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Importance: Antimicrobial resistance (AMR) is a serious public health threat. AMR bacteria and their resistance determinants in food can be transmitted to humans through the food chain and by direct contact and disseminate directly to the environment., Objective: This study examined the AMR characteristics and transferable R plasmids in Escherichia coli isolated from meat ducks raised in an open-house system., Methods: One hundred seventy-seven (n = 177) commensal E. coli were examined for their antimicrobial susceptibilities and horizontal resistance transfer. The plasmids were examined by PCR-based plasmid replicon typing (PBRT) and plasmid multi-locus sequence typing (pMLST)., Results: The highest resistance rate was found against ampicillin (AMP, 83.0%) and tetracycline (TET, 81.9%), and most isolates exhibited multidrug resistance (MDR) (86.4%). The R plasmids were conjugally transferred when TET (n = 4), AMP (n = 3), and chloramphenicol (n = 3) were used as a selective pressure. The three isolates transferred resistance genes either in AMP or TET. The bla CTX-M1 gene resided on conjugative plasmids. Five replicon types were identified, of which Inc FrepB was most common in the donors (n = 13, 38.4%) and transconjugants (n = 16, 31.2%). Subtyping F plasmids revealed five distinct replicons combinations, including F47:A-:B- (n = 2), F29:A-:B23 (n = 1), F29:A-:B- (n = 1), F18:A-B:- (n = 1), and F4:A-:B- (n = 1). The chloramphenicol resistance was significantly correlated with the other AMR phenotypes ( p < 0.05)., Conclusions and Relevance: The meat ducks harbored MDR E. coli and played an important role in the environmental dissemination of AMR bacteria and its determinants. This confirms AMR as a health issue, highlighting the need for routine AMR monitoring and surveillance of meat ducks., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Korean Society of Veterinary Science.)

Colistin is a last resort antibiotic for the treatment of carbapenemase producing Klebsiella pneumoniae. The disruption of the mgrB gene by insertion sequences (ISs) is a mechanism mediating colistin resistance. Plasmids encode mobilizable IS elements which integrate into the mgrB gene in K. pneumoniae causing gene inactivation and colistin resistance. The species prevalence of mgrB-gene disrupting insertion elements ISL3 (ISKpn25), IS5 (ISKpn26), ISKpn14, and IS903B present on plasmids were assessed. IS containing plasmids were also scanned for antimicrobial resistance genes, including carbapenem resistant genes. Plasmids encoding ISs are abundant in K. pneumoniae. IS903B was found in 28 unique Inc groups, while ISKpn25 was largely carried by IncFIB(pQil) plasmids. ISKpn26 and ISKpn14 were most often found associated with IncFII(pHN7A8) plasmids. Of the 34 unique countries which contained any of the IS elements, ISKpn25 was identified from 26. ISKpn26, ISKpn14, and IS903B ISs were identified from 89.3%, 44.9%, and 23.9% plasmid samples from China. Plasmids carrying ISKpn25, ISKpn14, and ISKpn26 IS have a 4.6-, 6.0-, and 6.6-fold higher carbapenemase gene count, respectively, relative to IS903B-carrying plasmids. IS903B bearing plasmids have a 20-, 5-, and 5-fold higher environmental source isolation count relative to ISKpn25, ISKpn14, and ISKpn26 bearing plasmids. ISKpn25 present on IncFIB(pQil) sourced from clinical settings is established across multiple countries, while ISKpn26, ISKpn14, and IS903B appear most often in China. Carbapenemase presence in tandem with IS elements may help promote an extensively drug resistant profile in K. pneumoniae limiting already narrow treatment options., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens., Competing Interests: CS, JH, CG, JS, AG, DH, SW, BK, SK No competing interests declared, AK, MT, CE, AP Antibodies disclosed in the manuscript are part of patent "Anti-staphylococcal antibodies" (US9873733B2 / EP2935324B1), assigned to the Universitaetsklinikum Tuebingen and MorphoSys AG. The authors have declared that no further conflict of interest exists, (© 2022, Slavetinsky et al.)

Corneal hysteresis and corneal resistance factor are parameters that reflect the dynamic biomechanical properties of the cornea and have been shown to be biomarkers of corneal disease. In this genome-wide association study of over 100 000 participants, we identified over 200 genetic loci, all but eight novel, significantly associated with either one or both of these traits. In addition to providing key insights into the genetic architecture underlying normal corneal function, these results identify many candidate loci in the study of corneal diseases that lead to severe visual impairment. Additionally, using Mendelian randomization, we were able to identify causal relationships between corneal biomechanics and intraocular pressure measurements, which help elucidate the relationship between corneal properties and glaucoma., (© The Author(s) 2020. Published by Oxford University Press.)

Despite the sporadic detection of fluoroquinolone-resistant Shigella in Asia in the early 2000s and the subsequent global spread of ciprofloxacin-resistant (cipR) Shigella sonnei from 2010, fluoroquinolones remain the recommended therapy for shigellosis 1-7 . The potential for cipR S. sonnei to develop resistance to alternative second-line drugs may further limit future treatment options 8 . Here, we aim to understand the evolution of novel antimicrobial resistant (AMR) S. sonnei variants after introduction into Vietnam. We found that cipR S. sonnei displaced the resident ciprofloxacin-susceptible (cipS) lineage while rapidly acquiring additional resistance to multiple alternative antimicrobial classes. We identified several independent acquisitions of extensively drug-resistant/multidrug-resistant-inducing plasmids, probably facilitated by horizontal transfer from commensals in the human gut. By characterizing commensal Escherichia coli from Shigella-infected and healthy children, we identified an extensive array of AMR genes and plasmids, including an identical multidrug-resistant plasmid isolated from both S. sonnei and E. coli in the gut of a single child. We additionally found that antimicrobial usage may impact plasmid transfer between commensal E. coli and S. sonnei. These results suggest that, in a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines.

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of bla IMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the bla IMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak.

Antimicrobial resistance (AMR) is currently one of the most important challenges to the treatment of bacterial infections. A critical issue to combat AMR is to restrict its spread. In several instances, bacterial plasmids are involved in the global spread of AMR. Plasmids belonging to the incompatibility group (Inc)HI are widespread in Enterobacteriaceae and most of them express multiple antibiotic resistance determinants. They play a relevant role in the recent spread of colistin resistance. We present in this report novel findings regarding IncHI plasmid conjugation. Conjugative transfer in liquid medium of an IncHI plasmid requires expression of a plasmid-encoded, large-molecular-mass protein that contains an Ig-like domain. The protein, termed RSP, is encoded by a gene (ORF R0009) that maps in the Tra2 region of the IncHI1 R27 plasmid. The RSP protein is exported outside the cell by using the plasmid-encoded type IV secretion system that is also used for its transmission to new cells. Expression of the protein reduces cell motility and enables plasmid conjugation. Flagella are one of the cellular targets of the RSP protein. The RSP protein is required for a high rate of plasmid transfer in both flagellated and nonflagellated Salmonella cells. This effect suggests that RSP interacts with other cellular structures as well as with flagella. These unidentified interactions must facilitate mating pair formation and, hence, facilitate IncHI plasmid conjugation. Due to its location on the outer surfaces of the bacterial cell, targeting the RSP protein could be a means of controlling IncHI plasmid conjugation in natural environments or of combatting infections caused by AMR enterobacteria that harbor IncHI plasmids., Competing Interests: The authors have declared that no competing interests exist.

Introduction: To characterize OXA-48 carbapenemase-producing Klebsiella pneumoniae strains isolated after an increase in carbapenem resistance in Catalonia., Methodology: K. pneumoniae identification, antimicrobial susceptibility studies, the Modified Hodge Test method, amplification of antimicrobial resistance genes (against β-lactamases, quinolones and aminoglycosides), molecular typing (by PFGE and MLST), conjugation assays, plasmid characterization (PBRT-PCR and Southern blot), a description of mobile genetic elements and statistical analysis were done., Results: OXA-48 was the only carbapenemase detected, with a prevalence of 1.9%. The bla OXA-48 gene was located in an IncL conjugative plasmid of 62kb and integrated into the transposons Tn1999.2 (91.7%) or Tn1999.1. Five PFGE profiles (A to E) were found, which exactly matched the MLST: ST101, ST17, ST1233, ST14 and ST405, respectively. ST1233 is described here for the first time. K. pneumoniae OXA-48-producing strains were also CTX-M-15 carriers, some producing OXA-1 and TEM-1 penicillinases. The acquired qnrB66 and qnrB1 and aac(3')-IIa, aac(6')-Ib genes were also identified., Conclusion: The K. pneumoniae ST405 clone has played an important role in the growing prevalence of OXA-48 in Catalonia. All clones described preserved the bla OXA-48 genetic environment and mobile genetic elements (Tn1999). Notably, the three strains with minor sequence types in this study are not multiresistant strains. These strains are expanding in elderly patients (average age of 76 years) with serious underlying diseases, mainly women (61.2%)., (Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Purpose: Multidrug-resistant Klebsiella pneumoniae strains are regularly involved in hospital outbreaks. This study describes an ESBL-producing K. pneumoniae clone (ST607-K25) responsible for a nosocomial outbreak in a neonatal intensive care unit., Methodology: Fourteen strains isolated from 13 patients were included. Antimicrobial susceptibility testing was performed by the agar diffusion method. A clonal link was first investigated by fingerprinting (ERIC-PCR and REP-PCR) then confirmed by MLST. Characterization was performed by molecular detection and identification of several drug resistance and virulence determinants., Results: All strains expressed the same antibiotype, combining ESBL production, fluoroquinolones and aminoglycoside resistance, except for one which remained susceptible to fluoroquinolones. Fingerprinting methods confirmed the clonal link and MLST identified a ST607 clone. Molecular investigations revealed: (I) genes encoding for two narrow-spectrum beta-lactamases (SHV-1 and TEM-1) and an ESBL (CTX-M-15); (II) absence of any chromosomal mutation in quinolone resistance-determining- regions (QRDR) of gyrA/gyrB and parC/parE genes; (III) genes encoding for three plasmid-mediated quinolone-resistance (PMQR) determinants: oqxAB (14/14), aac(6')-Ib-cr (14/14) and qnrB (13/14); (IV) production of a K25 capsule; and (V) carriage of three genes encoding for virulence factors: mrkD (type 3 fimbriae) (14/14), ybts (yersiniabactin) (12/14) and entB (enterobactin) (14/14)., Conclusion: We described a multidrug-resistant Kp ST607 clone responsible for a nosocomial outbreak in vulnerable and premature newborns. Molecular investigations allowed us to identify several resistance factors responsible for ESBL production (CTX-M-15) and quinolone resistance (three PMQR determinants). The detection of a gene (ybtS) belonging to the high-pathogenicity island yersiniabactin could partly explain its high colonization and diffusion potential.

Objectives: To identify the prevalence and the types of Neisseria gonorrhoeae (NG) resistance plasmids-mediated penicillin (PPNG) and tetracycline (TRNG), the ciprofloxacin resistance (CRNG), and related risk factors of each types of resistance., Methods: The beta-lactamase-producing plasmid types (Africa, Asia, and Toronto), tetM tetracycline resistance plasmid types (America and Dutch), and the determination of the Ser-91 mutation of GyrA were detected by specifics PCRs on 149 diagnosed NG positives samples followed by Hinf1 digestion for tetM and gyrA mutation., Results: 135 (90.1%) samples showed a profile of molecular resistance to at least one antibiotic with predominance of ciprofloxacin resistance. In fact, 36 (24.2%) and 69 (46.3%) cases harbored PPNG and TRNG, respectively, and 116 (77.9%) cases showed the mutation Ser-91 of GyrA (CRNG). From a total of 36 PPNG isolates, the Toronto, Asian, and Toronto/Asian types were detected in 13 (36.1%), 10 (27.8%), and 13 (36.1%) cases, respectively, whereas the African type was not detected. In addition, the American type of TRNG was detected in 92.8% (64/69) of cases, while the Dutch type was detected in 7.2% (5/69) of cases. The association of demographics and clinical variables with NG resistance to ciprofloxacin, penicillin, and tetracycline was studied and the risk factors have been determined., Conclusion: Resistance to penicillin, tetracycline, and ciprofloxacin among NG samples positives remained at high levels in Morocco as determined by molecular profile. So, the use of molecular tools for NG antimicrobial resistance detection can help in the management and spread limitation of this infection.

The aim of this study was to use single-molecule, nanopore sequencing to explore the genomic environment of the resistance determinants in a multidrug-resistant (MDR) strain of enteroaggregative Escherichia coli serotype O51 : H30, sequence type (ST) 38. Sequencing was performed on the MinION Flow cell MIN-106 R9.4. Nanopore raw FAST5 reads were base-called using Albacore v1.2.1, converted to FASTA and FASTQ formats using Poretools v0.6.0, and assembled using Unicycler v0.4.2, combining the long-read sequencing data with short-read data produced by Illumina sequencing. The genome was interrogated against an antimicrobial resistance (AMR) gene reference database using blast. The majority of the 12 AMR determinants identified were clustered together on the chromosome at three separate locations flanked by integrases and/or insertion elements [region 1 -catA, blaOXA-1, aac(6')-Ib-cr, tetA and blaCTX-M-15; region 2 - dfrA1 and aadA1; region 3 - catA, blaTEM-1, tetA and sul2]. AMR determinants located outside these three regions were a chromosomally encoded blaCMY-16, mutations in gyrA and parC, and two plasmid-encoded AMR determinants, blaOXA-181 and qnrS1 located on the same IncX3 plasmid. Long-read analysis of whole genome sequencing data identified mobile genetic elements on which AMR determinants were located and revealed the combination of different AMR determinants co-located on the same mobile element. These data contribute to a better understanding of the transmission of co-located AMR determinants in MDR E. coli causing gastrointestinal and extra-intestinal infections.

Objectives: Shewanella algae is an environmental marine bacterium and an emerging opportunistic human pathogen. Moreover, there are increasing reports of strains showing multidrug resistance, particularly carbapenem-resistant isolates. Although S. algae has been found in bivalve shellfish aquaculture, there is very little genome-wide data on resistance determinants in S. algae from shellfish. The aim of this study was to determine the whole genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone in Taiwan., Methods: Bacterial genomic DNA was sequenced using an Illumina MiSeq platform with 250-bp paired-end reads. De novo genome assembly was performed using Velvet v.1.2.07. The whole genome was annotated and several candidate genes for antimicrobial resistance were identified., Results: The genome size was calculated at 4751156bp, with a mean G+C content of 53.09%. A total of 4164 protein-coding sequences, 7 rRNAs, 85 tRNAs and 5 non-coding RNAs were identified. The genome contains genes associated with resistance to β-lactams, trimethoprim, tetracycline, colistin and quinolones. Multiple efflux pump genes were also detected., Conclusion: Small abalone is a potential source of foodborne drug-resistant S. algae. The genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone will provide valuable information for further study of the dissemination of resistance genes at the human-animal interface., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. All rights reserved.)

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer., Competing Interests: The authors have declared that no competing interests exist.

Objective: Colistin resistance has emerged worldwide and has been threatening the efficacy of one of the last-resort antimicrobials used for treatment of multidrug resistant Gram-negative bacteria. While five colistin resistance genes (mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5) have been described, few data are available on the prevalence of mcr-genes other than mcr-1 in human samples., Results: In this study, the presence of five currently described colistin resistance genes (mcr 1-5) in vaginal swabs of women undergoing infertility evaluation was reported. Most samples were found to be positive for the mcr-4 (12.7%), followed by two for the mcr-2 (1.5%), two for the mcr-3 (1.5%), one for the mcr-1 (0.7%), and one for the mcr-5 (0.7%). Phylogenetic comparison demonstrated identical (mcr-1, mcr-2, mcr-3, mcr-5) or similar (mcr-4) nucleotide sequences of human samples and those of animal origins from the same city, suggesting the potential transmission of mcr genes from animals to humans. This is the first detection of mcr-2, mcr-4 and mcr-5 genes in human samples, and warrants further research to determine the spread of the mcr genes and elucidate the full epidemiology of colistin resistance genes in humans.

In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.

The objective of this study was to investigate the molecular mechanisms explaining the multidrug-resistant (MDR) phenotype found in a novel clinical Shewanella sp. strain (Shew256) recovered from a diabetic patient. Whole-genome shotgun sequencing was performed using Illumina MiSeq-I and Nextera XT DNA library. De novo assembly was performed with SPAdes. RAST Server was used to predict the open-reading frames and the predictions were confirmed using BLAST. Further genomic analysis was carried out using average nucleotide identity (ANI), ACT (Artemis), OrthoMCL, ARG-ANNOT, ISfinder, PHAST, tRNAscan-SE, plasmidSPAdes, PlasmidFinder and MAUVE. PCR and plasmid extraction were also performed. Genomic analysis revealed a total of 456 predicted genes unique to Shew256 compared with other Shewanella genomes. Moreover, the presence of different resistance genes, including bla PER-2 , was found. A complex class 1 integron containing the ISCR1 gene, disrupted by two putative transposase genes, was identified. Furthermore, other resistance genes, a transposon containing aph(3'), insertion sequences, phages and non-coding RNAs were also found. In conclusion, evidence of acquisition of resistance genes and mobile elements that could explain the MDR phenotype were observed. This Shewanella sp. represents a prime example of how antibiotic resistance determinants can be acquired by uncommon pathogens., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

The enteropathogen Yersinia pseudotuberculosis causes gastrointestinal infections in humans. Although this species is usually susceptible to antibiotics active against Gram-negative bacteria, we identified three multidrug resistant (MDR) strains of Y. pseudotuberculosis that were isolated from the environment in Russia and from a patient in France. The resistance traits of the two Russian isolates were transferable at high frequencies (≈2×10 -1 /donor CFU) to Y. pseudotuberculosis. In contrast no transfer of the antibiotic resistances carried by the French strain was observed. Sequencing of the plasmid extracts of the Y. pseudotuberculosis transconjugants for the Russian isolates revealed the presence of conjugative plasmids of the IncN group that carried genes conferring resistance to four to six classes of antibiotics. The French strain harbored a large MDR plasmid of the IncHI2 group that carried resistance genes to six families of antibiotics, and contained a truncated set of transfer genes, accounting for the lack of plasmid transfer. All three Y. pseudotuberculosis plasmids were homologous to MDR plasmids found in various enterobacteria. A phylogenetic analysis showed that the two Russian strain plasmids were closely related to each other and were more distant from the French plasmid. To the best of our knowledge, this is the first molecular characterization of MDR plasmids in Y. pseudotuberculosis. Due to the propensity of this species to acquire exogenous plasmids, the risk of emergence of new MDR Y. pseudotuberculosis isolates should be seriously taken into consideration., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Background: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC β-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates., Methods: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC β-lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals., Results: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates., Conclusion: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC β-lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints., (Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Objectives: Quinolone antimicrobials are frequently misused due to self-medication and suboptimal dose administration, leading to the development of resistance as well as treatment failure. The present study aimed to characterise plasmid-mediated quinolone resistance (PMQR) determinants and their genetic selection in the presence of quinolone stress within members of the Enterobacteriaceae., Methods: A total of 209 non-duplicate Enterobacteriaceae isolates were collected from hospital and community health centres over the period July 2013-June 2014. Molecular characterisation of phenotypically screened quinolone-resistant isolates was done by multiplex PCR. Plasmids bearing the qnr and aac(6')-Ib-cr genes were transformed into Escherichia coli DH5α and were selected on Muller-Hinton agar plates containing 0.25μg/mL and 0.5μg/mL ciprofloxacin, norfloxacin, ofloxacin, levofloxacin and moxifloxacin. Conjugation experiments were performed to determine whether the aac(6')-Ib-cr- and qnr-carrying plasmids were self-transferable., Results: The transformation assay revealed that transformants carrying qnrA could be selected in media containing norfloxacin, ciprofloxacin and levofloxacin, whereas qnrB and aac(6')-Ib-cr were selected on media containing norfloxacin and ciprofloxacin. Transformed qnrD could be selected in media containing norfloxacin and ofloxacin, and qnrS was selected only in the presence of levofloxacin., Conclusions: The presence of qnr genes has been associated with an increase in quinolone minimum inhibitory concentrations (MICs) and therefore leads to treatment failure when quinolones are used as selective therapeutic drugs. Since PMQR determinants have a high prevalence, effective measures should be taken and surveillance should be performed in order to avoid treatment failures using this group of antimicrobials., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Escherichia coli (E. coli) commonly reside in human intestine and most E. coli strains are harmless, but some serotypes cause serious food poisoning. This study identified and molecularly characterized bla SHV genes from 490 E. coli strains with multi-drug resistance in a hospital population. PCR and molecular cloning and southern blot were performed to assess functions and localizations of this resistant E. coli gene and the pulsed-field gel electrophoresis (PFGE) was utilized to demonstrate the clonal relatedness of the positive E. coli strains. The data showed that 4 of these 490 E. coli strains (4/499, 0.8%) carried bla SHV genes that included EC D2485 (bla SHV-5 ), EC D2487 (bla SHV-5 ), EC D2684 (bla SHV-11 ) and EC D2616 (bla SHV-195, a novel bla SHV ). Analysis of bla SHV open-reading frame showed that bla SHV-5 had a high hydrolysis activity to the broad-spectrum penicillin (ampicillin or piperacillin), ceftazidime, ceftriaxone, cefotaxime and aztreonam. bla SHV-195 and bla SHV-11 had similar resistant characteristics with high hydrolysis activities to ampicillin and piperacillin, but low activities to cephalosporins. Moreover, the two bla SHV-5 genes were located on a transferable plasmid (23kb), whereas the other two bla SHV variants (bla SHV-11 and bla SHV-195 ) seemed to be located in the chromosomal material. Both EC D2485 and EC D2487 clones isolated in 2010 had the same DNA finger printing profile and they might be the siblings of clonal dissemination. The data from the current study suggest that the novel bla SHV and clonal dissemination may be developed, although bla SHV genes were infrequently identified in this hospital population. The results of the work demonstrate the necessity for molecular surveillance in tracking bla SHV -producing strains in large teaching hospital settings and emphasize the need for epidemiological monitoring., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by bla KPC2 , bla KPC3 , and bla KPC4 , which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation., Competing Interests: The authors declare no conflict of interest.

Antibiotic resistance is recognised as a major global threat to public health by the World Health Organization. Currently, several hundred thousand deaths yearly can be attributed to infections with antibiotic-resistant bacteria. The major driver for the development of antibiotic resistance is considered to be the use, misuse and overuse of antibiotics in humans and animals. Nonantibiotic compounds, such as antibacterial biocides and metals, may also contribute to the promotion of antibiotic resistance through co-selection. This may occur when resistance genes to both antibiotics and metals/biocides are co-located together in the same cell (co-resistance), or a single resistance mechanism (e.g. an efflux pump) confers resistance to both antibiotics and biocides/metals (cross-resistance), leading to co-selection of bacterial strains, or mobile genetic elements that they carry. Here, we review antimicrobial metal resistance in the context of the antibiotic resistance problem, discuss co-selection, and highlight critical knowledge gaps in our understanding., (© 2017 Elsevier Ltd. All rights reserved.)

This study identified a novel integrative and conjugative element (ICESsD9) carrying erm(B) and tet(O) resistance determinants in Streptococcus suis D9 and determined its prevalence in clinical isolates. Comparative genome analysis was performed using Mauve and Artemis Comparison Tool visualisation programs. Inverse PCR was utilised to detect its circular intermediate. The transfer capacity of ICESsD9 was evaluated by mating assays using S. suis A7 and Enterococcus faecalis JH2-2 as recipients. A genome walking approach was employed to analyse the characteristics of integration sites in transconjugants. A total of 118 clinical S. suis isolates were tested by PCR mapping assays to detect ICESsD9-like elements. MLST was performed on isolates containing ICESsD9 variants to determine their clonal relatedness. This 55 683-bp element can actively excise from the chromosome. Additionally, it was capable of transferring both into S. suis and E. faecalis with frequencies of 1.2×10 -4 and 5.8×10 -6 per donor, respectively. When investigating integration site features, it was found that ICESsD9 can enter S. suis and E. faecalis chromosomes by different sites, generating 15-bp and 3-bp direct repeat sequences, respectively. Twelve isolates mainly belonging to sequence types ST1, ST7 and ST28 were confirmed to harbour ICESsD9-like elements. In conclusion, this study provides the first description of an ICE in S. suis that is capable of transferring both into S. suis and E. faecalis. The presence of different ICESsD9 variants in clinical isolates suggests already wide dissemination of this family element in S. suis in China., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Antimicrobial resistance severely limits the therapeutic options for many clinically important bacteria. In Gram-negative bacteria, multidrug resistance is commonly facilitated by plasmids that have the ability to accumulate and transfer refractory genes amongst bacterial populations. The aim of this study was to isolate and identify bioactive compounds from the medicinal plant Mallotus philippensis (Lam.) Mull. Arg. with both direct antibacterial properties and the capacity to inhibit plasmid conjugal transfer. A chloroform-soluble extract of M. philippensis was subjected to bioassay-guided fractionation using chromatographic and spectrometric techniques that led to the isolation of the known compounds rottlerin [5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1,2-chromene] and the red compound (8-cinnamoyl-5,7-dihydroxy-2,2,6-trimethylchromene). Both compounds were characterised and elucidated using one-dimensional and two-dimensional nuclear magnetic resonance (NMR). Rottlerin and the red compound showed potent activities against a panel of clinically relevant Gram-positive bacteria, including meticillin-resistant Staphylococcus aureus (MRSA). No significant direct activities were observed against Gram-negative bacteria. However, both rottlerin and the red compound strongly inhibited conjugal transfer of the plasmids pKM101, TP114, pUB307 and R6K amongst Escherichia coli at a subinhibitory concentration of 100mg/L. Interestingly, despite the planar nature of the compounds, binding to plasmid DNA could not be demonstrated by a DNA electrophoretic mobility shift assay. These results show that rottlerin and the red compound are potential candidates for antibacterial drug lead development. Further studies are needed to elucidate the mode of inhibition of the conjugal transfer of plasmids., (Copyright © 2016. Published by Elsevier Ltd.)

With the emergence and spread of new methicilin resistant Staphylococcus aureus (MRSA) strains, control of dissemination, both in hospitals and in the community, requires the molecular characterization of the circulating strains in order to establish their dynamics and identify the sources of infection. During this study we analyzed the MRSA isolates by means of PCR-based methods in order to improve epidemiological surveillance and early application of prevention measures. The presence of mecA, nuc, lukF-PV and lukS-PV genes, as well as SCCmec types was assessed in relation to clinical characteristics and multidrug resistance (MDR) for 86 MRSA isolates and showed that 51% of MDR strains were carriers of mobile genetic elements SCCmec IV and the majority of non-MDR SCCmec type IV strains were PVL-positive (81.8%). Comparison of diagnostic methods showed that PBP2 detection represents an extremely useful alternative to PCR for the rapid screening of MRSA isolates, in laboratories that lack facilities necessary for molecular diagnosis, such as PFGE (Pulse Field Gel Electrophoresis), spa-typing and/or MLST (Multilocus Sequence Typing).

Infections caused by multidrug resistant Klebsiella pneumoniae have been increasingly reported in many parts of the world. A total of 93 Malaysian multidrug resistant K. pneumoniae isolated from patients attending to University of Malaya Medical Center, Kuala Lumpur, Malaysia from 2010-2012 were investigated for antibiotic resistance determinants including extended-spectrum beta-lactamases (ESBLs), aminoglycoside and trimethoprim/sulfamethoxazole resistance genes and plasmid replicons. CTX-M-15 (91.3%) was the predominant ESBL gene detected in this study. aacC2 gene (67.7%) was the most common gene detected in aminoglycoside-resistant isolates. Trimethoprim/sulfamethoxazole resistance (90.3%) was attributed to the presence of sul1 (53.8%) and dfrA (59.1%) genes in the isolates. Multiple plasmid replicons (1-4) were detected in 95.7% of the isolates. FIIK was the dominant replicon detected together with 13 other types of plasmid replicons. Conjugative plasmids (1-3 plasmids of ~3-100 kb) were obtained from 27 of 43 K. pneumoniae isolates. An ESBL gene (either CTX-M-15, CTX-M-3 or SHV-12) was detected from each transconjugant. Co-detection with at least one of other antibiotic resistance determinants [sul1, dfrA, aacC2, aac(6')-Ib, aac(6')-Ib-cr and qnrB] was noted in most conjugative plasmids. The transconjugants were resistant to multiple antibiotics including β-lactams, gentamicin and cotrimoxazole, but not ciprofloxacin. This is the first study describing the characterization of plasmids circulating in Malaysian multidrug resistant K. pneumoniae isolates. The results of this study suggest the diffusion of highly diverse plasmids with multiple antibiotic resistance determinants among the Malaysian isolates. Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of the multidrug resistant bacteria in healthcare settings.

Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

Aeromonas spp. are oxidase positive, gram-negative, facultative anaerobic bacilli that are widely distributed in aquatic environments. A.hydrophila, A.sobria and A.bestiarum may cause severe infections in both human and cold-blooded animals. Environmental persistance of quinolones that are widely used in both human and veterinary medicine plays an important role in the selection of resistant mutants. Plasmid-mediated resistance is one of the main mechanisms involved in quinolone resistance, and qnr, qepA, aac(6')-Ib-cr, oqxAB genes are identified as resistance determinants. Determination of various types of qnr gene in different bacteria mainly in Enterobacteriaceae, suggests that they are widely distributed in nature. Recently, plasmid-mediated quinolone resistance was defined among Aeromonas species isolated from water. The aim of this study was to investigate the presence of qnr genes among aquatic Aeromonas spp. in Turkey. A total of 45 Aeromonas strains isolated from water and fishes collected from three different geographical regions (Aegean, Mediterranean and Blacksea) in Turkey, were included in the study. The isolates were identified at species level by the use of 16S rDNA-RFLP (Restriction fragment length polymorphism) analysis and multiplex polymerase chain reaction (M-PCR). Among the isolates, 20 were identified as A.sobria, 10 as A.hydrophila, nine as A.salmonicida, four as A.bestiarum and two as A.veronii. The plasmid-mediated quinolone resistance determinants, qnrA, qnrB, qnrC and qnrS genes, were investigated by M-PCR, and sequence analysis was performed for nine qnr-positive isolates. According to the sequence analysis of the genes, qnr genes were characterized in six A.sobria, in two A.bestiarum and in one A.hydrophila isolate (9/45; 20%). When the sequence was compared with GenBank database, this gene was found as qnrS2. All qnrS-positive Aeromonas spp. isolates were ciprofloxacin-susceptible, while five of them were resistant to nalidixic acid. This study is the first research about the plasmid-mediated quinolone resistance and the presence of qnrS2 genes among Aeromonas spp. isolated from fishes and water in Turkey. In conclusion, various resistance genes of aquatic bacteria may constitute a potential risk for the transmission of those genes to other bacteria as well as clinical isolates.

Background & Objectives: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR), consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids) among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common β-lactamases encoding genes on these plasmids., Methods: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23, blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA) and metallo-β-lactamase (MBL) type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing., Results: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36%) carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance., Interpretation & Conclusions: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in A. baumannii clinical isolates. This indicates towards a need for preventive measures to avert the dissemination of plasmid resistance determinants in clinical environments.

We report here the complete nucleotide sequence of two IncR replicons encoding multidrug resistance determinants, including β-lactam (blaDHA-1, blaSHV-12), aminoglycoside (aphA1, strA, strB), and fluoroquinolone (qnrB4, aac6'-1b-cr) resistance genes. The plasmids have backbones that are similar to each other, including the replication and stability systems, and contain a wide variety of transposable elements carrying known antibiotic resistance genes. This study confirms the increasing clinical importance of IncR replicons as resistance gene carriers., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Antibiotic resistance increases costs for health care and causes therapy failure. An important mechanism for spreading resistance is transfer of plasmids containing resistance genes and subsequent selection. Yet the factors that influence the rate of transfer are poorly known. Rates of plasmid transfer were measured in co-cultures in chemostats of a donor and a acceptor strain under various selective pressures. To document whether specific mutations in either plasmid or acceptor genome are associated with the plasmid transfer, whole genome sequencing was performed. The DM0133 TetR tetracycline resistance plasmid was transferred between Escherichia coli K-12 strains during co-culture at frequencies that seemed higher at increased growth rate. Modeling of the take-over of the culture by the transformed strain suggests that in reality more transfer events occurred at low growth rates. At moderate selection pressure due to an antibiotic concentration that still allowed growth, a maximum transfer frequency was determined of once per 10(11) cell divisions. In the absence of tetracycline or in the presence of high concentrations the frequency of transfer was sometimes zero, but otherwise reduced by at least a factor of 5. Whole genome sequencing showed that the plasmid was transferred without mutations, but two functional mutations in the genome of the recipient strain accompanied this transfer. Exposure to concentrations of antibiotics that fall within the mutant selection window stimulated transfer of the resistance plasmid most., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Salmonella enterica serovar Heidelberg ranks among the most prevalent causes of human salmonellosis in the United States and Canada, although it has been infrequently reported in South American and European countries. Most Salmonella infections are self-limiting; however, some invasive infections require antimicrobial therapy. In this work we characterized an oxyimino-cephalosporin resistant S. Heidelberg isolate recovered from an inpatient in a Buenos Aires hospital. CMY-2 was responsible for the β-lactam resistance profile. S. Heidelberg contained a 97kb plasmid belonging to the Inc N group harboring blaCMY-2. ISEcp1 was located upstream blaCMY-2 driving its expression and mobilization. The isolate belonged to sequence type 15 and virotyping revealed the presence of sopE gene. In this study we identified the first CMY-2 producing isolate of S. Heidelberg in Argentina and even in South America., (Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.)

The complete sequence of the carbapenem-resistance-conferring conjugative plasmid pLD209 from a Pseudomonas putida clinical strain is presented. pLD209 is formed by 3 well-defined regions: an adaptability module encompassing a Tn402-like class 1 integron of clinical origin containing blaVIM-2 and aacA4 gene cassettes, partitioning and transfer modules, and a replication module derived from plasmids of environmental bacteria. pLD209 is thus a mosaic of modules originating in both the clinical and environmental (nonclinical) microbiota.

The novel lincosamide resistance gene lnu(E), truncated by insertion of an ISEnfa5-cfr-ISEnfa5 segment, was identified in Streptococcus suis. The gene lnu(E) encodes a 173-amino-acid protein with ≤69.4% identity to other lincosamide nucleotidyltransferases. The lnu(E) gene and its promoter region were de novo synthesized, and Staphylococcus aureus RN4220 carrying a shuttle vector with the cloned lnu(E) gene showed a 16-fold increase in the lincomycin MIC. Mass spectrometry experiments demonstrated that Lnu(E) catalyzed the nucleotidylation of lincomycin.

We describe here the sequence and gene organization of a new glycopeptide resistance operon (vanO) in Rhodococcus equi from soil. The vanO operon has low homology to enterococcal van operons and harbors a vanHOX cluster transcribed in the direction opposite that of the vanS-vanR regulatory system and composed of three open reading frames with unknown function. This finding has clinical interest, since glycopeptides are used to treat R. equi infections and resistance has been reported in clinical isolates.

Growing attention has been paid to the dissemination of antibiotic resistance genes (ARGs) in wastewater microbial communities. The application of membrane bioreactors (MBRs) in wastewater treatment is becoming increasingly widespread. We hypothesized that the transfer of ARGs among bacteria could occur in MBRs, which combine a high density of bacterial cells, biofilms, and antibiotic resistance bacteria or ARGs. In this study, the transfer discipline and dissemination of the RP4 plasmid in MBRs were investigated by the counting plate method, the MIDI microorganism identification system, and quantitative polymerase chain reaction (qPCR) techniques. The results showed that the average transfer frequency of the RP4 plasmid from the donor strain to cultivable bacteria in activated sludge was 2.76×10⁻⁵ per recipient, which was greater than the transfer frequency in wastewater and bacterial sludge reported previously. In addition, many bacterial species in the activated sludge had received RP4 by horizontal transfer, while the genera of Shewanella spp., Photobacterium spp., Pseudomonas spp., Proteus spp., and Vibrio spp. were more likely to acquire this plasmid. Interestingly, the abundance of the RP4 plasmid in total DNA remained at high levels and relatively stable at 10⁴ copies/mg of biosolids, suggesting that ARGs were transferred from donor strains to activated sludge bacteria in our study. Thus, the presence of ARGs in sewage sludge poses a potential health threat., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

To ensure their stable inheritance by daughter cells during cell division, bacterial low-copy-number plasmids make simple DNA segregating machines that use an elongating protein filament between sister plasmids. In the ParMRC system of the Escherichia coli R1 plasmid, ParM, an actinlike protein, forms the spindle between ParRC complexes on sister plasmids. By using a combination of structural work and total internal reflection fluorescence microscopy, we show that ParRC bound and could accelerate growth at only one end of polar ParM filaments, mechanistically resembling eukaryotic formins. The architecture of ParM filaments enabled two ParRC-bound filaments to associate in an antiparallel orientation, forming a bipolar spindle. The spindle elongated as a bundle of at least two antiparallel filaments, thereby pushing two plasmid clusters toward the poles.

The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in Escherichia coli isolated from food-producing animals and to characterize the PMQR-positive isolates. A total of 365 E. coli isolates which were either nalidixic acid resistant and ciprofloxacin susceptible (NAL(R)-CIP(S); n=185), or nalidixic acid and ciprofloxacin resistant (NAL(R)-CIP(R); n=180) were assessed for the presence of PMQR determinants by polymerase chain reaction. PMQR-positive isolates were further characterized by mutation analysis within the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE, phylogenetic group analysis, and pulsed-field gel electrophoresis (PFGE). Fourteen NAL(R)-CIP(S) (n=8) and NAL(R)-CIP(R) (n=6) E. coli isolates were positive for PMQR genes. Among them, qnrB4, qnrS1, and aac(6')-Ib-cr genes were detected in two (0.5%), eight (2.2%), and four (1.1%) isolates, respectively. None of the isolates harbored qnrA, qnrC, qnrD, and qepA genes. All but one PMQR-positive isolates harbored one or more point mutations in the QRDR of gyrA, and five of these isolates had additional mutations in the parC gene. Furthermore, one isolate each had additional substitutions in gyrB and parE genes, respectively. The most prevalent mutation was Ser83-Leu within the QRDR of gyrA. Phylogenetic analysis identified three major phylogenetic lineages, with phylogroups A (n=7) and D (n=4) being the most common phylogroups. None of the isolates belonged to virulent phylogroup B2. PFGE demonstrated that a combination of clonal and horizontal gene transmission is disseminating PMQR genes among the veterinary E. coli isolates in Korea. To our knowledge, this is the first report of occurrence of qnrB, qnrS, and aac(6')-Ib-cr genes in E. coli isolated from food-producing animals in Korea. Isolation of PMQR genes from food animals is a matter of concern since they could be transmitted to humans via food animals.

In this study, the prevalence of plasmid-mediated quinolone resistance (PMQR) was investigated in 495 Escherichia coli isolates from diseased food-producing animals in Guangdong province, China. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analysed for mutations in 55 isolates harbouring only oqxAB and all isolates harbouring other PMQR genes. Overall, 282 (57.0 %) E. coli isolates had at least one PMQR gene. oqxAB was detected in 215 isolates and predominated the PMQR genes, followed by qnrS (63 isolates), aac(6')-Ib-cr (56 isolates), qnrB (39 isolates) and qepA (18 isolates). qnrA, qnrC and qnrD were not found in any of the isolates. The rates of resistance to ciprofloxacin, enrofloxacin, levofloxacin and nalidixic acid were 75.2, 81.0, 70.5 and 97.4 %, respectively, among the 495 isolates. Eight types of mutation in gyrA were detected in 154 PMQR-positive isolates, and 147 isolates were found to have mutations in parC. PFGE analysis indicated that the PMQR-positive E. coli isolates were genetically diverse. This study demonstrated that the number of mutations in QRDRs of gyrA and/or parC was significantly associated with the MICs of quinolones (P<0.01). The rates of resistance to ciprofloxacin, enrofloxacin and nalidixic acid in PMQR-positive isolates were significantly higher than those in PMQR-negative isolates (P<0.05). In addition, the prevalence of oqxAB had significant Spearman correlation coefficients in relation to the MICs of all four tested quinolones (P<0.01).

Introduction: The frequency of aac(6')-Ib-cr gene in ESBL-producing strains of Klebsiella pneumoniae and Escherichia coli is unknown, in Chile., Methodology: The aac(6')-Ib and aac(6')-Ib-cr genes were investigated using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and sequencing, in strains isolated from 10 Chilean hospitals between 2008-2009., Results: The aac(6')-Ib-cr gene was detected in 54% of K. pneumoniae and 74% of E. coli strains. The CIM(50) of CIP was higher among strains harboring aac(6')-Ib-cr, 8 times higher in K. pneumoniae and 4 times higher in E. coli. Moreover, both aac(6')-Ib and aac(6')-Ib-cr were simultaneously found in 13 K. pneumoniae and 3 E. coli isolates., Conclusion: This is the first report of aac(6')-Ib-cr in ESBL-producing strains of K. pneumoniae and E. coli isolated from in-patients in Chilean hospitals located along an area of more than 2,800 Km., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

One hundred and seven Shigella spp. strains were isolated in our laboratory during the years 2000 to 2010. One Shigella sonnei harboured the genes that coded the β-lactamases TEM-1 and CTX-M-15, identifying the structure, ISEcp1+bla(CTX-M-15)+orf477, in their genetic environment. The strain also carried a class 2 integron with the gene cassettes dfrA1+sat+aadA1. A plasmid group IncI1 ST31 (CC-31) was detected and its mobilization by conjugation was demonstrated. We describe for the first time a S. sonnei strain producing a CTX-M-15 β-lactamase recovered from a Spanish patient who had not travelled abroad., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

In spite of a well-documented ability of Samonella enterica Typhi strains to receive R factors from Escherichia coli and other enterobacteria, epidemiological data show that Typhi is a rather poor host of antibiotic-resistance genes and in fact, of plasmids, suggesting that most of the plasmids naturally acquired by Typhi strains become unstable and eventually segregate. We have previously reported evidence that each of three plasmids conjugatively transferred to S. enterica Typhi experienced deletion-mediated loss of a resistance determinant before plasmid segregation occurred. We now report that in Typhi strains containing these unstable plasmids a superhelical DNA species of lower mobility is detected, probably representing plasmid dimer structures. Plasmid deletion is a RecA-dependent process since it is not detected in derivatives of a recA1 S. enterica Typhi strain containing the corresponding plasmids, and in such strains we were unable to detect either the low-mobility species. We propose that the deletable segments contain key information for plasmid stability in S. enterica Typhi, possibly a multimer resolution system., (Copyright © 2012 Elsevier Inc. All rights reserved.)