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July 7, 2019

Molecular evolution of a Klebsiella pneumoniae ST278 isolate harboring blaNDM-7 and involved in nosocomial transmission.

During 2013, ST278 Klebsiella pneumoniae with blaNDM-7 was isolated from the urine (KpN01) and rectum (KpN02) of a patient in Calgary, Canada. The same strain (KpN04) was subsequently isolated from another patient in the same unit. Interestingly, a carbapenem-susceptible K. pneumoniae ST278 (KpN06) was obtained 1 month later from the blood of the second patient. Next-generation sequencing (NGS) revealed that the loss of carbapenem-resistance in KpN06 was due to a 5-kb deletion on the blaNDM-7-harboring IncX3 plasmid. In addition, an IncFIB plasmid in KpN06 had a 27-kb deletion that removed genes encoding for heavy metal resistance. Phylogenetic analysis showed that the K. pneumoniae ST278 from patient 2 was likely a descendant of KpN02 and that KpN06 was a close progenitor of an environmental ST278. It is unclear whether KpN06 lost the blaNDM-7 gene in vivo. This study detailed the remarkable plasticity and speed of evolutionary changes in multidrug-resistant K. pneumoniae, demonstrating the highly recombinant nature of this species. It also highlights the ability of NGS to clarify molecular microevolutionary events within antibiotic-resistant organisms.© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

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July 7, 2019

Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems.

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. Here, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

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July 7, 2019

Comprehensive genomic and phenotypic metal resistance profile of Pseudomonas putida strain S13.1.2 isolated from a vineyard soil.

Trace metals are required in many cellular processes in bacteria but also induce toxic effects to cells when present in excess. As such, various forms of adaptive responses towards extracellular trace metal ions are essential for the survival and fitness of bacteria in their environment. A soil Pseudomonas putida, strain S13.1.2 has been isolated from French vineyard soil samples, and shown to confer resistance to copper ions. Further investigation revealed a high capacity to tolerate elevated concentrations of various heavy metals including nickel, cobalt, cadmium, zinc and arsenic. The complete genome analysis was conducted using single-molecule real-time (SMRT) sequencing and the genome consisted in a single chromosome at the size of 6.6 Mb. Presence of operons and gene clusters such as cop, cus, czc, nik, and asc systems were detected and accounted for the observed resistance phenotypes. The unique features in terms of specificity and arrangements of some genetic determinants were also highlighted in the study. Our findings has provided insights into the adaptation of this strain to accumulation and persistence of copper and other heavy metals in vineyard soil environment.

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July 7, 2019

Complete genome sequences of multidrug-resistant Campylobacter jejuni strain 14980A (turkey feces) and Campylobacter coli strain 14983A (housefly from a turkey farm), harboring a novel gentamicin resistance mobile element.

Multidrug resistance (MDR) in foodborne pathogens is a major food safety and public health issue. Here we describe whole-genome sequences of two MDR strains of Campylobacter jejuni and Campylobacter coli from turkey feces and a housefly from a turkey farm. Both strains harbor a novel chromosomal gentamicin resistance mobile element. Copyright © 2016 Miller et al.

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July 7, 2019

Comparative genomics and physiology of the butyrate-producing bacterium Intestinimonas butyriciproducens.

Intestinimonas is a newly described bacterial genus with representative strains present in the intestinal tract of human and other animals. Despite unique metabolic features including the production of butyrate from both sugars and amino acids, there is to date no data on their diversity, ecology, and physiology. Using a comprehensive phylogenetic approach, Intestinimomas was found to include at least three species that colonize primarily the human and mouse intestine. We focused on the most common and cultivable species of the genus, Intestinimonas butyriciproducens, and performed detailed genomic and physiological comparison of strains SRB521(T) and AF211, isolated from the mouse and human gut respectively. The complete 3.3-Mb genomic sequences of both strains were highly similar with 98.8% average nucleotide identity, testifying to their assignment to one single species. However, thorough analysis revealed significant genomic rearrangements, variations in phage-derived sequences, and the presence of new CRISPR sequences in both strains. Moreover, strain AF211 appeared to be more efficient than strain SRB521(T) in the conversion of the sugars arabinose and galactose. In conclusion, this study provides genomic and physiological insight into Intestinimonas butyriciproducens, a prevalent butyrate-producing species, differentiating strains that originate from the mouse and human gut.© 2016 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and JohnWiley & Sons Ltd.

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July 7, 2019

Complete sequencing of plasmids containing blaOXA-163 and blaOXA-48 in Escherichia coli ST131.

OXA-48-like enzymes have emerged as important extended-spectrum ß-lactamases/carbapenemases in E. coli ST131. We report the structure of the first fully sequenced blaOXA-163 plasmid, and of two other blaOXA-48 plasmids in this lineage. blaOXA-163 was located on a 71kb IncN plasmid with other resistance genes. blaOXA-48 was present on IncL/M plasmids, genetically similar to other blaOXA-48 plasmid sequences, and consistent with inter-species/inter-lineage spread. The presence of blaOXA-48-like genes on epidemic plasmids in ST131 is of concern. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Genomic analyses of multidrug resistant Pseudomonas aeruginosa PA1 resequenced by single-molecule real-time sequencing.

As a third-generation sequencing (TGS) method, single-molecule real-time (SMRT) technology provides long read length, and it is well suited for resequencing projects and de novo assembly. In the present study, Pseudomonas aeruginosa PA1 was characterized and resequenced using SMRT technology. PA1 was also subjected to genomic, comparative and pan-genomic analyses. The multidrug resistant strain PA1 possesses a 6,498,072 bp genome and a sequence type of ST-782. The genome of PA1 was also visualized, and the results revealed the details of general genome annotations, virulence factors, regulatory proteins (RPs), secretion system proteins, type II toxin-antitoxin (T-A) pairs and genomic islands. Whole genome comparison analysis suggested that PA1 exhibits similarity to other P. aeruginosa strains but differs in terms of horizontal gene transfer (HGT) regions, such as prophages and genomic islands. Phylogenetic analyses based on 16S rRNA sequences demonstrated that PA1 is closely related to PAO1, and P. aeruginosa strains can be divided into two main groups. The pan-genome of P. aeruginosa consists of a core genome of approximately 4,000 genes and an accessory genome of at least 6,600 genes. The present study presented a detailed, visualized and comparative analysis of the PA1 genome, to enhance our understanding of this notorious pathogen. © 2016 The Author(s).

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July 7, 2019

Clonal dissemination of Pseudomonas aeruginosa sequence type 235 isolates carrying blaIMP-6 and emergence of blaGES-24 and blaIMP-10 on novel genomic islands PAGI-15 and -16 in South Korea.

A total of 431 Pseudomonas aeruginosa clinical isolates were collected from 29 general hospitals in South Korea in 2015. Antimicrobial susceptibility was tested by the disk diffusion method, and MICs of carbapenems were determined by the agar dilution method. Carbapenemase genes were amplified by PCR and sequenced, and the structures of class 1 integrons surrounding the carbapenemase gene cassettes were analyzed by PCR mapping. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed for strain typing. Whole-genome sequencing was carried out to analyze P. aeruginosa genomic islands (PAGIs) carrying the blaIMP-6, blaIMP-10, and blaGES-24 genes. The rates of carbapenem-nonsusceptible and carbapenemase-producing P. aeruginosa isolates were 34.3% (148/431) and 9.5% (41/431), respectively. IMP-6 was the most prevalent carbapenemase type, followed by VIM-2, IMP-10, and GES-24. All carbapenemase genes were located on class 1 integrons of 6 different types on the chromosome. All isolates harboring carbapenemase genes exhibited genetic relatedness by PFGE (similarity > 80%); moreover, all isolates were identified as sequence type 235 (ST235), with the exception of two ST244 isolates by MLST. The blaIMP-6, blaIMP-10, and blaGES-24 genes were found to be located on two novel PAGIs, designated PAGI-15 and PAGI-16. Our data support the clonal spread of an IMP-6-producing P. aeruginosa ST235 strain, and the emergence of IMP-10 and GES-24 demonstrates the diversification of carbapenemases in P. aeruginosa in Korea. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

FDA-CDC antimicrobial resistance isolate bank: A publicly-available resource to support research, development and regulatory requirements.

The FDA-CDC Antimicrobial Resistance Isolate Bank was created in July 2015 as a publicly available resource to combat antimicrobial resistance. It is a curated repository of bacterial isolates with an assortment of clinically-important resistance mechanisms that have been phenotypically and genotypically characterized. In the first two years of operation, the Bank offered 14 panels comprising 496 unique isolates and had filled 486 orders from 394 institutions throughout the United States. New panels are being added. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Rhodobacter sp. Rb3, an aerobic anoxygenic phototroph which thrives in the polyextreme ecosystem of the Salar de Huasco, in the Chilean Altiplano.

The Salar de Huasco is an evaporitic basin located in the Chilean Altiplano, which presents extreme environmental conditions for life, i.e. high altitude (3800 m.a.s.l.), negative water balance, a wide salinity range, high daily temperature changes and the occurrence of the highest registered solar radiation on the planet (>?1200 W m-2). This ecosystem is considered as a natural laboratory to understand different adaptations of microorganisms to extreme conditions. Rhodobacter, an anoxygenic aerobic phototrophic bacterial genus, represents one of the most abundant groups reported based on taxonomic diversity surveys in this ecosystem. The bacterial mat isolate Rhodobacter sp. strain Rb3 was used to study adaptation mechanisms to stress-inducing factors potentially explaining its success in a polyextreme ecosystem. We found that the Rhodobacter sp. Rb3 genome was characterized by a high abundance of genes involved in stress tolerance and adaptation strategies, among which DNA repair and oxidative stress were the most conspicuous. Moreover, many other molecular mechanisms associated with oxidative stress, photooxidation and antioxidants; DNA repair and protection; motility, chemotaxis and biofilm synthesis; osmotic stress, metal, metalloid and toxic anions resistance; antimicrobial resistance and multidrug pumps; sporulation; cold shock and heat shock stress; mobile genetic elements and toxin-antitoxin system were detected and identified as potential survival mechanism features in Rhodobacter sp. Rb3. In total, these results reveal a wide set of strategies used by the isolate to adapt and thrive under environmental stress conditions as a model of polyextreme environmental resistome.

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July 7, 2019

Molecular preadaptation to antimony resistance in Leishmania donovani on the Indian subcontinent.

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (SbIII) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline SbIII susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIII resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to SbIII resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of SbIII The main driver of this preadaptation was shown to be MRPA, a gene involved in SbIII sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE The “antibiotic resistance crisis” is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century. Copyright © 2018 Dumetz et al.

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July 7, 2019

Emerging mechanisms of antimicrobial resistance in bacteria and fungi: advances in the era of genomics.

Bacteria and fungi continue to develop new ways to adapt and survive the lethal or biostatic effects of antimicrobials through myriad mechanisms. Novel antibiotic resistance genes such as lsa(C), erm(44), VCC-1, mcr-1, mcr-2, mcr-3, mcr-4, bla KLUC-3 and bla KLUC-4 were discovered through comparative genomics and further functional studies. As well, mutations in genes that hitherto were unknown to confer resistance to antimicrobials, such as trm, PP2C, rpsJ, HSC82, FKS2 and Rv2887, were shown by genomics and transcomplementation assays to mediate antimicrobial resistance in Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Saccharomyces cerevisae, Candida glabrata and Mycobacterium tuberculosis, respectively. Thus, genomics, transcriptomics and metagenomics, coupled with functional studies are the future of antimicrobial resistance research and novel drug discovery or design.

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July 7, 2019

Analysis of resistance genes of clinical Pannonibacter phragmitetus strain 31801 by complete genome sequencing.

To clarify the resistance mechanisms of Pannonibacter phragmitetus 31801, isolated from the blood of a liver abscess patient, at the genomic level, we performed whole genomic sequencing using a PacBio RS II single-molecule real-time long-read sequencer. Bioinformatic analysis of the resulting sequence was then carried out to identify any possible resistance genes. Analyses included Basic Local Alignment Search Tool searches against the Antibiotic Resistance Genes Database, ResFinder analysis of the genome sequence, and Resistance Gene Identifier analysis within the Comprehensive Antibiotic Resistance Database. Prophages, clustered regularly interspaced short palindromic repeats (CRISPR), and other putative virulence factors were also identified using PHAST, CRISPRfinder, and the Virulence Factors Database, respectively. The circular chromosome and single plasmid of P. phragmitetus 31801 contained multiple antibiotic resistance genes, including those coding for three different types of ß-lactamase [NPS ß-lactamase (EC 3.5.2.6), ß-lactamase class C, and a metal-dependent hydrolase of ß-lactamase superfamily I]. In addition, genes coding for subunits of several multidrug-resistance efflux pumps were identified, including those targeting macrolides (adeJ, cmeB), tetracycline (acrB, adeAB), fluoroquinolones (acrF, ceoB), and aminoglycosides (acrD, amrB, ceoB, mexY, smeB). However, apart from the tripartite macrolide efflux pump macAB-tolC, the genome did not appear to contain the complete complement of subunit genes required for production of most of the major multidrug-resistance efflux pumps.

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July 7, 2019

Transposon insertion sequencing elucidates novel gene involvement in susceptibility and resistance to phages T4 and T7 in Escherichia coli O157.

Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens.IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication. Copyright © 2018 Cowley et al.

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