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1.  CTX-M-2 and a New CTX-M-39 Enzyme Are the Major Extended-Spectrum Beta-Lactamases in Multiple Escherichia coli Clones Isolated in Tel Aviv, Israel 
Antimicrobial Agents and Chemotherapy  2005;49(11):4745-4750.
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.
doi:10.1128/AAC.49.11.4745-4750.2005
PMCID: PMC1280129  PMID: 16251320
2.  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▿ †  
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.
doi:10.1128/AAC.00171-08
PMCID: PMC2493136  PMID: 18505851
3.  Co-existence of beta-lactamases in clinical isolates of Escherichia coli from Kathmandu, Nepal 
BMC Research Notes  2014;7(1):694.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1756-0500-7-694
PMCID: PMC4197279  PMID: 25287013
ESBL producing Escherichia coli; Carbapenemases; Clinical isolates; Integron element
4.  Characterization of Extended-Spectrum β-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm 
Applied and Environmental Microbiology  2012;78(10):3668-3673.
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.
doi:10.1128/AEM.07507-11
PMCID: PMC3346353  PMID: 22407683
5.  Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel 
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.
doi:10.1128/AAC.49.3.1150-1156.2005
PMCID: PMC549242  PMID: 15728917
6.  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▿  
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 blaTEM, blaSHV, blaOXA, and blaCTX genes from clinical strains. Isolates were also screened for AmpC genes of the blaCMY, blaACT, blaFOX, and blaDHA families as well as the blaKPC 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, blaCTX-M-type β-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas blaSHV 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.
doi:10.1128/AAC.00883-08
PMCID: PMC2630642  PMID: 19015360
7.  Dominance of CTX-M-Type Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Isolated from Patients with Community-Onset and Hospital-Onset Infection in China 
PLoS ONE  2014;9(7):e100707.
Objective
To investigate CTX-M genotypes among extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) isolated from patients with community-onset and hospital-onset infections in China, their clonality and the distribution of CTX-M variants in different specimens of community-onset and hospital-onset infections.
Methods
ESBL-EC isolates were collected from general hospitals from 2011 to 2012 in China. Broth microdilution method antimicrobial susceptibility testing of 16 antibiotics was performed. Clinical data from community-onset and hospital-onset infections due to ESBL-EC were analyzed. ESBL-encoding genes were amplified by PCR and sequenced, and multilocus sequence typing (MLST) was performed for a random selection of predominant CTX-M type strains identified.
Results
A total of 1,168 ESBL-EC isolates were obtained from various clinical specimens, 41.7% of which were responsible for causing community-onset infections. The presence of urinary calculi was higher in community-onset infections, whereas malignancy, cardiovascular and cerebrovascular diseases, dementia, chronic renal disease, diabetes mellitus and surgical treatment were found to have higher proportions in hospital-onset infections. There was no significant difference in trauma between community-onset and hospital-onset infections. 96.2% of the isolates were detected to harbor blaCTX-M genes. blaCTX-M-1 group and blaCTX-M-9 group were detected at 40.7% and 48.7% respectively, and both positive group accounted for 10.6%. blaCTX-M-55 (24.8%) and blaCTX-M-15 (18.2%) were the major genotypes in blaCTX-M-1 group while blaCTX-M-14 (46.8%) was predominant in blaCTX-M-9 group. A comparison of blaCTX-M distribution in different specimens between ESBL-EC causing community-onset and hospital-onset infection showed no significant difference. A total of 229 isolates were tested for MLST. ST131 (14%) was the predominant type. ST648, ST405 and ST1193 were also detected.
Conclusions
Community-onset ESBL-EC has emerged as a common pathogen in China. CTX-M-14 is the most commonly encountered, CTX-M-55 and CTX-M-15 have spread rapidly. ST131 is the predominant clonal group, and the great diversity of CTX-M-producing isolates of E. coli has emerged in China.
doi:10.1371/journal.pone.0100707
PMCID: PMC4077569  PMID: 24983621
8.  Three Cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China 
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.
doi:10.1128/AAC.46.3.630-637.2002
PMCID: PMC127467  PMID: 11850241
9.  Using Nucleic Acid Microarrays To Perform Molecular Epidemiology and Detect Novel β-Lactamases: a Snapshot of Extended-Spectrum β-Lactamases throughout the World 
Journal of Clinical Microbiology  2012;50(5):1632-1639.
The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008–2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify blaESBL (blaSHV, blaTEM, and blaCTX-M genes of groups 1, 2, 9, and 8/25) and blaKPC genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the blaESBL and blaKPC genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one blaESBL gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of blaTEM (n = 5), blaSHV (n = 11), blaCTX-M (n = 19), and blaKPC (n = 3) were detected. A new blaSHV variant, blaSHV-129, and a new blaKPC variant, blaKPC-11, were also identified. The most common bla genes found in this study were blaCTX-M-15, blaCTX-M-14, and blaSHV-12. Using nucleic acid microarrays, we obtained a “molecular snapshot” of blaESBL genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants blaSHV-129 and blaKPC-11.
doi:10.1128/JCM.06115-11
PMCID: PMC3347121  PMID: 22322349
10.  Neonatal septicaemia caused by diverse clones of Klebsiella pneumoniae & Escherichia coli harbouring blaCTX-M-15 
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.
Methods:
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.
Results:
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.
PMCID: PMC3724262  PMID: 23703349
CTX-M-15; diverse clones; ESBLs; Escherichia coli; Klebsiella pneumoniae; neonatal sepsis; risk factor
11.  Molecular Characterization of CTX-M β-Lactamase and Associated Addiction Systems in Escherichia coli Circulating among Cattle, Farm Workers, and the Farm Environment 
Applied and Environmental Microbiology  2013;79(13):3898-3905.
A total of 84 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from cattle, farm workers, and the farm environment isolated from February to September 2008 in the Republic of Korea were investigated. All 84 ESBL-producing isolates carried blaCTX-M genes that belonged to the CTX-M-1 (n = 35) or CTX-M-9 (n = 49) family. The most predominant CTX-M type identified was CTX-M-14 (n = 49), followed by CTX-M-32 (n = 26). The blaCTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A blaCTX-M-14 gene was also detected in an E. coli isolate from a farmer's hand. Transfer of the blaCTX-M gene from 60 blaCTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from blaCTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) blaCTX-M genes possessed an insertion sequence, ISEcp1, upstream of the blaCTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The blaCTX-M genes were associated with seven kinds of addiction systems. Among them, pndAC, hok-sok, and srnBC were the most frequently identified addiction systems in both wild strains and transconjugants. The spread of blaCTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying blaCTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the blaCTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.
doi:10.1128/AEM.00522-13
PMCID: PMC3697548  PMID: 23584784
12.  Characterization of methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and extended-spectrum beta-lactamase-producing Escherichia coli in intensive care units in Canada: Results of the Canadian National Intensive Care Unit (CAN-ICU) study (2005–2006) 
BACKGROUND
Methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and vancomycin-resistant enterococci (VRE) are important hospital pathogens in Canada and worldwide.
OBJECTIVES
To genotypically and phenotypically characterize the isolates of MRSA, VRE and ESBL-producing E coli collected from patients in Canadian intensive care units (ICUs) in 2005 and 2006.
METHODS
Between September 1, 2005, and June 30, 2006, 19 medical centres participating in the Canadian National Intensive Care Unit (CAN-ICU) study collected 4133 unique patient isolates associated with infections in ICUs. Isolates of MRSA underwent mecA polymerase chain reaction (PCR) and Panton-Valentine leukocidin analysis; they were typed using pulsed-field gel electrophoresis. All isolates of E coli with ceftriaxone minimum inhibitory concentrations greater than or equal to 1 μg/mL were tested for the presence of an ESBL using the Clinical Laboratory Standards Institute double-disk diffusion method. Subsequently, PCR and sequence analysis were used to identify blaSHV, blaTEM and blaCTX-M. Isolates of VRE were tested for the presence of vanA and vanB genes by PCR.
RESULTS
Of the 4133 ICU isolates collected, MRSA accounted for 4.7% (193 of 4133) of all isolates. MRSA represented 21.9% (193 of 880) of all S aureus collected during the study; 90.7% were health care-associated MRSA strains and 9.3% were community-associated MRSA strains. Resistance rates for the isolates of MRSA were 91.8% to levofloxacin, 89.9% to clarithromycin, 76.1% to clindamycin and 11.7% to trimethoprim-sulfamethoxazole; no isolates were resistant to vancomycin, linezolid, tigecycline or daptomycin. ESBL-producing E coli accounted for 0.4% (18 of 4133) of all isolates and 3.7% (18 of 493) of E coli isolates. All 18 ESBL-producing E coli were PCR-positive for CTX-M, with blaCTX-M-15 occurring in 72% (13 of 18) of isolates. All ESBL-producing E coli displayed a multidrug-resistant phenotype (resistant to third-generation cephalosporins and one or more other classes of antimicrobials), with 77.8% of isolates resistant to ciprofloxacin, 55.6% resistant to trimethoprim-sulfamethoxazole, 27.8% resistant to gentamicin and 26.3% resistant to doxycycline; all isolates were susceptible to ertapenem, meropenem and tigecycline. VRE accounted for 0.4% (17 of 4133) of all isolates and 6.7% (17 of 255) of enterococci isolates; 88.2% of VRE had the vanA genotype. Isolated VRE that were tested were uniformly susceptible to linezolid, tigecycline and daptomycin.
CONCLUSIONS
MRSA isolated in Canadian ICUs in 2005 and 2006 was predominately health care-associated (90.7%), ESBL-producing E coli were all CTX-M producers (72% blaCTX-M-15) and VRE primarily harboured a vanA genotype (88.2%). MRSA, ESBL-producing E coli and VRE were frequently multidrug resistant.
PMCID: PMC2605872  PMID: 19412382
CAN-ICU; ESBL E coli; Intensive care; MRSA; Resistance; VRE
13.  Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel 
PLoS ONE  2012;7(9):e46329.
Objectives
We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.
Methods
ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The blaCTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the blaCTX-M-94 and blaCTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of blaCTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.
Results
The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. blaCTX-M-94 and blaCTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.
Conclusions
This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.
doi:10.1371/journal.pone.0046329
PMCID: PMC3458832  PMID: 23050014
14.  Detection of Escherichia coli and Associated β-Lactamases Genes from Diabetic Foot Ulcers by Multiplex PCR and Molecular Modeling and Docking of SHV-1, TEM-1, and OXA-1 β-Lactamases with Clindamycin and Piperacillin-Tazobactam 
PLoS ONE  2013;8(7):e68234.
Diabetic foot ulcer (DFU) is a common and devastating complication in diabetes. Antimicrobial resistance mediated by extended-spectrum β-lactamases (ESBLs) production by bacteria is considered to be a major threat for foot amputation. The present study deals with the detection of Escherichia coli and the prevalence of blaTEM, blaSHV and blaOXA genes directly from biopsy and swab of foot ulcers of diabetic patients. In total, 116 DFU patients were screened, of which 42 suffering with severe DFUs were selected for this study. Altogether 16 E. coli strains were successfully isolated from biopsy and/or swab samples of 15 (35.71%) patients. ESBL production was noted in 12 (75%) strains. Amplification of β-lactamase genes by multiplex PCR showed the presence of blaCTX-M like genes in 10 strains, blaTEM and blaOXA in 9 strains each, and blaSHV in 8 of the total 16 strains of E. coli. Out of the ten antibiotics tested, E. coli strains were found to be resistant to ampicillin (75%), cefoxitin (56.25%), cefazolin (50%), meropenem (37.5%), cefoperazone (25%), cefepime (31.25%), ceftazidime (56.25%), and cefotaxime (68.75%) but all showed sensitivity (100%) to clindamycin and piperacillin-tazobactam. 3D models of the most prevalent variants of β-lactamases namely TEM-1, SHV-1, OXA-1, and ESBL namely CTX-M-15 were predicted and docking was performed with clindamycin and piperacillin-tazobactam to reveal the molecular basis of drug sensitivity. Docking showed the best docking score with significant interactions, forming hydrogen bond, Van der Waals and polar level interaction with active site residues. Findings of the present study may provide useful insights for the development of new antibiotic drugs and may also prevent ESBLs-mediated resistance problem in DFU. The novel multiplex PCR assay designed in this study may be routinely used in clinical diagnostics of E. coli and associated blaTEM, blaSHV, and blaOXA like genes.
doi:10.1371/journal.pone.0068234
PMCID: PMC3701671  PMID: 23861873
15.  The Sudden Dominance of blaCTX–M Harbouring Plasmids in Shigella spp. Circulating in Southern Vietnam 
Background
Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.
Methodology
We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a blaCTX–M encoding plasmid.
Principal Findings
We show that two different blaCTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the blaCTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the blaCTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.
Significance
The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.
Author Summary
Shigellosis is a disease caused by bacteria belonging to Shigella spp. and is a leading cause of bacterial gastrointestinal infections in infants in unindustrialized countries. The Shigellae are dynamic and capable of rapid change when placed under selective pressure in a human population. Extended spectrum beta lactamases (ESBLs) are enzymes capable of degrading cephalosporins (a group of antimicrobial agents) and the genes that encode them are common in pathogenic E. coli and other related organisms in industrialized countries. In southern Vietnam, we have isolated multiple cephalosporin-resistant Shigella that express ESBLs. Furthermore, over two years these strains have replaced strains isolated from patients with shigellosis that cannot express ESBLs. Our work describes the genes responsible for this characteristic and we investigate one of the elements carrying one of these genes. These finding have implications for treatment of shigellosis and support the growing necessity for vaccine development. Our findings also may be pertinent for other countries undergoing a similar economic transition to Vietnam's and the corresponding effect on bacterial populations.
doi:10.1371/journal.pntd.0000702
PMCID: PMC2882334  PMID: 20544028
16.  Molecular Epidemiological Analysis of Escherichia coli Sequence Type ST131 (O25:H4) and blaCTX-M-15 among Extended-Spectrum-β-Lactamase-Producing E. coli from the United States, 2000 to 2009 
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.
doi:10.1128/AAC.05824-11
PMCID: PMC3346636  PMID: 22354301
17.  Development of a Multiplex PCR and SHV Melting-Curve Mutation Detection System for Detection of Some SHV and CTX-M β-Lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan 
Journal of Clinical Microbiology  2005;43(9):4486-4491.
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.
doi:10.1128/JCM.43.9.4486-4491.2005
PMCID: PMC1234143  PMID: 16145096
18.  Molecular Characteristics of Travel-Related Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates from the Calgary Health Region▿  
Extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli has recently emerged as a major risk factor for community-acquired, travel-related infections in the Calgary Health Region. Molecular characterization was done on isolates associated with infections in returning travelers using isoelectric focusing, PCR, and sequencing for blaCTX-Ms, blaTEMs, blaSHVs, blaOXAs, and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with pulsed-field gel electrophoresis using XbaI and multilocus sequence typing (MLST). A total of 105 residents were identified; 6/105 (6%) presented with hospital-acquired infections, 9/105 (9%) with health care-associated community-onset infections, and 90/105 (86%) with community-acquired infections. Seventy-seven of 105 (73%) of the ESBL-producing E. coli isolates were positive for blaCTX-M genes; 55 (58%) produced CTX-M-15, 13 (14%) CTX-M-14, six (6%) CTX-M-24, one (1%) CTX-M-2, one (1%) CTX-M-3, and one (1%) CTX-M-27, while 10 (10%) produced TEM-52, three (3%) TEM-26, 11 (11%) SHV-2, and four (4%) produced SHV-12. Thirty-one (30%) of the ESBL-producing E. coli isolates were positive for aac(6′)-Ib-cr, and one (1%) was positive for qnrS. The majority of the ESBL-producing isolates (n = 95 [90%]) were recovered from urine samples, and 83 (87%) were resistant to ciprofloxacin. The isolation of CTX-M-15 producers belonging to clone ST131 was associated with travel to the Indian subcontinent (India, Pakistan), Africa, the Middle East, and Europe, while clonally unrelated strains of CTX-M-14 and -24 were associated with travel to Asia. Our study suggested that clone ST131 coproducing CTX-M-15, OXA-1, TEM-1, and AAC(6′)-Ib-cr and clonally unrelated CTX-M-14 producers have emerged as important causes of community-acquired, travel-related infections.
doi:10.1128/AAC.00061-09
PMCID: PMC2687226  PMID: 19364876
19.  Epidemiology and Risk Factors for Isolation of Escherichia coli Producing CTX-M-Type Extended-Spectrum β-Lactamase in a Large U.S. Medical Center 
A case-case-control study was conducted to identify independent risk factors for recovery of Escherichia coli strains producing CTX-M-type extended-spectrum β-lactamases (CTX-M E. coli) within a large Southeastern Michigan medical center. Unique cases with isolation of ESBL-producing E. coli from February 2010 through July 2011 were analyzed by PCR for blaCTX-M, blaTEM, and blaSHV genes. Patients with CTX-M E. coli were compared to patients with E. coli strains not producing CTX-M-type ESBLs (non-CTX-M E. coli) and uninfected controls. Of 575 patients with ESBL-producing E. coli, 491 (85.4%) isolates contained a CTX-M ESBL gene. A total of 319 (84.6%) patients with CTX-M E. coli (282 [74.8%] CTX-M-15 type) were compared to 58 (15.4%) non-CTX-M E. coli patients and to uninfected controls. Independent risk factors for CTX-M E. coli isolation compared to non-CTX-M E. coli included male gender, impaired consciousness, H2 blocker use, immunosuppression, and exposure to penicillins and/or trimethoprim-sulfamethoxazole. Compared to uninfected controls, independent risk factors for isolation of CTX-M E. coli included presence of a urinary catheter, previous urinary tract infection, exposure to oxyimino-cephalosporins, dependent functional status, non-home residence, and multiple comorbid conditions. Within 48 h of admission, community-acquired CTX-M E. coli (n = 51 [16%]) and non-CTX-M E coli (n = 11 [19%]) strains were isolated from patients with no recent health care contacts. CTX-M E. coli strains were more resistant to multiple antibiotics than non-CTX-M E. coli strains. CTX-M-encoding genes, especially blaCTX-M-15 type, represented the most common ESBL determinants from ESBL-producing E. coli, the majority of which were present upon admission. Septic patients with risk factors for isolation of CTX-M E. coli should be empirically treated with appropriate agents. Regional infection control efforts and judicious antibiotic use are needed to control the spread of these organisms.
doi:10.1128/AAC.02516-12
PMCID: PMC3719715  PMID: 23752516
20.  Antibiotic-Resistant Escherichia coli Bacteria, Including Strains with Genes Encoding the Extended-Spectrum Beta-Lactamase and QnrS, in Waterbirds on the Baltic Sea Coast of Poland▿  
Applied and Environmental Microbiology  2010;76(24):8126-8134.
Individual cloacal swabs of mallards (Anas platyrhynchos) and of herring gulls (Larus argentatus), as well as samples of waterbird feces obtained in 2008 and 2009, were cultivated for Escherichia coli. Isolates of E. coli were tested for susceptibilities to 12 antimicrobial agents by the disk diffusion method. Moreover, the samples were subcultivated on MacConkey agar (MCA) containing cefotaxime (2 mg liter−1) to detect E. coli with extended-spectrum beta-lactamase (ESBL) and subsequently on MCA supplemented with ciprofloxacin (0.05 mg liter−1) and MCA with nalidixic acid (20 mg liter−1) to isolate fluoroquinolone-resistant E. coli. PCR was used to detect specific antibiotic resistance genes. We found 9 E. coli isolates producing ESBL with bla genes: blaCTX-M-1 (6 isolates), blaCTX-M-9 plus blaTEM-1b (1 isolate), blaCTX-M-15 plus blaOXA-1 (1 isolate), and blaSHV-12 (1 isolate). In the isolate with blaCTX-M-15, the gene aac(6)-Ib-cr was also detected. The bla genes were harbored by transferable plasmids of the IncN and IncI1 groups. Nine quinolone-resistant E. coli isolates with qnrS genes were found and characterized. The gene qnrS was associated with a Tn3-like transposon on the IncX1 plasmid together with blaTEM-1 in two isolates. The gene qnrS was also harbored by conjugative plasmids of the IncN and IncX2 groups. Even if populations of wild birds are not directly influenced by antibiotic practice, we have demonstrated that antibiotic-resistant E. coli strains, including strains with various ESBL and qnrS genes, are found in the feces of wild birds on the coast of the Baltic Sea in Poland.
doi:10.1128/AEM.01446-10
PMCID: PMC3008254  PMID: 20952638
21.  Nationwide high prevalence of CTX-M and an increase of CTX-M-55 in Escherichia coli isolated from patients with community-onset infections in Chinese county hospitals 
BMC Infectious Diseases  2014;14(1):659.
Background
In order to investigate the epidemiology, molecular characteristics, and distribution of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Escherichia coli from community-onset infections in Chinese county hospitals.
Methods
E. coli isolates were collected from patients with community-onset infections in 30 county hospitals. ESBL activity was confirmed by double-disc diffusion. Genetic confirmation and molecular typing of ESBL- and AmpC-producing isolates was determined by PCR and DNA sequencing. ESBL-positive isolates were further characterised by multi-locus sequence typing.
Results
Of 550 E. coli isolates, 256 (46.5%) carried ESBL genes and all were of the CTX-M type. The prevalence of ESBL-producing strains varied from 30.2% to 57.0% across different regions of China. Overall, 12 blaCTX-M subtypes were detected; the most abundant were blaCTX-M-14 (163/256 isolates, 64.5%), blaCTX-M-55(47/256, 18.4%), and blaCTX-M-15 (31/256, 12.1%). CMY-2-like AmpC β-lactamases were detected in 11 strains, three of which co-existed with blaCTX-M. A total of 64 sequence types (STs) were detected in 256 ESBL-producing strains, including nine that were new. ST131 was the most abundant type (27 isolates, 12.7%), followed by ST69 (14 isolates, 6.6%), ST405 (14 isolates, 6.6%), and ST38 (12 isolates, 5.6%).
Conclusions
This study revealed that the widespread prevalence of ESBLs among outpatient infections has reached a high level in county hospitals. The CTX-M genotype was most dominant, comprising a variety of subtypes. This is the first time the incidence of CTX-M-55 has exceeded that of CTX-M-15 in China. No predominant ST was detected, suggesting that ESBL-producing E. coli strains originate in different clones.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-014-0659-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12879-014-0659-0
PMCID: PMC4265337  PMID: 25466590
22.  Molecular Characteristics of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates Causing Bacteremia in the Calgary Health Region from 2000 to 2007: Emergence of Clone ST131 as a Cause of Community-Acquired Infections ▿  
A study was designed to characterize extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolates causing bacteremia over an 8-year period (2000 to 2007) in a large well-defined geographical region. Molecular characterization was done by using isoelectric focusing; PCR; and sequencing of the blaCTX-M-, blaTEM-, blaOXA-, blaSHV-, and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined by pulsed-field electrophoresis with XbaI and multilocus sequence typing. A total of 67 patients with incident bloodstream infections were identified, and the majority presented with community-acquired infections involving the urinary and biliary tracts. Of the 67 ESBL-producing E. coli isolates recovered, 60 (90%) were positive for blaCTX-M genes; 32 (48%) produced CTX-M-15, 25 (37%) produced CTX-M-14, 1 (2%) produced CTX-M-24, 1 (2%) produced CTX-M-2, and 1 (2%) produced CTX-M-3, while 2 (3%) produced TEM-52 and 5 (7%) produced SHV-2. Twenty-four (36%) isolates were positive for aac(6′)-Ib-cr. The majority of isolates were resistant to ciprofloxacin (60 [90%] isolates) and gentamicin (40 [60%] isolates). The occurrence of ESBL-producing isolates was stable during the first 5 years, but there was a substantial increase from 2005 to 2007, mostly due to clone ST131, which produces CTX-M-15 and CTX-M-14, in blood cultures submitted from the community. Our results illustrated that E. coli clone ST131, which coproduces CTX-M-15, OXA-1, TEM-1, and aac(6′)-Ib-cr, has emerged as an important cause of community-onset bacteremia caused by ESBL-producing E. coli isolates; and this is the first study to identify CTX-M-14 in E. coli clone ST131.
doi:10.1128/AAC.00247-09
PMCID: PMC2704701  PMID: 19380595
23.  Spread of Extended-Spectrum β-Lactamase CTX-M-Producing Escherichia coli Clinical Isolates in Community and Nosocomial Environments in Portugal▿  
Of the 181 unduplicated Escherichia coli strains isolated in nine different hospitals in three Portuguese regions, 119 were extended-spectrum β-lactamase (ESBL)-CTX-M producers and were selected for phenotype and genotype characterization. CTX-M producer strains were prevalent among community-acquired infections (56%), urinary tract infections (76%), and patients ≥60 years old (76%). In MIC tests, all strains were resistant to cefotaxime, 92% were resistant to ceftazidime, 93% were resistant to quinolones, 89% were resistant to aminoglycoside, and 26% were resistant to trimethoprim-sulfamethoxazole; all strains were sensitive to carbapenems, and 92% of the strains had a multidrug resistance phenotype. Molecular methods identified 109 isolates harboring a blaCTX-M-15 gene, 1 harboring the blaCTX-M-32 gene (first identification in the country), and 9 harboring the blaCTX-M-14 gene. All isolates presented the ISEcp1 element upstream from the blaCTX-M genes; one presented the IS903 element (downstream of blaCTX-M-14 gene), and none had the IS26 element; 85% carried blaTEM-1B, and 84% also carried a blaOXA-30. Genetic relatedness analysis based on pulsed-field gel electrophoresis defined five clusters and indicated that 76% of all isolates (from cluster IV) corresponded to a single epidemic strain. Of the 47 strains from one hospital, 41 belonged to cluster IV and were disseminated in three main wards. CTX-M-producing E. coli strains are currently a problem in Portugal, with CTX-M-15 particularly common. This study suggests that the horizontal transfer of blaCTX-M genes, mediated by plasmids and/or mobile elements, contributes to the dissemination of CTX-M enzymes to community and hospital environments. The use of extended-spectrum cephalosporins, quinolones, and aminoglycosides is compromised, leaving carbapenems as the therapeutic option for severe infections caused by ESBL producers.
doi:10.1128/AAC.01412-06
PMCID: PMC1891395  PMID: 17371815
24.  Increase in Resistance to Extended-Spectrum Cephalosporins in Salmonella Isolated from Retail Chicken Products in Japan 
PLoS ONE  2015;10(2):e0116927.
Extended-spectrum β-lactamase (ESBL)-producing Salmonella are one of the most important public health problems in developed countries. ESBL-producing Salmonella strains have been isolated from humans in Asian countries neighboring Japan, along with strains harboring the plasmid-mediated extended-spectrum cephalosporin (ESC)-resistance gene, ampC (pAmpC). However, only a few studies have investigated the prevalence of ESC-resistant Salmonella in chicken products in Japan, which are the main vehicle of Salmonella transmission. The aim of this study was to investigate the prevalence of ESBL-producing, pAmpC-harboring, or carbapenem-resistant Salmonella in chicken products in Japan. In total, 355 out of 779 (45.6%) chicken product samples collected from 1996–2010 contained Salmonella, resulting in 378 distinct isolates. Of these isolates, 373 were tested for resistance to ESCs, cephamycins, or carbapenems. Isolates that showed resistance to one or more of these antimicrobials were then examined by PCR and DNA sequence analysis for the presence of the blaCMY, blaCTX-M, blaTEM, and blaSHV resistance genes. Thirty-five resistant isolates were detected, including 26 isolates that contained pAmpC (blaCMY-2), and nine ESBL-producing isolates harboring blaCTX-M (n = 4, consisting of two blaCTX-M-2 and two blaCTX-M-15 genes), blaTEM (n = 4, consisting of one blaTEM-20 and three blaTEM-52 genes), and blaSHV (n = 1, blaSHV-12). All pAmpC-harboring and ESBL-producing Salmonella isolates were obtained from samples collected after 2005, and the percentage of resistant isolates increased significantly from 0% in 2004 to 27.9% in 2010 (P for trend = 0.006). This increase was caused in part by an increase in the number of Salmonella enterica subsp. enterica serovar Infantis strains harboring an approximately 280-kb plasmid containing blaCMY-2 in proximity to ISEcp1. The dissemination of ESC-resistant Salmonella containing plasmid-mediated blaCMY-2 in chicken products indicates the need for the development of continuous monitoring strategies in the interests of public health.
doi:10.1371/journal.pone.0116927
PMCID: PMC4314076  PMID: 25642944
25.  Infected foot ulcers in male and female diabetic patients: a clinico-bioinformative study 
Background
The study aimed at (i) characterizing the mode of transmission of blaCTX-M and blaTEM-1 among extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains isolated from infected diabetic foot ulcers, and (ii) identifying the risk factors for "sex-associated multidrug resistant Gram-negative bacterial (MDRGNB)-infection status" of the ulcers.
Methods
Seventy-seven diabetic patients having clinically infected foot ulcers were studied in a consecutive series. The E. coli strains isolated from the ulcers were screened for blaCTX-M, blaTEM-1, armA, rmtA and rmtB during the 2-year study-period. PCR amplified blaCTX-M genes were cloned and sequenced. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used for the analysis of genetic relatedness of the ESBL-producers. Risk factors for "sex-associated MDRGNB-infection status" of ulcers were assessed. Modeling was performed using Swiss-Model-Server and verified by Procheck and verify3D programmes. Discovery Studio2.0 (Accelrys) was used to prepare Ramachandran plots. Z-scores were calculated using 'WHAT IF'-package. Docking of cefotaxime with modeled CTX-M-15 enzyme was performed using Hex5.1.
Results
Among 51 E. coli isolates, 14 (27.5%) ESBL-producers were identified. Only 7 Class1 integrons, 2 blaCTX-M-15, and 1 blaTEM-1 were detected. Ceftazidime and cefotaxime resistance markers were present on the plasmidic DNA of both the blaCTX-M-15 positive strains and were transmissible through conjugation. The residues Asn132, Glu166, Pro167, Val172, Lys234 and Thr235 of CTX-M-15 were found to make important contacts with cefotaxime in the docked-complex. Multivariate analysis proved 'Glycemic control at discharge' as the single independent risk factor.
Conclusions
Male diabetic patients with MDRGNB-infected foot ulcers have poor glycemic control and hence they might have higher mortality rates compared to their female counterparts. Plasmid-mediated conjugal transfer, albeit at a low frequency might be the possible mechanism of transfer of blaCTX-M-15 resistance marker in the present setting. Since the docking results proved that the amino acid residues Asn132, Glu166, Pro167, Val172, Lys234 and Thr235 of CTX-M-15 (enzyme) make important contacts with cefotaxime (drug) in the 'enzyme-drug complex', researchers are expected to duly utilize this information for designing more potent and versatile CTX-M-inhibitors.
doi:10.1186/1476-0711-9-2
PMCID: PMC2821376  PMID: 20070911

Results 1-25 (1349953)