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

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

Predominance of Klebsiella pneumoniae ST14 carrying CTX-M-15 causing neonatal sepsis in Tanzania

Background: Klebsiella pneumoniae strains expressing ESBLs are a predominant cause of hospital acquired infections. Here we describe the molecular epidemiology of these isolates in a tertiary hospital in Tanzania, as potential pathogens for neonatal infections. Methods: Between April 2009 and March 2010 all Klebsiella pneumoniae isolates with phenotypic expression Extended Spectrum Beta Lactamase (ESBL) were collected and characterized. Identification was done using in house biochemical tests in case of ambiguous results confirmation was done using API 20E. Susceptibility testing was determined using the disc diffusion method followed by specific PCR and sequencing to determine ESBL genes. Phylogenetic analysis, Pulse field gel electrophoresis (PFGE) and Multi-Locus sequence typing (MLST) to PFGE clusters representative isolates were performed to determine clones of the isolates. Conjugation and hybridization were performed to determine the location of blaCTX-M-15 gene. Results: A total of 92 non- repetitive ESBL producing K. pneumoniae representing 50.3% of Klebsiella pneumoniae isolates were characterized. These isolates were from blood 61 (66%), wound swab 13 (14%), urine 12 (13%) and pus 6 (7%) were analyzed. Most blood culture strains originated from neonatal unit 39/61(64%) and 22 (36%) of the blood culture isolates were from neonatal ICU. All isolates were resistant to gentamicin and 54% were resistant to ciprofloxacin. Using a similarity index of 80%, the isolates were assigned to thirteen clusters based on PFGE patterns and contained sub-clusters with identical strains indicating clonal outbreaks. Cluster X5, X7 and X8, and X9 were grouped into ST48, ST14 and ST348 respectively. Based on gyrA PCR- RFLP phylogenetic analysis all isolates were grouped as KpI. The predominant ESBL allele detected was blaCTX-M-15 which was found in 76% of isolates, followed by blaTEM-104 (19%), blaSHV-11 (3.2%) and blaTEM-176 (2%). The blaCTX-M-15 gene was located in multiple conjugative IncF plasmids ranging from 25 kb-485 kb in size. Conclusion: The high prevalence of blaCTX-M-15 observed among ESBL producing K. pneumoniae in Tanzania, is possibly due to the spread of a common IncFII 145 kb plasmid and of certain clones such as ST14 and ST48

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

Enterobacter bugandensis: a novel enterobacterial species associated with severe clinical infection

Nosocomial pathogens can cause life-threatening infections in neonates and immunocompromised patients. E. bugandensis (EB-247) is a recently described species of Enterobacter, associated with neonatal sepsis. Here we demonstrate that the extended spectrum betha-lactam (ESBL) producing isolate EB-247 is highly virulent in both Galleria mellonella and mouse models of infection. Infection studies in a streptomycin-treated mouse model showed that EB-247 is as efficient as Salmonella Typhimurium in inducing systemic infection and release of proinflammatory cytokines. Sequencing and analysis of the complete genome and plasmid revealed that virulence properties are associated with the chromosome, while antibiotic-resistance genes are exclusively present on a 299 kb IncHI plasmid. EB-247 grew in high concentrations of human serum indicating septicemic potential. Using whole genome-based transcriptome analysis we found 7% of the genome was mobilized for growth in serum. Upregulated genes include those involved in the iron uptake and storage as well as metabolism. The lasso peptide microcin J25 (MccJ25), an inhibitor of iron-uptake and RNA polymerase activity, inhibited EB-247 growth. Our studies indicate that Enterobacter bugandensis is a highly pathogenic species of the genus Enterobacter. Further studies on the colonization and virulence potential of E. bugandensis and its association with septicemic infection is now warranted

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

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

Comparison of approaches for source attribution of ESBL-producing Escherichia coli in Germany

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli have been widely described as the cause of treatment failures in humans around the world. The origin of human infections with these microorganisms is discussed controversially and in most cases hard to identify. Since they pose a relevant risk to human health, it becomes crucial to understand their sources and the transmission pathways. In this study, we analyzed data from different studies in Germany and grouped ESBL-producing E. coli from different sources and human cases into subtypes based on their phenotypic and genotypic characteristics (ESBL-genotype, E. coli phylogenetic group and phenotypic antimicrobial resistance pattern). Then, a source attribution model was developed in order to attribute the human cases to the considered sources. The sources were from different animal species (cattle, pig, chicken, dog and horse) and also from patients with nosocomial infections. The human isolates were gathered from community cases which showed to be colonized with ESBL-producing E. coli. We used the attribution model first with only the animal sources (Approach A) and then additionally with the nosocomial infections (Approach B). We observed that all sources contributed to the human cases, nevertheless, isolates from nosocomial infections were more related to those from human cases than any of the other sources. We identified subtypes that were only detected in the considered animal species and others that were observed only in the human population. Some subtypes from the human cases could not be allocated to any of the sources from this study and were attributed to an unknown source. Our study emphasizes the importance of human-to-human transmission of ESBL-producing E. coli and the different role that pets, livestock and healthcare facilities may play in the transmission of these resistant bacteria. The developed source attribution model can be further used to monitor future trends. A One Health approach is necessary to develop source attribution models further to integrate also wildlife, environmental as well as food sources in addition to human and animal data.Peer Reviewe