Plasmid Location Research Articles

OXA-48-like carbapenemases in Proteus mirabilis - novel genetic environments and a challenge for detection

OXA-48-like enzymes represent the most frequently detected carbapenemases in Enterobacterales in Western Europe, North Africa and the Middle East. In contrast to other species, the presence of OXA-48-like in Proteus mirabilis leads to an unusually susceptible phenotype with low MICs for carbapenems and piperacillin-tazobactam, which is easily missed in the diagnostic laboratory. So far, there is little data available on the genetic environments of the corresponding genes, bla OXA-48-like, in P. mirabilis. In this study susceptibility phenotypes and genomic data of 13 OXA-48-like-producing P. mirabilis were investigated (OXA-48, n=9; OXA-181, n=3; OXA-162, n=1). Ten isolates were susceptible to meropenem and ertapenem and three isolates were susceptible to piperacillin-tazobactam. The gene bla OXA-48 was chromosomally located in 7/9 isolates. Thereof, in three isolates bla OXA-48 was inserted into a P. mirabilis genomic island. Of the three isolates harbouring bla OXA-181 one was located on an IncX3 plasmid and two were located on a novel MOBF plasmid, pOXA-P12, within the new transposon Tn7713. In 5/6 isolates with plasmidic location of bla OXA-48-like, the plasmids could conjugate to E. coli recipients in vitro. Vice versa, bla OXA-48-carrying plasmids could conjugate from other Enterobacterales into a P. mirabilis recipient. These data show a high diversity of bla OXA-48-like genetic environments compared to other Enterobacterales, where genetic environments are quite homogenous. Given the difficult-to-detect phenotype of OXA-48-like-producing P. mirabilis and the location of bla OXA-48-like on mobile genetic elements, it is likely that OXA-48-like-producing P. mirabilis can disseminate, escape most surveillance systems, and contribute to a hidden spread of OXA-48-like.

Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring bla NDM-1 and bla IMP-4 induce resistance transmission and fitness variation

ABSTRACT To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of bla IMP-4 and bla NDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-β-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that bla IMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of bla IMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that bla IMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, bla IMP-4 in different locations did not affect the gene stability. The bla NDM-1-harbouring plasmid contributed to the increased resistance to β-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

Molecular Characterization of a KPC-2- and NDM-1-Producing Klebsiella michiganensis Clinical Isolate in Cerebrospinal Fluid.

Klebsiella michiganensis is an emerging pathogen. In this context, we characterised a strain fxq isolated from a cerebrospinal fluid specimen of a patient with tentorial meningioma, and the K. michiganensis isolate produced carbapenemases of KPC and NDM types. The Phoenix 100 Automated Microbiology System, MALDI-TOF and whole-genome sequencing were used to identify the species. Anti-microbial susceptibility testing was also conducted with the Phoenix 100. The plasmid locations of the bla KPC-2 and bla NDM-1 genes were determined by S1-nuclease pulsed-field gel electrophoresis and Southern blot. The transfer capacity of plasmids carrying bla KPC-2 and bla NDM-1 was investigated by conjugation experiments, and the resistance plasmid stability was evaluated by culture and subculture. K. michiganensis subtypes were identified by multi-locus sequence typing. We performed whole-genome sequencing to confirm species, characterise plasmids and analyse core genes. fxq was originally identified as Klebsiella oxytoca and showed resistance to imipenem and meropenem, but whole-genome sequencing identified it to be K. michiganensis. The strain fxq belonged to the novel sequence type 202 (ST202) and carried the bla KPC-2 and bla NDM-1 genes located on the pB_KPC InFIA and pE_NDM IncU plasmids, respectively. The bla KPC-2-carrying plasmid was successfully transferred to Escherichia coli EC600 by conjugation, whereas the bla NDM-1 gene on the pE_NDM plasmid was not. The pB_KPC and pE_NDM plasmids demonstrated high stability. This work is the first report on a carbapenem-resistant clinical isolate K. michiganensis ST202 harbouring the bla KPC-2 and bla NDM-1 genes encoded by the IncFIA and IncU plasmids, respectively.

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical Shigella and Salmonella isolates in Belgium.

For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this will impact their spread differently. Whole-genome sequencing (WGS) is increasingly used for AMR surveillance. However, determining the genetic context of AMR genes using only short-read sequencing is complicated. The combination with long-read sequencing offers a potential solution, as it allows hybrid assemblies. Nevertheless, its use in surveillance has so far been limited. This study aimed to demonstrate its added value for AMR surveillance based on a case study of extended-spectrum beta-lactamases (ESBLs). ESBL genes have been reported to occur also on plasmids. To gain insight into the diversity and genetic context of ESBL genes detected in clinical isolates received by the Belgian National Reference Center between 2013 and 2018, 100 ESBL-producing Shigella and 31 ESBL-producing Salmonella were sequenced with MiSeq and a representative selection of 20 Shigella and six Salmonella isolates additionally with MinION technology, allowing hybrid assembly. The bla CTX-M-15 gene was found to be responsible for a rapid rise in the ESBL Shigella phenotype from 2017. This gene was mostly detected on multi-resistance-carrying IncFII plasmids. Based on clustering, these plasmids were determined to be distinct from the circulating plasmids before 2017. They were spread to different Shigella species and within Shigella sonnei between multiple genotypes. Another similar IncFII plasmid was detected after 2017 containing bla CTX-M-27 for which only clonal expansion occurred. Matches of up to 99 % to plasmids of various bacterial hosts from all over the world were found, but global alignments indicated that direct or recent ESBL-plasmid transfers did not occur. It is most likely that travellers introduced these in Belgium and subsequently spread them domestically. However, a clear link to a specific country could not be made. Moreover, integration of bla CTX-M in the chromosome of two Shigella isolates was determined for the first time, and shown to be related to ISEcp1. In contrast, in Salmonella, ESBL genes were only found on plasmids, of which bla CTX-M-55 and IncHI2 were the most prevalent, respectively. No matching ESBL plasmids or cassettes were detected between clinical Shigella and Salmonella isolates. The hybrid assembly data allowed us to check the accuracy of plasmid prediction tools. MOB-suite showed the highest accuracy. However, these tools cannot replace the accuracy of long-read and hybrid assemblies. This study illustrates the added value of hybrid assemblies for AMR surveillance and shows that a strategy where even just representative isolates of a collection used for hybrid assemblies could improve international AMR surveillance as it allows plasmid tracking.

Bacteriophages in the Dairy Industry: A Problem Solved?

Bacteriophages (or phages) represent one of the most persistent threats to food fermentations, particularly large-scale commercial dairy fermentations. Phages infecting lactic acid bacteria (LAB) that are used as starter cultures in dairy fermentations are well studied, and in recent years there have been significant advances in defining the driving forces of LAB-phage coevolution. The means by which different starter bacterial species defend themselves against phage predation and the chromosomal or plasmid location of the genes encoding these defense mechanisms have dictated the technological approaches for the development of robust starter cultures. In this review, we highlight recent advances in defining phage-host interactions and how phage resistance occurs in different bacterial species. Furthermore, we discuss how these insights continue to transform the dairy fermentation industry and how they also are anticipated to guide food fermentations involving plant-based alternatives in the future.

Enterococcus spp. from chicken meat collected 20 years apart overcome multiple stresses occurring in the poultry production chain: Antibiotics, copper and acids

Poultry meat has been a vehicle of antibiotic resistant bacteria and genes. Yet, the diversity of selective pressures associated with their maintenance in the poultry-production chain remains poorly explored. We evaluated the susceptibility of Enterococcus spp. from chicken meat collected 20 years apart to antibiotics, metals, acidic pH and peracetic acid-PAA. Contemporary chicken-meat samples (n = 53 batches, each including a pool of neck skin from 10 single carcasses) were collected in a slaughterhouse facility using PAA as disinfectant (March–August 2018, North of Portugal). Broilers were raised in intensive farms (n = 29) using CuSO4 and organic acids as feed additives. Data were compared with that of 67 samples recovered in the same region during 1999–2001. All 2018 samples had multidrug resistant-MDR isolates, with >45 % carrying Enterococcus faecalis, Enterococcus faecium or Enterococcus gallinarum resistant to tetracycline, erythromycin, ampicillin, quinupristin-dalfopristin, ciprofloxacin, chloramphenicol or aminoglycosides. Resistance rates were similar (P > 0.05) to those of 1999–2001 samples for all but five antibiotics. The decrease of samples carrying vancomycin-resistant isolates from 46 % to 0 % between 1999-2001 and 2018 was the most striking difference. Isolates from both periods were similarly susceptible to acid pH [minimum-growth pH (4.5-5.0), minimum-survival pH (3.0-4.0)] and to PAA (MIC90 = 100–120 mg/L/MBC90 = 140–160 mg/L; below concentrations used in slaughterhouse). Copper tolerance genes (tcrB and/or cueO) were respectively detected in 21 % and 4 % of 2018 and 1999-2001 samples. The tcrB gene was only detected in E. faecalis (MICCuSO4 > 12 mM), and their genomes were compared with other international ones of chicken origin (PATRIC database), revealing a polyclonal population and a plasmid or chromosomal location for tcrB. The tcrB plasmids shared diverse genetic modules, including multiple antimicrobial resistance genes (e.g. to tetracyclines, chloramphenicol, macrolide-lincosamide-streptogramin B-MLSB, aminoglycosides, bacitracin, coccidiostats). When in chromosome, the tcrB gene was co-located closely to merA (mercury) genes. Chicken meat remains an important vehicle of MDR Enterococcus spp. able to survive under diverse stresses (e.g. copper, acid) potentially contributing to these bacteria maintenance and flux among animal-environment-humans.

Fabrication and Characterization of pcDNA3.1(+) Location within Chitosan/Nanoparticles Complexes for Enhanced Gene Delivery.

Chitosan nanoparticles (CSNP) are becoming a popular alternative for delivering nucleic acids to tissues for gene transfer (gene therapy). The size and morphology of these biodegradable nano-carriers are adjustable, and their positive charge allows them to interact strongly with negatively charged nucleic acids. This study aimed to fabricate and characterize pcDNA3.1 (+) plasmid (pDNA) and CSNP complexes and determine the plasmid location in these vehicles. The characteristics of the pDNA/CSNP complex after production were investigated by SEM, XRD, DLS, TGA, and FTIR. The capacity of CSNP to form complexes with pDNA was investigated by labeling free plasmids with the fluorescent intercalating dye OliGreen. The stability of pDNA/CSNP in the presence of chitosanase was evaluated. Surface-Enhanced Raman Spectroscopy (SERS) for pDNA localization was performed, and absorption rate in BALB/c mice was assessed by real-time PCR. The optimum pDNA/CSNP ratio for plasmid complex formation was established to be 1:2 (w.w) by measuring spectroscopy. At these optimum complex formation ratios, spectroscopy, and gel digest experiments, SERS indicated that a part of the pDNA was present on the complex outer surface. The findings of plasmid absorption in mouse thigh tissue by real-time PCR revealed that the rate of gene uptake was significantly greater at a dose of 1:2 (w.w) of pDNA/CSNP than in other groups (P< 0.001). The findings of this study reveal exactly pDNA fits into polymer nanostructured delivery systems, allowing the formulation to be adjusted for selective distribution. This understanding will aid future research into the system's functioning in vitro and in vivo.

1247. Molecular Epidemiology of Multi-drug Resistant Klebsiella pneumoniae and K. quasipneumoniae in Qatar

BackgroundThe molecular epidemiology of carbapenem-resistant Klebsiella species is not well investigated in Qatar. The objective of this work was to characterize the genetic context of carbapenemase-producing Klebsiella isolates recovered from clinical specimens.Methods Klebsiella isolates (n=100) were collected at 7 tertiary hospitals from 2015-2017. Identification and susceptibility testing were performed using MALDI-TOF MS and BD Phoenix system, respectively. Whole Genome Sequencing was performed on the Illumina NextSeq platform. Phylogenomic analysis, screening of resistance and virulence genes, and comparison of genetic environment of carbapenemase were carried out.Results Klebsiella pneumoniae was common (80), followed by K. quasipneumoniae (16), K. aerogenes (3) and K. oxytoca (1). The most prevalent were genes encoding NDM-1 (39), OXA-48 (20), OXA-232 (10) and OXA-181 (12). KPC-2 (3) and KPC-3 (2) were also identified; no carbapenemase-encoding genes could be identified in 15 isolates. Plasmid locations of 24 carbapenemase-encoding genes were determined; blaNDM-1 was localized on IncFII replicon, while blaOXA-181 and blaOXA-232 were commonly associated with ColKP3 plasmids. pOXA-48-like plasmid was detected in 17/20 isolates harboring blaOXA-48. blaKPC-3 was located on a contig with ‘traditional’ Tn4401a mobile genetic element. Sequence types (STs) were diverse and the ‘traditional’ clonal group (CG) 258 was rare. K. pneumoniae ST147 was predominant (13), followed by ST231 (7) and ST11 (5). Nine K. quasipneumoniae isolates belonged to ST196 and were highly clonal. The virulence loci such as yersiniabactin (ybt) and rmpA were not detected within the study’s K. quasipneumoniae isolates. Amongst K. pneumoniae, there were 50 ybt+ isolates; 8 isolates had rmpA, and of these, 3 belonged to ST383. K. pneumoniae serotype K2, the capsular serotype associated with invasive liver abscess syndrome, was detected in 5 isolates. Genetic relationship of carbapenem-resistant Klebsiella pneumoniae and K. quasipneumoniae isolates in Qatar inferred from core genome SNPs. The tree is overlaid with predicted antimicrobial resistance genes and virulence factors for each isolate.ConclusionThe predominant carbapenemases among clinical Klebsiella species isolates in Qatar are NDM and OXA-48 like enzymes, disseminated through various plasmids. The detection of carbapenemase-producing isolate bearing rmpA and serotype K2 reflect the presence of both multidrug resistance and hypervirulence in K. pneumoniae.Disclosures Yohei Doi, MD, PhD, AstraZeneca (Speaker’s Bureau)bioMerieux (Consultant)FujiFilm (Advisor or Review Panel member, Speaker’s Bureau)Gilead (Consultant)GSK (Consultant)Meiji (Consultant)MSD (Consultant)Shionogi (Consultant) Yohei Doi, MD, PhD, Astellas (Individual(s) Involved: Self): Grant/Research Support; AstraZeneca (Individual(s) Involved: Self): Speakers’ bureau; bioMerieux (Individual(s) Involved: Self): Consultant, Speakers’ bureau; Chugai (Individual(s) Involved: Self): Consultant; Entasis (Individual(s) Involved: Self): Consultant; FujiFilm (Individual(s) Involved: Self): Advisor or Review Panel member; Gilead (Individual(s) Involved: Self): Consultant; GSK (Individual(s) Involved: Self): Consultant; Kanto Chemical (Individual(s) Involved: Self): Grant/Research Support; MSD (Individual(s) Involved: Self): Speaking Fee; Pfizer (Individual(s) Involved: Self): Grant/Research Support; Shionogi (Individual(s) Involved: Self): Grant/Research Support, Speakers’ bureau; Teijin Healthcare (Individual(s) Involved: Self): Speakers’ bureau; VenatoRx (Individual(s) Involved: Self): Consultant

Diseño Factorial para Elaborar, Seleccionar y Determinar una Cepa Va-cunal de Salmonella entérica Serotipo Enteritidis 82139

Factorial design allows the elaboration, selection and determination of a vaccine strain of enteric Salmonella Serotype Enteritidis 82139 with validation of dsbA, dle and dlp gene function with respect to mobility capacity and intracellular bacterial invasion due to Minimum Significant Difference (DMS) and with 99% of confidence level for mobility and 95% for bacterial intracellular invasion. Results at 0.025 DMS and significantly different, Dle, does show invasiveness in MDCK at 1 hour of bacterial-host cell interaction, intracellular adherence-internalization phase. Dlp, highly homologous protein, capable of complementing dsbA- mutation. Dle, it presents specific and limited function. In the absence of DsbA and Dlp, its invasive capacity decreases at the 1st, 2nd and 4th hours of bacterial interaction. DsbA is decisive in the 1st hour of interaction; Dlp of plasmid location has complementary activity to DsbA, with a higher activity potential at 4th hour. In the absence of DsbA, Dlp and Dle, S. enteritidis significantly loses its virulence capacity, an interrelated function under the same pathway, which are reoxidized by DsbB oxidatively. Say, it has no function in mobility. Salmonella ente-ritidis 82139 2C, lacking dsbA and dlp, is a genetic resource for the vaccine strain and 82139 2CI will require future research.

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor.

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.

Linezolid Resistance Genes in Enterococci Isolated from Sediment and Zooplankton in Two Italian Coastal Areas.

Linezolid is a last-resort antibiotic for the treatment of severe infections caused by multidrug-resistant Gram-positive organisms; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased in recent years due to worldwide spread of acquired resistance genes (cfr, optrA, and poxtA) in clinical, animal, and environmental settings. In this study, we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley agar plates were investigated for their carriage of optrA, poxtA, and cfr genes; optrA was found in one Enterococcus faecalis isolate, poxtA was found in three Enterococcus faecium isolates and two Enterococcus hirae isolates, and cfr was not found. Two of the three poxtA-carrying E. faecium isolates and the two E. hirae isolates showed related pulsed-field gel electrophoresis (PFGE) profiles. Two E. faecium isolates belonged to the new sequence type 1710, which clustered in clonal complex 94, encompassing nosocomial strains. S1 PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and a plasmid location of optrA Whole-genome sequencing revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA was flanked by two copies of IS1216 in both plasmids. In mating experiments, all but one strain (E. faecalis EN3) were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid-resistant strains to humans from environmental reservoirs.IMPORTANCE Linezolid is one of the few antimicrobials available to treat severe infections due to drug-resistant Gram-positive bacteria; therefore, the emergence of linezolid-resistant enterococci carrying transferable resistance determinants is of great concern for public health. Linezolid resistance genes (cfr, optrA, and poxtA), often plasmid located, can be transmitted via horizontal gene transfer and have the potential to spread globally. This study highlights the detection of enterococci carrying linezolid resistance genes from sediment and zooplankton samples from two coastal urban areas in Italy. The presence of clinically relevant resistant bacteria, such as linezolid-resistant enterococci, in marine environments could reflect their spillover from human and/or animal reservoirs and could indicate that coastal seawaters also might represent a source of these resistance genes.

Genome analysis of enterobacteriaceae with non-wild type susceptibility to third-generation cephalosporins recovered from diseased dogs and cats in Europe

BackgroundExtended-spectrum-β-lactamases (ESBL) and plasmid-mediated cephalosporinases (pAmpC)-producing Enterobacteriaceae isolates are now reported worldwide in humans, animals, and in the environment. We identified the determinants of resistance to β-lactams and associated resistance genes as well as phylogenetic diversity of 53 ESBL- or pAmpC-producing Enterobacteriaceae isolated from dogs and cats in Europe. Materials/methodsOf a collection of 842 Enterobacteriaceae isolates that were recovered in 2013 and 2014 from 842 diseased and untreated dogs and cats, for 242 ampicillin or amoxicillin resistant isolates (MIC ≥ 16 mg/L), cefotaxime (CTX) and ceftazidime (CAZ) MICs were determined. Isolates with CTX and/or CAZ MIC ≥ 1 mg/L (n = 63) were selected, and their genomes were fully sequenced using Illumina Technology. Genomic data were explored to identify the resistance determinants, the plasmid incompatibility groups, and the sequence types (STs). Plasmid location of blaESBL and blaAmpC was evaluated for all isolates based on the co-localization of resistance and plasmid incompatibility group genes on the same contig. Phylogenetic trees were constructed using core-genome MLST. ResultsOf the 63 sequenced isolates, 53 isolates harbored a blaESBL or blaAmpC gene. Ten CTX and/or CAZ non-wild type isolates had neither blaESBL nor blaAmpC. Among the 63 isolates, 44 (69.8 %) were Escherichia coli, 11 (17.5 %) were Klebsiella pneumoniae, and 8 (12.7 %) were Proteus mirabilis. Fifty-one (80.9 %) isolates originated from dogs and 12 (19.1 %) from cats. Isolates were sampled from urinary tract (n = 36), skin and soft tissue (n = 22) and respiratory tract infections (n = 5). Thirty-two isolates (32/53, 60.4 %) carried blaESBL genes, including blaCTX-M-15 (n = 12), blaCTX-M-14 (n = 6), blaCTX-M-1 (n = 5), blaCTX-M-2 (n = 3), blaCTX-M-27 (n = 3), blaSHV-28 (n = 4), blaSHV-12 (n = 2), and blaVEB-6 (n = 1). Four isolates of K. pneumoniae had both blaCTX-M-15 and blaSHV-28. Twenty-one isolates (21/53, 39.6 %) carried genes encoding pAmpC, including blaCMY-2 (n = 19) and blaDHA-1 (n = 2). Thirteen E. coli isolates harbored both blaESBL or blaAmpC genes and plasmids of incompatibility groups IncIB (9/13), IncI1 (8/13), and IncFII (6/13). In addition to the reduced susceptibility to CTX and/or CAZ, reduced susceptibility or evidence of acquired resistance to at least one other relevant class of antibiotics was observed for all 63 isolates. E. coliisolates clustered in 23 STs, including B2 virulent clones from humans such as ST131 (n = 5), K. pneumoniae isolates mostly clustered in 3 STs: ST11 (n = 4), ST307 (n = 3), and ST16 (n = 2). Phylogenetic analysis identified the spread of E. coli ST131 blaCTX-M-27, and of K. pneumoniae ST307 harboring blaCTX-M-15 and blaSHV-28 or ST11 blaCTX-M-15. ConclusionsWe report here a 6.3 % prevalence of ESBL/pAmpC producing Enterobacteriaceae in diseased dogs and cats. This EU survey confirms that dogs and cats can be infected with epidemic multidrug resistant clones that may also spread in humans.

Host-specific competitiveness to form nodules in Rhizobium leguminosarum symbiovar viciae.

Summary Fabeae legumes such as pea and faba bean form symbiotic nodules with a large diversity of soil Rhizobium leguminosarum symbiovar viciae (Rlv) bacteria. However, bacteria competitive to form root nodules (CFN) are generally not the most efficient to fix dinitrogen, resulting in a decrease in legume crop yields. Here, we investigate differential selection by host plants on the diversity of Rlv.A large collection of Rlv was collected by nodule trapping with pea and faba bean from soils at five European sites. Representative genomes were sequenced. In parallel, diversity and abundance of Rlv were estimated directly in these soils using metabarcoding. The CFN of isolates was measured with both legume hosts. Pea/faba bean CFN were associated to Rlv genomic regions.Variations of bacterial pea and/or faba bean CFN explained the differential abundance of Rlv genotypes in pea and faba bean nodules. No evidence was found for genetic association between CFN and variations in the core genome, but variations in specific regions of the nod locus, as well as in other plasmid loci, were associated with differences in CFN.These findings shed light on the genetic control of CFN in Rlv and emphasise the importance of host plants in controlling Rhizobium diversity.

An Escherichia coli ST131 pangenome atlas reveals population structure and evolution across 4,071 isolates

Escherichia coli ST131 is a major cause of infection with extensive antimicrobial resistance (AMR) facilitated by widespread beta-lactam antibiotic use. This drug pressure has driven extended-spectrum beta-lactamase (ESBL) gene acquisition and evolution in pathogens, so a clearer resolution of ST131’s origin, adaptation and spread is essential. E. coli ST131’s ESBL genes are typically embedded in mobile genetic elements (MGEs) that aid transfer to new plasmid or chromosomal locations, which are mobilised further by plasmid conjugation and recombination, resulting in a flexible ESBL, MGE and plasmid composition with a conserved core genome. We used population genomics to trace the evolution of AMR in ST131 more precisely by extracting all available high-quality Illumina HiSeq read libraries to investigate 4,071 globally-sourced genomes, the largest ST131 collection examined so far. We applied rigorous quality-control, genome de novo assembly and ESBL gene screening to resolve ST131’s population structure across three genetically distinct Clades (A, B, C) and abundant subclades from the dominant Clade C. We reconstructed their evolutionary relationships across the core and accessory genomes using published reference genomes, long read assemblies and k-mer-based methods to contextualise pangenome diversity. The three main C subclades have co-circulated globally at relatively stable frequencies over time, suggesting attaining an equilibrium after their origin and initial rapid spread. This contrasted with their ESBL genes, which had stronger patterns across time, geography and subclade, and were located at distinct locations across the chromosomes and plasmids between isolates. Within the three C subclades, the core and accessory genome diversity levels were not correlated due to plasmid and MGE activity, unlike patterns between the three main clades, A, B and C. This population genomic study highlights the dynamic nature of the accessory genomes in ST131, suggesting that surveillance should anticipate genetically variable outbreaks with broader antibiotic resistance levels. Our findings emphasise the potential of evolutionary pangenomics to improve our understanding of AMR gene transfer, adaptation and transmission to discover accessory genome changes linked to novel subtypes.

Inference of Convergent Gene Acquisition Among Pseudomonas syringae Strains Isolated From Watermelon, Cantaloupe, and Squash.

Pseudomonas syringae sensu stricto (phylogroup 2; referred to as P. syringae) consists of an environmentally ubiquitous bacterial population associated with diseases of numerous plant species. Recent studies using multilocus sequence analysis have indicated the clonal expansion of several P. syringae lineages, located in phylogroups 2a and 2b, in association with outbreaks of bacterial spot disease of watermelon, cantaloupe, and squash in the United States. To investigate the evolutionary processes that led to the emergence of these epidemic lineages, we sequenced the genomes of six P. syringae strains that were isolated from cucurbits grown in the United States, Europe, and China over a period of more than a decade, as well as eight strains that were isolated from watermelon and squash grown in six different Florida counties during the 2013 and 2014 seasons. These data were subjected to comparative analyses along with 42 previously sequenced genomes of P. syringae stains collected from diverse plant species and environments available from GenBank. Maximum likelihood reconstruction of the P. syringae core genome revealed the presence of a hybrid phylogenetic group, comprised of cucurbit strains collected in Florida, Italy, Serbia, and France, which emerged through genome-wide homologous recombination between phylogroups 2a and 2b. Functional analysis of the recombinant core genome showed that pathways involved in the ATP-dependent transport and metabolism of amino acids, bacterial motility, and secretion systems were enriched for recombination. A survey of described virulence factors indicated the convergent acquisition of several accessory type 3 secreted effectors (T3SEs) among phylogenetically distinct lineages through integrative and conjugative element and plasmid loci. Finally, pathogenicity assays on watermelon and squash showed qualitative differences in virulence between strains of the same clonal lineage, which correlated with T3SEs acquired through various mechanisms of horizontal gene transfer (HGT). This study provides novel insights into the interplay of homologous recombination and HGT toward pathogen emergence and highlights the dynamic nature of P. syringae sensu lato genomes.

Carbapenem-resistant Acinetobacter baumannii: Current status of the problem in four Bulgarian university hospitals (2014-2016).

A total of 226 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates was collected during 2014-2016 from inpatients (age range 5-88 years) in four Bulgarian university hospitals (H1-H4) to assess their antimicrobial susceptibility and to explore carbapenem resistance mechanisms as well as the molecular epidemiology of the isolates. Antimicrobial susceptibility testing, multiplex PCR, DNA sequencing and electrotransformation experiments were performed. Epidemiological typing by random amplification of polymorphic DNA (RAPD)-PCR was also performed. The resistance rates were as follows: imipenem, 90.7%; meropenem, 98.2%; doripenem, 100%; amikacin, 92.9%; gentamicin, 87.2%; tobramycin, 55.8%; levofloxacin, 98.2%; trimethoprim/sulfamethoxazole, 86.3%; tigecycline, 22.1%; colistin, 0%; and ampicillin/sulbactam, 41.6%. Intrinsic blaOXA-51-like genes were found in all of the isolates. The majority of the A. baumannii isolates harboured either blaOXA-23-like associated with the upstream-located ISAba1 (26.1%) or blaOXA-40/24-like (46.7%), 45 isolates (19.9%) harboured both genes, and 1 isolate harboured blaOXA-58-like surrounded by ISAba3C upstream and ISAba3 downstream. The blaOXA-58 gene was transferable by electroporation indicating its plasmid location. Epidemiological typing revealed the dissemination of nosocomial CRAB with high clonal relatedness (70% similarity threshold) belonging to six, four, three and two clusters in H1, H2, H3, and H4 hospitals, respectively. The A. baumannii isolates studied were problematic nosocomial pathogens. Their multidrug resistance greatly limits therapeutic options. The persistence of endemic clones comprised of OXA carbapenemase-producing multidrug-resistant A. baumannii in the monitored hospitals over a period of ca. 3 years is of concern and requires continuous detailed investigations in the future.

A sequence database analysis of 5-nitroimidazole reductase and related proteins to expand knowledge on enzymes responsible for metronidazole inactivation

nim genes are associated, in combination with other factors, with acquired resistance to metronidazole (MTZ) in anaerobes. These genes encode 5-nitroimidazole reductase enzymes (Nim proteins) that reduce MTZ into an inactive compound. Eleven variants (nimA to nimK) are currently described in anaerobes with either a chromosomal or a plasmidic location. Mostly found in members of the Bacteroides fragilis group, nim genes were demonstrated in anaerobic taxa outside the phylum Bacteroidetes. Nitroreductase enzymes, weakly related to those found in Bacteroidetes but associated with MTZ inactivation, were also characterized both in anaerobic and non-anaerobic taxa.Published data only poorly reflect the growing number of data from cultivation-independent studies and sequences deposited in databases. Considering this limitation, we performed herein an analysis of the sequence databases with the aim to increase the current knowledge on Nim protein distribution and diversity. The 250 sequences the most closely related to the 11 known Nim proteins were selected and analyzed for identity level and phylogenetic relationships with Nim A to K proteins.The analysis revealed a larger diversity of anaerobic species harboring known Nim proteins than that currently described in the literature. Putative new variants of known Nim proteins and novel Nim proteins were found. In addition, nitroreductase proteins and homologs related to the pyridoxamine 5′-phosphate oxidase family were found in highly diverse anaerobic and aerobic taxa of human but also animal and environmental origin. On the other hand, we found a very low number of sequences recovered from metagenomic studies. Considering the different databases currently available to identify antimicrobial resistance genes (ARG) among metagenomic sequences, we hypothesized that this may, at least in part, be related to the incompleteness of ARG databases because none of them includes the 11 described nim genes at the time of our study.Both the wide distribution of proteins with potential MTZ inactivation ability within the bacterial world and a wider diversity of Nim determinants than expected from published literature is underlined in this sequence database analysis.

Structure analysis of transposons carrying the aac(6′)-aph(2″) gene in Enterococcus faecalis isolated in Beijing, China, and comparison of their transfer efficiency

Transfer of aac(6')-aph(2″) transposons mediating high-level gentamicin resistance (HLGR) in Enterococcus faecalis is a serious problem in the clinic. However, factors affecting the transfer of aac(6')-aph(2″) have not yet been elucidated. The current study aimed to examine the genetic and molecular basis of HLGR in E. faecalis strains isolated in Beijing (China) and to clarify the relationship between transfer efficiency of aac(6')-aph(2″) transposons and the transposon structure/location. A total of five transposon structures were identified by PCR mapping of the corresponding transposon regions, including a Tn5281-like non-truncated transposon and four truncated transposons. A plasmid location study of aac(6')-aph(2″) by Southern blot following S1-PFGE and filter mating conjugation experiments demonstrated that plasmid location rates correlated with conjugation-positive rates. Chromosome walking to identify the sequence upstream of a representative type III truncated transposon found a truncated aph(2″)-Ia region, and further PCR analysis of this region among strains from different groups revealed similar a positive rate trend as the transposon plasmid location rate and conjugation-positive rate. In conclusion, aac(6')-aph(2″) transposons were of different structures in E. faecalis strains from Beijing, with two new transposon structures that have not been reported elsewhere. Presence of the truncated aph(2″)-Ia region upstream of some truncated transposons suggests recombination between aminoglycoside-modifying enzyme genes. Possible links exist among plasmid location, conjugation and the presence of truncated aph(2″)-Ia upstream of the transposon.

PirA & B toxins discovered in archived shrimp pathogenic Vibrio campbellii isolated long before EMS/AHPND outbreaks

Toxins PirvpA and PirvpB were first described in 2014 from Vibrio parahaemolyticus (VP) isolates discovered in 2013 as the cause of acute hepatopancreatic necrosis disease (AHPND) that emerged as a new disease of Chinese cultivated shrimp in 2009. It was subsequently reported from Thailand in 2012. By using PirvpA and PirvpB specific monoclonal antibodies (MAbs) together with reference VPAHPND isolates to screen archived bacterial cultures, we discovered 3 isolates of Vibrio (previously identified as V. harveyi) including VH 639, VH 1526 and VH Surat that were positive for both PirA and PirB toxins, even though the isolates were collected in Thailand over 10 years prior to reported Thai AHPND outbreaks. Western blot analysis revealed that the PirA/B toxins from these Vibrio isolates matched the molecular masses of the PirvpA/B toxins of previously described VPAHPND isolates. PCR analysis using VPAHPND detection methods that target the PirvpA and PirvpB toxin genes (AP4 and TUMSAT-Vp3) gave amplicons of the expected size from the 3 Vibrio isolates, and sequence analysis of the amplicons revealed 99% identity with the sequences of the PirvpA and PirvpB genes of previously described VPAHPND isolates. Pathogenicity tests performed by reverse gavage and immersion challenge tests demonstrated the pattern of AHPND pathology. Species confirmation of these 3 Vibrio isolates by multilocus sequence analysis (MLSA) of 16S rRNA, rpoD, rctB, and toxR genes revealed that they belong to V. campbellii clade. The results revealed that V. campbellii isolates capable of producing PirA and PirB toxins were present in Thailand up to 7 years before AHPND was reported from China and up to a decade before it was reported from Thailand. The plasmid or chromosomal location of the PirA and PirB toxin genes in these archived V. campbellii isolates and their phylogenetic relationship to current VPAHPND isolates are being determined.