Extended-spectrum Cephalosporinase Research Articles

Impact of extended-spectrum chromosomal AmpC (ESAC) of Escherichia coli on susceptibility to cefiderocol.

We showed here that chromosomally encoded intrinsic extended-spectrum cephalosporinases of Escherichia coli may impact susceptibility not only to ceftazidime and cefepime but also to cefiderocol.

High-risk lineages of extended spectrum cephalosporinase producing Escherichia coli from Eurasian griffon vultures (Gyps fulvus) foraging in landfills in north-eastern Spain

Extended-spectrum cephalosporinase producing (ESC) E. coli are regarded as key indicator microorganisms of antimicrobial resistance (AMR), calling for a One Health integrated global surveillance strategy. Wildlife is exposed to antibiotic contaminants and/or resistant bacteria that have been released into the environment, potentially acting as reservoirs and spreaders of resistance genes as well as sentinels of anthropogenic pressure. Monitoring AMR in wildlife has become crucial in determining anthropogenic environmental impacts as well as transmission routes. In this study, we determined the occurrence and potential sources of ESC E. coli in 218 Eurasian griffon vultures (Gyps fulvus) foraging regularly on human waste disposed at a dumpsite in north-eastern Spain. Minimal inhibitory concentration for 14 different antimicrobials was performed to evaluate the phenotype of the isolates, and whole genome sequencing was carried out to investigate lineages and plasmids harbouring ESC genes. Our sequences were compared to previously published Spanish sequences of human, animal, and wildlife origin. We report a high prevalence of CTX-M-15, as well as the presence of other resistance genes such as OXA-10, CTX-M-27, and CTX-M-65 which are rarely described in European livestock, suggesting a human origin. The isolates also carried a diverse range of additional AMR genes for a broad spectrum of drug families, with the majority being multi-drug resistant. The phylogenomic analyses suggests the transmission of high-risk lineages from humans to vultures, with 49 % of our isolates matching the most common extraintestinal pathogenic E. coli (ExPEC) lineages described in humans worldwide, including ST131, ST10 and ST58. We conclude that anthropogenically altered habitats, such as landfills, are hotspots for the acquisition and spread of high-risk ESC E. coli lineages associated with hospital infections. Measures must be implemented to limit their spread into natural environments.

Antimicrobial Resistance on Farms: A Review Including Biosecurity and the Potential Role of Disinfectants in Resistance Selection.

Resistance to therapeutic antimicrobial agents is recognized as a growing problem for both human and veterinary medicine, and the need to address the issue in both of these linked domains is a current priority in public policy. Efforts to limit antimicrobial resistance (AMR) on farms have so far focused on control of the supply and use of antimicrobial drugs, plus husbandry measures to reduce infectious disease. In the United Kingdom and some other countries, substantial progress has been made recently against targets on agricultural antimicrobial drug use. However, evidence suggests that resistant pathogenic and commensal bacteria can persist and spread within and between premises despite declining or zero antimicrobial drug use. Reasons for this are likely complex and varied but may include: bacterial adaptations to ameliorate fitness costs associated with maintenance and replication of resistance genes and associated proteins, horizontal transmission of genetic resistance determinants between bacteria, physical transfer of bacteria via movement (of animals, workers, and equipment), ineffective cleaning and disinfection, and co-selection of resistance to certain drugs by use of other antimicrobials, heavy metals, or biocides. Areas of particular concern for public health include extended-spectrum cephalosporinases and fluoroquinolone resistance among Enterobacteriaceae, livestock-associated methicillin-resistant Staphylococcus aureus, and the emergence of transmissible colistin resistance. Aspects of biosecurity have repeatedly been identified as risk factors for the presence of AMR on farm premises, but there are large gaps in our understanding of the most important risk factors and the most effective interventions. The present review aims to summarize the present state of knowledge in this area, from a European perspective.

High level of multidrug-resistant Escherichia coli in young dairy calves in southern Vietnam

This study investigated the occurrence of antimicrobial-resistant Escherichia coli in dairy calves in southern Vietnam. Fecal samples were taken directly from the rectum of 84 calves from 41 smallholder dairy farms, when newborn and at 14 days of age for isolation of E. coli. Escherichia coli strains were isolated from 144 of the 168 fecal samples tested. Of the 144 E. coli isolates, 40% were found to be susceptible to all 12 antimicrobial drugs tested and 53% of the E. coli isolates were resistant to at least three antimicrobials. Calves were colonized with antimicrobial-resistant E. coli already on the day of birth. Resistance to tetracycline was most common, followed by resistance to sulfamethoxazole, ampicillin, trimethoprim, and ciprofloxacin. Four isolates carried a gene encoding for extended-spectrum cephalosporinases (ESC), and these genes belonged to blaCTX-M group 1 (2 isolates), blaCTX-M group 9 (1 isolate), and blaCMY-2 (1 isolate). Thirty-three isolates had a plasmid-mediated quinolone resistance (PMQR) phenotype, and 30 of these carried the qnrS gene. These results are of importance for management routines of dairy cattle to prevent the spread of antimicrobial resistance.

A metabonomic analysis on the response of Enterobacter cloacae from coastal outfall for land-based pollutant under phoxim stress

Enterobacter cloacae is an opportunistic pathogen widely distributed in human and animal intestinal systems. The secretion of extended-spectrum β-lactamases (ESBLs) and cephalosporinase (AmpC) endows E. cloacae with strong drug resistance. In a previous study by our group, protein expression of E. cloacae under phoxim stress was measured by two-dimensional electrophoresis. Here, nuclear magnetic resonance was used to detect differences in E. cloacae metabonomics when under phoxim stress. We determined that there are 29 types of metabolites that differ between phoxim stress and normal culture conditions. Among these, 6 types of metabolites were upregulated in the phoxim stress group, and 23 types of metabolites were inhibited. Though enrichment analysis, seven pathways were identified by different expression levels of metabolites, which were involved in DNA and RNA synthesis, DNA damage repair, antioxidation and functions of the cell membrane and cell wall. The mechanism underlying how phoxim affects E. cloacae was determined by studying the results of both two-dimensional electrophoresis in our prior work and the analysis of E. cloacae metabonomic changes under phoxim stress.

Unexpected common occurrence of transferable extended spectrum cephalosporinase-producing Escherichia coli in Swedish surface waters used for drinking water supply

The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans, food-producing animals and food world-wide. However, the occurrence and impact of these so-called extended spectrum cephalosporinase (ESC)-producing Enterobacteriaceae in aquatic environments are poorly documented. This study investigated the occurrence, concentrations and characteristics of ESC-producing E. coli (ESC-Ec) in samples of surface water collected at five Swedish water treatment plants that normally have relatively high prevalence and concentration of E. coli in surface water. ESC-Ec was found in 27 of 98 surface water samples analysed. All but two positive samples were collected at two of the water treatment plants studied. The ESC-Ec concentration, 1–3cfu/100mL, represented approximately 4% of the total amount of E. coli in the respective surface water sample. In total, 74% of the isolates were multi-resistant, but no isolate was resistant to carbapenems. Six types of ESBL/pAmpC genes were found in the 27 E. coli isolates obtained from the positive samples, of which four (blaCTX-M-15, blaCMY-2, blaCTX-M-1 and blaCTX-M-14) were found during the whole sampling period, in samples taken at more than one water treatment plant. In addition, the genes were situated on various types of plasmids and most E. coli isolates were not closely related with regard to MLST types. The combinations of ESBL/pAmpC genes, plasmids and E. coli isolates were generally similar to those found previously in healthy and sick individuals in Sweden. In conclusion, the occurrence of ESC-Ec in Swedish surface water shows that resistant bacteria of clinical concern are present in aquatic environments even in a low-prevalence country such as Sweden.

Molecular Characterization of Extended-Spectrum-Cephalosporin-Resistant Enterobacteriaceae from Wild Kelp Gulls in South America

Extended-spectrum-cephalosporin-resistant Enterobacteriaceae are a public health concern due to limited treatment options. Here, we report on the occurrence and the molecular characteristics of extended-spectrum-cephalosporin-resistant Enterobacteriaceae recovered from wild birds (kelp gulls). Our results revealed kelp gulls as a reservoir of various extended-spectrum cephalosporinase genes associated with different genetic platforms. In addition, we report for the first time the presence of a known epidemic clone of Salmonella enterica serotype Heidelberg (JF6X01.0326/XbaI.1966) among wild birds.

Structural and Functional Aspects of Extended-Spectrum AmpC Cephalosporinases.

β-lactam antibiotics have revolutionized modern medicine, but resistance to these drugs is a major public health crisis. Traditionally, class C β-lactamases were referred to as cephalosporinases due to their substrate preference for this particular class of β-lactams. However, the emergence of AmpC enzymes with extended-spectrum activity (extended-spectrum cephalosporinases or ESACs) is particularly worrisome, especially given that most clinical β-lactamase inhibitors are ineffective against these enzymes. This review summarizes structures of several extended spectrum class C β-lactamases and analyzes the structure-function relationship observed among them.

Comparative Genomics of Two ST 195 Carbapenem-Resistant Acinetobacter baumannii with Different Susceptibility to Polymyxin Revealed Underlying Resistance Mechanism.

Acinetobacter baumannii is a Gram-negative nosocomial pathogen of importance due to its uncanny ability to acquire resistance to most antimicrobials. These include carbapenems, which are the drugs of choice for treating A. baumannii infections, and polymyxins, the drugs of last resort. Whole genome sequencing was performed on two clinical carbapenem-resistant A. baumannii AC29 and AC30 strains which had an indistinguishable ApaI pulsotype but different susceptibilities to polymyxin. Both genomes consisted of an approximately 3.8 Mbp circular chromosome each and several plasmids. AC29 (susceptible to polymyxin) and AC30 (resistant to polymyxin) belonged to the ST195 lineage and are phylogenetically clustered under the International Clone II (IC-II) group. An AbaR4-type resistance island (RI) interrupted the comM gene in the chromosomes of both strains and contained the blaOXA−23 carbapenemase gene and determinants for tetracycline and streptomycin resistance. AC29 harbored another copy of blaOXA−23 in a large (~74 kb) conjugative plasmid, pAC29b, but this gene was absent in a similar plasmid (pAC30c) found in AC30. A 7 kb Tn1548::armA RI which encodes determinants for aminoglycoside and macrolide resistance, is chromosomally-located in AC29 but found in a 16 kb plasmid in AC30, pAC30b. Analysis of known determinants for polymyxin resistance in AC30 showed mutations in the pmrA gene encoding the response regulator of the two-component pmrAB signal transduction system as well as in the lpxD, lpxC, and lpsB genes that encode enzymes involved in the biosynthesis of lipopolysaccharide (LPS). Experimental evidence indicated that impairment of LPS along with overexpression of pmrAB may have contributed to the development of polymyxin resistance in AC30. Cloning of a novel variant of the blaAmpC gene from AC29 and AC30, and its subsequent expression in E. coli also indicated its likely function as an extended-spectrum cephalosporinase.

The use of third and fourth generation cephalosporins affects the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli in Danish pig herds

Extended-spectrum cephalosporinase resistance is currently the fastest emerging antimicrobial resistance problem worldwide; however, evidence documenting the effect of potential risk factors is limited. The main objective of this study was to investigate the effect of using third and fourth generation cephalosporins on the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli (ESC-Ec) in Danish pig herds.Conventional, integrated, medium to large herds were selected based on information from the Danish Central Husbandry Register and two groups were formed based on the use of third and fourth generation cephalosporins within a specified period, namely, 20 herds with no cephalosporin use (non-exposed) and 19 herds with frequent use (exposed). Data on prescribed antimicrobials were obtained from the National database (VetStat). Management data were obtained through a questionnaire. At the herd level, three pooled faecal samples were collected from sows with their piglets (farrowing pens), weaners, and finishers. ESC-Ec were then identified using selective enrichment. Because several of the herds only had a low number of weaners and/or finishers, analysis was only performed on samples from the farrowing pens.Logistic regression showed a significant effect of using cephalosporins-III/IV on the occurrence of ESC-Ec in the farrowing pens, even when adjusted for use of other antimicrobials 1 year prior to sampling. No confounding effect was identified in relation to management data. The relative risk ESC-Ec in exposed compared to non-exposed was 4.7 (95% confidence interval 2.0–11.5), confirming that regular use of cephalosporins-III/IV was a significant risk factor for the occurrence of ESC-Ec.

Spread of extended spectrum cephalosporinase-producing Escherichia coli clones and plasmids from parent animals to broilers and to broiler meat in a production without use of cephalosporins.

This study investigated the occurrence of extended spectrum cephalosporinase (ESC)-producing Escherichia coli in a broiler production with no cephalosporin use and a low use of antimicrobials in general. Furthermore, it investigated whether the current consumption of aminopenicillins selects for ESC-producing E. coli and whether certain clones or plasmids spread from imported parent flocks to the meat. ESC-producing E. coli was isolated using MacConkey broth with 1 mg/L of ceftriaxone. ESC genes were identified using polymerase chain reaction and sequencing. Isolates with blaCMY-2 were subtyped by pulsed-field gel electrophoresis (PFGE), phylotyping, and antimicrobial susceptibility testing. Selected isolates were used as donors in filter-mating experiments, multilocus sequence typing (MLST), and plasmid replicons were typed. Aminopenicillin use at the farm (not flock) level was obtained from VetStat, a database for mandatory registration of veterinary prescriptions in Denmark. ESC-producing E. coli occurred in 93% (27/29) of broiler parent farms in 2011, 27% (53/197) of broiler flocks in 2010, and 3.3% (4/121) of Danish retail broiler meat in 2009 and 8.6% (16/187) in 2010. The ESC producing E. coli contained blaCMY-2, blaSHV-2 or blaCTX-M-1. Isolates with blaCMY-2 represented 35 PFGE groups. One group dominated (39 isolates) and included isolates with indistinguishable PFGE patterns from parents, broilers, and meat. Most blaCMY-2 isolates were susceptible to non-β-lactams, and blaCMY-2 was mostly present on horizontally transferable incI1 or incK plasmids. Phylogroup D was most common and E. coli MLST types previously found in humans were observed. Broiler farms with registered aminopenicillin use had significantly higher occurrence of ESC E. coli. ESC-producing E. coli from flocks of imported broiler parents spread clonally and horizontally to broiler meat (including potentially human pathogenic types) even in a country with no cephalosporin use. Use of aminopenicillins may influence the persistence of ESC-producing E. coli in the broiler production, but other factors should be investigated.

Interactions of Oximino-Substituted Boronic Acids and β-Lactams with the CMY-2-Derived Extended-Spectrum Cephalosporinases CMY-30 and CMY-42

CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY-30) and Ser (CMY-42) for Val211 in the Ω-loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino-β-lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oximino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the Ω-loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site (Ω-loop-Q120 loop-R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the covalently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adaptation of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).

Voluntary ban on cephalosporin use in Danish pig production has effectively reduced extended-spectrum cephalosporinase-producing Escherichia coli in slaughter pigs

To measure the effect of a voluntary ban on cephalosporin usage in the Danish pig production on the prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in pigs and pork. Data on cephalosporin consumption were obtained from the VetStat database. For detection of ESC-producing E. coli, three sampling types were included: at slaughter, caecal samples were collected from pigs in 2009 and 2010 (June) before and in two periods (2010 and 2011) after a voluntary ban on cephalosporins was effected (July 2010); at farm level, pools of five stool samples from different pigsties were collected in 2010 and in 2011; and samples from pork were collected randomly at retail stores and outlets from 2009 to 2011. ESC-producing E. coli was isolated after selective enrichment in MacConkey broth with 1 mg/L ceftriaxone. ESC genes were detected using PCR, microtube array and sequencing. From July 2010 the consumption of cephalosporins approximated zero. The occurrence of ESC-producing E. coli in pigs at slaughter was not significantly different (P=0.7) between 2009 [10.8% (85/786)] and 2010 [11.8% (48/407)], but in 2011 the occurrence [3.6% (28/777)] decreased significantly (P<0.001). A significant decrease (P=0.002) in occurrence of ESC-producing E. coli at pig farm level from 2010 [11% (11/99)] to 2011 (0/78) was also observed. The bla(CTX-M-1) gene was most often detected (63%), but bla(CTX-M-14) and bla(CTX-M-15) were also found. Occurrence in pork was between 1.3% and 0.9%. The discontinuation of an already low use of cephalosporins in pig production has significantly reduced the occurrence of ESC-producing E. coli.

Klebsiella pneumoniae Carbapenemases in Enterobacteriaceae: History, Evolution, and Microbiology Concerns

Since the discovery of penicillin 80 years ago, gram-negative bacteria have become proficient at evading the lethal activity of β-lactam antibiotics, principally through the production of β-lactamases. The rapid emergence of penicillinases in both gram-positive and gram-negative bacteria led to the development of cephalosporin β-lactam antibiotics, but production of plasmid-mediated extended-spectrum cephalosporinases (or extended-spectrum β-lactamases) and AmpC enzymes resulted in resistance to this drug class. Because carbapenems were the only β-lactam agents active against such extended-spectrum β-lactamase-producing strains, appropriate and inappropriate use soon resulted in Enterobacteriaceae resistance. As a result, two distinct types of carbapenemases-the metallo-β-lactamases and Klebsiella pneumoniae carbapenemases (KPCs)-were soon identified. The KPCs comprise 10 variants that differ from one another by one to three amino acid substitutions (KPC-2 to KPC-11). The KPC-producing Enterobacteriaceae are not only multidrug resistant but are also difficult to detect routinely in the clinical microbiology laboratory. Tigecycline, polymyxins (colistin and polymyxin B), and aminoglycosides are possible candidate therapies for infections caused by KPC-producing organisms, although well-conducted clinical trials are required to fully define their roles in patient management. The shortage of new antimicrobial agents on the immediate horizon suggests that enhanced adherence with infection prevention procedures and antimicrobial stewardship programs are needed to curb patient-to-patient transmission and to reduce the selection of multidrug-resistant bacteria.

Prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in Danish slaughter pigs and retail meat identified by selective enrichment and association with cephalosporin usage

To investigate the prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in pigs at slaughter and retail meat, and possible associations with the consumption of third- and fourth-generation cephalosporins. During 2009, faecal samples from Danish pigs (n=786) were collected at slaughter, and 866 meat samples [Danish: pork (153), broiler meat (121) and beef (142); and imported: pork (173), broiler meat (193) and beef (84)] were randomly collected in retail stores and outlets. E. coli was isolated after enrichment in MacConkey broth with ceftriaxone (1 mg/L). ESC genotypes were detected using PCR, microtube array and sequencing. The MIC of cefotaxime was determined for 150 E. coli from the pigs and 606 E. coli from meat isolated without selective enrichment. Eleven percent (86/786) of slaughter pigs contained ESC E. coli and a significantly higher prevalence was observed among pigs originating from farms with registered cephalosporin consumption in slaughter pigs (P=0.034). Among ESC E. coli from pigs, 66% contained bla(CTX-M-1). From meat, a high prevalence of ESC E. coli was found in imported broiler meat (36%) compared with 0.7%-3.3% in other meat types. ESC E. coli from imported broiler meat (n=69) contained bla(CMY-2) (48%), bla(CTX-M-1) (25%) and bla(SHV-12) (16%). Without selective enrichment, no ESC E. coli from pigs and only 4.1% from imported broiler meat were found. The usage of cephalosporins for slaughter pigs may increase the prevalence of ESC E. coli in slaughter pigs. Meat may be a source of ESCs in humans, especially imported broiler meat. Selective enrichment should be considered as a supplementary surveillance method.

Molecular Characterization and Antimicrobial Susceptibility Testing of Escherichia coli Isolates from Patients with Urinary Tract Infections in 20 Chinese Hospitals

A total of 222 urinary Escherichia coli isolates from 20 tertiary hospitals in 15 different provinces and 4 municipalities in mainland China were characterized by antimicrobial susceptibility, phylogrouping, and the presence of plasmid-mediated quinolone resistance genes. A subset of 138 suspected extended-spectrum cephalosporinase (ESC) producers were examined for genes encoding cephalosporin resistance. Forty-three isolates harboring bla(CTX-M-14) or bla(CTX-M-15) were analyzed by pulsed-field gel electrophoresis (PFGE), and plasmids containing these genes were typed using PCR-based replicon typing (PBRT). Thirteen phylogroup B2 bla(CTX-M-14)- and bla(CTX-M-15)-positive isolates were analyzed by multilocus sequence typing (MLST). A frequent occurrence of resistance (>46%) was observed toward cephalosporins, gentamicin, and fluoroquinolones. Among the 222 isolates, 4 qnrS1, 4 qepA, and 16 aac(6')-Ib-cr genes were confirmed. Four major phylogroups (A, B1, B2, and D) and nontypeable isolates (NTs) were found among the isolates, with phylogroup D (54%) being the most common phylogroup. A total of 110 (80%) of the 138 screened isolates harbored bla(CTX-M) genes, with bla(CTX-M-14) (71%) and bla(CTX-M-15) (24%) being the most prevalent of these genes. Nine of the 13 CTX-M-15- or CTX-M-14-containing B2 isolates belonged to ST131. PFGE typing showed a high level of diversity, and plasmid analysis indicated a very large pool of different resistance plasmids mediating the spread of bla(CTX-M) genes in mainland China. An equally very high frequency of resistance and equally high levels of diversity in phylogroups, PFGE types, and plasmids were observed among community- and hospital-acquired E. coli isolates, indicating the presence of a large reservoir in the community and a long-term spread of cephalosporin resistance in China.

The Salmonella Genomic Island 1 Is Specifically Mobilized In Trans by the IncA/C Multidrug Resistance Plasmid Family

BackgroundThe Salmonella genomic island 1 (SGI1) is a Salmonella enterica-derived integrative mobilizable element (IME) containing various complex multiple resistance integrons identified in several S. enterica serovars and in Proteus mirabilis. Previous studies have shown that SGI1 transfers horizontally by in trans mobilization in the presence of the IncA/C conjugative helper plasmid pR55.Methodology/Principal FindingsHere, we report the ability of different prevalent multidrug resistance (MDR) plasmids including extended-spectrum β-lactamase (ESBL) gene-carrying plasmids to mobilize the multidrug resistance genomic island SGI1. Through conjugation experiments, none of the 24 conjugative plasmids tested of the IncFI, FII, HI2, I1, L/M, N, P incompatibility groups were able to mobilize SGI1 at a detectable level (transfer frequency <10−9). In our collection, ESBL gene-carrying plasmids were mainly from the IncHI2 and I1 groups and thus were unable to mobilize SGI1. However, the horizontal transfer of SGI1 was shown to be specifically mediated by conjugative helper plasmids of the broad-host-range IncA/C incompatibility group. Several conjugative IncA/C MDR plasmids as well as the sequenced IncA/C reference plasmid pRA1 of 143,963 bp were shown to mobilize in trans SGI1 from a S. enterica donor to the Escherichia coli recipient strain. Depending on the IncA/C plasmid used, the conjugative transfer of SGI1 occurred at frequencies ranging from 10−3 to 10−6 transconjugants per donor. Of particular concern, some large IncA/C MDR plasmids carrying the extended-spectrum cephalosporinase bla CMY-2 gene were shown to mobilize in trans SGI1.Conclusions/SignificanceThe ability of the IncA/C MDR plasmid family to mobilize SGI1 could contribute to its spread by horizontal transfer among enteric pathogens. Moreover, the increasing prevalence of IncA/C plasmids in MDR S. enterica isolates worldwide has potential implications for the epidemic success of the antibiotic resistance genomic island SGI1 and its close derivatives.

Development of High-Level Carbapenem Resistance inKlebsiella pneumoniaeAmong Patients with Prolonged Hospitalization and Carbapenem Exposure

An increasing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections has been reported worldwide. The aim of this study was to investigate the mechanism underlying carbapenem resistance and its relationship to antibiotic exposure. Sixteen isolates with various carbapenem susceptibilities recovered from five patients between 2003 and 2006 were subjected to molecular study. The medical records of the patients were also reviewed. All of the patients were admitted for complicated respiratory illness, had a prolonged hospital stay, and were exposed to antibiotics. Carbapenems were prescribed before the emergence of the CRKP. Various combinations of extended-spectrum cephalosporinase genes belonging to the SHV, CTX-M, and AmpC groups were found among the isolates. Other carbapenem resistance-associated genes, such as bla(IMP), bla(VIM), bla(OXA), and bla(KPC), were not found. OmpK35 was not expressed in any of the isolates, and additional loss of OmpK36 was observed in all CRKP isolates. Two insertion elements, ISPa13 or IS5, were found inserted into OmpK36 in the isolates derived from three patients. These IS elements were also identified in their parental carbapenem-susceptible isolates, suggesting that an internal transposition into OmpK36 resulted in resistance. OmpK36 loss represents the major mechanism for the development of CRKP in extended-spectrum cephalosporinase-producing isolates. A prolonged hospital stay and recent carbapenem exposure may predispose patients to CRKP, impacting the clinical outcome.

Molecular Characterization of Extended-Spectrum Cephalosporinase-Producing Salmonella enterica Serovar Choleraesuis Isolates from Patients in Thailand and Denmark

The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla(CMY-2) containing incA/C or bla(CTX-M-14) containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the bla(CTX-M-14) gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A "travel-associated" spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.

Molecular Epidemiology and Mechanisms of Carbapenem Resistance in Pseudomonas aeruginosa

The contributions of different mechanisms of resistance to carbapenems among a collection of imipenem- and meropenem-nonsusceptible Pseudomonas aeruginosa isolates were investigated. This screening included the recently reported extended-spectrum cephalosporinases (ESACs) weakly hydrolyzing carbapenems. Eighty-seven percent of the studied isolates were resistant to imipenem. Genes encoding metallo-beta-lactamases or carbapenem-hydrolyzing oxacillinases were not identified. The main mechanism associated with imipenem resistance was the loss of outer membrane protein OprD. Identification of overexpressed ESACs and loss of OprD were observed for 65% of the isolates, all being fully resistant to imipenem. Resistance to meropenem was observed in 78% of the isolates, with all but one also being resistant to imipenem. Overexpression of the MexAB-OprM, MexXY-OprM, or MexCD-OprJ efflux systems was observed in 60% of the isolates, suggesting the contribution of efflux mechanisms in resistance to meropenem. The loss of porin OprD and the overproduction of ESACs were observed in 100% and 92% of the meropenem-resistant isolates, respectively. P. aeruginosa can very often accumulate different resistance mechanisms, including ESAC production, leading to carbapenem resistance.