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July 7, 2019

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates.

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630?erm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut. Copyright © 2017 Elsevier GmbH. All rights reserved.

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July 7, 2019

Prevalence of colistin resistance gene (mcr-1) containing Enterobacteriaceae in feces of patients attending a tertiary care hospital and detection of a mcr-1 containing, colistin susceptible E. coli.

The emergence of the plasmid-mediated mcr colistin resistance gene in the community poses a potential threat for treatment of patients, especially when hospitalized. The aim of this study was to determine the prevalence of all currently known mcr mediated colistin resistance gene in fecal samples of patients attending a tertiary care hospital. From November 2014 until July 2015, fecal samples of patients attending the Leiden University Medical Center were collected and screened for presence of mcr using real-time PCR. Two of 576 patients were positive for mcr-1, resulting in a prevalence of 0.35%, whereas no mcr-2 was found. One of these samples was culture negative, the second sample contained a blaCMY-2 and mcr-1 containing E.coli. This strain belonged to Sequence Type 359 and serotype O177:H21. The mcr-1 containing E.coli was phenotypically susceptible to colistin with a MIC of = 0.25mg/l, due to a 1329bp transposon IS10R inserted into the mcr-1 gene as identified by WGS. This prevalence study shows that mcr-1 is present in low levels patients out of the community attending a hospital. Furthermore the study underlines the importance of phenotypical confirmation of molecular detection of a mcr-1 gene.

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July 7, 2019

Complete genome sequence of Dolosigranulum pigrum from a patient with interstitial lung disease using single-molecule real-time sequencing technology.

The whole genome sequence of Dolosigranulum pigrum isolated from the blood of a patient with interstitial lung disease was sequenced with the Pacific Biosciences RS II platform. The genome size is 2.1 Mb with 2,127 annotated coding sequences; it contained two clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems. Copyright © 2017 Mukhopadhyay et al.

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July 7, 2019

Whole genome and core genome multilocus sequence typing and single nucleotide polymorphism analyses of Listeria monocytogenes associated with an outbreak linked to cheese, United States, 2013.

Epidemiological findings of a listeriosis outbreak in 2013 implicated Hispanic-style cheese produced by Company A, and pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed on clinical isolates and representative isolates collected from Company A cheese and environmental samples during the investigation. The results strengthened the evidence for cheese as the vehicle. Surveillance sampling and WGS three months later revealed that the equipment purchased by Company B from Company A yielded an environmental isolate highly similar to all outbreak isolates. The whole genome and core genome multilocus sequence typing and single nucleotide polymorphism (SNP) analyses were compared to demonstrate the maximum discriminatory power obtained by using multiple analyses, which were needed to differentiate outbreak-associated isolates from a PFGE-indistinguishable isolate collected in a non-implicated food source in 2012. This unrelated isolate differed from the outbreak isolates by only 7 to 14 SNPs, and as a result, minimum spanning tree by the whole genome analyses and certain variant calling approach and phylogenetic algorithm for core genome-based analyses could not provide the differentiation between unrelated isolates. Our data also suggest that SNP/allele counts should always be combined with WGS clustering generated by phylogenetically meaningful algorithms on sufficient number of isolates, and SNP/allele threshold alone is not sufficient evidence to delineate an outbreak. The putative prophages were conserved across all the outbreak isolates. All outbreak isolates belonged to clonal complex 5 and serotype 1/2b, had an identical inlA sequence, which did not have premature stop codons.IMPORTANCE In this outbreak, multiple analytical approaches were used for maximum discriminatory power. A PFGE-matched, epidemiologically unrelated isolate had high genetic similarity to the outbreak-associated isolates, with as few as only 7 SNP differences. Therefore, the SNP/allele threshold should not be used as the only evidence to define the scope of an outbreak. It is critical that the SNP/allele counts be complemented by WGS clustering generated by phylogenetically meaningful algorithms to distinguish outbreak-associated isolates from epidemiologically unrelated isolates. Careful selection of a variant calling approach and phylogenetic algorithm is critical for core genome-based analyses. The whole genome-based analyses were able to construct the highly resolved phylogeny needed to support the findings of the outbreak investigation. Ultimately, epidemiologic evidence and multiple WGS analyses should be combined to increase the confidence in outbreak investigations. Copyright © 2017 Chen et al.

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July 7, 2019

Evolutionary origin of the staphylococcal cassette chromosome mec (SCCmec).

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the ß-lactam resistance gene mecA However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Antibiotic resistance markers in Burkholderia pseudomallei strain Bp1651 identified by genome sequence analysis.

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ß-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A ß-lactamase and was previously implicated in resistance to ß-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Zinc resistance within swine associated methicillin resistant staphylococcus aureus (MRSA) Isolates in the USA is associated with MLST lineage.

Zinc resistance in livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) sequence type (ST) 398 is primarily mediated by the czrC gene co-located with the mecA gene, encoding methicillin resistance, within the type V SCCmec element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of in feed zinc as an antidiarrheal agent has the potential to contribute to the emergence and spread of MRSA in swine through increased selection pressure to maintain the SCCmec element in isolates obtained from pigs. In this study we report the prevalence of the czrC gene and phenotypic zinc resistance in US swine associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and US swine associated LA-MRSA ST398 isolates. We demonstrate that the prevalence of zinc resistance in US swine associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact, swine associated LA-MRSA ST398 isolates, and previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the US swine population. Additionally, our data indicate that zinc resistance is associated with MLST lineage suggesting a potential link between genetic lineage and carriage of resistance determinants.Importance Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) ST5 in the US swine population. Additionally, our data indicate that zinc resistance is more associated with multi locus sequence type (MLST) lineage suggesting a potential link between genetic lineage and carriage of resistance markers. This information is important to public health professionals, veterinarians, producers, and consumers. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequence of a Legionella longbeachae serogroup 1 strain isolated from a patient with Legionnaires’ disease.

Legionella longbeachae serogroup 1, predominantly found in soil and composted plant material, causes the majority of cases of Legionnaires’ disease (LD) in New Zealand. Here, we report the complete genome sequence of an L. longbeachae serogroup 1 (sg1) isolate derived from a patient hospitalized with LD in Christchurch, New Zealand. Copyright © 2017 Slow et al.

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July 7, 2019

An L-threonine transaldolase is required for L-threo-ß-hydroxy-a-amino acid assembly during obafluorin biosynthesis.

ß-Lactone natural products occur infrequently in nature but possess a variety of potent and valuable biological activities. They are commonly derived from ß-hydroxy-a-amino acids, which are themselves valuable chiral building blocks for chemical synthesis and precursors to numerous important medicines. However, despite a number of excellent synthetic methods for their asymmetric synthesis, few effective enzymatic tools exist for their preparation. Here we report cloning of the biosynthetic gene cluster for the ß-lactone antibiotic obafluorin and delineate its biosynthetic pathway. We identify a nonribosomal peptide synthetase with an unusual domain architecture and an L-threonine:4-nitrophenylacetaldehyde transaldolase responsible for (2S,3R)-2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate biosynthesis. Phylogenetic analysis sheds light on the evolutionary origin of this rare enzyme family and identifies further gene clusters encoding L-threonine transaldolases. We also present preliminary data suggesting that L-threonine transaldolases might be useful for the preparation of L-threo-ß-hydroxy-a-amino acids.

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July 7, 2019

Genetic and functional characterization of blaCTX-M-199, a novel tazobactam and sulbactam resistance-encoding gene located in a conjugative mcr-1-bearing IncI2 plasmid.

The study reported the genetic and functional characterization of a novel CTX-M-199 ß-lactamase, which was encoded by a blaCTX-M-64 variant gene found in a conjugative mcr-1-bearing IncI2 plasmid and exhibited resistance to ß-lactamase inhibitors, tazobactam and sulbactam. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Benzalkonium tolerance genes and outcome in Listeria monocytogenes meningitis.

Listeria monocytogenes is a food-borne pathogen that can cause meningitis. The listerial genotype ST6 has been linked to increasing rates of unfavourable outcome over time. We investigated listerial genetic variation and the relation with clinical outcome in meningitis.We sequenced 96 isolates from adults with listerial meningitis included in two prospective nationwide cohort studies by whole genome sequencing, and evaluated associations between bacterial genetic variation and clinical outcome. We validated these results by screening listerial genotypes of 445 cerebrospinal fluid and blood isolates from patients over a 30-year period from the Dutch national surveillance cohort.We identified a bacteriophage, phiLMST6 co-occurring with a novel plasmid, pLMST6, in ST6 isolates to be associated with unfavourable outcome in patients (p 2.83e-05). The plasmid carries a benzalkonium chloride tolerance gene, emrC, conferring decreased susceptibility to disinfectants used in the food-processing industry. Isolates harbouring emrC were growth inhibited at higher levels of benzalkonium chloride (median 60 mg/L versus 15 mg/L; p <0.001), and had higher MICs for amoxicillin and gentamicin compared with isolates without emrC (both p <0.001). Transformation of pLMST6 into naive strains led to benzalkonium chloride tolerance and higher MICs for gentamicin.These results show that a novel plasmid, carrying the efflux transporter emrC, is associated with increased incidence of ST6 listerial meningitis in the Netherlands. Suggesting increased disease severity, our findings warrant consideration of disinfectants used in the food-processing industry that select for resistance mechanisms and may, inadvertently, lead to increased risk of poor disease outcome. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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July 7, 2019

Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-ß-lactamase found in a Pseudomonas aeruginosa clinical isolate.

To characterize a novel subclass B1 metallo-ß-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate.The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat .P. aeruginosa NRZ-03096 was resistant to all tested ß-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to ß-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem.The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .

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July 7, 2019

Genomic epidemiology of NDM-1-encoding plasmids in Latin American clinical isolates reveals insights into the evolution of multidrug resistance

Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-ß-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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