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April 21, 2020

Novel trimethoprim resistance gene dfrA34 identified in Salmonella Heidelberg in the USA.

Trimethoprim/sulfamethoxazole is a synthetic antibiotic combination recommended for the treatment of complicated non-typhoidal Salmonella infections in humans. Resistance to trimethoprim/sulfamethoxazole is mediated by the acquisition of mobile genes, requiring both a dfr gene (trimethoprim resistance) and a sul gene (sulfamethoxazole resistance) for a clinical resistance phenotype (MIC =4/76?mg/L). In 2017, the CDC investigated a multistate outbreak caused by a Salmonella enterica serotype Heidelberg strain with trimethoprim/sulfamethoxazole resistance, in which sul genes but no known dfr genes were detected.To characterize and describe the molecular mechanism of trimethoprim resistance in a Salmonella Heidelberg outbreak isolate.Illumina sequencing data for one outbreak isolate revealed a 588?bp ORF encoding a putative dfr gene. This gene was cloned into Escherichia coli and resistance to trimethoprim was measured by broth dilution and Etest. Phylogenetic analysis of previously reported dfrA genes was performed using MEGA. Long-read sequencing was conducted to determine the context of the novel dfr gene.The novel dfr gene, named dfrA34, conferred trimethoprim resistance (MIC =32?mg/L) when cloned into E. coli. Based on predicted amino acid sequences, dfrA34 shares less than 50% identity with other known dfrA genes. The dfrA34 gene is located in a class 1 integron in a multiresistance region of an IncC plasmid, adjacent to a sul gene, thus conferring clinical trimethoprim/sulfamethoxazole resistance. Additionally, dfrA34 is associated with ISCR1, enabling easy transmission between other plasmids and bacterial strains.

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April 21, 2020

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020

One Aeromonas salmonicida subsp. salmonicida isolate with a pAsa5 variant bearing antibiotic resistance and a pRAS3 variant making a link with a swine pathogen.

The Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is an aquatic pathogen which causes furunculosis to salmonids, especially in fish farms. The emergence of strains of this bacterium exhibiting antibiotic resistance is increasing, limiting the effectiveness of antibiotherapy as a treatment against this worldwide disease. In the present study, we discovered an isolate of A. salmonicida subsp. salmonicida that harbors two novel plasmids variants carrying antibiotic resistance genes. The use of long-read sequencing (PacBio) allowed us to fully characterize those variants, named pAsa5-3432 and pRAS3-3432, which both differ from their classic counterpart through their content in mobile genetic elements. The plasmid pAsa5-3432 carries a new multidrug region composed of multiple mobile genetic elements, including a Class 1 integron similar to an integrated element of Salmonella enterica. With this new region, probably acquired through plasmid recombination, pAsa5-3432 is the first reported plasmid of this bacterium that bears both an essential virulence factor (the type three secretion system) and multiple antibiotic resistance genes. As for pRAS3-3432, compared to the classic pRAS3, it carries a new mobile element that has only been identified in Chlamydia suis. Hence, with the identification of those two novel plasmids harboring mobile genetic elements that are normally encountered in other bacterial species, the present study puts emphasis on the important impact of mobile genetic elements in the genomic plasticity of A. salmonicida subsp. salmonicida and suggests that this aquatic bacterium could be an important reservoir of antibiotic resistance genes that can be exchanged with other bacteria, including human and animal pathogens. Copyright © 2019 Elsevier B.V. All rights reserved.

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April 21, 2020

Multidrug-Resistant Bovine Salmonellosis Predisposing for Severe Human Clostridial Myonecrosis.

BACKGROUND The overuse of antibiotics in animals promotes the development of multidrug-resistance predisposing for severe polymicrobial human infections. CASE REPORT We describe a case of spontaneous clostridial myonecrosis due to ulcerative colonic infection with multidrug-resistant Salmonella enterica subsp. enterica, serotype 4,[5],12: i: -. Serotyping of the colonic Salmonella isolate in the index case and the bovine farm outbreak isolates from where the patient worked indicated they were both serotype I 4,[5],12: i: -, which is linked with a multitude of large reported disease outbreaks. Further analysis revealed that they are highly genetically related and antibiotic susceptibility testing indicated that they are phenotypically identical. CONCLUSIONS Enteritis due to human acquisition of multidrug-resistant Salmonella from cattle led to the invasion and dissemination of Clostridium septicum resulting in devastating myonecrotic disease. This highlights the ramifications of co-existence and evolution of pathogenic bacteria in animals and humans and lends support to reducing the use of antibiotics in animals.

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April 21, 2020

Carbohydrate catabolic capability of a Flavobacteriia bacterium isolated from hadal water.

Flavobacteriia are abundant in many marine environments including hadal waters, as demonstrated recently. However, it is unclear how this flavobacterial population adapts to hadal conditions. In this study, extensive comparative genomic analyses were performed for the flavobacterial strain Euzebyella marina RN62 isolated from the Mariana Trench hadal water in low abundance. The complete genome of RN62 possessed a considerable number of carbohydrate-active enzymes with a different composition. There was a predominance of GH family 13 proteins compared to closely related relatives, suggesting that RN62 has preserved a certain capacity for carbohydrate utilization and that the hadal ocean may hold an organic matter reservoir distinct from the surface ocean. Additionally, RN62 possessed potential intracellular cycling of the glycogen/starch pathway, which may serve as a strategy for carbon storage and consumption in response to nutrient pulse and starvation. Moreover, the discovery of higher glycoside hydrolase dissimilarities among Flavobacteriia, compared to peptidases and transporters, suggested variation in polysaccharide utilization related traits as an important ecophysiological factor in response to environmental alterations, such as decreased labile organic carbon in hadal waters. The presence of abundant toxin exporting, transcription and signal transduction related genes in RN62 may further help to survive in hadal conditions, including high pressure/low temperature.Copyright © 2019 Elsevier GmbH. All rights reserved.

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April 21, 2020

Genetic characterization and potential molecular dissemination mechanism of tet(31) gene in Aeromonas caviae from an oxytetracycline wastewater treatment system.

Recently, the rarely reported tet(31) tetracycline resistance determinant was commonly found in Aeromonas salmonicida, Gallibacterium anatis, and Oblitimonas alkaliphila isolated from farming animals and related environment. However, its distribution in other bacteria and potential molecular dissemination mechanism in environment are still unknown. The purpose of this study was to investigate the potential mechanism underlying dissemination of tet(31) by analysing the tet(31)-carrying fragments in A. caviae strains isolated from an aerobic biofilm reactor treating oxytetracycline bearing wastewater. Twenty-three A. caviae strains were screened for the tet(31) gene by polymerase chain reaction (PCR). Three strains (two harbouring tet(31), one not) were subjected to whole genome sequencing using the PacBio RSII platform. Seventeen A. caviae strains carried the tet(31) gene and exhibited high resistance levels to oxytetracycline with minimum inhibitory concentrations (MICs) ranging from 256 to 512?mg/L. tet(31) was comprised of the transposon Tn6432 on the chromosome of A. caviae, and Tn6432 was also found in 15 additional tet(31)-positive A. caviae isolates by PCR. More important, Tn6432 was located on an integrative conjugative element (ICE)-like element, which could mediate the dissemination of the tet(31)-carrying transposon Tn6432 between bacteria. Comparative analysis demonstrated that Tn6432 homologs with the structure ISCR2-?phzF-tetR(31)-tet(31)-?glmM-sul2 were also carried by A. salmonicida, G. anatis, and O. alkaliphila, suggesting that this transposon can be transferred between species and even genera. This work provides the first report on the identification of the tet(31) gene in A. caviae, and will be helpful in exploring the dissemination mechanisms of tet(31) in water environment.Copyright © 2018. Published by Elsevier B.V.

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April 21, 2020

Iron-associated protein interaction networks reveal the key functional modules related to survival and virulence of Pasteurella multocida.

Pasteurella multocida causes respiratory infectious diseases in a multitude of birds and mammals. A number of virulence-associated genes were reported across different strains of P. multocida, including those involved in the iron transport and metabolism. Comparative iron-associated genes of P. multocida among different animal hosts towards their interaction networks have not been fully revealed. Therefore, this study aimed to identify the iron-associated genes from core- and pan-genomes of fourteen P. multocida strains and to construct iron-associated protein interaction networks using genome-scale network analysis which might be associated with the virulence. Results showed that these fourteen strains had 1587 genes in the core-genome and 3400 genes constituting their pan-genome. Out of these, 2651 genes associated with iron transport and metabolism were selected to construct the protein interaction networks and 361 genes were incorporated into the iron-associated protein interaction network (iPIN) consisting of nine different iron-associated functional modules. After comparing with the virulence factor database (VFDB), 21 virulence-associated proteins were determined and 11 of these belonged to the heme biosynthesis module. From this study, the core heme biosynthesis module and the core outer membrane hemoglobin receptor HgbA were proposed as candidate targets to design novel antibiotics and vaccines for preventing pasteurellosis across the serotypes or animal hosts for enhanced precision agriculture to ensure sustainability in food security. Copyright © 2018. Published by Elsevier Ltd.

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April 21, 2020

Integration of two pKPX-2-derived antibiotic resistance islands in the genome of an ESBL-producing Klebsiella pneumoniae ST3483 from Lebanon.

Contamination of fresh water with clinically important Gram-negative bacteria in Lebanon is being investigated in-depth, especially with evidence of dissemination into clinical settings. This study aimed to report the draft genome sequence of a Klebsiella pneumoniae strain with an integrated plasmid segment harbouring two antibiotic resistance islands (ARI). It is believed that this is the first report of plasmid antibiotic resistance islands integration in the genome of K. pneumoniae.Whole genome sequencing of the isolate was performed using Sequel platform. The genome was assembled using HGAP4. Analysis was conducted by uploading the sequence to the online databases from the Center for Genomic Epidemiology.The strain had a newly assigned ST 3483 with a genome size of 5385844 bp. The investigation of the antibiotic resistance islands suggested integration of two DNA segments from a previously identified IncFIA plasmid. The results revealed that the integration could have been accomplished either as a single-step integration event, with the two segments being integrated as a whole transposon mediated by the flanking IS26, or through two separate integration events involving the two segments, but independently.The sequenced genome revealed interesting aspects related to antibiotic resistance dissemination. The ARI are more stable in the genome and the chance of losing it is less probable, with the possibility of the described transposon to re-integrate in other plasmids, facilitating the dissemination of such resistance determinants.Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.

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April 21, 2020

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation.

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp. Copyright © 2017 Elsevier Inc. All rights reserved.

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April 21, 2020

Epidemiologic and genomic insights on mcr-1-harbouring Salmonella from diarrhoeal outpatients in Shanghai, China, 2006-2016.

Colistin resistance mediated by mcr-1-harbouring plasmids is an emerging threat in Enterobacteriaceae, like Salmonella. Based on its major contribution to the diarrhoea burden, the epidemic state and threat of mcr-1-harbouring Salmonella in community-acquired infections should be estimated.This retrospective study analysed the mcr-1 gene incidence in Salmonella strains collected from a surveillance on diarrhoeal outpatients in Shanghai Municipality, China, 2006-2016. Molecular characteristics of the mcr-1-positive strains and their plasmids were determined by genome sequencing. The transfer abilities of these plasmids were measured with various conjugation strains, species, and serotypes.Among the 12,053 Salmonella isolates, 37 mcr-1-harbouring strains, in which 35 were serovar Typhimurium, were detected first in 2012 and with increasing frequency after 2015. Most patients infected with mcr-1-harbouring strains were aged <5?years. All strains, including fluoroquinolone-resistant and/or extended-spectrum ß-lactamase-producing strains, were multi-drug resistant. S. Typhimurium had higher mcr-1 plasmid acquisition ability compared with other common serovars. Phylogeny based on the genomes combined with complete plasmid sequences revealed some clusters, suggesting the presence of mcr-1-harbouring Salmonella outbreaks in the community. Most mcr-1-positive strains were clustered together with the pork strains, strongly suggesting pork consumption as a main infection source.The mcr-1-harbouring Salmonella prevalence in community-acquired diarrhoea displays a rapid increase trend, and the ESBL-mcr-1-harbouring Salmonella poses a threat for children. These findings highlight the necessary and significance of prohibiting colistin use in animals and continuous monitoring of mcr-1-harbouring Salmonella.Copyright © 2019. Published by Elsevier B.V.

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April 21, 2020

Genomic Characterization of a Newly Isolated Rhizobacteria Sphingomonas panacis Reveals Plant Growth Promoting Effect to Rice

This article reports the full genome sequence of Sphingomonas panacis DCY99T (=KCTC 42347T =JCM30806T), which is a Gram-negative rod-shaped, non-spore forming, motile bacterium isolated from rusty ginseng root in South Korea. A draft genome of S. panacis DCY99T and a single circular plasmid were generated using the PacBio platform. Antagonistic activity experiment showed S. panacis DCY99T has the plant growth promoting effect. Thus, the genome sequence of S. panacis DCY99T may contribute to biotechnological application of the genus Sphingomonas in agriculture.

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April 21, 2020

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.

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April 21, 2020

Dysbiosis and Variation in Predicted Functions of the Granulation Tissue Microbiome in HPV Positive and Negative Severe Chronic Periodontitis.

Retrospective analysis has already shown correlation between severe Chronic Periodontitis (CP) cases with human papiloma virus (HPV). Hence, we aimed to explore deep-seated infected granulation tissue removed during periodontal flap surgery procedures for residential bacterial species between HPV+ and HVP- CP cases, which may serve as good predisposition marker for oral cancer. All CP-granulation samples showed the prominence of Firmicutes, Proteobacteria, and Bacteroidetes phyla with an abundance of gram negative anaerobes, except Streptococcus. In Beta diversity nonmetric multidimensional scaling plot, the random distribution of species was observed between HPV+ and HPV- CP granulation-samples. However, an abundance of Capnocytophaga ochracea was observed in HPV+ CP samples (p<0.05), while Porphyromonas endodontalis, Macellibacteroides fermentas, Treponema phagedenis, and Campylobacter rectus species were highly abundant in HPV- CP samples (p<0.05). The differential species richness leads altered functions related to mismatch-repair and nucleotide excision-repair and cytoskeleton-proteins. Hence, differential abundance of gram negative bacterial species between HPV+ and HPV- granulation-samples under anaerobic conditions may release virulence factors which may alter pathways favouring carcinogenesis. Hence, these species may serve as good predisposition marker for oral-cancer.

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April 21, 2020

Complete Genome Sequence of Photobacterium damselae Subsp. damselae Strain SSPD1601 Isolated from Deep-Sea Cage-Cultured Sebastes schlegelii with Septic Skin Ulcer.

Photobacterium damselae subsp. damselae (PDD) is a Gram-negative bacterium that can infect a variety of aquatic organisms and humans. Based on an epidemiological investigation conducted over the past 3 years, PDD is one of the most important pathogens causing septic skin ulcer in deep-sea cage-cultured Sebastes schlegelii in the Huang-Bohai Sea area and present throughout the year with high abundance. To further understand the pathogenicity of this species, the pathogenic properties and genome of PDD strain SSPD1601 were analyzed. The results revealed that PDD strain SSPD1601 is a rod-shaped cell with a single polar flagellum, and the clinical symptoms were replicated during artificial infection. The SSPD1601 genome consists of two chromosomes and two plasmids, totaling 4,252,294?bp with 3,751 coding sequences (CDSs), 196 tRNA genes, and 47 rRNA genes. Common virulence factors including flagellin, Fur, RstB, hcpA, OMPs, htpB-Hsp60, VasK, and vgrG were found in strain SSPD1601. Furthermore, SSPD1601 is a pPHDD1-negative strain containing the hemolysin gene hlyAch and three putative hemolysins (emrA, yoaF, and VPA0226), which are likely responsible for the pathogenicity of SSPD1601. The phylogenetic analysis revealed SSPD1601 to be most closely related to Phdp Wu-1. In addition, the antibiotic resistance phenotype indicated that SSPD1601 was not sensitive to ceftazidime, pipemidic, streptomycin, cefalexin, bacitracin, cefoperazone sodium, acetylspiramycin, clarithromycin, amikacin, gentamycin, kanamycin, oxacillin, ampicillin, and trimethoprim-sulfamethoxazole, but only the bacitracin resistance gene bacA was detected based on Antibiotic Resistance Genes Database. These results expand our understanding of PDD, setting the stage for further studies of its pathogenesis and disease prevention.

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