Transposon Tn5 Research Articles

Prevalence of blaKPC-2, blaKPC-3 and blaKPC-30-Carrying Plasmids in Klebsiella pneumoniae Isolated in a Brazilian Hospital.

Klebsiella pneumoniae carbapenemase (KPC) actively hydrolyzes carbapenems, antibiotics often used a last-line treatment for multidrug-resistant bacteria. KPC clinical relevance resides in its widespread dissemination. In this work, we report the genomic context of KPC coding genes blaKPC-2, blaKPC-3 and blaKPC-30 in multidrug-resistant Klebsiella pneumoniae isolates from Brazil. Plasmids harboring blaKPC-3 and blaKPC-30 were identified. Fifteen additional carbapenem-resistant K. pneumoniae isolates were selected from the same tertiary hospital, collected over a period of 8 years. Their genomes were sequenced in order to evaluate the prevalence and dissemination of blaKPC–harboring plasmids. We found that blaKPC genes were mostly carried by one of two isoforms of transposon Tn4401 (Tn4401a or Tn4401b) that were predominantly located on plasmids highly similar to the previously described plasmid pKPC_FCF3SP (IncN). The identified pKPC_FCF3SP-like plasmids carried either blaKPC-2 or blaKPC-30. Two K. pneumoniae isolates harbored pKpQIL-like (IncFII) plasmids, only recently identified in Brazil; one of them harbored blaKPC-3 in a Tn4401a transposon. Underlining the risk of horizontal spread of KPC coding genes, this study reports the prevalence of blaKPC-2 and the recent spread of blaKPC-3, and blaKPC-30, in association with different isoforms of Tn4401, together with high synteny of plasmid backbones among isolates studied here and in comparison with previous reports.

An Update on Wastewater Multi-Resistant Bacteria: Identification of Clinical Pathogens Such as Escherichia coli O25b:H4-B2-ST131-Producing CTX-M-15 ESBL and KPC-3 Carbapenemase-Producing Klebsiella oxytoca.

Wastewater treatment plants (WWTPs) are significant reservoirs of bacterial resistance. This work aims to identify the determinants of resistance produced by Gram-negative bacteria in the influent and effluent of two WWTPs in Portugal. A total of 96 wastewater samples were obtained between 2016 and 2019. The numbers of total aerobic and fecal contamination bacteria were evaluated, and genomic features were searched by polymerase chain reaction (PCR) and Next-Generation Sequencing (NGS). Enterobacteriaceae corresponded to 78.6% (n = 161) of the 205 isolates identified by 16sRNA. The most frequent isolates were Escherichia spp. (57.1%, n = 117), followed by Aeromonas spp. (16.1%, n = 33) and Klebsiella spp. (12.7%, n = 26). The remaining 29 isolates (14.1%) were distributed across 10 different genera. Among the 183 resistant genes detected, 54 isolates produced extended spectrum β-lactamases (ESBL), of which blaCTX-M-15 was predominant (37 isolates; 68.5%). A KPC-3 carbapenemase-producing K. oxytoca was identified (n = 1), with blaKPC-3 included in a transposon Tn4401 isoform b. A higher number of virulence genes (VG) (19 genes) was found in the E. coli 5301 (O25b-ST131-B2) isolate compared with a commensal E. coli 5281 (O25b-ST410-A) (six genes). Both shared five VG [Enterobactin; Aerobactin, CFA/1 (clade α); Type1 (clade γ1); Type IV]. In conclusion, this work highlights the role of relevant clinical bacteria in WWTPs, such as KPC-3-producing K. oxytoca, and, for the first time, a CTX-M-15-producing Ochromobactrum intermedium, a human opportunistic pathogen, and a SED-1-producing Citrobacter farmeri, an uncommon CTX-M-type extended-spectrum beta-lactamase.

Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals

BackgroundEnterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations.MethodsWe performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012–2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination.ResultsOn average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination.ConclusionsIn related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance.

The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19

BackgroundCarbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel blaOXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance.MethodsA. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN.ResultsMICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene blaOXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer blaOXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants.ConclusionsOur investigation showed that A. johnsonii M19 is a source and disseminator of blaOXA-23 and carbapenem resistance. The ability to transfer blaOXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance.Graphic abstractThe carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Pathogenicity of a Vibrio owensii strain isolated from Fenneropenaeus chinensis carrying pirAB genes and causing AHPND

An emergent shrimp disease, acute hepatopancreatic necrosis disease (AHPND), has been causing great economic losses to the shrimp farming industry. The original causing agent of AHPND was regarded as Vibrio parahaemolyticus, with a plasmid named pVA1 carrying two Photorhabdus insect-related (Pir) toxin genes pirA and pirB. In the present study, a Vibrio owensii strain named FcYS03 was isolated from the Chinese shrimp Fenneropenaeus chinensis, which caused a high mortality in shrimp with typical AHPND signs. This strain carried the binary toxin genes pirAB and could cause AHPND both in Fenneropenaeus chinensis and Penaeus vannamei (previously named Litopenaeus vannamei). The pathogenicity of FcYS03 to P. vannamei exhibited apparent temperature and salinity dependence. Through genome sequence analysis among FcYS03 and AHPND-causing V. parahaemolyticus strain 3HP, we found that a segmental rearrangement phenomenon existed in the plasmid of FcYS03. The toxin genes pirAB happened to be located at the rearranged segment, and two transposases for transposon Tn903 were distributed at the two ends of the rearranged segment, which indicated that transposon Tn903 might be related to the transfer of the toxin genes among the bacteria. The study will provide valuable information for the understanding and prevention of AHPND in shrimp aquaculture.

TN5-BASED TRANSPOSON MUTAGENESIS OF STREPTOMYCES GHANAENSIS ATCC14672: SEARCHING FOR NOVEL REGULATORS OF MOENOMYCIN PRODUCTION

Streptomyces ghanaensis ATCC14672 is the only genetically characterized natural producer of moenomycin A (MmA), a unique phosphoglycolipid antibiotic capable of direct inhibition of peptidoglycan glycosyltransferases. At the moment there are no drugs on the market that would target this step of peptidoglycan formation. There is much interest in development of MmA-based drugs to combat growing threat of multidrug resistant Gram-positive infections. Consequently, there is also major interest in development of microbiological approaches towards production of this natural compound via submerged cultivation. Currently available ATCC14672 strains accumulate minute quantities of MmA, which fueled the investigations of genetic control of its biosynthesis over the last decade. Much of our understanding of the regulation of MmA production stems from homology-based approaches. Namely, homologs of known pleiotropic regulators of antibiotic production were searched in ATCC14672 genome and functionally characterized. Although this approach was largely successful, it left out of the focus those regulators of MmA production that share no similarity to the regulators identified in model streptomycetes. In this work we report an attempt to discover novel genes involved in regulation of moenomycin biosynthesis via the use of Streptomyces codon-adapted transposon Tn5, which represents an unbiased way to find genuinely new regualtors. After a primary screening of transposon library we were able to identify five Tn mutants that differed in morphology and/or total antibiotic production. We identified Tn5 insertion sites in these mutants through sequencing and re-analyzed their moenomycin production levels, revealing two strains with increased total antibiotic activity. The genes affected in Tn5 mutants were cloned into expression vectors and introduced back into wild type (ATCC14672) in order to study the effects of their increased dosage on the strain. One of the genes, ssfg_04565, exerted negative effects on antibiotic activity when introduced on the plasmid. Possible functions of the identified genes in the context of secondary metabolism are discussed.

Characterization of the Riemerella anatipestifer M949_RS00050 gene

Based on its causing ever-increasing heavy economic losses, Riemerella anatipestifer has been viewed as an important bacterial pathogen in the duck industry worldwide. However, the molecular mechanisms regarding its pathogenicity are poorly understood. In our previous study, we have built a random mutagenesis library of Riemerella anatipestifer CH3 using transposon Tn4351. In this study, we screened the library by determining bacterial median lethal dose in ducklings. A mutant strain showed about 376-fold attenuated virulence in comparison with the wild-type strain CH3 was obtained. Subsequently, the Tn4351 inserted gene was identified as M949_RS00050, which encodes a putative protein containing an outer membrane protein beta-barrel domain by genome walking and sequence analyses. Southern blot analysis indicated a single Tn4351 insertion in the CH3 chromosomal DNA. Inactivation of M949_RS00050 gene did not affect bacterial metabolic activity and the silver stained lipopolysaccharide pattern. However, the bacterial sensitivity to normal duck sera killing and bacterial hydrophobicity were dramatically enhanced in the M949_RS00050 gene inactivated mutant strain, compared to its wild-type strain CH3. Moreover, bacterial adherence and invasion abilities, bacterial capsular polysaccharide quantity, biofilm formation capacity and the bacterial virulence of the mutant strain were obviously decreased, compared to the wild-type strain CH3. Thus, our finding demonstrates that the M949_RS00050 gene functions on multiple bacterial biological properties and virulence in Riemerella anatipestifer.

Small Klebsiella pneumoniae Plasmids: Neglected Contributors to Antibiotic Resistance.

Klebsiella pneumoniae is the causative agent of community- and, more commonly, hospital-acquired infections. Infections caused by this bacterium have recently become more dangerous due to the acquisition of multiresistance to antibiotics and the rise of hypervirulent variants. Plasmids usually carry genes coding for resistance to antibiotics or virulence factors, and the recent sequence of complete K. pneumoniae genomes showed that most strains harbor many of them. Unlike large plasmids, small, usually high copy number plasmids, did not attract much attention. However, these plasmids may include genes coding for specialized functions, such as antibiotic resistance, that can be expressed at high levels due to gene dosage effect. These genes may be part of mobile elements that not only facilitate their dissemination but also participate in plasmid evolution. Furthermore, high copy number plasmids may also play a role in evolution by allowing coexistence of mutated and non-mutated versions of a gene, which helps to circumvent the constraints imposed by trade-offs after certain genes mutate. Most K. pneumoniae plasmids 25-kb or smaller replicate by the ColE1-type mechanism and many of them are mobilizable. The transposon Tn1331 and derivatives were found in a high percentage of these plasmids. Another transposon that was found in representatives of this group is the blaKPC-containing Tn4401. Common resistance determinants found in these plasmids were aac(6′)-Ib and genes coding for β-lactamases including carbapenemases.

Draft genome of a macrolide resistant XDR Salmonella enterica serovar Paratyphi A strain using a shotgun sequencing approach.

Salmonella enterica serovar Paratyphi A, the causative pathogen of enteric fever, is a major public-health concern affecting millions of people around the world. We conducted whole-genome sequencing and analysis of a novel macrolide-resistant Salmonella Paratyphi A strain isolated from Karachi, Pakistan. Genomic DNA of Salmonella Paratyphi A strain JRCGR-AK14 was sequenced on a MiSeq platform. Read quality was evaluated and paired-end reads were assembled into contigs and scaffolds. The quality of contigs and scaffolds was evaluated and assembled contigs were annotated. Virulence genes, antimicrobial resistance genes (ARGs), tRNAs, rRNAs, coding sequences and clustered regularly interspaced short palindromic repeats (CRISPRs) were identified. ARGs and mutations in quinolone-resistance determining regions (QRDRs) were identified by Antimicrobial Resistance Identification By Assembly (ARIBA) and ResFinder. Known and unknow mutations in the QRDRs were predicted. The genome of Salmonella Paratyphi A was calculated at 4529866 bp with 4381 genes and 1088 hypothetical proteins. Several putative genes coding for multidrug efflux pumps were identified. In addition, gene mutations conferring resistance to nitrofurantoin (e.g. marA, mdsC, Escherichia coli soxS), pulvomycin (e.g. H-NS, cpxA, E. coli EF-Tu) and fosfomycin (CRP, kdpE, E. coli glpT) were also identified. Several ARGs along with the mobile genetic element transposon Tn10 were also identified. It is evident from the results that diverse redundant mechanisms are involved in regulation of drug resistance in this strain. The current findings provide valuable data for understanding the multidrug resistance and pathogenic characteristics of clinical Salmonella Paratyphi A isolates.

<p>Genetic characterization of two vancomycin-resistant <em>Staphylococcus aureus</em> isolates in Kerman, Iran</p>

AimThe aim of this study was the genetic characterization of two clinical vancomycin-resistant Staphylococcus aureus (VRSA) isolates.Materials and methodsResistance to vancomycin was determined by phenotypic method. PCR was used for detection of mecA, vanA, ermA, ermB, ermC, msrA/B, aph(2”)-Ic, aph(3ʹ)-IIIa, pvl, Immune Evasion Cluster [sea, sep, chip, sak and scn] genes and biofilm operon icaABCD. On the other hand, multilocus sequence typing and agr typing methods were performed for the determination of clonal relationship and van operon was detected and sequenced.ResultsVancomycin-resistant Staphylococcus aureus strain 1 (VRSA-1) was positive for vanA, ermA, ermC, aph(2”)-Ic, aph(3ʹ)-IIIa, sea, sep, icaD genes, belonging to agr type I; SCCmec type III; spa type t030; and ST239. However, the genetic characterization of Vancomycin-resistant Staphylococcus aureus strain 2 (VRSA-2) revealed the presence of various types of resistance genes vanA, ermA, ermC, aph(2”)-Ic, aph(3ʹ)-IIIa, sea, icaD, relating to agr type I; SCCmec type III; spa type t459; and ST239. The presence of transposon Tn1546 was determined by PCR sequencing.The Basic Local Alignment Search Tool analysis of van operon in the VRSA isolates showed 99.6% sequence homology to Tn1546 in vancomycin-resistant enterococci, indicating the vanA operon has an enterococcal origin.ConclusionIn conclusion, the ST239 is one of the most common clones of MRSA isolates which involved the hospital-associated infections, therefore, the emergence of VRSA isolates with ST239 increased the spread of resistance to vancomycin in the hospital settings.

The Gene Encoding NAD-Dependent Epimerase/Dehydratase, wcaG, Affects Cell Surface Properties, Virulence, and Extracellular Enzyme Production in the Soft Rot Phytopathogen, Pectobacterium carotovorum.

Pectobacterium carotovorum is a gram-negative bacterium that, together with other soft rot Enterobacteriaceae causes soft rot disease in vegetables, fruits, and ornamental plants through the action of exoproteins including plant cell wall-degrading enzymes (PCWDEs). Although pathogenicity in these bacteria is complex, virulence levels are proportional to the levels of plant cell wall-degrading exoenzymes (PCWDEs) secreted. Two low enzyme-producing transposon Tn5 mutants were isolated, and compared to their parent KD100, the mutants were less virulent on celery petioles and carrot disks. The inactivated gene responsible for the reduced virulence phenotype in both mutants was identified as wcaG. The gene, wcaG (previously denoted fcl) encodes NAD-dependent epimerase/dehydratase, a homologue of GDP-fucose synthetase of Escherichia coli. In Escherichia coli, GDP-fucose synthetase is involved in the biosynthesis of the exopolysaccharide, colanic acid (CA). The wcaG mutants of P. carotovorum formed an enhanced level of biofilm in comparison to their parent. In the hydrophobicity test the mutants showed more hydrophobicity than the parent in hexane and hexadecane as solvents. Complementation of the mutants with extrachromosomal copies of the wild type gene restored these functions to parental levels. These data indicate that NAD-dependent epimerase/dehydratase plays a vital rule in cell surface properties, exoenzyme production, and virulence in P. carotovorum.

Highly variable vancomycin-resistant enterococci in the north-eastern part of the Czech Republic.

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens of increasing medical importance. This study involved 121 VRE selectively obtained from a representative set of 1464 samples collected from various sources in the north-eastern part of the Czech Republic. In total, 119 VRE belonged to Enterococcus faecium and two to Enterococcus faecalis. All isolates of E. faecium were resistant to at least three antibiotic classes. The resistance genes vanA, erm(B), tet(M), tet(L), aac(3)-IIIa and aac(6')-aph(2'') were detected. We assigned the E. faecium to sequence types ST5, ST18, ST38, ST64, ST92, ST273, ST549 and ST640. In E. faecium isolates, we identified the presence of replicases rep20p LG 1 , rep2p RE 25 , rep17p RUM , rep21p VEF 1/2 and rep14p RI 1 , as well as relaxases relp EF 1 , relp LG 1 , relp CIZ 2 , relp RE 25 and relp RUM . The presence of the toxin-antitoxin system axe-txe was detected mainly among isolates of hospital origin. The A and D types of transposon Tn1546 were those occurring most frequently. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first extensive study of vancomycin-resistant enterococci of diverse origin in a single well-defined area of the Czech Republic. The isolates were investigated for their antibiotic resistance, epidemiological characteristics and plasmid characteristics. Based on the results obtained, we can make assumptions as to the ways that vancomycin resistance is disseminated throughout the environment including humans and animals.

Antimicrobial Agent Susceptibility and Typing of Staphylococcal Isolates from Subclinical Mastitis in Ewes.

Objective was to study susceptibility to antimicrobial agents of 142 staphylococcal isolates from subclinical mastitis in ewes. In total, 41.5% of these were resistant and 5.6% multidrug resistant. More coagulase-negative staphylococci (47.0%) were resistant than Staphylococcus aureus (18.5%) isolates. Resistance was greater to penicillin (22.5%), tetracycline, or ampicillin (18.3%). More biofilm-forming (20.6%) isolates were resistant to tetracycline than nonbiofilm-forming (0.0%) ones. Presence of tetK was associated with presence of icaA in the same strains. Further, 76.6% of resistant isolates versus 57.7% of susceptible ones were recovered immediately postpartum and 23.4% of resistant isolates versus 9.9% of susceptible ones were recovered in farms that practiced routine administration of antimicrobial agents at the end of a lactation period. Most S. aureus (59.3%) were classified in ST133 and most Staphylococcus epidermidis were classified in ST100, ST142, or ST152 (19.0% each). There was no association of sequence types with resistance. Whole genome sequencing showed that, in a Staphylococcus lentus strain, the ermB gene was part of transposon Tn917 integrated into the chromosome; also, a small plasmid was observed in an ermC-carrying Staphylococcus hominis strain and, finally, in an S. aureus and an S. epidermidis strains, small tetK-carrying plasmids (pSau-2716Lar, pSau-3893Lar) of 4.439 kb were found.

First detection of an optrA-positive, linezolid-resistant ST16 Enterococcus faecalis from human in Greece.

Until now, in Greece, the resistance of enterococci to linezolid was associated with mutations of domain V of 23S ribosomal RNA (G2576T). Here we report the first linezolid-resistant optrA-positive Enterococcus faecalis sequence type (ST) 16 isolated from a patient with a urinary tract infection (UTI). No travels overseas, contact with food-producing animals or previous treatment with linezolid were reported. Plasmid analysis suggested the chromosomal location of optrA gene. Additionally, whole genome sequencing data revealed the association of optrA with transposon Tn554 and the coexistence with fexA, spc and ermA-like resistance genes. A similar genetic structure has been previously identified in an ST767 E. faecalis from Taiwan.

TETyper: a bioinformatic pipeline for classifying variation and genetic contexts of transposable elements from short-read whole-genome sequencing data.

Much of the worldwide dissemination of antibiotic resistance has been driven by resistance gene associations with mobile genetic elements (MGEs), such as plasmids and transposons. Although increasing, our understanding of resistance spread remains relatively limited, as methods for tracking mobile resistance genes through multiple species, strains and plasmids are lacking. We have developed a bioinformatic pipeline for tracking variation within, and mobility of, specific transposable elements (TEs), such as transposons carrying antibiotic-resistance genes. TETyper takes short-read whole-genome sequencing data as input and identifies single-nucleotide mutations and deletions within the TE of interest, to enable tracking of specific sequence variants, as well as the surrounding genetic context(s), to enable identification of transposition events. A major advantage of TETyper over previous methods is that it does not require a genome reference. To investigate global dissemination of Klebsiella pneumoniae carbapenemase (KPC) and its associated transposon Tn4401, we applied TETyper to a collection of over 3000 publicly available Illumina datasets containing blaKPC. This revealed surprising diversity, with over 200 distinct flanking genetic contexts for Tn4401, indicating high levels of transposition. Integration of sample metadata revealed insights into associations between geographic locations, host species, Tn4401 sequence variants and flanking genetic contexts. To demonstrate the ability of TETyper to cope with high-copy-number TEs and to track specific short-term evolutionary changes, we also applied it to the insertion sequence IS26 within a defined K. pneumoniae outbreak. TETyper is implemented in python and is freely available at https://github.com/aesheppard/TETyper.

Detection of the sul2-strA-strB gene cluster in an ice core from Dome Fuji Station, East Antarctica.

Bacteria harbouring antimicrobial resistance genes (ARGs) have been isolated from various locations, including ancient microbiomes, indicating that these genes pre-date the discovery of antibiotics. To gain further information regarding ARGs in the pre-antibiotic era, ice samples derived from Dome Fuji Station, Eastern Antarctica, were examined. DNA was extracted from firn or ice core samples (n=3; 1200-1400ybp, 1700-2100ybp and 2200-2800ybp, respectively) under sterile conditions. Whole-genome amplification and PCR analyses were utilised to detect ARGs. A 2764-bp gene cluster containing the type II dihydropteroate synthase gene sul2 and the aminoglycoside phosphotransferase genes strA and strB was detected in the 1200-1400-year-old Antarctic ice core (DF-63.5). The sul2-strA-strB gene cluster is frequently associated with plasmid RSF1010 and transposon Tn5393; however, these elements were not detected in sample DF-63.5. The gene cluster exhibited a high level of sequence identity to sequences harboured in present-day bacteria, although there were sequence polymorphisms in the strA gene. Furthermore, expression of this gene cluster in Escherichia coli resulted in reduced susceptibility to dihydrostreptomycin and sulfamethoxazole. The results of this study provide further evidence that certain ARGs existed in the pre-antibiotic era. Because the sul2 gene confers resistance to the synthetic compound sulfamethoxazole, these findings suggest that ARGs against synthetic antimicrobials emerged in bacteria during the pre-antibiotic era.

Spread of Carbapenem and Colistin-Resistant Klebsiella pneumoniae ST512 Clinical Isolates in Israel: A Cause for Vigilance.

Genomic and phenotypic characterization of resistance mechanisms to carbapenems and colistin in Klebsiella pneumoniae strains isolated from the Shaare Zedek Medical Center (Jerusalem, Israel). The 15 K. pneumoniae isolates studied present a high level of resistance to the antibiotics tested. Microbiological tests revealed production of carbapenemase enzymes by all isolates. ABI SOLiD sequencing of K. pneumoniae genomes generated between 5,033,665 and 8,876,861 million reads per genome. The genomic study revealed that carbapenem resistance was mediated by production of the KPC-3 enzyme in 13 isolates and NDM-1 enzyme in the remaining 2 isolates. In addition, colistin resistance was induced either by a missense mutation in the mgrB gene or inactivation of mgrB by an IS5-like insertion sequence. The mobile genetic element, transposon Tn4401, was identified in all genomes harboring the blaKPC gene. The 15 K. pneumoniae strains were assigned to 4 different sequence types, including ST16, ST76, ST258, and ST512. In this study, we report the usefulness of whole-genome sequencing in detection of antibiotic resistance mechanisms and in highlighting the emergence of the carbapenem and colistin-resistant K. pneumoniae clone ST512 in Israeli hospitals.

Isolation, characterization, genomic sequencing, and GFP-marked insertional mutagenesis of a high-performance nitrogen-fixing bacterium, Kosakonia radicincitans GXGL-4A and visualization of bacterial colonization on cucumber roots.

A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology. The analyses of average nucleotide identity based on BLAST+ (ANIb) and correlation indexes of tetra-nucleotide signatures (Tetra) showed that the NF isolate GXGL-4A is closely related to the Kosakonia radicincitans type strain DSM 16656. Therefore, the isolate GXGL-4A was eventually classified into the species of Kosakonia radicincitans and designated K. radicincitans GXGL-4A. A high consistency in composition and gene arrangement of nitrogen-fixing gene cluster I (nif cluster I) was found between K. radicincitans GXGL-4A and other Kosakonia NF strains. The mutants tagged with green fluorescence protein (GFP) were obtained by transposon Tn5 mutagenesis, and then, the colonization of gfp-marked K. radicincitans GXGL-4A cells on cucumber seedling root were observed under fluorescence microscopy. The preferential sites of the labeled GXGL-4A cell population were the lateral root junctions, the differentiation zone, and the elongation zone. All these results should benefit for the deep exploration of nitrogen fixation mechanism of K. radicincitans GXGL-4A and will definitely facilitate the genetic modification process of this NF bacterium in sustainable agriculture.

Wild corvid birds colonized with vancomycin-resistant Enterococcus faecium of human origin harbor epidemic vanA plasmids

The most prevalent type of acquired vancomycin resistance in Enterococcus faecium (VREfm) is encoded by the vanA transposon Tn1546, mainly located on transferable plasmids. vanA plasmids have been characterized in VREfm from a variety of sources but not wild birds. The aim of this study was to analyse the genetic context of VREfm strains recovered from wild corvid birds and to compare their plasmid and strain characteristics with human strains. To achieve that, 75 VREfm isolates, including strains from wild birds recovered during wide surveillance studies performed in Europe, Canada and the United States (2010−2013), and clinical and wastewater strains from Czech Republic, a region lacking data about vanA plasmids, were analysed. Their population structure, presence of major putative virulence markers and characterization of vanA transposons and plasmids were established. VREfm from wild birds were mainly associated with major human lineages (ST18 and ST78) circulating in hospitals worldwide and were enriched in putative virulence markers that are highly associated with clinical E. faecium from human infections. They also carried plasmids of the same families usually found in the clinical setting [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18]. The clinically widespread IS1251-carrying Tn1546 type “F” was predominant and Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18- or pLG1-like (Europe) plasmids. VREfm from hospitals and wastewaters carried Tn1546-vanA in different plasmid types including mosaic pRUM-Inc18 plasmids, not identified in wild birds. This is the first characterization of vanA plasmids obtained from wild birds. A similar plasmid pool seems to exist in different clonal E. faecium backgrounds of humans and wild birds. The isolation of VREfm strains from wild birds that belong to human E. faecium adapted lineages and carry virulence genes, Tn1546 and plasmid variants widespread in the clinical setting is of concern and highlight their role as potential drivers of the global dissemination of vancomycin resistance.

Vancomycin resistance in Enterococcus faecium isolated from Danish chicken meat is located on a pVEF4-like plasmid persisting in poultry for 18 years

The occurrence of vancomycin-resistant Enterococcus faecium (VREfm) in food is relevant to public health as foodborne VREfm may colonize the gut of consumers and transfer vancomycin resistance genes to the indigenous gut microbiota. Therefore, we determined occurrence and elucidated genetic traits of VREfm in Danish retail chicken meat. Three out of 40 samples (7.5%) from two slaughterhouses yielded VREfm (vancomycin MIC > 32 mg/L). This is the first report of VREfm in Danish retail poultry meat since 2010 (DANMAP). All three VREfm belonged to the sequence type ST32, cluster type CT1068. Using whole genome sequencing, we detected transposon Tn1546 harbouring the vanA operon encoding vancomycin resistance. The vanA operon was located on a 43.4 kb plasmid highly similar (99.9% identity across 97.5% of the sequence) to pVEF4, which was observed in VREfm in Norwegian poultry in 1998 and in Danish poultry in 2010. The remarkable persistence of a pVEF4-like plasmid in enterococcal populations may be explained by the presence of two independent plasmid stability systems, the ω/ε/ζ toxin-antitoxin system and the prgOPN gene cluster. Filter mating experiments showed that the pVEF4-like plasmid could transfer between E. faecium strains in vitro and that transfer occurred concomitantly with a larger, co-residing plasmid. The data presented here indicate that poultry meat constitutes a reservoir of VREfm and further investigations are needed to assess the risk of foodborne transmission to humans.