Here, we sequenced multidrug resistance plasmid pT3, which encodes mcr-3.5 and other resistance genes. The cooccurrence of mcr genes and other resistance genes on a single plasmid is of concern.Copyright © 2019 Zurfluh et al.
Raoultella terrigena is a bacterial species associated with soil and aquatic environments; however, sporadic cases of opportunistic disease in humans have been reported. Here, we report the first two complete genome sequences from clinical strains isolated from human sources that have been deposited in the National Collection of Type Cultures (NCTC). © Crown copyright 2019.
Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.Copyright © 2019 American Society for Microbiology.
Here, we report the full-genome sequence of an NAD-hemin-independent Avibacterium paragallinarum serovar C-2 strain, FARPER-174, isolated from layer hens in Peru. This genome contained 12 potential genomic islands that include ribosomal protein-coding genes, a nadR gene, hemocin-coding genes, sequences of fagos, an rtx operon, and drug resistance genes. Copyright © 2019 Tataje-Lavanda et al.
Introduction: Emergence of resistance determinants of blaNDM and mcr-1 has undermined the antimicrobial effectiveness of the last line drugs carbapenems and colistin. Aim: This work aimed to assess the prevalence of blaNDM and mcr-1 in E. coli strains collected from food in Shenzhen, China, during the period 2015 to 2017. Methods: Multidrug-resistant E. coli strains were isolated from food samples. Plasmids encoding mcr-1 or blaNDM genes were characterised and compared with plasmids found in clinical isolates.ResultsAmong 1,166 non-repeated cephalosporin-resistant E. coli strains isolated from 2,147 food samples, 390 and 42, respectively, were resistant to colistin and meropenem, with five strains being resistant to both agents. The rate of resistance to colistin increased significantly (p?
Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.Copyright © 2019 American Society for Microbiology.
Acinetobacter baumannii is an important Gram-negative pathogen in hospital-related infections. However, treatment options for A. baumannii infections have become limited due to multidrug resistance. Bacterial virulence is often associated with capsule genes found in the K locus, many of which are essential for biosynthesis of the bacterial envelope. However, the roles of other genes in the K locus remain largely unknown. From an in vitro evolution experiment, we obtained an isolate of the virulent and multidrug-resistant A. baumannii strain MDR-ZJ06, called MDR-ZJ06M, which has an insertion by the ISAba16 transposon in gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase), a gene found in the K locus. The isolate showed an increased resistance toward tigecycline, whereas the MIC decreased in the case of carbapenems, cephalosporins, colistin, and minocycline. By using knockout and complementation experiments, we demonstrated that gnaA is important for the synthesis of lipooligosaccharide and capsular polysaccharide and that disruption of the gene affects the morphology, drug susceptibility, and virulence of the pathogen.Copyright © 2019 American Society for Microbiology.
Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum ß-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.Copyright © 2019 Mahérault et al.
Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.
The novel 12,932-bp nonconjugative multiresistance transposon Tn6674 was identified in the chromosomal DNA of a porcine Enterococcus faecalis strain. Tn6674 belongs to the Tn554 family of transposons. It shares the same arrangement of the transposase genes tnpA, tnpB, and tnpC with Tn554 However, in addition to the Tn554-associated resistance genes spc and erm(A), Tn6674 harbored the resistance genes fexA and optrA Circular forms of Tn6674 were detected and suggest the functional activity of this transposon.Copyright © 2019 American Society for Microbiology.
The spread of antimicrobial resistance genes (ARGs) among Gram-negative pathogens, including Acinetobacter baumannii, is primarily mediated by transferable plasmids; however, ARGs are frequently integrated into its chromosome. How ARG gets horizontally incorporated into the chromosome of A. baumannii, and whether it functions as a cause for further spread of ARG, remains unknown. Here, we demonstrated intercellular prophage-mediated transfer of chromosomal ARGs without direct cell-cell interaction in A. baumannii We prepared ARG-harboring extracellular DNA (eDNA) components from the culture supernatant of a multidrug-resistant (MDR) A. baumannii NU-60 strain and exposed an antimicrobial-susceptible (AS) A. baumannii ATCC 17978 strain to the eDNA components. The antimicrobial-resistant (AR) A. baumannii ATCC 17978 derivatives appeared to acquire various ARGs, originating from dispersed loci of the MDR A. baumannii chromosome, along with their surrounding regions, by homologous recombination, with the ARGs including armA (aminoglycoside resistance), blaTEM-1 (ß-lactam resistance), tet(B) (tetracycline resistance), and gyrA-81L (nalidixic acid resistance) genes. Notably, the eDNAs conferring antimicrobial resistance were enveloped in specific capsid proteins consisting of phage particles, thereby protecting the eDNAs from detergent and DNase treatments. The phages containing ARGs were likely released into the extracellular space from MDR A. baumannii, thereby transducing ARGs into AS A. baumannii, resulting in the acquisition of AR properties by the recipient. We concluded that the generalized transduction, in which phages were capable of carrying random pieces of A. baumannii genomic DNAs, enabled efficacious intercellular transfer of chromosomal ARGs between A. baumannii strains without direct cell-cell interaction. Copyright © 2019 American Society for Microbiology.
Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae (n?=?23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases. Copyright © 2019 Mathers et al.
Nontyphoidal Salmonella enterica (NTS) poses a major public health risk worldwide that is amplified by the existence of antimicrobial-resistant strains, especially those resistant to quinolones and extended-spectrum cephalosporins (ESC). Little is known on the dissemination of plasmids harboring the acquired genetic determinants that confer resistance to these antimicrobials across NTS serotypes from livestock in the United States. NTS isolates (n?=?183) from U.S. swine clinical cases retrieved during 2014 to 2016 were selected for sequencing based on their phenotypic resistance to enrofloxacin (quinolone) or ceftiofur (3rd-generation cephalosporin). De novo assemblies were used to identify chromosomal mutations and acquired antimicrobial resistance genes (AARGs). In addition, plasmids harboring AARGs were identified using short-read assemblies and characterized using a multistep approach that was validated by long-read sequencing. AARGs to quinolones [qnrB15, qnrB19, qnrB2, qnrD, qnrS1, qnrS2, and aac(6′)Ib-cr] and ESC (blaCMY-2, blaCTX-M-1, blaCTX-M-27, and blaSHV-12) were distributed across serotypes and were harbored by several plasmids. In addition, chromosomal mutations associated with resistance to quinolones were identified in the target enzyme and efflux pump regulation genes. The predominant plasmid harboring the prevalent qnrB19 gene was distributed across serotypes. It was identical to a plasmid previously reported in S. enterica serovar Anatum from swine in the United States (GenBank accession number KY991369.1) and similar to Escherichia coli plasmids from humans in South America (GenBank accession numbers GQ374157.1 and JN979787.1). Our findings suggest that plasmids harboring AARGs encoding mechanisms of resistance to critically important antimicrobials are present in multiple NTS serotypes circulating in swine in the United States and can contribute to resistance expansion through horizontal transmission.Copyright © 2019 American Society for Microbiology.
Four Enterobacteriaceae clinical isolates bearing mcr-1 gene-harboring plasmids were characterized. All isolates demonstrated the ability to transfer colistin resistance to Escherichia coli; plasmids were stable in conjugants after multiple passages on nonselective media. mcr-1 was located on an IncX4 (n?=?3) or IncN (n?=?1) plasmid. The IncN plasmid harbored 13 additional antimicrobial resistance genes. Results indicate that the mcr-1-bearing plasmids in this study were highly transferable in vitro and stable in the recipients.This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
In 2011, VIM-1-producing Salmonella enterica serovar Infantis and Escherichia coli were isolated for the first time in four German livestock farms. In 2015/2016, highly related isolates were identified in German pig production. This raised the issue of potential reservoirs for these isolates, the relation of their mobile genetic elements, and potential links between the different affected farms/facilities. In a piglet-producing farm suspicious for being linked to some blaVIM-1 findings in Germany, fecal and environmental samples were examined for the presence of carbapenemase-producing Enterobacteriaceae and Salmonella spp. Newly discovered isolates were subjected to Illumina whole-genome sequencing (WGS) and S1 pulsed-field gel electrophoresis (PFGE) hybridization experiments. WGS data of these isolates were compared with those for the previously isolated VIM-1-producing Salmonella Infantis isolates from pigs and poultry. Among 103 samples, one Salmonella Goldcoast isolate, one Salmonella Infantis isolate, and one Enterobacter cloacae isolate carrying the blaVIM-1 gene were detected. Comparative WGS analysis revealed that the blaVIM-1 gene was part of a particular Tn21-like transposable element in all isolates. It was located on IncHI2 (ST1) plasmids of ~290 to 300?kb with a backbone highly similar (98 to 100%) to that of reference pSE15-SA01028. SNP analysis revealed a close relationship of all VIM-1-positive S Infantis isolates described since 2011. The findings of this study demonstrate that the occurrence of the blaVIM-1 gene in German livestock is restricted neither to a certain bacterial species nor to a certain Salmonella serovar but is linked to a particular Tn21-like transposable element located on transferable pSE15-SA01028-like IncHI2 (ST1) plasmids, being present in all of the investigated isolates from 2011 to 2017.IMPORTANCE Carbapenems are considered one of few remaining treatment options against multidrug-resistant Gram-negative pathogens in human clinical settings. The occurrence of carbapenemase-producing Enterobacteriaceae in livestock and food is a major public health concern. Particularly the occurrence of VIM-1-producing Salmonella Infantis in livestock farms is worrisome, as this zoonotic pathogen is one of the main causes for human salmonellosis in Europe. Investigations on the epidemiology of those carbapenemase-producing isolates and associated mobile genetic elements through an in-depth molecular characterization are indispensable to understand the transmission of carbapenemase-producing Enterobacteriaceae along the food chain and between different populations to develop strategies to prevent their further spread.Copyright © 2019 Roschanski et al.
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