Encoding Extended-spectrum Beta-lactamases Research Articles

CTX-M-15/27-positive Escherichia coli and VIM-2-producing Pseudomonas putida in free-living pigeons (Columba livia) in Tunisia

ObjectivesWild birds are vectors of antimicrobial resistance. Birds living in close contact with humans or other animals, like feral pigeons (Columba livia), might be especially prone to acquire resistance genes such as those encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. MethodsCloacal samples (n = 206) of free-living feral pigeons (C. livia) were collected in Sousse and Monastir, Tunisia. Antimicrobial susceptibility profiles were determined by disc-diffusion, and resistant isolates were short- and long-read whole-genome sequenced. Sequence analysis was performed using tools of the Centre for Genomic Epidemiology, and Phylogenetic analysis was performed based on the core-genome MLST. ResultsFourteen (14/206, 6.8%) pigeons harboured Enterobacterales resistant to last-generations cephalosporins, of which 10 were CTX-M-15- or CTX-M-27-producers, while two (1.0%) carried a VIM-2-producing Pseudomonas putida. Positive pigeons lived on four different livestock farms. Three STs (ST206, ST5584, ST8149) were identified among E. coli, of which ST5584 and ST8149 were found in two different farms. Genetic diversity was also observed in Enterobacter cloacae and P. putida isolates. The blaCTX−M-27 genes were chromosomally encoded, while the blaCTX−M-15 genes were carried on highly similar IncF/F-:A-:B53 plasmids. The blaVIM-2 gene was located on a class 1 integron co-harbouring several resistance genes. ConclusionPigeons living on livestock farms carried clinically important resistance genes encoding ESBLs and carbapenemases. Our results evidenced that both clonal (ST8149 and ST5584) and plasmidic (IncF/F-:A-:B53) transfers played a role in the spread of resistance genes among pigeons. Further studies are needed to identify factors favouring the transfer and persistence of resistance genes within the pigeon communities.

Detection of extended-spectrum beta-lactamases and carbapenemases in clinical isolates of Stenotrophomonas maltophilia in the northeast of Iran

PurposeStenotrophomonas maltophilia has emerged as an important pathogen associated with significant mortality in pneumonia and bacteremia of severely immunocompromised, hospitalized patients. The main objectives of the present study are to describe the surveillance of antibiotic resistance and to detect the genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemase enzymes in clinical S. maltophilia isolates from blood cultures in the northeast of Iran. MethodsA total of 114 clinical human isolates of S. maltophilia were collected from blood cultures of patients admitted at three teaching hospitals in Mashhad, northeast of Iran. Antimicrobial susceptibility was determined by the Kirby-Bauer disk diffusion test. The extended-spectrum beta-lactamase (ESBL) production was confirmed by the combination disk method. The PCR was applied for molecular epidemiology of ESBLs and carbapenemase coding genes. ResultsOverall, 10 % and 8 % of isolates were resistant to co-trimoxazole and levofloxacin, respectively. The prevalence of the blaTEM, blaSHV, and blaCTX-M-15 genes in the 69 ESBL-producing isolates was 2.9 %, 5.8 %, and 10.14 %, respectively. Moreover, the prevalence of the blaKPC-1, blaIMP, blaVIM, blaNDM, and blaOXA-48 genes was 18.42 % (n = 21), 4.39 % (n = 5), 6.14 % (n = 7), 15.79 % (n = 18), and 14.91 % (n = 17), respectively. ConclusionFor the first time in Iran, ESBLs and carbapenemase coding genes have been identified in human clinical strains of S. maltophilia. Circulation of these resistant strains with transferable genes is a sign of flow of resistome genes from other bacterial genera to S. maltophilia, most probably leading to the evolution of the superbugs of this bacterium.

Distribution of resistance genes encoding Extended Spectrum Beta-Lactamase (ESBL) in Proteus mirabilis species isolated from selected Hospitals in Jigawa State, Nigeria

Distribution of resistance genes encoding Extended Spectrum Beta-Lactamase (ESBL) in Proteus mirabilis species isolated from selected Hospitals in Jigawa State, Nigeria

In vitro activity of imipenem/relebactam, meropenem/vaborbactam and comparators against Enterobacterales from patients with intra-abdominal infections: Results of the study for Monitoring Antimicrobial Resistance Trends (SMART) in Taiwan, 2020

Multi-drug resistant Enterobacterales is a growing health threat. Imipenem/relebactam and meropenem/vaborbactam, are not clinically used in Taiwan and the susceptibility is lack from routine laboratory tests. Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints criteria. Isolates that were not susceptible to imipenem (MIC ≥ 2 mg/L), imipenem/relebactam (MIC ≥ 2 mg/L), or ceftolozane-tazobactam (MIC ≥ 4 mg/L) were selected for further molecular testing for genes encoding extended-spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases by multiplex PCR assays. A total of 290 Enterobacterales isolates from 9 participating hospitals were collected in 2020. Escherichia coli (n=135, 46.6%) and Klebsiella pneumoniae (n=88, 30.3%) were two leading pathogens of all Enterobacterales isolates. The antimicrobial agents with susceptibility rates more than 90% included amikacin (99.3%, 288/290), ertapenem (90.0%, 261/290), meropenem (97.2%, 282/290), imipenem/relebactam (94.8%, 275/290) and meropenem/vaborbactam (99.3%, 288/290). K. pneumoniae isolates were less susceptible to ertapenem, imipenem, meropenem, piperacillin-tazobactam and ceftozolane/tazobactam than E. coli (all p <0.05). ESBL, AmpC, and carbapenemase were detected in 40.5% (17/42), 45.2% (19/42) and 11.9% (5/42) among tested isolates, respectively. The 5 carbapenemase genes included 4 bla KPC and 1 bla IMP . The imipenem-non-susceptible isolates (n = 30) had higher susceptibility rates to meropenem/vaborbactam (93.3%, 28/30) than imipenem/relebactam (50%, 12/30) ( p < 0.05). Imipenem/relebactam and meropenem/vaborbactam had excellent efficacy against Enterobacterales isolates . Meropenem/vaborbactam allowed better salvage therapy for carbapenem-resistant Enterobacterales infections.

Whole genome global insight of antibiotic resistance gene repertoire and virulome of high - risk multidrug-resistant Uropathogenic Escherichiacoli

Elucidation of genetic determinants via whole genome sequence (WGS) analyses can help understand the high risk multidrug-resistant (MDR) Uropathogenic Escherichia coli (UPEC) associated with urinary tract infections (UTI) and its evasion strategies from treatment. We investigated the WGS of 30 UPEC strains from UTI samples across the world (2016–2019) and found 25 UPEC strains carrying 2–23 antibiotic resistance genes (ARGs) scattered across 1–3 plasmids per strain. Different ARGs (blaTEM, blaCTXM, blaNDM, blaOXA, blaCMY) encoding extended-spectrum beta-lactamases (TEM, CTXM, CMY) and carbapenemases (NDM, OXA) were found in 24/30, ARGs encoding aminoglycoside modifying enzymes (AAC, APH, AAD) variants in 23/30, trimethoprim ARGs (dfrA17, dfrA12, dfrA5, dfrB4 variants) encoding dihydrofolate reductase in 19/30 and sulfonamide ARGs (sul1, sul2, sul3) encoding dihydropteroate synthase and macrolide ARGs (mph1) encoding macrolide 2’ phosphotransferase in 15/30 UPEC strains. Collectively the ARGs were distributed in different combinations in 40 plasmids across UPEC strains with 20 plasmids displaying co-occurrence of multiple ARGs conferring resistance to beta lactam, aminoglycoside, sulfonamide, trimethoprim and macrolide antibiotics. These resistance plasmids belonged to seven incompatibility groups (IncF, IncI, IncC, IncH, IncN, IncB and Col), with IncFI and IncFII being the predominant resistance plasmids. Additionally, we observed co-occurrence of specific mutation pattern in quinolone resistance determining region (QRDR) viz., DNA gyrase (gyrA: S83L, D87N), and topoisomerase IV (parC: S80I, E84V; parE: I529L) in 18/30 strains. The strains also harbored diverse virulence genes, such as fimH, gad, iss, iha, ireA, iroN, cnf1 and san. Multilocus sequence typing (MLST) reconfirmed ST131(n = 10) as the predominant global high-risk clonal strain causing UTI. In summary, our findings contribute to better understand the plasmid mediated ARGs and its encoded enzymes that may contribute in antibiotic inactivation/modification or alteration in the antibiotic target site in high risk MDR hypervirulent UPEC strains causing UTI. The study reinforces the need to characterize and design appropriate inhibitors to counterattack different enzymes and devise strategies to curtail resistance plasmid.

A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids.

Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

Combining stool and stories: exploring antimicrobial resistance among a longitudinal cohort of international health students

BackgroundAntimicrobial resistance (AMR) is a global public health concern that requires transdisciplinary and bio-social approaches. Despite the continuous calls for a transdisciplinary understanding of this problem, there is still a lack of such studies. While microbiology generates knowledge about the biomedical nature of bacteria, social science explores various social practices related to the acquisition and spread of these bacteria. However, the two fields remain disconnected in both methodological and conceptual levels. Focusing on the acquisition of multidrug resistance genes, encoding extended-spectrum betalactamases (CTX-M) and carbapenemases (NDM-1) among a travelling population of health students, this article proposes a methodology of ‘stool and stories’ that combines methods of microbiology and sociology, thus proposing a way forward to a collaborative understanding of AMR.MethodsA longitudinal study with 64 health students travelling to India was conducted in 2017. The study included multiple-choice questionnaires (n = 64); a collection of faecal swabs before travel (T0, n = 45), in the first week in India (T1, n = 44), the second week in India (T2, n = 41); and semi-structured interviews (n = 11). Stool samples were analysed by a targeted metagenomic approach. Data from semi-structured interviews were analysed using the method of thematic analysis.ResultsThe incidence of ESBL- and carbapenemase resistance genes significantly increased during travel indicating it as a potential risk; for CTX-M from 11% before travel to 78% during travel and for NDM-1 from 2% before travel to 11% during travel. The data from semi-structured interviews showed that participants considered AMR mainly in relation to individual antibiotic use or its presence in a clinical environment but not to travelling.ConclusionThe microbiological analysis confirmed previous research showing that international human mobility is a risk factor for AMR acquisition. However, sociological methods demonstrated that travellers understand AMR primarily as a clinical problem and do not connect it to travelling. These findings indicate an important gap in understanding AMR as a bio-social problem raising a question about the potential effectiveness of biologically driven AMR stewardship programs among travellers. Further development of the ‘stool and stories’ approach is important for a transdisciplinary basis of AMR stewardship.

Molecular Characterization of Antimicrobial Resistance and Virulence Genes of Bacterial Pathogens from Bovine and Caprine Mastitis in Northern Lebanon.

Mastitis is an infectious disease encountered in dairy animals worldwide that is currently a growing concern in Lebanon. This study aimed at investigating the etiology of the main mastitis-causing pathogens in Northern Lebanon, determining their antimicrobial susceptibility profiles, and identifying their antimicrobial resistance (AMR) genes. A total of 101 quarter milk samples were collected from 77 cows and 11 goats presenting symptoms of mastitis on 45 dairy farms. Bacterial identification was carried out through matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibility was tested by disc diffusion and broth microdilution methods. Molecular characterization included polymerase chain reaction (PCR) screening for genes encoding extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC among Enterobacterales isolates, and virulence factors among Staphylococcus isolates. Escherichia coli isolates were subjected to phylogenetic typing by a quadruplex PCR method. The most frequently identified species were Streptococcus uberis (19.2%), Streptococcus agalactiae (15.1%), E. coli (12.3%), and Staphylococcus aureus (10.96%). Gram-positive bacteria were resistant to macrolides and tetracycline, whereas gram-negative bacteria displayed resistance to ampicillin and tetracycline. Two ESBL genes, blaTEM (83.3%) and blaOXA (16.7%), and one AmpC beta-lactamase gene, blaCMY-II (16.7%), were detected among six E. coli isolates, which mainly belonged to phylogenetic group B1. Among Staphylococcus spp., the mecA gene was present in three isolates. Furthermore, four isolates contained at least one toxin gene, and all S. aureus isolates carried the ica operon. These findings revealed the alarming risk of AMR in the Lebanese dairy chain and the importance of monitoring antimicrobial usage.

Characterization of carbapenem-resistant gram-negative bacterial isolates from Nigeria by whole genome sequencing

This study characterized the mechanisms of carbapenem resistance in gram-negative bacteria isolated from patients in Yola, Nigeria. Whole genome sequencing (WGS) was performed on 66 isolates previously identified phenotypically as carbapenem-non–susceptible.The patterns of beta-lactamase resistance genes identified were primarily species-specific. However, blaNDM-7 and blaCMY-4 were detected in all Escherichia coli and most Providencia rettgeri isolates; blaNDM-7 was also detected in 1 Enterobacter cloacae. The E. coli and E. cloacae isolates also shared blaOXA-1, while blaOXA-10 was found in all P. rettgeri, one Pseudomonas aeruginosa and 1 E. coli. Except for Stenotrophomonas maltophilia isolates, which only contained blaL1, most species carried multiple beta-lactamase genes, including those encoding extended-spectrum beta-lactamases, AmpC and OXA in addition to a carbapenemase gene. Carbapenemase genes were either class B or class D beta-lactamases. No carbapenemase gene was detected by WGS in 13.6% of isolates.

Genotypic and phenotypic characterisation of the Klebsiella oxytoca complex

Klebsiella spp. are associated with 3 to 7% of nosocomial infections and can be responsible for a range of conditions including pneumonia, bloodstream infections, meningitis, and necrotizing enterocolitis in infants. The role of Klebsiella pneumoniae in causing disease is well-characterised but, to date, the closely related species Klebsiella oxytoca has not received the same attention, despite often encoding extended-spectrum beta-lactamases and carbapenemases in clinical settings. K. oxytoca is the causative agent of Clostridiodes difficile-negative antibiotic-associated haemorrhagic colitis, a rare condition seen in some individuals receiving antibiotics. Whole-genome sequence analyses have shown K. oxytoca to be a complex comprising at least six species (K. oxytoca, K. michiganensis, K. grimontii, K. huaxiensis, ‘K. pasteurii’, ‘K. spallanzanii’). Our study aims to better characterise the K. oxytoca complex using a polyphasic approach. Preliminary investigations into the genomes of three K. michiganensis clinical isolates revealed the presence of a plasmid-borne ccdABlocus. ccdAB is a toxin-antitoxin (TA) system known to maintain plasmids in other pathogenic enterobacteria. We aim to functionally validate this TA system by cloning and conducting toxicity assays on the CcdB toxin, and cloning and assessing the ability of CcdA to function as an antidote. We also aim to sequence and generate Illumina/Oxford Nanopore hybrid genome assemblies of a larger collection of K. oxytoca complex clinical isolates and investigate their plasmids and TA systems in the same manner.

Multidrug-Resistant and Clinically Relevant Gram-Negative Bacteria Are Present in German Surface Waters.

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored blaVIM–1. Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.

Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut.

Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation.IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.

Discriminatory Power of Three Typing Techniques in Determining Relatedness of Nosocomial Klebsiella pneumoniae Isolates from a Tertiary Hospital in India

Purpose: The purpose of this study was to evaluate the discriminatory power of two DNA-based technique and a protein-based technique for the typing of nosocomial isolates of Klebsiella pneumoniae. A second objective was to determine the antimicrobial susceptibility pattern and characterise the presence of genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. Materials and Methods: Forty-six K. pneumoniae isolates from patients with bloodstream infections at a tertiary care hospital in India between December 2014 and December 2015 were studied. All isolates were typed using enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) analysis and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility profiles and ESBLs were detected using the BD Phoenix system. The types of ESBL and carbapenemase genes present were detected using PCR. Results: Isolates were subtyped into 31, 30 and 33 distinct genotypes by ERIC-PCR, RAPD and MALDI-TOF, respectively. Several isolates displaying identical DNA fingerprints were binned into different branches based on their proteomic fingerprint. Antimicrobial susceptibility tests revealed that 33/46 strains were multidrug resistant (MDR); a majority of the strains (83%) were sensitive to colistin. PCR-based analysis demonstrated 19 strains to harbour two or more ESBL and carbapenemase genes. Conclusion: ERIC-PCR was the most reproducible method for typing K. pneumoniae isolates and could not be substituted by MALDI-TOF for clonality analysis. A high degree of genetic diversity and the presence of MDR genes highlight the challenges in treating K. pneumoniae-associated infections.

First Report of Extended-Spectrum β-Lactamases Among Clinical Isolates of Klebsiella pneumoniae in Gaza Strip, Palestine.

Sixteen broad-spectrum cephalosporin-resistant Klebsiella pneumoniae isolates were recovered between April and June 2013 from Palestinian hospitals, Gaza Strip. Genes encoding extended-spectrum beta-lactamases (ESBLs) and other resistance genes were characterized by polymerase chain reaction and sequencing. The following β-lactamase genes were detected: blaCTX-M-15+ blaSHV1+ blaTEM-1 (six isolates), blaCTX-M-15+ blaSHV5+ blaOXA-1 (two isolates), blaCTX-M-14a (two isolates), blaCTX-M-15+ blaSHV33 (one isolate), blaCTX-M-15+ blaTEM-1 (one isolate), blaCTX-M-15+ blaSHV12+ blaOXA-1(one isolate), blaCTX-M-15+ blaSHV5 (one isolate), blaCTX-M-15+ blaSHV1 (one isolate), and blaCTX-M-3 (one isolate). The ISEcp1 (in four cases truncated by IS26), orf477, or IS903 sequences were found upstream or downstream of blaCTX-M genes. The aac(6')-Ib-cr gene was found in seven isolates. The qnrS1 and qnrB1 genes were detected in five isolates and two isolates, respectively. Seven isolates contained class 1 integrons with four gene cassette arrangements: dfrA5 (three isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). A high clonal diversity was also observed among studied isolates by pulsed-field gel electrophoresis (12 unrelated profiles). This study demonstrates for the first time the emergence and polyclonal spread of multidrug-resistant ESBL-producing K. pneumoniae isolates among patients in a hospital setting in Gaza Strip, Palestine.

Frequency of BKC-1-Producing Klebsiella Species Isolates.

BKC-1 is a new class A serine carbapenemase that was recently identified in Klebsiella pneumoniae clinical isolates. The principal objective of this study was to evaluate the frequency of blaBKC-1 by testing a collection of Klebsiella isolates. Only 2 of 635 Klebsiella isolates (0.3%) carried blaBKC-1 The two BKC-1-producing isolates belonged to clonal complex 442 and possessed identical pulsed-field gel electrophoresis patterns. The blaBKC-1 gene was inserted into a 10-kb plasmid that was identical to the previously reported plasmid, p60136. The BKC-producing K. pneumoniae isolates presented also possessed other mechanisms for beta-lactam resistance, such as genes encoding extended-spectrum beta-lactamases and mutations in the genes ompK35 and ompK36, encoding the major porins.

Community-acquired Extended-spectrum and Plasmid-mediated ampC Beta-lactamase-producing Multidrug-resistant Enterobacter cloacae Septicaemia in a Cat with Euthyroid Sick Syndrome

A 7-year-old castrated male Korean Shorthair cat was referred with lethargy and anorexia. Laboratory examination revealed moderate degenerative changes of peripheral neutrophils on blood smear examination and decreased levels of free and total thyroxine (T₄) as well as bacterial growth on blood culture. Molecular analyses of the 16S ribosomal RNA gene and heat shock protein 60 gene confirmed the bacterium as Enterobacter cloacae. A minimal inhibitory concentration test showed multidrug resistance of the bacterium against 16 antibiotics. Polymerase chain reaction (PCR) and subsequent sequencing specifically for bla TEM , bla SHV , bla CTX-M , and plasmid-mediated ampC genes revealed positive results to bla TEM-1 , bla CTX-M-15 , and plasmid-mediated bla ACT-1 genes, indicating that the isolated bacterium contains plasmids containing genes encoding extended-spectrum beta-lactamase and plasmid-mediated ampC beta-lactamase. After 1 month of treatment with antibiotics and levothyroxine, the cat’s condition improved; both the thyroid function test and the blood culture showed no abnormalities. This is the first report of community-acquired multidrug-resistant E. cloacae-induced euthyroid sick syndrome in a cat. By the prompt diagnostic procedures and properly selected antibiotic therapy, the cat was recovered from the multidrug-resistant bacterium-induced septicaemia.

High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4.

Horizontal dissemination of the genes encoding extended spectrum beta-lactamases (ESBLs) via conjugative plasmids is facilitating the increasingly widespread resistance of pathogens to beta-lactam antibiotics. However, there is relatively little known about the regulatory factors and mechanisms that govern the spread of these plasmids. Here, we carried out a high-throughput, transposon insertion site sequencing analysis (TnSeq) to identify genes that enable the maintenance and transmission of pESBL, an R64 (IncI1)-related resistance plasmid that was isolated from Escherichia coli O104:H4 linked to a recent large outbreak of gastroenteritis. With a few exceptions, the majority of the genes identified as required for maintenance and transmission of pESBL matched those of their previously defined R64 counterparts. However, our analyses of the high-density transposon insertion library in pESBL also revealed two very short and linked regions that constitute a previously unrecognized regulatory system controlling spread of IncI1 plasmids. In addition, we investigated the function of the pESBL-encoded M.EcoGIX methyltransferase, which is also encoded by many other IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in expanding the plasmid's host range. Collectively, our work illustrates the power of the TnSeq approach to enable rapid and comprehensive analyses of plasmid genes and sequences that facilitate the dissemination of determinants of antibiotic resistance.

Determination of extended spectrum beta-lactamases, metallo-beta-lactamases and AmpC-beta-lactamases among carbapenem resistant Pseudomonas aeruginosa isolated from burn patients

BackgroundPseudomonas aeruginosa is an important cause of morbidity and mortality in patients with burns. MethodA total of 214 nonduplicated burn wound isolates of P. aeruginosa were recovered from burn patients. Identification of carbapenem resistant isolates and their antimicrobial susceptibility pattern was carried out using the phenotypic methods. The presence of genes encoding extended spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) enzymes were determined by PCR. The genetic relationships between carbapenem resistant isolates were determined by Random Amplified Polymorphic DNA (RAPD)-PCR. ResultsOf 214 investigated P. aeruginosa isolates, 100 (46.7%) were carbapenem resistant. All carbapenem resistant P. aeruginosa were resistant to imipenem, meropenem, ertapenem, carbenicillin, aztreonam, gentamicin and ciprofloxacin but susceptible to polymyxin B. Among 100 carbapenem resistant P. aeruginosa isolates, 3%, 65% and 52% were identified as ESBLs, carbapenemase and AmpC overproduction positive isolates respectively. The most prevalent ESBLs and MBLs genes included blaOXA-10 (97%), blaTEM (61%), blaVIM (55%), blaPER (13%), blaIMP (3%) and blaAIM (1%). RAPD analysis yielded 13 distinct profiles among 92 isolates. A dominant RAPD type was designated as A that consisting of 80 isolates. ConclusionThis is the first report of Adelaide IMipenmase (AIM) MBLs producing P. aeruginosa from Iran and also of the high prevalence of AmpC overproduction isolates. According to the results of current study, P. aeruginosa isolates producing OXA-10, TEM, VIM, PER and IMP beta-lactamases are frequent and the population structures of these isolates are highly similar.

CTX-M from community-acquired urinary tract infections in Algeria

Thirty-eight Enterobacteriaceae isolates including seventeen Klebsiella pneumoniae,thirteen Escherichia coli and eight Enterobacter cloacae resistant to broad-spectrum cephalosporins were recovered from community-acquired urinary-tract infections at four private-laboratories in Bejaia, Algeria, between March 2007 and May 2009. Antimicrobial susceptibility testing of these isolates to 23 antimicrobials was performed by disk diffusionaccording to the recommendations of the Antibiogram Committee of the French Society for Microbiology. Results of antimicrobial testing showed that, all isolates are resistant to ticarcillin, piperacillin, cefotaxime, Cefpirome. Decreased susceptibility was observed against others agents. They remained susceptible toward imipenem. Genes encoding extended-spectrum-beta-lactamases (ESBL) and plasmid-mediated quinolone resistance (PMQR) were characterized by PCR and sequencing. All isolates were found to produce CTX-M-type ESBL: CTX-M-15 was identified in thirty-three isolates and CTX-M-3 in five isolates. In addition, nine isolates were positive for the qnr gene, including 5 of qnrB1 and 4 of qnrS1. Ten isolates were found to produce aac(6’)lb-cr resistance determinants.Molecular typing showed a clonal relatedness between isolates.   Key words: Urinary tract infection, community, Enterobacteriaceae, CTX-M, plasmid-mediated quinolone resistance, Algeria.

Evaluation of an autogenous vaccine against E. coli bearing the CTX-M-14 plasmid Roberto M. La Ragione, Sabrina Lotz, Hannah Cockrem, Martin J. Woodward and Nick Coldham Department of Bacteriology, Animal Health and Veterinary Laboratories Agency (Weybridge), UK.

Event Abstract Back to Event Evaluation of an autogenous vaccine against E. coli bearing the CTX-M-14 plasmid Roberto La Ragione1* 1 University of Surrey, Veterinary Laboratories Agency - Department of Bacteriology, United Kingdom Roberto M. La Ragione, Sabrina Lotz, Hannah Cockrem, Martin J. Woodward and Nick Coldham Department of Bacteriology, Animal Health and Veterinary Laboratories Agency (Weybridge), UK. Over the past decade enteric bacteria in Europe, Africa, and Asia have become increasingly resistant to cephalosporin antibiotics. This is largely due to the spread of genes encoding extended-spectrum beta-lactamase (ESBL) enzymes that can inactivate 3rd and 4th generation cephalosporins. The recent emergence of bacteria carrying ESBLs has highlighted their zoonotic potential and the requirement to prevent their dissemination through the food chain. One such approach to controlling bacterial disease is the use of autogenous vaccines. Autogenous vaccines are manufactured from the specific pathogenic bacteria isolated from the diseased animal. The studies presented here aimed to establish if there is any scientific evidence to support the use of autogenous vaccines to prevent the colonisation of cattle by ESBL harbouring bacteria. Preliminary studies established a bovine colonisation and persistence model for E. coli O33 bearing the blaCTX-M-14 plasmid. An autogenous vaccine study was then set up whereby thirty 5-6 week old cross bred calves were randomly divided into two groups, one receiving the vaccine and the other a placebo intramuscularly. At 12 weeks of age all calves were orally challenged with 1 x 1010 CFU of E. coli O33 and faecal shedding monitored daily. Total E. coli and E. coli O33 shedding rates were determined using selective media. Serial post-mortem examinations were also performed to evaluate colonisation levels together with detailed histopathological studies. Blood samples were taken throughout the study for immunological assays. The results indicated good colonisation of the bovine GIT by the ESBL bearing E. coli O33. However, there was no significant protection afforded by the autogenous vaccine as assessed by faecal shedding and enumeration of ESBLs from tissues sampled at post mortem examination. Interestingly though, an elevated immune response to E. coli O33 was detected in the serum of the vaccinated animals. Future studies aim to evaluate the effect of the vaccine on plasmid transfer and the identification of ESBL specific antigens that may be appropriate as novel vaccine candidates. Keywords: Autogenous vaccine, Calves, ESBL harbouring bacteria Conference: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination, Ghent, Belgium, 2 Jul - 5 Jul, 2011. Presentation Type: Oral Presentation Topic: Abstracts Citation: La Ragione R (2012). Evaluation of an autogenous vaccine against E. coli bearing the CTX-M-14 plasmid . Front. Immunol. Conference Abstract: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination. doi: 10.3389/conf.fimmu.2012.01.00010 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Jan 2012; Published Online: 12 Jan 2012. * Correspondence: Prof. Roberto La Ragione, University of Surrey, Veterinary Laboratories Agency - Department of Bacteriology, Surrey, GU2 7XH, United Kingdom, R.Laragione@surrey.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Roberto La Ragione Google Roberto La Ragione Google Scholar Roberto La Ragione PubMed Roberto La Ragione Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.