Emergence of carbapenem non‐susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103B and 92B harboring OXA‐type carbapenemases and metallo‐β‐lactamases in Southern India

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP‐PCR) and multi‐locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA‐carbapenemases, metallo‐β‐lactamases (MBLs) and efflux pumps. REP‐PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103^B. Second most prevalent ST belonged to clonal complex (CC) 92^B which is also referred to as international clone II. Most of the isolates were multi‐drug resistant, being susceptible only to polymyxin‐B and newer quinolones. Class D β‐lactamases such as bla_{OXA‐51‐like} (100%), bla_{OXA‐23‐like} (56.8%) and bla_{OXA‐24‐like} (14.8%) were found to be predominant, followed by a class B β‐lactamase, namely bla_IMP‐1 (40.7%); none of the isolates had bla_OXA‐58 like, bla_NDM‐1 or bla_SIM‐1. Genes of efflux‐pump adeABC were predominant, most of isolates being biofilm producers that were PCR‐positive for autoinducer synthase gene (>94%). Carbapenem non‐susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA‐type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.     

Antimicrobial susceptibility profile of Acinetobacter species isolated from blood cultures in two Japanese university hospitals

Carbapenem‐resistant Acinetobacter baumannii has rapidly spread worldwide. This study investigated antibiotic susceptibility and genotypic resistance of 123 consecutive blood culture isolates of Acinetobacter species collected between 2003 and 2011 in two Japanese hospitals. The isolates were assigned to 13 species. Carbapenem resistance was detected in four isolates. Only one A. baumannii isolate had bla_OXA‐23 together with ISAba1; the remaining three isolates had IMP‐1 metallo‐β‐lactamase. Quinolone resistance was detected in five isolates that had point mutations in the quinolone resistance‐determining region. The predominance of various non‐A. baumannii species and low prevalence of carbapenem resistance among blood culture isolates of Acinetobacter species in two Japanese hospitals were confirmed.     

<Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> is Able to Gain and Maintain a Plasmid Harbouring In35 Found in <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> Isolates From Argentina

The aim of this study was to determine the presence of bla _CTX-M-2 in our A. baumannii population and their putative role as an alternative mechanism of resistance to third-generation cephalosporins in this species. The bla _CTX-M-2 gene is widespread among the Enterobacteriaceae isolates from our country; however, it was not found in 76 isolates A. baumannii non-epidemiologically related clinical isolates resistant to third-generation cephalosporins isolated since 1982 in hospitals from Buenos Aires City. A plasmid isolated from Proteus mirabilis that possesses the complex class 1 integron In35::ISCR1::bla _CTX-M-2 was used to transform the natural competent A. baumannii clinical strain A118. PCR, plasmid extraction, DNA restriction, and susceptibility test confirmed that A118 could gain and maintain the plasmid possessing In35::ISCR1::bla _CTX-M-2, the genetic platform where the bla _CTX-M-2 gene is dispersing in Argentina.     

Characterization of IS26‐composite transposons and multidrug resistance in conjugative plasmids from Enterobacter cloacae

SHV‐12 is the most widespread resistance determinant of Enterobacter cloacae in Taiwan; however, bla_SHV‐12 has rarely been mobilized. Six multidrug‐resistant E. cloacae isolates were collected. After conjugal transfer, plasmid profiling and analysis of incompatibility groups was performed to characterize the genetic context of bla_SHV‐12‐containing fragments. The presence of mobile genetic elements was demonstrated by PCR, cloning, sequencing and bioinformatics analyses. Four different β‐lactamase genes (bla_TEM‐1, bla_SHV‐12, bla_CTX‐M‐3 and/or bla_CTX‐M‐14) were observed in the conjugative plasmids belonging to the IncHI2 (n = 4), IncI1 or IncP incompatibility groups. The IS26‐bla_SHV‐12‐IS26 locus was located in five different genetic environments. A novel structural organization of a class 1 integron with the aac(6')‐IIc cassette truncated by IS26 was identified in one isolate. Thus, bla_SHV‐12 was obtained from different plasmids through IS26‐mediated homologous recombination. IS26 plays a vital role in the distribution of mobile resistance elements between different plasmids found in multidrug‐resistant E. cloacae isolates.     

Identification of carbapenem‐resistant Acinetobacter baumannii clones using infrared spectroscopy

In this work we assessed the discriminatory ability of Fourier‐transform Infrared Spectroscopy (FTIR) in 22 representative isolates from a collection of 318 carbapenem‐hydrolyzing class D β ‐lactamases (CHDL)‐producing Acinetobacter spp. (5 hospitals; 2001–2008) previously characterized by DNA‐based typing methods. FTIR spectra were acquired with a Bruker spectrometer and analyzed with support of several chemometric tools. The results showed that FTIR spectroscopy was able to distinguish the main CHDL‐producing Acinetobacter baumannii lineages causing infection in Portugal, the ST103 carrying bla_OXA‐58, ST98 carrying bla_OXA‐24/40and ST92 carrying bla_OXA‐23. Moreover, this study revealed distinctive phenotypic features of A. baumannii lineages causing infections that might justify different epidemic potential. Spectroscopy may arise as a low cost and easily to perform alternative for typing A. baumannii isolates. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Transition of bla OXA-58-like to bla OXA-23-like in Acinetobacter baumannii Clinical Isolates in Southern China: An 8-Year Study

Background

The prevalence of carbapenem-resistant Acinetobacter baumannii in hospitals has been increasing worldwide. This study aims to investigate the carbapenemase genes and the clonal relatedness among A. baumannii clinical isolates in a Chinese hospital.

Methods

Carbapenemase genes and the upstream locations of insertion sequences were detected by polymerase chain reaction (PCR), and the clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.

Results

A total of 231 nonduplicate carbapenemase gene-harboring A. baumannii clinical isolates recovered from Shenzhen People’s Hospital, were investigated between 2002 and 2009. bla _OXA-23-like, bla _OXA-58-like, bla _OXA-40-like, and ISAba1-bla _OXA-51-like were identified in 119, 107, 1, and 4 isolates, respectively. IS1008-ΔISAba3, ISAba3, and ISAba1 were detected upstream of the bla _OXA-58-like gene in 69, 35, and 3 isolates, respectively. All bla _OXA-23-like genes but one had an upstream insertion of ISAba1. bla _OXA-58-like was the most common carbapenemase gene in A.baumannii before 2008, thereafter bla _OXA-23-like became rapidly prevalent and replaced bla _OXA-58-like in 2009. The majority of bla _OXA-58-like-carrying isolates showed lower level of resistance to imipenem and meropenem (minimum inhibitory concentrations (MICs), 1 μg/ml to 16 μg/ml), compared with the majority of bla _OXA-23-like-carrying isolates (MICs, 16 μg/ml to 64 μg/ml for both imipenem and meropenem). All 231 bla _OXA carbapenemase gene-harboring isolates belonged to 14 PFGE types (A–N), and three dominant clones A, J, and H accounted for 43.3%, 42.0%, and 8.2% of the tested isolates, respectively. Clone A (sequence type ST92/ST208) with bla _OXA-58-like was the most prevalent before 2008. Clone H (ST229) with bla _OXA-23-like became striking between 2007 and 2008. Clone J (ST381) with bla _OXA-23-like rapidly spread and replaced clones A and H in 2009.

Conclusion

This study is the first to reveal that the distinct bla _OXA-23-like-carrying A. baumannii ST381 displaced the previously prevalent bla _OXA-58-like-carrying A. baumannii ST92/ST208, resulting in the rapidly increasing resistance to carbapenems in A. baumannii in Shenzhen People’s Hospital in 2009.     

Characterization of a Novel Plasmid Type and Various Genetic Contexts of bla OXA-58 in Acinetobacter spp. from Multiple Cities in China

Background/Objective

Several studies have described the epidemiological distribution of bla _OXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of bla _OXA-58-carrying plasmids and the genetic context surrounding bla _OXA-58 in Acinetobacter spp. in China.

Methodology/Principal Findings

Twelve non-duplicated bla _OXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of bla _OXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of bla _OXA-58-harboring plasmids. The genetic structure surrounding bla _OXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both bla _OXA-58 and bla _OXA-23. bla _OXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in bla _OXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of bla _OXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-bla _OXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-bla _OXA-58 were carbapenem susceptible.

Conclusion

The study revealed the unique features of bla _OXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of bla _OXA-58 contributed to various antibiotics resistance profiles.     

Influence of agricultural practice on mobile bla genes: IncI1‐bearing CTX‐M,SHV, CMY and TEM in Escherichia coli from intensive farming soils

Many calls have been made to address antibiotic resistance in an environmental perspective. With this study, we showed the widespread presence of high‐level antibiotic resistant isolates on a collection of non‐susceptible Gram‐negative bacteria (n = 232) recovered from soils. Bacteria were selected using amoxicillin, cefotaxime and imipenem, from sites representing different agricultural practices (extensive, intensive and organic). Striking levels of non‐susceptibility were noticed in intensive soils for norfloxacin (74%), streptomycin (50.7%) and tetracycline (46.6%); indeed, the exposure to intensive agricultural practices constituted a risk factor for non‐susceptibility to many antibiotics, multidrug resistance and production of extended‐spectrum β‐lactamases (ESBL). Analyses of non‐susceptibility highlighted that environmental and clinical bacteria from the same species might not share the same intrinsic resistance patterns, raising concerns for therapy choices in environment‐borne infections. The multiple sequence‐type IncI1‐driven spread of penicillinases (bla_TEM‐1, bla_TEM‐135), ESBL (bla_SHV‐12 and bla_CTX‐M‐1) and plasmid‐mediated AmpC β‐lactamases (bla_CMY‐2), produced by isolates that share their molecular features with isolates from humans and animals, suggests contamination of agricultural soils. This is also the first appearance of IncI1/ST28‐harbouring bla_CTX‐M‐1, which should be monitored to prevent their establishment as successfully dispersed plasmids. This research may help disclose paths of contamination by mobile antibiotic resistance determinants and the risks for their dissemination.     

Occurrence of the genes encoding carbapenemases,ESBLs and class 1 integron-integrase among fermenting and non-fermenting bacteria from retail goat meat

The present study was planned to detect the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among bacteria obtained from retail goat meat. Fermenting and non-fermenting bacterial isolates (n = 57), recovered from 61 goat meat samples, were identified by 16S rRNA gene sequencing. Antimicrobial susceptibility of isolates was tested by the broth dilution method using ceftazidime, cefotaxime, meropenem and imipenem. Plasmids were isolated and tested for their physical characters. Plasmids were subjected to screening of carbapenemase, ESBL and intI1 gene. Conjugation assay was performed using bla_NDM-positive isolates as the donor, and Escherichia coli HB101 as the recipient. Isolates showed the high rates of resistance to ceftazidime (77·2%), cefotaxime (70·2%), meropenem (22·8%) and imipenem (17·5%). They showed variability in number and size (~1 to >20 kb) of plasmids. Among all, 1, 4, 13 and 31 isolates showed the bla_KPC, bla_NDM, bla_SHV and bla_TEM genes, respectively. The bla_KPC-2 gene was observed in one E. coli isolate. The bla_NDM-1 gene was detected in Stenotrophomonas maltophilia (n = 2), Acinetobacter baumannii (n = 1) and Ochrobactrum anthropi (n = 1) isolates. These isolates co-harboured the bla_TEM and bla_SHV genes. The intI1 gene was detected in 22 (38·6%) isolates, and 16 of these isolates showed the carbapenemase and/or ESBL genes. The conjugative movement of bla_NDM gene could not be proved after three repetitive mating experiments. The presence of genes encoding carbapenemases and ESBLs in bacteria from goat meat poses public health risks.     

Antimicrobial‐resistant faecal Escherichia coli in wild mammals in central Europe: multiresistant Escherichia coli producing extended‐spectrum beta‐lactamases in wild boars

Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli in populations of wild mammals in the Czech Republic and Slovakia. Methods and Results: Rectal swabs or faeces collected during 2006–2008 from wild mammals were spread on MacConkey agar and MacConkey agar containing 2 mg l^?1 of cefotaxime. From plates with positive growth, one isolate was recovered and identified as E. coli. Susceptibility to 12 antibiotics was tested using the disk diffusion method. Resistance genes, class 1 and 2 integrons and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). Extended‐spectrum beta‐lactamases (ESBL) were further characterized by DNA sequencing, macrorestriction profiling and determination of plasmid sizes. Plasmid DNA was subjected to EcoRV digestion, transferability by conjugation and incompatibility grouping by multiplex PCR. The prevalence of resistant isolates was 2% in small terrestrial mammals (rodents and insectivores, n_E. coli = 242), 12% in wild ruminants and foxes (n_E. coli = 42), while no resistant isolates were detected in brown bears (n_E. coli = 16). In wild boars (Sus scrofa) (n_E. coli = 290), the prevalence of resistant isolates was 6%. Class 1 and 2 integrons with various gene cassettes were recorded in resistant isolates. From wild boars, five (2%, n_{rectal smears} = 293) multiresistant isolates producing ESBL were recovered: one isolate with bla_CTX‐M‐1 + bla_TEM‐1, three with bla_CTX‐M‐1 and one with bla_TEM‐52b. The bla_CTX‐M‐1 genes were carried on approx. 90 kb IncI1 conjugative plasmids. Conclusions: Antibiotic‐resistant E. coli occured in populations of wild mammals in various prevalences. Significance and Impact of the Study: Wild mammals are reservoirs of antibiotic‐resistant E. coli including ESBL‐producing strains which were found in wild boars.     

Occurrence and molecular characteristics of antimicrobial resistance of Escherichia coli from broilers,pigs and meat products in Thailand and Cambodia provinces

Nine hundred and forty‐one samples were collected in Sa Keao, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) from July 2014 to January 2015. A total of 667 Escherichia coli isolates (381 isolates from Sa Keao and 286 isolates from Banteay Meanchey) were obtained and examined for antimicrobial susceptibility, class 1 integrons, ESBL genes and horizontal transfer of resistance determinants. Prevalence of E. coli in pig and broiler carcass samples from slaughterhouses and fresh markets was 36–85% in Sa Keao and 11–69% in Banteay Meanchey. The majority of these isolates were multidrug resistant (75.3%). Class 1 integrons were common in both Thai (47%) and Cambodian (62%) isolates, of which four resistance gene cassette arrays including aadA1, dfrA1‐aadA1, dfrA12‐aadA2 and aadA2‐linF were identified. Class 1 integrons in two broiler isolates from Sa Keao (dfrA12‐aadA2) and one broiler isolate from Banteay Meanchey (dfrA1‐aadA1) were horizontally transferable. Sixteen isolates were confirmed to be ESBL‐producing strains with ESBL gene bla_CTX‐M‐15, broad spectrum β‐lactamase gene bla_TEM‐1 and the AmpC gene bla_CMY‐2 being detected. The bla_TEM‐1 gene was most prevalent and located on a conjugative plasmid.     

Molecular characteristics and resistant mechanisms of imipenem-resistant <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> isolates in Shenyang,China

The investigation was carried out to elucidate the molecular characteristics and resistant mechanisms of imipenem-resistant Acinetobacter baumannii. Thirty-seven isolates were collected from January 2007 to December 2007. The homology of the isolates was analyzed by both pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genes of β-lactamases, adeB, and class 1 integron were polymerase chain reaction amplified. Genotype analysis of the 37 A. baumannii isolates by PFGE revealed the circulation of four PFGE types (A-D); the A- and B-type accounted for 48.6% and 40.5%, respectively. MLST showed the existence of three allelic profiles. The agar dilution method was carried out to determine the MIC of imipenem, in the absence or presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μg/ml). The MICs of the strains to imipenem were between 16 μg/ml and 128 μg/ml. When CCCP was added, a MIC decrease of at least four-fold was observed in 20 isolates, which belonged to the A- or C-type. AdeB and bla _PER-1 genes were each detected in 35 isolates, bla _OXA-23 gene in 34 isolates and bla _OXA-58-like gene in 24 isolates. All isolates harbored bla _OXA-51-like genes. No isolates carried the bla _IMP-1 gene. Integron was detected in 25 isolates, which mediated the resistance to aminoglycosides and rifampin. The epidemiologic data suggested that the increasing infection of A. baumannii in our hospital was mainly caused by the inter-hospital spread of two epidemic clones. The AdeABC efflux system may be the important factor that leads to the high level of imipenem-resistance in PFGE A-type.     

Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil

The aim of the present study was to investigate the resistance profile, to detect the presence of beta-lactam resistance genes, phenotypic expression of efflux pump systems and class 1 integrons in Pseudomonas spp. strains obtained from untreated hospital effluents. Effluent samples were collected from four hospitals in Porto Alegre, RS, Brazil. Pseudomonas were isolated on MacConkey agar plates and the identification was confirmed by 16S rRNA PCR and biochemical tests. Susceptibility testing was determined by disk-diffusion method using 11 different beta-lactams and MIC assays were performed on isolates resistant to imipenem and ceftazidime. The beta-lactamase genes bla _IMP, bla _VIM, bla _SPM-1, bla _OXA-23-like, bla _OXA-24-like, bla _OXA-51-like and the intl1 gene from class 1 integron were analysed by PCR. One hundred and twenty-four isolates were recovered and the most common species was Pseudomonas pseudoalcaligenes. The resistance found among the isolates was considered high, 62 (50%) isolates were multiresistant. No isolate carrying the beta-lactamase genes tested was found among the strains. Seven isolates showed reduction of MIC for imipenem and ceftazidime in the presence of cyanide m-chlorophenylhydrazone, indicating the hyper expression of efflux pumps. From the 124 isolates, 52 (41.9%) were identified as carrying the class 1 integron gene, intI1. Untreated hospital effluents could be a source of environmental contamination due to discharge of antimicrobial resistant bacteria which can carry integron class 1 and act as a reservoir of resistance genes and have efflux pump systems.     

Cephalosporin resistant Escherichia coli from cancer patients in Cairo,Egypt

Cephalosporin‐resistant Escherichia coli has been increasingly reported worldwide. In this study, 32 cephalosporin resistant E. coli isolates identified from cancer patients in Cairo, Egypt in 2009–2010 were analyzed. Twenty‐three were of phylogenetic group D, seven A and one each B1 and B2. By rep‐PCR 15 phylogroup D isolates were grouped in four clusters, one with sequence type (ST) 405 and three ST68. Seventeen isolates showed single patterns. bla_CTX‐M‐15 and aac(6')‐Ib‐cr were the most common resistance determinants. bla_OXA‐48 and bla_VIM were also detected. Multidrug resistant E. coli seriously affects healthcare, especially in immunocompromised hosts, such as cancer patients.     

Inverse PCR for subtyping of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> carrying IS<Emphasis Type="Italic">Aba</Emphasis>1

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla_OXA-51-like, bla_OXA-23-like, and bla_ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes — bla_ampC, bla_OXA-66-like, and csuD — were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.     

Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital

Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla _CTX-M-15 (12 isolates), bla _CTX-M-14a (one isolate), and bla _CTX-M-14b (one isolate). The bla _OXA-1 gene was detected in 13 bla _CTX-M-producing strains and a bla _TEM-1 gene in 6 of them. The ISEcp1 sequence was found upstream of bla _CTX-M genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17–aadA5 being the most common. One of the strains (bla _CTX-M-14a-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6′)-Ib and cmlA1 genes and it was linked to the bla _CTX-M-14a gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.     

Selection of broad‐spectrum cephalosporin‐resistant Escherichia coli in the feces of healthy dogs after administration of first‐generation cephalosporins

Although antimicrobial products are essential for treating diseases caused by bacteria, antimicrobial treatment selects for antimicrobial‐resistant (AMR) bacteria. The aim of this study was to determine the effects of administration of first‐generation cephalosporins on development of resistant Escherichia coli in dog feces. The proportions of cephalexin (LEX)‐resistant E. coli in fecal samples of three healthy dogs treated i.v. with cefazolin before castration and then orally with LEX for 3 days post‐operation (PO) were examined using DHL agar with or without LEX (50 µg/mL). LEX‐resistant E. coli were found within 3 days PO, accounted for 100% of all identified E. coli 3–5 days PO in all dogs, and were predominantly found until 12 days PO. LEX‐resistant E. coli isolates on DHL agar containing LEX were subjected to antimicrobial susceptibility testing, pulsed‐field gel electrophoresis (PFGE) genotyping, β‐lactamase typing and plasmid profiling. All isolates tested exhibited cefotaxime (CTX) resistance (CTX minimal inhibitory concentration ≥4 µg/mL). Seven PFGE profiles were classified into five groups and three β‐lactamase combinations (bla_CMY‐4‐bla_TEM‐1, bla_TEM‐1‐bla_CTX‐M‐15 and bla_TEM‐1‐bla_CTX‐M‐15‐bla_CMY‐4). All isolates exhibited identical PFGE profiles in all dogs on four days PO and subsequently showed divergent PFGE profiles. Our results indicate there are two selection periods for AMR bacteria resulting from the use of antimicrobials. Thus, continuing hygiene practices are necessary to prevent AMR bacteria transfer via dog feces after antimicrobial administration.     

Extended-spectrum beta-lactamase-producing Escherichia coli in turkey meat production farms in the Czech Republic: national survey reveals widespread isolates with bla(SHV-12) genes on IncFII plasmids

Aim: The occurrence and epidemiology of extended‐spectrum beta‐lactamase (ESBL)‐producing Escherichia coli in the environment of turkey farms in the Czech Republic were studied. Methods and Results: Extended‐spectrum beta‐lactamase‐producing E. coli isolates were found on 8 (20%) of 40 turkey farms surveyed. A total of 200 environmental smears were examined, and a total of 25 ESBL‐producing E. coli were isolated. These isolates were analysed using XbaI pulsed‐field gel electrophoresis and divided into nine pulsotypes. Most of the isolates harboured the gene bla_SHV‐12 on a 40‐kb plasmid of the IncFII group with an identical EcoRV restriction profile. Indistinguishable or clonally related SHV‐12‐producing isolates belonging to the same pulsotypes were found at some unrelated farms. Conclusions: Widespread occurrence of ESBL‐producing E. coli isolates with bla_SHV‐12 carried on IncFII plasmids in meat production flocks in the Czech Republic was demonstrated. Significance and Impact of the Study: Results indicate vertical transmission of ESBL‐producing E. coli within the turkey production pyramid. The study shows the risk of multiresistant ESBL‐producing bacteria and antibiotic‐resistance genes being transmitted to humans via the food chain.     

Molecular Characterization of CTX-M β-Lactamase and Associated Addiction Systems in Escherichia coli Circulating among Cattle,Farm Workers,and the Farm Environment

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 bla_CTX-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 bla_CTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A bla_CTX-M-14 gene was also detected in an E. coli isolate from a farmer''s hand. Transfer of the bla_CTX-M gene from 60 bla_CTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from bla_CTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) bla_CTX-M genes possessed an insertion sequence, ISEcp1, upstream of the bla_CTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The bla_CTX-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 bla_CTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying bla_CTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the bla_CTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.     

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

Aims: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla_TEM‐52‐carrying plasmid (lactose‐negative mutant of B1‐54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Methods and Results: Fresh faeces from a healthy volunteer, negative for cephalosporin‐resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed‐field gel electrophoresis profiles (PFGE). The avian extended‐spectrum β‐lactamase‐positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1‐54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla_TEM‐52‐carrying plasmid. Conclusions: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. Significance and Impact of the Study: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.