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April 21, 2020

The history, genome and biology of NCTC 30: a non-pandemic Vibrio cholerae isolate from World War One.

The sixth global cholera pandemic lasted from 1899 to 1923. However, despite widespread fear of the disease and of its negative effects on troop morale, very few soldiers in the British Expeditionary Forces contracted cholera between 1914 and 1918. Here, we have revived and sequenced the genome of NCTC 30, a 102-year-old Vibrio cholerae isolate, which we believe is the oldest publicly available live V. cholerae strain in existence. NCTC 30 was isolated in 1916 from a British soldier convalescent in Egypt. We found that this strain does not encode cholera toxin, thought to be necessary to cause cholera, and is not part of V. cholerae lineages responsible for the pandemic disease. We also show that NCTC 30, which predates the introduction of penicillin-based antibiotics, harbours a functional ß-lactamase antibiotic resistance gene. Our data corroborate and provide molecular explanations for previous phenotypic studies of NCTC 30 and provide a new high-quality genome sequence for historical, non-pandemic V. cholerae.

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April 21, 2020

Plasmid analysis of Escherichia coli isolates from South Korea co-producing NDM-5 and OXA-181 carbapenemases.

Recently, Escherichia coli isolates co-producing New Delhi metallo-ß-lactamase (NDM)-5 and oxacillinase (OXA)-181 were identified in a tertiary-care hospital of South Korea. Isolate CC1702-1 was collected from urine in January 2017 and isolate CC1706-1 was recovered from a transtracheal aspirate of a hospitalized patient in May 2017. Carbapenemase genes were identified by multiplex PCR and sequencing, and whole genome sequencing was performed subsequently using the PacBio RSII system. Both E. coli isolates belonged to the same clone (ST410) and were resistant to all ß-lactams including carbapenems. We obtained whole plasmid sequences of the isolates: pCC1702-NDM-5 from CC1702-1 and pCC1706-NDM-5 and pCC1706-OXA-181 from CC1706-1. The two E. coli isolates belonged to the same clone (ST410) and they were completely resistant to all ß-lactams, as well as carbapenems. Two blaNDM-5-harboring plasmids belonged to the same incompatibility group, IncFIA/B, and consisted of 79,613?bp and 111,890?bp with 87 and 130 coding sequences, respectively. The genetic structures of the two blaNDM-5-bearing plasmids, which were distinct from the blaNDM-5-bearing plasmids from the Klebsiella pneumoniae isolates previously transmitted from the United Arab Emirates (UAE) to South Korea, differed from each other. While pCC1702-NDM-5 showed high degree of identity with the plasmid from a multidrug-resistant isolate of Citrobacter fruendii P5571 found in China, pCC1706-NDM-5 was very similar to the plasmid from a multidrug-resistant isolate of E. coli AMA1176 found in Denmark. pCC1706-OXA-181, which was a 51?kb, self-transmissible IncX3 plasmid, was identical to the E. coli plasmids pAMA1167-OXA-181 from Denmark and pOXA-181-WCHEC14828 from China. Plasmids harboring blaNDM-5 in E. coli isolates might not be transferred from K. pneumoniae isolates co-producing NDM-5 and OXA-181. They probably originated from multiple sources.Copyright © 2019 Elsevier Inc. All rights reserved.

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April 21, 2020

Characterization of a clinical Clostridioides difficile isolate with markedly reduced fidaxomicin susceptibility and a V1143D mutation in rpoB.

The identification and characterization of clinical Clostridioides difficile isolates with reduced fidaxomicin susceptibility.Agar dilution assays were used to determine fidaxomicin MICs. Genome sequence data were obtained by single-molecule real-time (SMRT) sequencing in addition to amplicon sequencing of rpoB and rpoC alleles. Allelic exchange was used to introduce the identified mutation into C. difficile 630?erm. Replication rates, toxin A/B production and spore formation were determined from the strain with reduced fidaxomicin susceptibility.Out of 50 clinical C. difficile isolates, isolate Goe-91 revealed markedly reduced fidaxomicin susceptibility (MIC >64?mg/L). A V1143D mutation was identified in rpoB of Goe-91. When introduced into C. difficile 630?erm, this mutation decreased fidaxomicin susceptibility (MIC >64?mg/L), but was also associated with a reduced replication rate, low toxin A/B production and markedly reduced spore formation. In contrast, Goe-91, although also reduced in toxin production, showed normal growth rates and only moderately reduced spore formation capacities. This indicates that the rpoBV1143D allele-associated fitness defect is less pronounced in the clinical isolate.To the best of our knowledge, this is the first description of a pathogenic clinical C. difficile isolate with markedly reduced fidaxomicin susceptibility. The lower-than-expected fitness burden of the resistance-mediating rpoBV1143D allele might be an indication for compensatory mechanisms that take place during in vivo selection of mutants.

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April 21, 2020

Genome and plasmid diversity of Extended-Spectrum ß-Lactamase-producing Escherichia coli ST131 – tracking phylogenetic trajectories with Bayesian inference.

Clonal lineages of ESBL (Extended-Spectrum ß-Lactamase)-producing E. coli belonging to sequence type 131 (ST131) have disseminated globally during the last 30 years, leading to an increased prevalence of resistance to fluoroquinolones and extended-spectrum cephalosporins in clinical isolates of E. coli. We aimed to study if Swedish ESBL-producing ST131 isolates originated from single or multiple introductions to the population by assessing the amount of genetic variation, on chromosomal and plasmid level, between Swedish and international E. coli ST131. Bayesian inference of Swedish E. coli ST131 isolates (n?=?29), sequenced using PacBio RSII, together with an international ST131 dataset showed that the Swedish isolates were part of the international ST131 A, C1 and C2 clades. Highly conserved plasmids were identified in three clusters although they were separated by several years, which indicates a strong co-evolution between some ST131 lineages and specific plasmids. In conclusion, the tight clonal relationship observed within the ST131 clades, together with highly conserved plasmids, challenges investigation of strain transmission events. A combination of few SNPs on a genome-wide scale and an epidemiological temporospatial link, are needed to track the spread of the ST131 subclones.

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April 21, 2020

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

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April 21, 2020

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

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April 21, 2020

Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection.

Recurrent urinary tract infections (rUTIs) are extremely common, with ~?25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.

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April 21, 2020

Sequencing and Genomic Diversity Analysis of IncHI5 Plasmids.

IncHI plasmids could be divided into five different subgroups IncHI1-5. In this study, the complete nucleotide sequences of seven blaIMP- or blaVIM-carrying IncHI5 plasmids from Klebsiella pneumoniae, K. quasipneumoniae, and K. variicola were determined and compared in detail with all the other four available sequenced IncHI5 plasmids. These plasmids carried conserved IncHI5 backbones composed of repHI5B and a repFIB-like gene (replication), parABC (partition), and tra1 (conjugal transfer). Integration of a number of accessory modules, through horizontal gene transfer, at various sites of IncHI5 backbones resulted in various deletions of surrounding backbone regions and thus considerable diversification of IncHI5 backbones. Among the accessory modules were three kinds of resistance accessory modules, namely Tn10 and two antibiotic resistance islands designated ARI-A and ARI-B. These two islands, inserted at two different fixed sites (one island was at one site and the other was at a different site) of IncHI5 backbones, were derived from the prototype Tn3-family transposons Tn1696 and Tn6535, respectively, and could be further discriminated as various intact transposons and transposon-like structures. The ARI-A or ARI-B islands from different IncHI5 plasmids carried distinct profiles of antimicrobial resistance markers and associated mobile elements, and complex events of transposition and homologous recombination accounted for assembly of these islands. The carbapenemase genes blaIMP-4, blaIMP-38 and blaVIM-1 were identified within various class 1 integrons from ARI-A or ARI-B of the seven plasmids sequenced in this study. Data presented here would provide a deeper insight into diversification and evolution history of IncHI5 plasmids.

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April 21, 2020

FDA-ARGOS is a database with public quality-controlled reference genomes for diagnostic use and regulatory science.

FDA proactively invests in tools to support innovation of emerging technologies, such as infectious disease next generation sequencing (ID-NGS). Here, we introduce FDA-ARGOS quality-controlled reference genomes as a public database for diagnostic purposes and demonstrate its utility on the example of two use cases. We provide quality control metrics for the FDA-ARGOS genomic database resource and outline the need for genome quality gap filling in the public domain. In the first use case, we show more accurate microbial identification of Enterococcus avium from metagenomic samples with FDA-ARGOS reference genomes compared to non-curated GenBank genomes. In the second use case, we demonstrate the utility of FDA-ARGOS reference genomes for Ebola virus target sequence comparison as part of a composite validation strategy for ID-NGS diagnostic tests. The use of FDA-ARGOS as an in silico target sequence comparator tool combined with representative clinical testing could reduce the burden for completing ID-NGS clinical trials.

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April 21, 2020

Multidrug Resistant Uropathogenic Escherichia coli ST405 With a Novel, Composite IS26 Transposon in a Unique Chromosomal Location.

Escherichia coli ST405 is an emerging urosepsis pathogen, noted for carriage of blaCTX-M, blaNDM, and a repertoire of virulence genes comparable with O25b:H4-ST131. Extraintestinal and multidrug resistant E. coli ST405 are poorly studied in Australia. Here we determined the genome sequence of a uropathogenic, multiple drug resistant E. coli ST405 (strain 2009-27) from the mid-stream urine of a hospital patient in Sydney, Australia, using a combination of Illumina and SMRT sequencing. The genome of strain 2009-27 assembled into two unitigs; a chromosome comprising 5,287,472 bp and an IncB/O plasmid, pSDJ2009-27, of 89,176 bp. In silico and phenotypic analyses showed that strain 2009-27 is a serotype O102:H6, phylogroup D ST405 resistant to ampicillin, azithromycin, kanamycin, streptomycin, trimethoprim, and sulphafurazole. The genes encoding resistance to these antibiotics reside within a novel, mobile IS26-flanked transposon, identified here as Tn6242, in the chromosomal gene yjdA. Tn6242 comprises four modules that each carries resistance genes flanked by IS26, including a class 1 integron with dfrA17 and aadA5 gene cassettes, a variant of Tn6029, and mphA. We exploited unique genetic signatures located within Tn6242 to identify strains of ST405 from Danish patients that also carry the transposon in the same chromosomal location. The acquisition of Tn6242 into yjdA in ST405 is significant because it (i) is vertically inheritable; (ii) represents a reservoir of resistance genes that can transpose onto resident/circulating plasmids; and (iii) is a site for the capture of further IS26-associated resistance gene cargo.

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April 21, 2020

Discovery and Characterization of Mycobacterium basiliense sp. nov., a Nontuberculous Mycobacterium Isolated From Human Lungs.

Bacteria belonging to the genus Mycobacterium are predominantly responsible for pulmonary diseases; most notably Mycobacterium tuberculosis causes granulomatous pulmonary infections. Here we describe a novel slow growing mycobacterial species isolated from respiratory samples from five patients, four with underlying pulmonary disease. The isolates were characterized by biochemical and molecular techniques, including whole genome sequencing. Biochemical characteristics generally match those of M. marinum and M. ulcerans; however, the most striking difference of the new species is its ability to grow at 37°C. The new species was found to grow in human macrophages, but not amoebae, suggesting a pathogenic rather than an environmental lifestyle. Phylogenetic analysis reveals a deep-rooting relationship to M. marinum and M. ulcerans. A complete genome sequence was obtained through combining short and long-read sequencing, providing a genome of 5.6 Mb. The genome appears to be highly intact, syntenic with that of M. marinum, with very few insertion sequences. A vast array of virulence factors includes 283 PE/PPE surface-associated proteins, making up 10% of the coding capacity, and 22 non-ribosomal peptide synthase clusters. A comparison of six clinical isolates from the five patients shows that they differ by up to two single nucleotide polymorphisms, suggesting a common source of infection. Our findings are in accordance with the recognition of a new taxonomic entity. We propose the name M. basiliense, as all isolates were found in patients from the Basel area of Switzerland.

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April 21, 2020

Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1.

Klebsiella pneumoniae is a major threat to public health with the emergence of isolates resistant to most, if not all, useful antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae collected from patients resident in a region of Pakistan, during the period 2010-2012, when the now globally-distributed carbapenemase bla-NDM-1 was being acquired by Klebsiella. We observed two dominant lineages, but neither the overall resistance profile nor virulence-associated factors, explain their evolutionary success. Phenotypic analysis of resistance shows few differences between the acquisition of resistance genes and the phenotypic resistance profile, including beta-lactam antibiotics that were used to treat ESBL-positive strains. Resistance against these drugs could be explained by inhibitor-resistant beta-lactamase enzymes, carbapenemases or ampC type beta-lactamases, at least one of which was detected in most, but not all relevant strains analysed. Complete genomes for six selected strains are reported, these provide detailed insights into the mobile elements present in these isolates during the initial spread of NDM-1. The unexplained success of some lineages within this pool of highly resistant strains, and the discontinuity between phenotypic resistance and genotype at the macro level, indicate that intrinsic mechanisms contribute to competitive advantage and/or resistance.

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April 21, 2020

Characterization of Five Escherichia coli Isolates Co-expressing ESBL and MCR-1 Resistance Mechanisms From Different Origins in China

Present study characterized five Escherichia coli co-expressing ESBL and MCR-1 recovered from food, food-producing animals, and companion animals in China. Antimicrobial susceptibility tests, conjugation experiments, and plasmid typing were performed. Whole genome sequencing (WGS) was undertaken for all five isolates using either PacBio RS II or Illumina HiSeq 2500 platforms. The cefotaxime and colistin resistance encoded by blaCTX-M and mcr-1 genes, respectively, was transferable by conjugation either together or separately for all five strains. Interestingly, the ESBL and mcr-1 genes could be co-selected by cefotaxime, while the colistin only selected the mcr-1-carrying plasmids during the conjugation experiments. Five E. coli sequence types (ST88, ST93, ST602, ST162, and ST457) were detected. Although diverse plasmid profiles were identified, IncI2, IncFIB, and IncFII plasmid types were predominant. These five clonally unrelated isolates harbored the mcr-1 gene located on similar plasmid backbones, which showed high nucleotide similarity to plasmid pHNSHP45. The mcr-1 gene can be co-transmitted with blaCTX-Mgenes through IncI2 plasmids with or without ISApl1 in our study. Characterization of these co-existence ESBL and mcr-1 isolates extends our understanding on the dissemination of these resistance markers among bacteria of diverse origins.

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April 21, 2020

Arcobacter cryaerophilus Isolated From New Zealand Mussels Harbor a Putative Virulence Plasmid.

A wide range of Arcobacter species have been described from shellfish in various countries but their presence has not been investigated in Australasia, in which shellfish are a popular delicacy. Since several arcobacters are considered to be emerging pathogens, we undertook a small study to evaluate their presence in several different shellfish, including greenshell mussels, oysters, and abalone (paua) in New Zealand. Arcobacter cryaerophilus, a species associated with human gastroenteritis, was the only species isolated, from greenshell mussels. Whole-genome sequencing revealed a range of genomic traits in these strains that were known or associated virulence factors. Furthermore, we describe the first putative virulence plasmid in Arcobacter, containing lytic, immunoavoidance, adhesion, antibiotic resistance, and gene transfer traits, among others. Complete genome sequence determination using a combination of long- and short-read genome sequencing strategies, was needed to identify the plasmid, clearly identifying its benefits. The potential for plasmids to disseminate virulence traits among Arcobacter and other species warrants further consideration by researchers interested in the risks to public health from these organisms.

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April 21, 2020

Occurrence and Characterization of mcr-1-Positive Escherichia coli Isolated From Food-Producing Animals in Poland, 2011-2016.

The emergence of plasmid-mediated colistin resistance (mcr genes) threatens the effectiveness of polymyxins, which are last-resort drugs to treat infections by multidrug- and carbapenem-resistant Gram-negative bacteria. Based on the occurrence of colistin resistance the aims of the study were to determine possible resistance mechanisms and then characterize the mcr-positive Escherichia coli. The research used material from the Polish national and EU harmonized antimicrobial resistance (AMR) monitoring programs. A total of 5,878 commensal E. coli from fecal samples of turkeys, chickens, pigs, and cattle collected in 2011-2016 were screened by minimum inhibitory concentration (MIC) determination for the presence of resistance to colistin (R) defined as R > 2 mg/L. Strains with MIC = 2 mg/L isolated in 2014-2016 were also included. A total of 128 isolates were obtained, and most (66.3%) had colistin MIC of 2 mg/L. PCR revealed mcr-1 in 80 (62.5%) isolates recovered from 61 turkeys, 11 broilers, 2 laying hens, 1 pig, and 1 bovine. No other mcr-type genes (including mcr-2 to -5) were detected. Whole-genome sequencing (WGS) of the mcr-1-positive isolates showed high diversity in the multi-locus sequence types (MLST) of E. coli, plasmid replicons, and AMR and virulence genes. Generally mcr-1.1 was detected on the same contig as the IncX4 (76.3%) and IncHI2 (6.3%) replicons. One isolate harbored mcr-1.1 on the chromosome. Various extended-spectrum beta-lactamase (blaSHV-12, blaCTX-M-1, blaCTX-M-15, blaTEM-30, blaTEM-52, and blaTEM-135) and quinolone resistance genes (qnrS1, qnrB19, and chromosomal gyrA, parC, and parE mutations) were present in the mcr-1.1-positive E. coli. A total of 49 sequence types (ST) were identified, ST354, ST359, ST48, and ST617 predominating. One isolate, identified as ST189, belonged to atypical enteropathogenic E. coli. Our findings show that mcr-1.1 has spread widely among production animals in Poland, particularly in turkeys and appears to be transferable mainly by IncX4 and IncHI2 plasmids spread across diverse E. coli lineages. Interestingly, most of these mcr-1-positive E. coli would remain undetected using phenotypic methods with the current epidemiological cut-off value (ECOFF). The appearance and spread of mcr-1 among various animals, but notably in turkeys, might be considered a food chain, and public health hazard.

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