Complete genome sequence of C130_2, a novel myovirus infecting pathogenic Escherichia coli and Shigella strains

The genome sequence of a novel virulent bacteriophage, named “ C130_2”, that is morphologically a member of the family Myoviridae is reported. The 41,775-base-pair double-stranded DNA genome of C130_2 contains 59 ORFs but exhibits overall low sequence similarity to bacteriophage genomes for which sequences are publicly available. Phylogenetic analysis indicated that C130_2 represents a new phage type. C130_2 could be propagated well on enterohemorrhagic Escherichia coli (EHEC) O157:H7 and other pathogenic E. coli strains, as well as on strains of various Shigella species

Draft Genome Sequences of Pandrug-Resistant Serratia marcescens Clinical Isolates Harboring bla NDM-1

The draft genome sequences of two clonal, pandrug-resistant Serratia marcescens clinical isolates were determined. The resistance phenotype was plasmid driven, as 14 of 17 resistance genes were present on large IncFIB(K), IncHI2, and IncA/C2 plasmids indicating a large pool of transmissible antibiotic resistance genes

Complete Genome Sequence of blaCTX-M-27-Encoding Escherichia coli Strain H105 of Sequence Type 131 Lineage C1/H30R

Escherichia coli sequence type 131 (ST131) is the most frequent antimicrobial-resistant lineage of E. coli, propagating extended-spectrum ß-lactamases (ESBL) worldwide. Recently, an alarming rate of increase in isolates of the sublineage C1/H30R-blaCTX-M-27 of ST131 in geographically distant countries was reported. Here, we present the complete genome sequence of the ST131 sublineage C1/H30R E. coli isolate harboring blaCTX-M-27 from Germany

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

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

Insights into a Novel blaKPC-2-Encoding IncP-6 Plasmid Reveal Carbapenem-Resistance Circulation in Several Enterobacteriaceae Species from Wastewater and a Hospital Source in Spain

Untreated wastewater, particularly from hospitals and other healthcare facilities, is considered to be a reservoir for multidrug-resistant bacteria. However, its role in the spread of antibiotic resistances in the human population remains poorly investigated. We used whole genome sequencing (WGS) to analyze 25 KPC-2-producing Enterobacteriaceae isolates from sewage water collected during a 3-year period and three clinical Citrobacter freundii isolates from a tertiary hospital in the same collection area in Spain. We detected a common, recently described, IncP-6 plasmid carrying the gene blaKPC-2 in 21 isolates from both sources. The plasmid was present in diverse environmental bacterial species of opportunistic pathogens such as C. freundii, Enterobacter cloacae, Klebsiella oxytoca, and Raoultella ornithinolytica. The 40,186 bp IncP-6 plasmid encoded 52 coding sequences (CDS) and was composed of three uniquely combined regions that were derived from other plasmids recently reported in different countries of South America. The region harboring the carbapenem resistance gene (14 kb) contained a Tn3 transposon disrupted by an ISApu-flanked element and the core sequence composed by ISKpn6 / blaKPC-2 / ?blaTEM-1 / ISKpn27. We document here the presence of a novel promiscuous blaKPC-2 plasmid circulating in environmental bacteria in wastewater and human populations

Molecular epidemiology and characterization of an outbreak causing Klebsiella pneumoniae clone carrying chromosomally located bla CTX-M-15 at a German University-Hospital

Background: Multi-drug resistant Klebsiella pneumoniae strains are a common cause of health care associated infections worldwide. Clonal spread of Klebsiella pneumoniae isolates carrying plasmid mediated CTX-M-15 have been commonly reported. Limited data is available regarding dissemination of chromosomally encoded CTX-M-15 in Klebsiella pneumoniae worldwide. Results: We examined 23 non-repetitive ESBL-producing Klebsiella pneumoniae strains isolated from clinical specimens over a period of 4 months in a German University Hospital. All isolates were characterized to determine their genetic relatedness using Pulsed-Field Gel Electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). PFGE revealed three clusters (B1, B2, and B3) with a sub-cluster (A3) comprising of 10 isolates with an identical PFGE pattern. All strains of the cluster B3 with similar PFGE patterns were typed as ST101, indicating an outbreak situation. The ESBL allele bla CTX-M-15 was identified in 16 (69.6 %) of all isolates, including all of the outbreak strains. Within the A3 sub-cluster, the CTX-M-15 allele could not be transferred by conjugation. DNA hybridization studies suggested a chromosomal location of bla CTX-M-15. Whole genome sequencing located CTX-M-15 within a complete ISEcp-1 transposition unit inserted into an ORF encoding for a putative membrane protein. PCR-based analysis of the flanking regions demonstrated that insertion into this region is unique and present in all outbreak isolates. Conclusion: This is the first characterization of a chromosomal insertion of bla CTX-M-15 in Klebsiella pneumonia ST101, a finding suggesting that in Enterobacteriaceae, chromosomal locations may also act as reservoirs for the spread of bla CTX-M-15 encoding transposition units

Multiresistant extended-spectrum beta-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany

BACKGROUND:Multiresistant Gram-negative bacteria producing extended-spectrum beta-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of beta-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses. RESULTS:In this study 153 (83.6%) of the human isolates (n=183) and 163 (91.6%) of the animal isolates (n=178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac(´6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates. CONCLUSIONS:Isolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations

Retrospective Analysis of Bacterial Cultures Sampled in German Chicken- Fattening Farms During the Years 2011–2012 Revealed Additional VIM-1 Carbapenemase-Producing Escherichia coli and a Serologically Rough Salmonella enterica Serovar Infantis

Carbapenems are last-resort antibiotics used in human medicine. The increased detection of carbapenem-resistant Enterobacteriaceae (CRE) is therefore worrying. In 2011 we reported the first livestock-associated VIM-1-producing Salmonella (S.) enterica serovar Infantis (R3) isolate from dust, sampled in a German chicken fattening farm. Due to this observation we retrospectively investigated more than 536 stored bacterial cultures, isolated from 45 chicken fattening farms during the years 2011 and 2012. After a non-selective overnight incubation, the bacteria were transferred to selective media. Escherichia (E.) coli and Salmonella growing on these media were further investigated, including antibiotic susceptibility testing, carbapenemase gene screening and whole genome sequencing (WGS). In total, four CRE were found in three out of 45 investigated farms: Besides R3, one additional Salmonella (G-336-1a) as well as two E. coli isolates (G-336-2, G-268-2). All but G-268-2 harbored the blaVIM-1 gene. Salmonella isolates R3 and G-336-1 were closely related although derived from two different farms. All three blaVIM-1-encoding isolates possessed identical plasmids and the blaVIM-1- containing transposon showed mobility at least in vitro. In isolate G-268-2, the AmpC beta-lactamase gene blaCMY-2 but no known carbapenemase gene was identified. However, a transfer of the phenotypic resistance was possible. Furthermore, G-268-2 contained the mcr-1 gene, combining phenotypical carbapenem- as well as colistin resistance in one isolate. Carbapenem-resistant Enterobacteriaceae have been found in three out of 45 investigated chicken flocks. This finding is alarming and emphasizes the importance of intervention strategies to contain the environmental spread of resistant bacteria in animals and humans

Predictors of blaCTX-M-15 in varieties of Escherichia coli genotypes from humans in community settings in Mwanza, Tanzania

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae commonly cause infections worldwide. Bla CTX-M-15 has been commonly detected in hospital isolates in Mwanza, Tanzania. Little is known regarding the faecal carriage of ESBL isolates and bla CTX-M-15 allele among humans in the community in developing countries. A cross-sectional study involving 334 humans from the community settings in Mwanza City was conducted between June and September 2014. Stool specimens were collected and processed to detect ESBL producing enterobacteriaceae. ESBL isolates were confirmed using disc approximation method, commercial ESBL plates and VITEK-2 system. A polymerase chain reaction and sequencing based allele typing for CTX-M ESBL genes was performed to 42 confirmed ESBL isolates followed by whole genome sequence of 25 randomly selected isolates to detect phylogenetic groups, sequence types plasmid replicon types. Of 334 humans investigated, 55 (16.5 %) were found to carry ESBL-producing bacteria. Age, history of antibiotic use and history of admission were independent factors found to predict ESBL-carriage. The carriage rate of ESBL-producing Escherichia coli was significantly higher than that of Klebsiella pneumoniae (15.1 % vs. 3.8 %, p = 0.026). Of 42 ESBL isolates, 37 (88.1 %) were found to carry the bla CTX-M-15 allele. Other transferrable resistance genes were aac(6')Ib-cr, aac(3)-IIa, aac(3)-IId, aadA1, aadA5, strA, strB and qnrS1. Eight multi-locus sequence types (ST) were detected in 25 E. coli isolates subjected to genome sequencing. ST-131 was detected in 6 (24 %), ST-38 in 5 (20 %) and 5 (20 %) clonal complex - 10(ST-617, ST-44) of isolates. The pathogenic phylogenetic groups D and B2 were detected in 8/25 (32 %) and 6/25 (24 %) of isolates respectively. BlaCTX-M-15 was found to be located in multiple IncY and IncF plasmids while in 13/25(52 %) of cases it was chromosomally located. The overlap of multi-drug resistant bacteria and diversity of the genotypes carrying CTX-M-15 in the community and hospitals requires an overall approach that addresses social behaviour and activity, rationalization of the antibiotic stewardship policy and a deeper understanding of the ecological factors that lead to persistence and spread of such alleles

Tracking bacterial virulence: global modulators as indicators

The genomes of Gram-negative bacteria encode paralogues and/or orthologues of global modulators. The nucleoid-associated H-NS and Hha proteins are an example: several enterobacteria such as Escherichia coli or Salmonella harbor H-NS, Hha and their corresponding paralogues, StpA and YdgT proteins, respectively. Remarkably, the genome of the pathogenic enteroaggregative E. coli strain 042 encodes, in addition to the hha and ydgT genes, two additional hha paralogues, hha2 and hha3. We show in this report that there exists a strong correlation between the presence of these paralogues and the virulence phenotype of several E. coli strains. hha2 and hha3 predominate in some groups of intestinal pathogenic E. coli strains (enteroaggregative and shiga toxin-producing isolates), as well as in the widely distributed extraintestinal ST131 isolates. Because of the relationship between the presence of hha2/hha3 and some virulence factors, we have been able to provide evidence for Hha2/Hha3 modulating the expression of the antigen 43 pathogenic determinants. We show that tracking global modulators or their paralogues/orthologues can be a new strategy to identify bacterial pathogenic clones and propose PCR amplification of hha2 and hha3 as a virulence indicator in environmental and clinical E. coli isolatesThe authors acknowledge funding from the Spanish MICINN-FEDER (BFU2010-21836-C02-01) and Mineco (BIO2013-49148-C2-1-R and BIO2015-69085-REDC). Work in the LREC-USC-laboratory was financed by the grant CN2012/303 from Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and The European Regional Development Fund (ERDF)S