Occurrence of <em>Vibrio</em> and <em>Salmonella</em> species in mussels (<em>Mytilus</em> <em>galloprovincialis</em>) collected along the Moroccan Atlantic coast

This study reports the occurrence of different Vibrio and Salmonella species in 52 samples of Mytilus galloprovincialis collected from four sites along the Atlantic coast between Agadir and Essaouira (Anza, Cap Ghir, Imssouane and Essaouira). The level of Escherichia coli (E. coli) was also determined to evaluate the degree of microbial pollution in the investigated areas. In this study three methods were used : AFNOR NF EN ISO 6579 V08-013 for Salmonella spp., the provisional method routinely used by several laboratories (Institut Pasteur, Paris,…) for Vibrio cholerae and Vibrio parahaemolyticus in the seafood, and the most probable number method (MPN) using Norm ISO/TS 16649–3 (2005) for E. coli. The most frequently isolated Vibrios were Vibrio alginolyticus (90.4% of samples), followed by V. cholerae non O1 non O139 (15.4%) and V. parahaemolyticus (7.7%). Salmonella spp. was found in 15% of the samples. The number of E. coli ranged between 0.2/100 g and 1.8 10(3) /100 g of mussel soft tissues. This study indicates the potential sanitary risk associated with the presence of pathogenic bacteria in cultivated mussels in the two populous regions of southern Morocco, where shellfish production and maritime tourism are important to the local economy

Vibrios from the Norwegian marine environment: Characterization of associated antibiotic resistance and virulence genes

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Revealing Genomic Insights of the Unexplored Porcine Pathogen Actinobacillus pleuropneumoniae Using Whole Genome Sequencing

Actinobacillus pleuropneumoniae (APP) is the causative agent of pleuropneumonia in pigs, one of the most relevant bacterial respiratory diseases in the swine industry. To date, 19 serotypes have been described based on capsular polysaccharide typing with significant virulence dissimilarities. In this study, 16 APP isolates from Spanish origin were selected to perform antimicrobial susceptibility tests and comparative genomic analysis using whole genome sequencing (WGS). To obtain a more comprehensive worldwide molecular epidemiologic analyses, all APP whole genome assemblies available at the National Center for Biotechnology Information (NCBI) at the time of the study were also included. An in-house in silico PCR approach enabled the correct serotyping of unserotyped or incorrectly serotyped isolates and allowed for the discrimination between serotypes 9 and 11. A pangenome analysis identified the presence or absence of gene clusters to be serotype specific, as well as virulence profile analyses targeting the apx operons. Antimicrobial resistance genes were correlated to the presence of specific plasmids. Altogether, this study provides new insights into the genetic variability within APP serotypes, correlates phenotypic tests with bioinformatic analyses and manifests the benefits of populated databases for a better assessment of diversity and variability of relatively unknown pathogens. Overall, genomic comparative analysis enhances the understanding of transmission and epidemiological patterns of this species and suggests vertical transmission of the pathogen, including the resistance genes, within the Spanish integrated systems.info:eu-repo/semantics/publishedVersio

Transoceanic spreading of pathogenic strains of <i>Vibrio parahaemolyticus</i> with distinctive genetic signatures in the<i> recA</i> gene

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090-96, Peru, 1996), the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%), and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3) identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family

mcr-Colistin resistance genes mobilized by IncX4, IncHI2, and IncI2 plasmids in Escherichia coli of pigs and white stork in Spain

Colistin has become the last-line antimicrobial for the treatment of multidrug resistant (MDR) Enterobacterales in human medicine. To date, several colistin resistance genes have been described. Of them mcr-1 is disseminated worldwide in Escherichia coli of human and animal origin. The aim of this study was to characterize mcr-mediated resistance plasmids from E. coli of animal origin in Spain. From our strain collection, 70 E. coli of pig origin collected between 2005 and 2014 (10 per year, except for years 2009-2010-2013) were randomly selected and screened for the presence of mcr-genes. Additionally, 20 E. coli isolated in 2011 from white storks (Ciconia ciconia) from the same urban household waste landfill associated colony were also included. Whole genome sequencing of mcr-positive isolates was carried out on a MiSeq (Illumina). Hybrid whole genome sequencing strategy combining nanopore and Illumina technologies were performed in a selection of isolates to close the genomes and plasmids and identify the presence of antimicrobial resistance genes. Minimum inhibitory concentration (MIC) was used to assess the susceptibility to colistin. Mating experiments were carried out to evaluate transferability of the mcr-genes. A total of 19 mcr-1 and one mcr-4 positive isolates were detected, 15 from pigs distributed during the study period, and five from storks collected in 2011. No other mcr-variants were found. The MICs for colistin ranged between 4 and >4 mg/L. High diversity of STs were detected among the mcr-1 positive E. coli isolates, with only ST-10 shared between pigs and white storks. Except for one isolate, all were genotypic and phenotypically MDR, and five of them also harbored cephalosporin resistance genes (bla CTX-M- 14, bla SHV- 12, and three bla CMY- 2). mcr-1 genes were mobilizable by conjugation, associated with IncX4, IncHI2, and IncI2 plasmids. In our study, mcr-1 genes have been circulating in pig farms since 2005 harbored by a variety of E. coli clones. Its persistence may be driven by co-selection since plasmids containing mcr-1 also exhibit resistance to multiple drugs used in veterinary medicine. Furthermore, this is the first report of the presence of mcr-1 gene in isolates from white storks in Spain. This finding highlights the potential importance of wildlife that forage at urban household waste landfills in the transmission and spread of colistin resistance genes.info:eu-repo/semantics/publishedVersio

mcr -Colistin Resistance Genes Mobilized by IncX4, IncHI2, and IncI2 Plasmids in Escherichia coli of Pigs and White Stork in Spain

Colistin has become the last-line antimicrobial for the treatment of multidrug resistant (MDR) Enterobacterales in human medicine. To date, several colistin resistance genes have been described. Of them mcr -1 is disseminated worldwide in Escherichia coli of human and animal origin. The aim of this study was to characterize mcr -mediated resistance plasmids from E. coli of animal origin in Spain. From our strain collection, 70 E. coli of pig origin collected between 2005 and 2014 (10 per year, except for years 2009-2010-2013) were randomly selected and screened for the presence of mcr -genes. Additionally, 20 E. coli isolated in 2011 from white storks (Ciconia ciconia) from the same urban household waste landfill associated colony were also included. Whole genome sequencing of mcr -positive isolates was carried out on a MiSeq (Illumina). Hybrid whole genome sequencing strategy combining nanopore and Illumina technologies were performed in a selection of isolates to close the genomes and plasmids and identify the presence of antimicrobial resistance genes. Minimum inhibitory concentration (MIC) was used to assess the susceptibility to colistin. Mating experiments were carried out to evaluate transferability of the mcr -genes. A total of 19 mcr -1 and one mcr -4 positive isolates were detected, 15 from pigs distributed during the study period, and five from storks collected in 2011. No other mcr -variants were found. The MICs for colistin ranged between 4 and >4 mg/L. High diversity of STs were detected among the mcr-1 positive E. coli isolates, with only ST-10 shared between pigs and white storks. Except for one isolate, all were genotypic and phenotypically MDR, and five of them also harbored cephalosporin resistance genes (bla , bla , and three bla ). mcr -1 genes were mobilizable by conjugation, associated with IncX4, IncHI2, and IncI2 plasmids. In our study, mcr -1 genes have been circulating in pig farms since 2005 harbored by a variety of E. coli clones. Its persistence may be driven by co-selection since plasmids containing mcr -1 also exhibit resistance to multiple drugs used in veterinary medicine. Furthermore, this is the first report of the presence of mcr -1 gene in isolates from white storks in Spain. This finding highlights the potential importance of wildlife that forage at urban household waste landfills in the transmission and spread of colistin resistance genes