Characterization of CTX‐M‐producing Escherichia coli by repetitive sequence‐based PCR and real‐time PCR‐based replicon typing of CTX‐M‐15 plasmids

The emergence of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae is a major global concern. CTX‐M is the dominating ESBL type worldwide, and CTX‐M‐15 is the most widespread CTX‐M type. The dissemination of CTX‐M appears to be in part due to global spread of the Escherichia coli clone O25b‐ST131. However, the gene‐encoding CTX‐M is mainly located on mobile genetic elements, such as plasmids, that also promote the horizontal dissemination of the CTX‐M genes. In this study, 152 CTX‐M‐producing E. coli isolated in 1999–2008 in Örebro County, Sweden, were typed using a commercial repetitive sequence‐based PCR (the DiversiLab system), and the prevalence of ST131 was investigated by pabB PCR. Real‐time PCR‐based plasmid replicon typing was performed on 82 CTX‐M‐15‐producing E. coli isolates. In general, the CTX‐M‐producing E. coli population was genetically diverse; however, ST131 was highly prevalent (27%), and the dominating clone in our area. The bla_CTX‐M‐15 gene was mainly located on IncF plasmids (69%), but a relatively high proportion of IncI1 plasmids (29%) were also detected among E. coli with diverse rep‐PCR patterns, indicating that horizontal transmission of IncI1 plasmids carrying bla_CTX‐M‐15 may have occurred between different E. coli strains.     

Dissemination of metallo‐β‐lactamase in Pseudomonas aeruginosa isolates in Egypt: mutation in blaVIM‐4

This study was designed to investigate the prevalence of metallo‐β‐lactamase (MBL) in Pseudomonas aeruginosa isolates collected from Suez Canal University Hospital in Ismailia, Egypt. Antibiotic susceptibility testing and phenotypic and genotypic screening for MBLs were performed on 147 isolates of P. aeruginosa. MICs were determined by agar dilution method for carbapenem that was ≥2 μg/mL for meropenem. MBL genes were detected by multiplex and monoplex PCR for P. aeruginosa‐harbored plasmids. Mutation profile of sequenced MBL genes was screened using online software Clustal Omega. Out of 147 P. aeruginosa, 39 (26.5%) were carbapenem‐resistant isolates and 25 (64%) were confirmed to be positive for MBLs. The susceptibility rate of P. aeruginosa toward polymyxin B and norfloxacin was 99% and 88%, respectively. Identification of collected isolates by API analysis and constructed phylogenetic tree of 16S rRNA showed that the isolates were related to P. aeruginosa species. The frequency of blaGIM‐1, blaSIM‐1, and blaSPM‐1 was 52%, 48%, and 24%, respectively. BlaVIM and blaIMP‐like genes were 20% and 4% and the sequences confirm the isolate to be blaVIM‐1, blaVIM‐2, blaVIM‐4, and blaIMP‐1. Three mutations were identified in blaVIM‐4 gene. Our study emphasizes the high occurrence of multidrug‐resistant P. aeruginosa‐producing MBL enzymes.     

Survival in the environment is a possible key factor for the expansion of Escherichia coli strains producing extended‐spectrum β‐lactamases

Acquired resistance to cephalosporins in Enterobacteriaceae is a global problem. After an outbreak at Uppsala University Hospital of extended‐spectrum β‐lactamase (ESBL)‐positive Klebsiella pneumoniae producing CTX‐M‐15, there was a shift from AmpC to ESBL production among Escherichia coli isolates. To explore the basis for this epidemiological shift, 46 E. coli isolates (ESBLs, n = 23; AmpC, n = 23) were characterized with regard to genetic relatedness, β‐lactamase, replicon and integron types, antibiotic resistance profiles, and genes encoding virulence factors. In addition, the survival in the environment and on hospital‐associated materials was analysed. CTX‐M‐15 was the most frequent ESBL (78%). Only three (13%) of the AmpC enzymes were harboured on plasmids (CMY‐2, DHA‐1). Independent of plasmid‐mediated beta‐lactamase, IncF plasmids predominated and only class I integrons were detected. The ESBL producers carried more virulence genes (p = 0.04), exhibited a broader resistance phenotype (p = 0.01) and survived significantly longer (p = 0.03) on different materials than the AmpC‐producing isolates. In conclusion, ESBL‐producing isolates had properties which are likely to augment their competitiveness. Apart from antibiotic resistance and virulence factors, extended survival in the environment could be a selective trait for successful ESBL‐producing E. coli strains.     

Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae

Background: Detecting plasmid-mediated AmpC (pAmpC) β-lactamase-producing organism is important for optimal infection control and providing accurate and effective treatment option for physicians. Objectives: The aim of this study was to investigate the prevalence of pAmpC β-lactamase and compare the results of boronic acid (BA) disk test with other phenotypic tests detecting AmpC positive isolates. Materials and Methods: A total of 273 clinical isolates of Klebsiella pneumoniae (n: 82) and Escherichia coli (n: 191) were analysed. The presence of pAmpC β-lactamase was determined by BA disk test, cefoxitin (FOX) screening test, modified three dimensional test (M3DT), and multiplex polymerase chain reaction (PCR). Pulsed-field gel electrophoresis was performed to evaluate the genetic similarities between isolates. To detect extended spectrum β-lactamases (ESBL) in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution. Results: Of the 273 strains tested, 127 strains were found FOX resistant, 114 were positive by M3DT, 108 were positive in BA disk test, and the multiplex PCR detected 24 pAmpC β-lactamase-positive isolate. The prevalence of AmpC-producing strains was 10.9% in E. coli and 3.6% in K. pneumoniae in the tested population by PCR. CIT and MOX group genes were predominant type in these strains. Conclusion: These results emphasize that clinical laboratories should consider testing the presence of pAmpC enzymes particularly in FOX-resistant isolates, and BA disk test will improve detection of this emerging resistance phenotype.     

Prevalence and molecular epidemiology of acquired AmpC β-lactamases and carbapenemases in Enterobacteriaceae isolates from 35 hospitals in Spain

The purpose of this investigation was to determine the prevalence of plasmid-mediated AmpC (pAmpC) and carbapenemases in Enterobacteriaceae collected from 35 hospitals in Spain and to establish their epidemiological relationships. We conducted a prospective multi-centre study on pAmpC- or carbapenemase-producing Enterobacteriaceae isolates from clinical samples collected from February to July 2009. The strains suspected to carry pAmpC were resistant or showed intermediate susceptibility to co-amoxiclav and second- or third-generation cephalosporins. Strains suspected to carry a carbapenemase were selected because they showed a minimum inhibitory concentration (MIC) to imipenem >1 mg/L. Polymerase chain reaction (PCR) and a sequencing strategy were used to characterise the enzymes. The clonal relationships between isolates was analysed by pulsed field gel electrophoresis (PFGE). Among 100,132 Enterobacteriaceae isolates collected, 1,654 were compatible with the production of pAmpC or carbapenemases. We found a prevalence of 0.64 % of pAmpC (n?=?635) and 0.04 % of carbapenemases (n?=?43). The most prevalent pAmpC enzymes were CMY-type (78.3 %), DHA-type (19.5 %), ACC-type (1.6 %) and FOX-type (0.6 %). The CMY-type was the most frequent in Escherichia coli and Proteus mirabilis species, whereas the DHA-type was mainly found in Klebsiella spp. The enzymes involved in carbapenem resistance were VIM-1, IMP-22 and the new IMP-28. Nine new bla genes were described: bla _CMY-54, bla _CMY-55, bla _CMY-56, bla _CMY-57, bla _CMY-96, bla _DHA-6, bla _DHA-7, bla _FOX-8 and bla _IMP-28. The prevalence of pAmpC or carbapenemases found is not negligible. The CMY-types were the predominant pAmpC, whereas the VIM or IMP enzymes were the predominant carbapenemases. Furthermore, we observed a great genetic diversity among pAmpC-producing strains and a close clonal relationship between carbapenemase-producing strains.     

Predominance of IncL/M and IncF plasmid types among CTX‐M‐ESBL‐producing Escherichia coli and Klebsiella pneumoniae in Bulgarian hospitals

Our objective was to investigate the plasmid replicon‐types involved in spread of ESBLs among Bulgarian Klebsiella pneumoniae and Escherichia coli. Sixty‐three isolates, with transferable beta‐lactam resistance determinants, collected between 2007 and 2009 in six medical institutions, were analysed with respect to their antimicrobial susceptibility, ESBL‐, RAPD‐, and plasmid replicon‐type. Phylogenetic typing and screening for the O25b‐ST131 lineage were carried out for E. coli. The predominant ESBLs were CTX‐M‐15 (81%) among E. coli and CTX‐M‐3 (58%) among K. pneumoniae. Other sporadically found ESBLs were SHV‐12 and TEM‐139, and for the first time in Bulgaria, CTX‐M‐1 and CTX‐M‐14. Replicon typing revealed that plasmids carrying bla_CTX‐M‐3 exclusively belonged to IncL/M‐type, while bla_CTX‐M‐15 was predominantly (94%) associated with IncF‐type plasmids. Among E. coli, 59% of the isolates were clonally related. Isolates of that cluster produced CTX‐M‐15, belonged to the O25b‐ST131 lineage, predominantly harboured plasmids with the FIA replicon, and were found in five centres. Among CTX‐M‐3‐producing K. pneumoniae, two prevailing RAPD‐types were found, one remained restricted to one centre and the second was found in three centres. The incompatibility groups IncN and IncA/C linked with bla_SHV‐12 respectively bla_TEM‐139 were found only once. To the best of our knowledge, this is the first detailed investigation of plasmids carrying ESBL genes among Bulgarian isolates demonstrating wide distribution of conjugative IncF plasmids among CTX‐M‐15‐producing E. coli and IncL/M plasmids among CTX‐M‐3 positive K. pneumoniae isolates.     

Wild boars as reservoirs of extended‐spectrum beta‐lactamase (ESBL) producing Escherichia coli of different phylogenetic groups

ESBL‐producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10.4%) of wild boars in Portugal. The ESBL types identified by PCR and sequencing were bla_CTX‐M‐1 (6 isolates) and bla_CTX‐M‐1 + bla_TEM1‐b (2 isolates). Further resistance genes detected included tet (A) or tet (B) (in three tetracycline‐resistant isolates), aad A (in three streptomycin‐resistant isolates), cml A (in one chloramphenicol‐resistant isolate), sul 1 and/or sul 2 and/or sul 3 (in all sulfonamide‐resistant isolates). The intI 1 gene encoding class 1 integrase was detected in all ESBL‐producing E. coli isolates. One isolate also carried the intI 2 gene, encoding class 2 integrase. The ESBL‐producing E. coli isolates could be assigned to phylogenetic groups B1 (3 isolates), B2 (3 isolates) or A (2 isolates). Amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in three nalidixic acid‐resistant and ciprofloxacin‐susceptible isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in two nalidixic acid‐ and ciprofloxacin‐resistant isolates. As evidenced by this study wild boars could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Emergence of co-production of plasmid-mediated AmpC beta-lactamase and ESBL in cefoxitin-resistant uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Plasmid-mediated AmpC (pAmpC) and ESBL co-production was detected in Escherichia coli a major etiologic agent of urinary tract infection. Isolates resistant to cefoxitin by CLSI methodology were tested for pAmpC beta-lactamase using phenylboronic acid and ESBLs by combined disk diffusion method. pAmpC/ESBL genes were characterized by PCR and sequencing. Transconjugation experiments were done to study the transfer of pAmpC and ESBL production from clinical isolates as donor to E. coli J53 AziR as recipient. Incompatibility groups of transmissible plasmids were classified by PCR-based replicon typing (PBRT). Among 148 urine culture positive isolates, E. coli was reported in 39.86 % (59/148), with 93.22 % (55/59) of cefoxitin resistance. pAmpC production was detected in 25, with varied distribution of blaCMY-2 and blaDHA-1type genes alone (n?=?13 and 7 respectively) or in combination (n?=?5). ESBL co-production was observed in 88 % (22/25) of pAmpC producing isolates with predominance of blaTEM (n?=?20). Twenty-three transconjugants showed transmission of pAmpC-and ESBL-resistant genes with co-carriage of blaCMY-2 and blaTEM (n?=?15) in plasmids of IncF type (n?=?9) being predominant, followed by IncI1 (n?=?4) and IncH1 (n?=?2) in combination. All clinical isolates were clonally diverse. Resistance against different beta-lactams in uropathogenic E. coli has been an emerging concern in resource- poor countries such as India. Knowledge on the occurrence of AmpC beta-lactamases and ESBL amongst this pathogen and its transmission dynamics may aid in hospital infection control.     

Antibiotic resistance and molecular epidemiology of the beta‐lactamase‐producing Haemophilus influenzae isolated in Chongqing,China

This study aimed to determine the antibiotic resistance and molecular epidemiology of Haemophilus influenzae isolated from children with acute respiratory infection in Chongqing, China. To this end, 1967 H. influenzae isolates from 2006 to 2009 were analysed regarding β‐lactamase production and antibiotic resistance. Ninety‐nine β‐lactamase‐producing H. influenzae isolates from 2010 were analysed for antibiotic resistance and promoter regions of bla_TEM‐1. β‐lactamase production was found in 35.8% (705/1967) of the strains. All ninety‐nine β‐lactamase‐producing strains from 2010 were of the TEM‐1 type as determined by PCR but did not produce the predicted 1075 bp product. According to PCR‐SSCP and DNA sequencing, the promoter regions of bla_TEM‐1 were categorized into 6 genotypes as SSCP1 (Pdel), SSCP2 (Pa/Pb), SSCP3 (P4), SSCP4 (Prpt.b), SSCP5 (2Prpt) and SSCP6 (P3.b). The Pdel, Pa/Pb and Prpt.b were common promoters of bla_TEM‐1 for H. influenzae isolated from children in Chongqing. Strains with Prpt.b were more resistant to ampicillin (AMP) than strains with Pdel, Pa/Pb and P4 (p < 0.05). Therefore, bla_TEM‐1 β‐lactamase is the main mechanism for resistance of H. influenzae to ampicillin in Chongqing. Furthermore, the Prpt.b promoters may be related to the high resistance of H. influenzae to AMP.     

The molecular characteristics of cefepime-susceptible Escherichia coli and Klebsiella spp. isolates with a positive β-lactamase screening test result but negative confirmation

A negative extended-spectrum β-lactamase (ESBL) phenotypic confirmation test result obtained after a positive ESBL screening test result but which was cefepime-susceptible (NCPSCS) using the Clinical and Laboratory Standards Institute (CLSI) methods has been observed among isolates of Escherichia coli and Klebsiella spp. in the antimicrobial surveillance program in Shanghai, China. Among isolates collected from Huashan Hospital in 2005, NCPSCS strains were observed in 2.5% of 433 E. coli isolates and in 1.2% of 562 Klebsiella spp. isolates. We then selected 11 E. coli isolates and seven Klebsiella spp. NCPSCS isolates. Transmission electron microscopy (TEM), SHV, plasmid-borne AmpC, and CTX-M type ESBL genes were detected by the polymerase chain reaction (PCR) method. We found that all except two K. pneumoniae strains of NCPSCS isolates producing ESBL and AmpC harbored a plasmid-borne CMY-2 or DHA-1 type AmpC enzyme. The majority of NCPSCS E. coli and K. pneumoniae strains from Shanghai harbor plasmid-borne AmpC enzyme, and we recommend that, when NCPSCS strains are identified, further work such as the PCR detection of ESBL genes is necessary to determine whether they will produce ESBLs. The ESBL-positive strains should be reported as resistant to cefepime according to the CLSI guidelines.     

Isolation of Enterobacter aerogenes carrying blaTEM‐1 and blaKPC‐3 genes recovered from a hospital Intensive Care Unit

Enterobacter aerogenes has recently emerged as an important hospital pathogen. In this study, we showed the emergence of E. aerogenes isolates carrying the bla_KPC gene in patients colonized by carbapenem‐resistant Klebsiella pneumoniae strains. Two multiresistant E. aerogenes isolates were recovered from bronchial aspirates of two patients hospitalized in the Intensive Care Unit at the “Santa Maria della Scaletta” Hospital, Imola. The antimicrobial susceptibility test showed the high resistance to carbapenems and double‐disk synergy test confirmed the phenotype of KPC and AmpC production. Other investigation revealed that ESBL and bla_KPC genes were carried on the conjugative pKpQIL plasmid. This is a relevant report in Italy that describes a nosocomial infection due to the production of KPC beta‐lactamases by an E. aerogenes isolate in patients previously colonized by K. pneumoniae carbapenem‐resistant. In conclusion, it's necessary a continuous monitoring of multidrug‐resistant strains for the detection of any KPC‐producing bacteria that could expand the circulation of carbapenem‐resistant pathogens.     

Clonal dissemination of multilocus sequence type 11 Klebsiella pneumoniae carbapenemase – producing K. pneumoniae in a Chinese teaching hospital

Klebsiella pneumoniae carbapenemase (KPC)‐producing K. pneumoniae has disseminated rapidly in China. We aimed to analyze the molecular epidemiology of four KPC‐producing K. pneumoniae strains isolated from a suspected clonal outbreak during a 3‐month period and to track the dissemination of KPC‐producing K. pneumonia retrospectively. We created antimicrobial susceptibility profiles using an automated broth microdilution system and broth microdilution methods. We screened carbapenemase and KPC phenotypes using the modified Hodge test and meropenem–boronic acid (BA) disk test, respectively. We identified β‐lactamase genes with PCR and sequencing. We investigated clonal relatedness for epidemiological comparison using pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates expressed multidrug resistance and yielded positive results for the modified Hodge and meropenem‐BA disk tests. The isolates all carried bla_KPC‐2, and coproduced CTX‐M–type extended‐spectrum β‐lactamase. PFGE and MLST showed that the isolates were clonally related. The PFGE patterns of these isolates had ≥90% similarity. We found a single clone, sequence type (ST) 11, and its typical dissemination mode resembled clonal spread. The dissemination of KPC‐producing K. pneumoniae is clonally related and there is probable local transmission of a successful ST11 clone.     

Low‐level resistance and clonal diversity of Pseudomonas aeruginosa among chronically colonized cystic fibrosis patients

A prospective study was conducted in Brazil to evaluate antimicrobial resistance patterns and molecular epidemiology of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients with chronic lung infection. All isolates were obtained between May 2009 and June 2010 from 75 patients seen in four reference centers in Brazil: HCPA (20 patients) and HEOM (15 patients), located in southern and northeastern Brazil, respectively; IFF (20 patients) and HUPE (20 patients), both in southwestern Brazil. Antimicrobial susceptibility testing, PCR for detection of carpapenemases, and pulsed‐field gel electrophoresis (PFGE) were performed in 274 isolates. A total of 224 PFGE types were identified and no clones were found circulating among the centers or within the same center. Despite the chronic infection, most patients were colonized by intermittent clones. Only three patients (4%) maintained the same clone during the study. The resistance rates were lower than 30% for the majority of antimicrobials tested in all centers and only 17% of isolates were multiresistant. Isolates (n = 54) with reduced susceptibility to imipenem and/or meropenem presented negative results for bla_SPM‐1, bla_IMP?1, bla_VIM, and bla_KPCgenes. Our results indicate an unexpected low level of antimicrobial resistance and a high genotypic diversity among P. aeruginosa from Brazilian chronic CF patients.     

Unreliable Extended-Spectrum β-Lactamase Detection in the Presence of Plasmid-Mediated AmpC in Escherichia coli Clinical Isolates

The emergence of extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC (pAmpC) enzymes in Escherichia coli raises concern regarding accurate laboratory detection and interpretation of susceptibility testing results. Twenty-six cefpodoxime ESBL screen-positive, cefoxitin-resistant E. coli clinical isolates were subjected to clavulanate ESBL confirmatory testing employing disk augmentation, Etest, and the BD Phoenix NMC/ID-132 panel. Phenotypic pAmpC production was assessed by boronic acid disk augmentation. ESBL and pAmpC genes were detected by gene amplification and sequencing. ESBL genes (SHV and/or CTX-M-type genes) were detected in only 7/26 ESBL screen-positive isolates. Of 23 aminophenylboronic acid screen-positive isolates, pAmpC genes were detected in 20 (CMY-2 or FOX-5 genes). High incidences of false-positive ESBL confirmatory results were observed for both clavulanate disk augmentation (9/19) and BD Phoenix (5/19). All were associated with the presence of pAmpC genes with or without TEM-1. Etest performed poorly, as the majority of interpretations were nondeterminable. In addition, false-negative ESBL confirmatory results were observed in isolates possessing concomitant ESBL and pAmpC genes for Etest (four of five), BD Phoenix (three of five), and disk augmentation (one of five). The results indicate poor performance of currently employed ESBL confirmatory methods in the setting of concomitant pAmpC. Some isolates with pAmpC and ESBL genes fell within the susceptible category to extended-spectrum cephalosporins, raising concern over currently employed breakpoints.     

Antimicrobial resistance,prevalence of resistance genes,and molecular characterization in intestinal Bacteroides fragilis group isolates

Since the level of antimicrobial resistance in Bacteroides fragilis has increased, monitoring the antimicrobial susceptibility could be necessary. The objectives of this study were to (i) investigate the prevalence of species, the occurrence of reduced antimicrobial susceptibility (E‐test method), and antibiotic resistance genes in the B. fragilis group and (ii) evaluate the prevalence of enterotoxigenic B. fragilis and the distribution of bft gene subtypes in hospitalized patients. As many as 475 isolates out of 250 stool samples were detected to be B. fragilis group by using conventional biochemical tests (API‐32A system) and multiplex‐PCR. In addition, 48.2%, 13.9%, 76.6%, and 1.2% of B. fragilis group isolates were resistant (according to EUCAST breakpoint) to piperacillin‐tazobactam, meropenem, clindamycin, and metronidazole, respectively. Six metronidazole‐resistant strains were isolated; B. fragilis (n: 3), B. thetaiotaomicron, B. vulgates, and B. ovatus. The presence of the cfiA, cepA, ermF, and nim genes was observed in 3.8%, 15.9%, 34.1%, and 0.7% of the B. fragilis isolates, respectively. One hundred thirty‐two B. fragilis isolates (27.8%)and 21 B  fragilis isolates (15.9%) turned out to be bft gene positive by multiplex‐PCR; eleven isolates (52.4%) harbored bft‐1, eight isolates (38%) harbored bft‐2 isotypes, and two isolates (9.5%) harbored bft‐3 isotype (16.66%). These bacteria harbor antimicrobial resistance genes that could be transferred to other susceptible intestinal strains. Further investigations on lineage analysis are needed for a better understanding of these bacteria in Iran.     

Molecular characterization and antibiotic resistance of clinical isolates of methicillin‐resistant Staphylococcus aureus obtained from Southeast of Iran (Kerman)

Staphylococcus aureus infections, particularly infections caused by methicillin‐resistant S. aureus (MRSA) strains, are emerging as a major public health problem. The aim of this study was to determine the prevalence of MRSA, antibiotic resistance profile and staphylococcal cassette chromosome mec (SCCmec) type of MRSA isolates obtained from clinical samples. Totally, 162 S. aureus isolates were obtained from clinical samples at three university hospitals in Kerman, Iran from March 2011 to February 2012. All isolates were identified as S. aureus by phenotypic methods and confirmed by PCR amplification of the nuc gene . MRSA isolates were screened by phenotypic tests and confirmed by presence of mecA gene. The minimum inhibitory concentrations (MICs) of the MRSA isolates against antibacterial agents were determined by E‐test. All isolates were analyzed by PCR for the presence of mecA and pvl genes. SCCmec typing of MRSA isolates was performed by multiplex PCR assay. Strain typing was carried out with REP‐PCR. Using mecA gene PCR and phenotypic methods, 56.8% of the isolates were identified as MRSA. All MRSA isolates were susceptible to vancomycin and linezolid. The sensitivity of MRSA isolates to trimethoprim‐sulfamethoxazole, clindamycin, ciprofloxacin, gentamicin, and erythromycin was 70.66, 66.53, 42.4, 38.05, and 29.35%, respectively. The most frequent SCCmec types were type III (48.31%) followed by type V (19.1%), type I (16.85%), and type IV (3.37%). The pvl gene was detected in 3.08% of isolates (two MRSA and three MSSA isolates). REP‐PCR typing divided the 92 MRSA isolates into 10 distinct clusters. Our results indicate that vancomycin and linezolid are the most effective antibacterial agents against MRSA isolates and SCCmec type III is predominant in MRSA strains in this area.     

Extended spectrum cephalosporin resistance among clinical isolates of Enterobacteriaceae in West Norway during 2006–2013; a prospective surveillance study

Routine surveillance of resistance to broad‐spectrum cephalosporins in Enterobacteriaceae and phenotypic identification of underlying mechanisms using a simple strategy was commenced in 2006 at our laboratory, serving West Norway. This report focuses on the results until 2013. The classical plasmid‐mediated extended spectrum beta‐lactamase (ESBL_A) among clinically relevant Escherichia coli isolates showed an increase from 0.6% to 4.3% during the surveillance period, while prevalence for other mechanisms remained stable, below 0.7%. ESBL_A in Klebsiella pneumoniae had similar prevalence in 2006 (0.6%) and 2013 (4.4%), but in between it peaked to 3.9% in 2008 and to 9.3% in 2011. Within the other species, the numbers of clinically relevant isolates and isolates‐producing ESBL_A were much lower. An increasing resistance due to hyperproduction of AmpC enzymes was seen in Enterobacter and Citrobacter, with prevalence increasing from 18% and 12.2% in 2006 to 27.5% and 26.1% in 2013, respectively. Hyperproduction of KOXY enzyme in Klebsiella oxytoca remained below 9.5% and did not show an increasing trend. The overall increase in the proportions of isolates‐producing ESBL_A in E. coli/K. pneumoniae and hyperproduction of AmpC in Enterobacter/Citrobacter necessitates measures to hinder the spread of resistant bacteria and vigilant antibiotic stewardship.     

Detection of OXA-type carbapenemases and integrons among carbapenem-resistant Acinetobactor baumannii in a teaching hospital in China

The increasing trend of carbapenem‐resistance (CR) and multi‐drug resistance (MDR) in A. baumannii worldwide has limited the therapeutic effectiveness of antibiotic therapy. The study was conducted to determine the prevalence of carbapenemases and integrons among the isolates of imipenem‐resistant A. baumannii (IRAB). A total of 71 non‐repetitive imipenem‐ resistant A. baumannii isolates were collected and tested for susceptibility to 17 antimicrobials. The modified Hodge test and EDTA‐disc synergy test were performed for the screening of carbapenemases and metallo‐β ‐lactamases (MBLs) production, respectively. Isolates were then subjected to multiplex‐PCR targeting genes encoding for OXA‐type carbapenemase, MBLs and integrases. Random amplified polymorphic DNA (RAPD) genotyping was performed to assess genetic relatedness. All isolates exhibited multi‐drug resistant phenotype. Colistin was the most active antimicrobial agent tested. Seventy‐one isolates (100%) demonstrated positive in the modified Hodge test. Thirty‐nine isolates showed positive in the EDTA‐disc synergy test, however, no MBL genes were detected. All strains possessed a bla_OXA‐51‐like gene. The co‐exis‐tence of bla_OXA‐51‐like/bla_OXA‐23‐like/intI1, bla_OXA‐51‐like/bla_OXA‐23‐like, bla_OXA‐51‐like/bla_OXA‐24‐like was detected in 91.6% (n = 65), 5.6% (n = 4), 2.8% (n = 2), respectively. Analysis of the genetic con‐text of bla_OXA‐23 showed the presence of ISAba1 upstream of bla_OXA‐23. No ISAba1 was detected upstream of bla_OXA‐51. Two different gene cassettes were found in these strains, and a high prevalence of aacA4, aadA1 and catB8 genes was observed. RAPD of 71 isolates showed 7 genotypes. The strains were mainly recovered from patients in intensive care unit, neurosurgery and department of respiratory disease. These ?ndings show that multi‐drug resistance in A. baumannii is a common problem. This study also shows a high distribution of bla_OXA‐23‐like and intI1 genes in imipenem‐resistant A. baumannii isolates. The clonal spread played an important role in the increase of OXA‐23 producing IRABs in the hospital environment. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Clonal dissemination of multilocus sequence type ST15 KPC‐2‐producing Klebsiella pneumoniae in Bulgaria

A total of 36 consecutive clinical and two fecal‐screening carbapenem‐resistant Klebsiella pneumoniae isolates from two Bulgarian university hospitals (Varna and Pleven) were investigated. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta‐lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and multilocus sequence typing (MLST). Most of the isolates demonstrated multidrug resistance profile. Amikacin and tigecycline retained good activity with susceptibility rates of 95 and 87%, respectively. The resistance rate to colistin was 63%. Six RAPD‐ and MLST‐types were identified: the dominating MLST‐type was ST15 (27 isolates), followed by ST76 (six isolates), and ST1350 (two isolates). ST101, ST258, and ST151 were detected once. All except one of the K. pneumoniae produced KPC‐2, mostly in combination with CTX‐M‐15, while for one isolate (ST101) the enzymes OXA‐48 and CTX‐M‐14 were found. All KPC‐2‐producing transconjugants revealed the presence of IncFII plasmid. The OXA‐48‐ and CTX‐M‐14‐producing isolate showed the presence of L/M replicon type. The dissemination of KPC‐2‐producing K.pneumoniae in Bulgaria is mainly due to the sustained spread of successful ST15 clone and to a lesser extent of ST76 clone. This is the first report of OXA‐48 producing ST101 K. pneumoniae in Bulgaria.     

Outbreak of CTX‐M‐15‐producing Klebsiella pneumoniae of sequence type 199 in a Latvian teaching hospital

Dumpis U, Iversen A, Balode A, Saule M, Mikla?evi?s E, Giske CG. Outbreak of CTX‐M‐15‐producing Klebsiella pneumoniae of sequence type 199 in a Latvian teaching hospital. APMIS 2010; 118: 713–6. Previous studies on the epidemiology of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae in Latvia are lacking. ESBL‐producing Klebsiella pneumoniae (n = 32) were subjected to pulsed‐field gel electrophoresis (PFGE) and selected isolates to multi‐locus sequence typing (MLST). Species identification and susceptibility testing were performed using VITEK2, and sequencing of bla_CTX‐M was performed in selected isolates. PFGE revealed one major clone (n = 23), with most of the isolates derived from the ICU. The clone harboured bla_CTX‐M‐15, was sequence type 199 and comprised two ertapenem non‐susceptible isolates. This is the first report of an ESBL outbreak in Latvia, and calls for increased epidemiological typing of ESBL‐producing Enterobacteriaceae, as well as improved infection control routines.