed by blaCTX-M-14a, blaCTX-M-14b, blaCTX-M-24a, and blaCTX-M-24b. Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed. blaCTX-M genes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M gene blaCTX-M-105 was flanked by two hypothetical protein sequences, partial ISEcp1 upstream and truncated IS903D, iroN, orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer of blaCTX-M genes mediated by mobile elements and the clonal spread of CTX-M-producing E. coli isolates contributed to the dissemination of blaCTX-M in the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.
The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was blaCTX-M; 16 isolates carried blaCTX-M-2 and three carried a new ESBL gene designated blaCTX-M-39. Three strains carried blaSHV (two blaSHV-12 and one blaSHV-5), and two strains carried inhibitor-resistant ESBL genes, blaTEM-33 and blaTEM-30; 18 strains carried blaTEM-1 and eight strains carried blaOXA-2. Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that blaCTX-M-2 is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated blaCTX-M-39, which revealed 99% homology with blaCTX-M-26, with a substitution of arginine for glutamine at position 225.
Data on CTX-M type extended-spectrum β-lactamase (ESBL) produced by Gram-negative bacteria by molecular methods are limited from India. This study was conducted to investigate the prevalence of CTX-M type ESBL producing Escherichia coli and Klebsiella pneumoniae from nosocomial isolates in a tertiary care hospital in southern India. A total of 179 clinical isolates of K. pneumoniae (n = 72) and E. coli (n = 107) were obtained in a period of 3 months and assessed for ESBL production phenotypically. Associated resistance to a panel of antibiotics and Minimum Inhibitory Concentration for 3rd generation cephalosporins was determined. Phenotypically ESBL positive isolates were subjected to PCR for blaCTX-M gene using two sets of primers for the simultaneous detection of all the five major groups of CTX-M types. All the positive isolates were then subjected to a group specific PCR to detect the prevalent group. Out of 179 isolates, 156 (87.1%) were positive for ESBL phenotypically, which includes 39.2% of K. pneumoniae and 60.8% of E. coli. All of them were examined by PCR using two primers for the presence of blaCTX-M genes. Among the 156 phenotypic positive isolates, 124 (79.4%) were positive for blaCTX-M genes, of which 45 (36.2%) were K. pneumoniae, 79 (63.7%) were E. coli. When the 124 positive clinical isolates were further tested with CTX-M group-specific primers, all were positive for the CTX-M-1 group. Our findings document evidence of the high prevalence of multidrug resistant CTX-M group 1 type ESBL among nosocomial isolates in this region. High co-resistance to other non-β-lactam antibiotics is a major challenge for management of ESBL infections. This is alarming and calls for the judicious use of carbapenems, especially in developing countries. This has significant implications for patient management, and indicates the need for increased surveillance and for further molecular characterization of these isolates.
Extended spectrum β-lactamase; CTX-M types; Antibiotic resistance; Nosocomial isolates; South India
Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK.
Enterobacteriaceae isolates were studied from two major centres in India—Chennai (south India), Haryana (north India)—and those referred to the UK's national reference laboratory. Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene blaNDM-1 was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing. Case data for UK patients were reviewed for evidence of travel and recent admission to hospitals in India or Pakistan.
We identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those from UK and Chennai were readily transferable whereas those from Haryana were not conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within the past year, or had links with these countries.
The potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed.
European Union, Wellcome Trust, and Wyeth.
Of 15 extended-spectrum β-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae collected from the First Municipal People's Hospital of Guangzhou, in the southern part of the People's Republic of China, 9 were found to produce CTX-M ESBLs, 3 produced SHV-12, and 3 produced both CTX-M and SHV-12. Eleven isolates produced either TEM-1B or SHV-11, in addition to an ESBL. Nucleotide sequence analysis of the 12 isolates carrying blaCTX-M genes revealed that they harbored three different blaCTX-M genes, blaCTX-M-9 (5 isolates), blaCTX-M-13 (1 isolate), and blaCTX-M-14 (6 isolates). These genes have 98% nucleotide homology with blaToho-2. The blaCTX-M genes were carried on plasmids that ranged in size from 35 to 150 kb. Plasmid fingerprints and pulsed-field gel electrophoresis showed the dissemination of the blaCTX-M genes through transfer of different antibiotic resistance plasmids to different bacteria, suggesting that these resistance determinants are highly mobile. Insertion sequence ISEcp1, found on the upstream region of these genes, may be involved in the translocation of the blaCTX-M genes. This is the first report of the occurrence of SHV-12 and CTX-M ESBLs in China. The presence of strains with these ESBLs shows both the evolution of blaCTX-M genes and their dissemination among at least three species of the family Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, isolated within a single hospital. The predominance of CTX-M type enzymes seen in this area of China appears to be similar to that seen in South America but is different from those seen in Europe and North America, suggesting different evolutionary routes and selective pressures. A more comprehensive survey of the ESBL types from China is urgently needed.
Background & objectives:
Information about the genetic diversity of the extended-spectrum β-lactamases (ESBLs) and the clonal relationship of the organisms causing neonatal infections is limited, particularly from India where neonatal mortality is high. This study was undertaken to investigate the molecular epidemiology and risk factors associated with neonatal septicaemia caused by ESBL-producing Klebsiella pneumoniae and Escherichia coli.
Bloodstream isolates (n=26) of K. pneumoniae (n=10) and E. coli (n=16) from the neonates admitted in a tertiary care hospital in New Delhi during January to May 2008 were characterized. Antimicrobial susceptibility tests were carried out and ESBL production was assessed phenotypically. PCR was carried out for ESBL and ampC genes. Genotyping was performed by pulsed-field gel electrophoresis (PFGE). Conjugation experiments were done to determine the mobility of ESBL genes. Risk factors associated with ESBL-producing K. pneumoniae and E. coli infections were analysed.
Resistance rates to most of the antibiotics tested were high, except for imipenem. Among the isolates tested, 60 per cent of K. pneumoniae and 75 per cent of E. coli were ESBL producers. PFGE of the isolates demonstrated a vast diversity of genotypes with no epidemic clones. Despite the clonal diversity, blaCTX-M-15 was detected in 100 per cent of ESBL-positive isolates. The other genes present in ESBL-positive isolates were blaTEM-1, blaSHV-1, blaSHV-28, blaSHV-11, and blaSHV-12. Class 1 integrons were detected in 7 of 18 ESBL-positive isolates. Moreover, the plasmid carrying blaCTX-M-15, in E. coli and K. pneumoniae were self transferable. Feeding through an enteral tube was identified as the only risk factor for sepsis by ESBL-producing organisms.
Interpretation & conclusions:
The study emphasises the presence of blaCTX-M-15 in clonally diverse isolates indicating probable horizontal transfer of this gene. The widespread dissemination of CTX-M-15 is of great concern as it further confines the limited therapeutic interventions available for neonates.
CTX-M-15; diverse clones; ESBLs; Escherichia coli; Klebsiella pneumoniae; neonatal sepsis; risk factor
The aim of this study was to investigate the frequency of betalactamase producing EAEC isolates among young children with diarrhea in Zanjan, Iran.
Entero aggregative Escherichia coli (EAEC) is an emerging enteric pathogen associated with acute and persistent diarrhea and the evolution and spread of acquired extended spectrum betalactamases (ESBLs) among these strains has become a serious problem in the management of infectious diseases in developing countries.
Patients and methods
During the period from March 2011 to January 2012, 140 isolates of E. coli from diarrheal children aged 0–60 months and 90 isolates from age-matched controls without diarrhea were investigated for EAEC using PCR. Antimicrobial susceptibility testing was performed as CLSI guidelines and betalactamase genes, including bla
VIM and bla
NDM-1 investigated in EAEC isolates.
In this study, EAEC was detected with slightly higher frequency in children with (8%) than in children without (4.6%) diarrhea (P > 0.05). Diarrheagenic E. coli exhibited high level resistance to aztreonam (80.7%), amoxicillin (74.4%) and tetracycline (69.3%). Also, 86.4% of E. coli isolates were resistant to at least three different classes of antimicrobial agents and considered as multidrug resistance. Molecular characterization of betalactamase genes showed that bla
TEM was the most frequently isolated betalactamase. It was detected in 78.9% of ESBL producing EAEC isolates. Also, the frequency of bla
CTX-M was 63.1% (12/19) and 8 (42.1%) isolates carried the bla
TEM and bla
CTX-M, simultaneously. None MBL producing EAEC was detected in our study.
Our results indicate that ESBLs especially bla
TEM and bla
CTX-M are widespread among EAEC isolates and appropriate surveillance and control measures are essential to prevent further dissemination of betalactamases in our country.
Antibiotic resistance; Diarrhea; EAEC; ESBL
The purpose of this study was to identify the genes coding for resistance to ceftazidime and imipenem and describe the molecular epidemiology of A. baumannii strains isolated from a clinical center in Colombia. Twenty isolates of imipenem-resistant A. baumannii from an equal number of patients with nosocomial infections were obtained. Primers were used to amplify genes blaIMP, blaVIM, blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-51 and blaADC-7. To detect insertion sequences ISAba1/blaOXA-23,
ISAba1/blaOXA-51 and ISAba1/blaADC-7, mapping by PCR using combinations of reverse primers ISAba1 and reverse primers of blaOXA-23, blaOXA-51 and blaADC-7 were used. The amplification products were purified and cloned into PCR 2.1-TOPO vector and transformed into chemically competent Escherichia coli TOP10. These amplicons were then sequenced. PFGE was performed on DNA of A. baumannii isolates digested with ApaI. Results. The DNA profiles obtained included 9 clusters with, four 2–7 isolates per profile, and 5 single-isolate profiles. Of the 20 isolates resistant to imipenem, 15 carried blaOXA-23 gene, 4 contained ISAba1 upstream of blaOXA-51 gene, and 6 contained ISAba1 upstream of blaOXA-23 gene. Eighteen of these isolates carried the blaADC-7 gene, with 9 of the isolates having ISAba1 located upstream of this gene. This is the first report of the ISAba1/ADC-7 associated with OXAs genes in A. baumannii isolates from Colombia.
Nosocomial pathogens; Antimicrobial resistance; PFGE
Context: Urinary tract infections (UTIs) are amongst the most common infections described in outpatient settings. Increased antimicrobial resistance (AMR) of urinary tract pathogens is a matter of global public health concern. Treatment of UTI depends on both prevalence and antimicrobial resistance (AMR) of causative bacteria at any specific geographical location.
Aim: This study was undertaken to compare the prevalence of uropathogens and their AMR profile in two different geographical parts of India.
Materials and Methods: Clean-catch mid-stream urine samples were collected from adult patients, bacterial flora isolated from human urine was evaluated for antimicrobial susceptibility profile using Kirby Bauer’s disc diffusion method among patients from Hyderabad (Southern India), Rajasthan and Punjab (Northern India). The data were analysed using Chi-square (χ2) test, confidence interval (CI), odds ratio (OR) analysis and p-value using SPSS 16 software.
Escherichia coli (55.1%) were the most prevalent isolates followed by Enterococcus faecalis (15.8%). Amikacin was the most active antimicrobial agents which showed low resistance rate of 14%. The present study revealed the geographical difference in prevalence of uropathogens with Klebsiella pneumoniae being the second most common uropathogen followed by E. faecalis in the states from northern India while no K. pneumoniae was seen in samples from southern India but E. faecalis was the second most prevalent organism.
Conclusion: Therefore, development of regional surveillance programs is highly recommended for implementation of national CA-UTI guidelines in Indian settings.
Antimicrobial resistance; Bacteriuria and antibiotics; Community-acquired urinary yract infections; Uropathogens
Extended-spectrum beta-lactamase (ESBL) producing bacteria have an important role in nosocomial infections. Due to the limited availability of information about the molecular epidemiology of ESBL producing bacteria in Mashhad, we decided to investigate about TEM, CTX and SHV ESBLs among urinary Escherichia coli isolates in Mashhad, a city in northeast Iran.
Materials and Methods
One hundred and eleven clinical isolates of E. coli were diagnosed from hospitalized patients in 2009. After performing antibiogram and phenotypic confirmation test, polymerase chain reaction (PCR) was performed by blaTEM, blaSHV and blaCTX primers and restriction digestion was carried out using PstI and TaqI (Fermentas-Lithuania) for confirmation.
ESBL producers of E. coli isolates were 33.3%. Among 37 ESBL-producing isolates, 35 (94.6%), 21 (56.8%) and 5 (13.5%) were shown to have blaCTX, blaTEM and blaSHV, genes respectively. Co-resistance to non-beta lactam antibiotics was observed more with ESBL producers (P < 0.05).
The results showed that the studied ESBL genes are found with high prevalence and among them blaCTX is more widespread in urine E. coli isolates in Mashhad.
Antibiotic resistance; Escherichia coli; Extended-spectrum beta-lactamase; Urinary tract infection
Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.
The extended-spectrum beta-lactamase (ESBL)-producing phenotype is frequent among Enterobacter isolates at the Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. We examined the clonal relatedness and characterized the ESBLs of a collection of these strains. Clonal relatedness was determined by pulsed-field gel electrophoresis. Isoelectric focusing (IEF) and transconjugation experiments were performed. ESBL gene families were screened by colony hybridization and PCR for blaTEM, blaSHV, blaCTX-M, blaIBC, blaPER, blaOXA, blaVEB, and blaSFO; and the PCR products were sequenced. The 17 Enterobacter isolates studied comprised 15 distinct genotypes. All isolates showed at least one IEF band (range, one to five bands) whose appearance was suppressed by addition of clavulanate; pIs ranged from 5.4 to ≥8.2. Colony hybridization identified at least one family of beta-lactamase genes in 11 isolates: 10 harbored blaTEM and 9 harbored blaSHV. PCR screening and sequence analysis of the PCR products for blaTEM, blaSHV, and blaCTX-M identified TEM-1 in 11 isolates, SHV-12 in 7 isolates, SHV-1 in 1 isolate, a CTX-M-2-like gene in 2 isolates, and CTX-M-26 in 1 isolate. In transconjugation experiments with four isolates harboring blaTEM-1 and blaSHV-12, both genes were simultaneously transferred to the recipient strain Escherichia coli HB101. Plasmid mapping, PCR, and Southern analysis with TEM- and SHV-specific probes demonstrated that a single transferred plasmid carried both the TEM-1 and the SHV-12 genes. The widespread presence of ESBLs among Enterobacter isolates in Tel Aviv is likely due not to clonal spread but, rather, to plasmid-mediated transfer, at times simultaneously, of genes encoding several types of enzymes. The dominant ESBL identified was SHV-12.
The diversity and evolution of the class A OXY β-lactamase from Klebsiella oxytoca were investigated and compared to housekeeping gene diversity. The entire blaOXY coding region was sequenced in 18 clinical isolates representative of the four K. oxytoca β-lactamase gene groups blaOXY-1 to blaOXY-4 and of two new groups identified here, blaOXY-5 (with four isolates with pI 7.2 and one with pI 7.7) and blaOXY-6 (with four isolates with pI 7.75 and three with pI 8.1). Genes blaOXY-5 and blaOXY-6 showed 99.8% within-group nucleotide similarity but differed from each other by 4.2% and from blaOXY-1, their closest relative, by 2.5% and 2.9%, respectively. Antimicrobial susceptibility to β-lactams was similar among OXY groups. Nucleotide sequence diversity of the 16S rRNA (1,454 bp), rpoB (940 bp), gyrA (383 bp), and gapDH (573 bp) genes was in agreement with the β-lactamase gene phylogeny. Strains with blaOXY-1, blaOXY-2, blaOXY-3, blaOXY-4, and blaOXY-6 genes formed five phylogenetic groups, named KoI, KoII, KoIII, KoIV, and KoVI, respectively. Isolates harboring blaOXY-5 appeared to represent an emerging lineage within KoI. We estimated that the blaOXY gene has been evolving within K. oxytoca for approximately 100 million years, using as calibration the 140-million-year estimation of the Escherichia coli-Salmonella enterica split. These results show that the blaOXY gene has diversified along K. oxytoca phylogenetic lines over long periods of time without concomitant evolution of the antimicrobial resistance phenotype.
The presence of multidrug-resistant bacterial pathogens in the environment poses a serious threat to public health. The opportunistic Acinetobacter spp. are among the most prevalent causes of nosocomial infections. Here, we performed complete genome sequencing of the Acinetobacter calcoaceticus strain XM1570, which was originally cultivated from the sputum of a patient diagnosed with pneumonia in Xiamen in 2010. We identified carbapenem resistance associated gene blaNDM-1 located on a 47.3-kb plasmid. Three methods – natural reproduction, sodium dodecyl sulfate treatment and nalidixic acid treatment – were used to eliminate the blaNDM-1-encoding plasmid, which achieved elimination rates of 3.32% (10/301), 83.78% (278/332), and 84.17% (298/354), respectively. Plasmid elimination dramatically increased antibiotic sensitivity, reducing the minimum bacteriostatic concentration of meropenem from 256 µg/ml in the clinical strain to 0.125 µg/ml in the plasmid-eliminated strain. Conjugation transfer assays showed that the blaNDM-1-containing plasmid could be transferred into Escherichia coli DH5α:pBR322 in vitro as well as in vivo in mice. The blaNDM-1 genetic environment was in accordance with that of other blaNDM-1 genes identified from India, Japan, and Hong-Kong. The multilocus sequence type of the isolate was identified as ST-70. Two novel genes encoding intrinsic OXA and ADC were identified and named as OXA-417 and ADC-72. The finding of blaNDM-1 in species like A. calcoaceticus demonstrates the wide spread of this gene in gram-negative bacteria which is possible by conjugative plasmid transfer. The results of this study may help in the development of a treatment strategy for controlling NDM-1 bacterial infection and transmission.
Infection by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify blaSHV, blaCTX-M-3-like, and blaCTX-M-14-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent blaSHV genes (blaSHV-1, blaSHV-2, blaSHV-2a, blaSHV-5, blaSHV-11, and blaSHV-12) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the blaSHV and blaCTX-M genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the blaCTX-M-3-like (CTX-M-15) and blaCTX-M-14-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.