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July 19, 2019

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10?kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC-E. coli strains, we identified substantial strain diversity (n?=?21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (=9 replicon types); and substantial bla KPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM, bla CTX-M) suggests that it may become similarly prevalent.

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July 19, 2019

Discovery and biosynthesis of gladiolin: A Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

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July 19, 2019

Monitoring microevolution of OXA-48-producing Klebsiella pneumoniae ST147 in a hospital setting by SMRT sequencing.

Carbapenemase-producing Klebsiella pneumoniae pose an increasing risk for healthcare facilities worldwide. A continuous monitoring of ST distribution and its association with resistance and virulence genes is required for early detection of successful K. pneumoniae lineages. In this study, we used WGS to characterize MDR blaOXA-48-positive K. pneumoniae isolated from inpatients at the University Medical Center Göttingen, Germany, between March 2013 and August 2014.Closed genomes for 16 isolates of carbapenemase-producing K. pneumoniae were generated by single molecule real-time technology using the PacBio RSII platform.Eight of the 16 isolates showed identical XbaI macrorestriction patterns and shared the same MLST, ST147. The eight ST147 isolates differed by only 1-25 SNPs of their core genome, indicating a clonal origin. Most of the eight ST147 isolates carried four plasmids with sizes of 246.8, 96.1, 63.6 and 61.0?kb and a novel linear plasmid prophage, named pKO2, of 54.6?kb. The blaOXA-48 gene was located on a 63.6?kb IncL plasmid and is part of composite transposon Tn1999.2. The ST147 isolates expressed the yersinabactin system as a major virulence factor. The comparative whole-genome analysis revealed several rearrangements of mobile genetic elements and losses of chromosomal and plasmidic regions in the ST147 isolates.Single molecule real-time sequencing allowed monitoring of the genetic and epigenetic microevolution of MDR OXA-48-producing K. pneumoniae and revealed in addition to SNPs, complex rearrangements of genetic elements.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 19, 2019

Genomic analysis of hospital plumbing reveals diverse reservoir of bacterial plasmids conferring carbapenem resistance.

The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization ofblaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections withblaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission ofLeclerciasp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings.IMPORTANCECarbapenemase-producing organisms (CPOs) are a global concern because of the morbidity and mortality associated with these resistant Gram-negative bacteria. Horizontal plasmid transfer spreads the resistance mechanism to new bacteria, and understanding the plasmid ecology of the hospital environment can assist in the design of control strategies to prevent nosocomial infections. A 5-year genomic and epidemiological survey was undertaken to study the CPOs in the patient-accessible environment, as well as in the plumbing system removed from the patient. This comprehensive survey revealed a vast, unappreciated reservoir of CPOs in wastewater, which was in contrast to the low positivity rate in both the patient population and the patient-accessible environment. While there were few patient-environmental isolate associations, there were plasmid backbones common to both populations. These results are relevant to all hospitals for which CPO colonization may not yet be defined through extensive surveillance.

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July 19, 2019

A high-throughput approach for identification of nontuberculous mycobacteria in drinking water reveals relationship between water age and Mycobacterium avium.

Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTMrpoBgenes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age (i.e., combined distribution system residence time and home plumbing stagnation time) was associated with a greater relative abundance ofMycobacterium aviumsubsp.avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, includingMycobacterium abscessusandMycobacterium chelonaeOverall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM.IMPORTANCEAn extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTMrpoBgenes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). The extraction procedure recovered, on average, eight times as much NTM DNA and three times as much total DNA from DW as two widely used commercial DNA extraction kits. The combined DNA extraction and sequencing approach allowed high-throughput screening of DW samples to identify NTM, revealing that the relative abundance ofMycobacterium aviumsubsp.aviumincreased with water age. Furthermore, the two-step barcoding approach developed as part of the PacBio sequencing method makes this procedure highly adaptable, allowing it to be used for other target genes and species. Copyright © 2018 Haig et al.

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July 19, 2019

Survey on the use of whole-genome sequencing for infectious diseases surveillance: Rapid expansion of European national capacities, 2015-2016.

Whole-genome sequencing (WGS) has become an essential tool for public health surveillance and molecular epidemiology of infectious diseases and antimicrobial drug resistance. It provides precise geographical delineation of spread and enables incidence monitoring of pathogens at genotype level. Coupled with epidemiological and environmental investigations, it delivers ultimate resolution for tracing sources of epidemic infections. To ascertain the level of implementation of WGS-based typing for national public health surveillance and investigation of prioritized diseases in the European Union (EU)/European Economic Area (EEA), two surveys were conducted in 2015 and 2016. The surveys were designed to determine the national public health reference laboratories’ access to WGS and operational WGS-based typing capacity for national surveillance of selected foodborne pathogens, antimicrobial-resistant pathogens, and vaccine-preventable diseases identified as priorities for European genomic surveillance. Twenty-eight and twenty-nine out of the 30 EU/EEA countries participated in the survey in 2015 and 2016, respectively. National public health reference laboratories in 22 and 25 countries had access to WGS-based typing for public health applications in 2015 and 2016, respectively. Reported reasons for limited or no access were lack of funding, staff, and expertise. Illumina technology was the most frequently used followed by Ion Torrent technology. The access to bioinformatics expertise and competence for routine WGS data analysis was limited. By mid-2016, half of the EU/EEA countries were using WGS analysis either as first- or second-line typing method for surveillance of the pathogens and antibiotic resistance issues identified as EU priorities. The sampling frame as well as bioinformatics analysis varied by pathogen/resistance issue and country. Core genome multilocus allelic profiling, also called cgMLST, was the most frequently used annotation approach for typing bacterial genomes suggesting potential bioinformatics pipeline compatibility. Further capacity development for WGS-based typing is ongoing in many countries and upon consolidation and harmonization of methods should enable pan-EU data exchange for genomic surveillance in the medium-term subject to the development of suitable data management systems and appropriate agreements for data sharing.

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July 7, 2019

A novel Tn3-like composite transposon harboring blaVIM-1 in Klebsiella pneumoniae spp. pneumoniae isolated from river water.

We present a new plasmid (pOW16C2) with a novel Tn3-like transposon harboring blaVIM-1 from a Klebsiella pneumoniae strain isolated from river water in Switzerland.Complete nucleotide sequence of pOW16C2 was obtained using a Pacific Biosciences SMRT sequencing approach and coding sequences were predicted.The 59,228?bp sequence included a typical IncN-like backbone and a mosaic structure with blaVIM-1, aacA4, aphA15, aadA1, catB2, qnrS1, sul1, and dfrA14 conferring resistance to carbapenems and other ß-lactam antibiotics, aminoglycosides, chloramphenicol, quinolones, sulfonamides, and trimethoprim, respectively. Most of these resistance genes were inserted in a class 1 integron that was embedded in a novel Tn3-like composite transposon.IncN plasmids carrying carbapenemases are frequently isolated from K. pneumoniae strains in clinical settings. The dissemination of K. pneumoniae harboring blaVIM-1 in surface water is a cause for increased concern to public health.

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July 7, 2019

Accumulation-associated protein enhances Staphylococcus epidermidis biofilm formation under dynamic conditions and is required for infection in a rat catheter model.

Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae at a single institution: insights into endemicity from whole-genome sequencing.

The global emergence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) multilocus sequence type ST258 is widely recognized. Less is known about the molecular and epidemiological details of non-ST258 K. pneumoniae in the setting of an outbreak mediated by an endemic plasmid. We describe the interplay of blaKPC plasmids and K. pneumoniae strains and their relationship to the location of acquisition in a U.S. health care institution. Whole-genome sequencing (WGS) analysis was applied to KPC-Kp clinical isolates collected from a single institution over 5 years following the introduction of blaKPC in August 2007, as well as two plasmid transformants. KPC-Kp from 37 patients yielded 16 distinct sequence types (STs). Two novel conjugative blaKPC plasmids (pKPC_UVA01 and pKPC_UVA02), carried by the hospital index case, accounted for the presence of blaKPC in 21/37 (57%) subsequent cases. Thirteen (35%) isolates represented an emergent lineage, ST941, which contained pKPC_UVA01 in 5/13 (38%) and pKPC_UVA02 in 6/13 (46%) cases. Seven (19%) isolates were the epidemic KPC-Kp strain, ST258, mostly imported from elsewhere and not carrying pKPC_UVA01 or pKPC_UVA02. Using WGS-based analysis of clinical isolates and plasmid transformants, we demonstrate the unexpected dispersal of blaKPC to many non-ST258 lineages in a hospital through spread of at least two novel blaKPC plasmids. In contrast, ST258 KPC-Kp was imported into the institution on numerous occasions, with other blaKPC plasmid vectors and without sustained transmission. Instead, a newly recognized KPC-Kp strain, ST941, became associated with both novel blaKPC plasmids and spread locally, making it a future candidate for clinical persistence and dissemination. Copyright © 2015, Mathers et al.

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July 7, 2019

Gut symbionts from distinct hosts exhibit genotoxic activity via divergent colibactin biosynthetic pathways.

Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is “colibactin”, a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway, inflicting DNA damage upon eukaryotic cells and contributing to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees, and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Ceftriaxone-resistant Salmonella enterica serotype Typhimurium sequence type 313 from Kenyan patients is associated with the blaCTX-M-15 gene on a novel IncHI2 plasmid.

Multidrug-resistant bacteria pose a major challenge to the clinical management of infections in resource-poor settings. Although nontyphoidal Salmonella (NTS) bacteria cause predominantly enteric self-limiting illness in developed countries, NTS is responsible for a huge burden of life-threatening bloodstream infections in sub-Saharan Africa. Here, we characterized nine S. Typhimurium isolates from an outbreak involving patients who initially failed to respond to ceftriaxone treatment at a referral hospital in Kenya. These Salmonella enterica serotype Typhimurium isolates were resistant to ampicillin, chloramphenicol, cefuroxime, ceftriaxone, aztreonam, cefepime, sulfamethoxazole-trimethoprim, and cefpodoxime. Resistance to ß-lactams, including to ceftriaxone, was associated with carriage of a combination of blaCTX-M-15, blaOXA-1, and blaTEM-1 genes. The genes encoding resistance to heavy-metal ions were borne on the novel IncHI2 plasmid pKST313, which also carried a pair of class 1 integrons. All nine isolates formed a single clade within S. Typhimurium ST313, the major clone of an ongoing invasive NTS epidemic in the region. This emerging ceftriaxone-resistant clone may pose a major challenge in the management of invasive NTS in sub-Saharan Africa. Copyright © 2015, Kariuki et al.

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July 7, 2019

Complete genome sequence of a carbapenem-resistant extraintestinal pathogenic Escherichia coli strain belonging to the sequence type 131 H30R subclade.

Here, we report the completed genome sequence of a carbapenem-resistant extraintestinal pathogenic Escherichia coli sequence type 131 (ST131) isolate, MNCRE44. The isolate was obtained in 2012 in Minnesota, USA, from a sputum sample from a hospitalized patient with multiple comorbidities, and it belongs to the H30R sublineage. Copyright © 2015 Johnson et al.

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July 7, 2019

What caused the outbreak of ESBL-producing Klebsiella pneumoniae in a neonatal intensive care unit, Germany 2009 to 2012? Reconstructing transmission with epidemiological analysis and whole-genome sequencing.

We aimed to retrospectively reconstruct the timing of transmission events and pathways in order to understand why extensive preventive measures and investigations were not sufficient to prevent new cases.We extracted available information from patient charts to describe cases and to compare them to the normal population of the ward. We conducted a cohort study to identify risk factors for pathogen acquisition. We sequenced the available isolates to determine the phylogenetic relatedness of Klebsiella pneumoniae isolates on the basis of their genome sequences.The investigation comprises 37 cases and the 10 cases with ESBL (extended-spectrum beta-lactamase)-producing K. pneumoniae bloodstream infection. Descriptive epidemiology indicated that a continuous transmission from person to person was most likely. Results from the cohort study showed that ‘frequent manipulation’ (a proxy for increased exposure to medical procedures) was significantly associated with being a case (RR 1.44, 95% CI 1.02 to 2.19). Genome sequences revealed that all 48 bacterial isolates available for sequencing from 31 cases were closely related (maximum genetic distance, 12 single nucleotide polymorphisms). Based on our calculation of evolutionary rate and sequence diversity, we estimate that the outbreak strain was endemic since 2008. Epidemiological and phylogenetic analyses consistently indicated that there were additional, undiscovered cases prior to the onset of microbiological screening and that the spread of the pathogen remained undetected over several years, driven predominantly by person-to-person transmission. Whole-genome sequencing provided valuable information on the onset, course and size of the outbreak, and on possible ways of transmission. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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July 7, 2019

Complete genome sequence of endophytic nitrogen-fixing Klebsiella variicola strain DX120E.

Klebsiella variicola strain DX120E (=CGMCC 1.14935) is an endophytic nitrogen-fixing bacterium isolated from sugarcane crops grown in Guangxi, China and promotes sugarcane growth. Here we summarize the features of the strain DX120E and describe its complete genome sequence. The genome contains one circular chromosome and two plasmids, and contains 5,718,434 nucleotides with 57.1% GC content, 5,172 protein-coding genes, 25 rRNA genes, 87 tRNA genes, 7 ncRNA genes, 25 pseudo genes, and 2 CRISPR repeats.

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July 7, 2019

Draft genome sequence of Raoultella terrigena R1Gly, a diazotrophic endophyte.

Raoultella terrigena R1Gly is a diazotrophic endophyte isolated from surface-sterilized roots of Nicotiana tabacum. The whole-genome sequence was obtained to investigate the endophytic characteristics of this organism at the genetic level, as well as to compare this strain with its close relatives. To our knowledge, this is the first genome obtained from the Raoultella terrigena species and only the third genome from the Raoultella genus, after Raoultella ornitholytic and Raoultella planticola. This genome will provide a foundation for further comparative genomic, metagenomic, and functional studies of this genus. Copyright © 2015 Schicklberger et al.

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