In-Depth Genomic Characterization of a Meropenem-nonsusceptible Pseudomonas otitidis Strain Contaminating Chicken Carcass

Background: The indiscriminate use of antibiotics in food-animal production has a major impact on public health, particularly in terms of contributing to the emergence and dissemination of antimicrobial resistant bacteria in the food-animal production chain. Although Pseudomonas species are recognized as important spoilage organisms in foodstuff, they are also known as opportunistic pathogens associated with hospital-acquired infections. Furthermore, Pseudomonas can play a role as potential reservoirs of antimicrobial resistance genes, which may be horizontally transferred to other bacteria. Considering that cephalosporins (3rd and higher generations) and carbapenems are critically important beta-lactam antimicrobials in human medicine, this study reports the occurrence and genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain recovered from a chicken carcass in Brazil.Materials, Methods & Results: During the years 2018-2019, 72 frozen chicken carcasses were purchased on the retail market from different regions in Brazil. Aliquots from individual carcass rinses were screened for Extended Spectrum Beta-lactamase (ESBL)-producing bacteria in MacConkey agar supplemented with 1mg.L-1 cefotaxime. Phenotypically resistant isolates were further tested for resistance to other antimicrobials and confirmed as ESBL-producers by means of disk-diffusion method using Müller-Hinton agar. Only one meropenen-nonsusceptible isolate was detected and submitted to whole genome sequencing (WGS) in Illumina Miseq. The strain was identified as Pseudomonas otitidis by local alignment of the 16S rRNA sequence using BLASTn and confirmed by Average Nucleotide Identity (ANI) analysis using JspeciesWS database. Genes encoding for antimicrobial resistance were detected by means of Resfinder and Comprehensive Antibiotic Resistance Database (CARD) databases. The phenotypic non-susceptibility to meropenen was attributed to the gene blaPOM-1. A total of 192 different genes encoding for quorum sensing system, antiphagocytosis, iron uptake, efflux pump, endotoxin and toxin, adherence, and secretion systems were detected by means of Virulence Factor Database (VFDB). Pseudomonas otitidis-pan genome was built using Roary-rapid large-scale prokaryote pan genome analysis using the present strain (K_25) and other two P. otitidis genomes (PAM-1, DSM 17224) publicly available at the NCBI. The core genome analysis of the two human strains resulted in similar percentages.Discussion: Carbapenems are critically important drugs for human health and bacterial strains resistant to these antimicrobials pose a public health problem. The blaPOM-1 gene harbored by the Pseudomonas otitidis K_25 strain encodes a metallo-beta-lactamase (MBL) conferring resistance to carbapenems. Pseudomonas otitidis was the first confirmed pathogenic Pseudomonas species expressing MBL constitutively in the absence of inducible beta-lactamase genes. Furthermore, the several virulence genes associated with the capacity of the P. otitidis K_25 to colonize, evade the immune system and cause lesions in the human host confirm this strain as a potential opportunistic pathogen contaminating foodstuff. These reinforce the need to address antimicrobial resistance in a One Health perspective, in which resistant bacteria and resistance determinants circulate among environment, animals and humans

Genome Sequence of the Fish Brain Bacterium Clostridium tarantellae

Eubacterium tarantellae was originally cultivated from the brain of fish affected by twirling movements. Here, we present the draft genome sequence of E. tarantellae DSM 3997, which consists of 3,982,316\u2009bp. Most protein-coding genes in this strain are similar to genes of Clostridium bacteria, supporting the renaming of E. tarantellae as Clostridium tarantella

In-depth genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain contaminating chicken carcass

Background: The indiscriminate use of antibiotics in food-animal production has a major impact on public health, particularly in terms of contributing to the emergence and dissemination of antimicrobial resistant bacteria in the food-animal production chain. Although Pseudomonas species are recognized as important spoilage organisms in foodstuff, they are also known as opportunistic pathogens associated with hospital-acquired infections. Furthermore, Pseudomonas can play a role as potential reservoirs of antimicrobial resistance genes, which may be horizontally transferred to other bacteria. Considering that cephalosporins (3rd and higher generations) and carbapenems are critically important beta-lactam antimicrobials in human medicine, this study reports the occurrence and genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain recovered from a chicken carcass in Brazil. Materials, Methods & Results: During the years 2018-2019, 72 frozen chicken carcasses were purchased on the retail market from different regions in Brazil. Aliquots from individual carcass rinses were screened for Extended Spectrum Beta-lactamase (ESBL)-producing bacteria in MacConkey agar supplemented with 1mg.L-1 cefotaxime. Phenotypically resistant isolates were further tested for resistance to other antimicrobials and confirmed as ESBL-producers by means of disk-diffusion method using Müller-Hinton agar. Only one meropenen-nonsusceptible isolate was detected and submitted to whole genome sequencing (WGS) in Illumina Miseq. The strain was identified as Pseudomonas otitidis by local alignment of the 16S rRNA sequence using BLASTn and confirmed by Average Nucleotide Identity (ANI) analysis using JspeciesWS database. Genes encoding for antimicrobial resistance were detected by means of Resfinder and Comprehensive Antibiotic Resistance Database (CARD) databases. The phenotypic non-susceptibility to meropenen was attributed to the gene blaPOM-1. A total of 192 different genes encoding for quorum sensing system, antiphagocytosis, iron uptake, efflux pump, endotoxin and toxin, adherence, and secretion systems were detected by means of Virulence Factor Database (VFDB). Pseudomonas otitidis-pan genome was built using Roary-rapid large-scale prokaryote pan genome analysis using the present strain (K_25) and other two P. otitidis genomes (PAM-1, DSM 17224) publicly available at the NCBI. The core genome analysis of the two human strains resulted in similar percentages. Discussion: Carbapenems are critically important drugs for human health and bacterial strains resistant to these antimicrobials pose a public health problem. The blaPOM-1 gene harbored by the Pseudomonas otitidis K_25 strain encodes a metallo-beta-lactamase (MBL) conferring resistance to carbapenems. Pseudomonas otitidis was the first confirmed pathogenic Pseudomonas species expressing MBL constitutively in the absence of inducible beta-lactamase genes. Furthermore, the several virulence genes associated with the capacity of the P. otitidis K_25 to colonize, evade the immune system and cause lesions in the human host confirm this strain as a potential opportunistic pathogen contaminating foodstuff. These reinforce the need to address antimicrobial resistance in a One Health perspective, in which resistant bacteria and resistance determinants circulate among environment, animals and humans

gcType : a high-quality type strain genome database for microbial phylogenetic and functional research

Taxonomic and functional research of microorganisms has increasingly relied upon genome-based data and methods. As the depository of the Global Catalogue of Microorganisms (GCM) 10K prokaryotic type strain sequencing project, Global Catalogue of Type Strain (gcType) has published 1049 type strain genomes sequenced by the GCM 10K project which are preserved in global culture collections with a valid published status. Additionally, the information provided through gcType includes >12 000 publicly available type strain genome sequences from GenBank incorporated using quality control criteria and standard data annotation pipelines to form a high-quality reference database. This database integrates type strain sequences with their phenotypic information to facilitate phenotypic and genotypic analyses. Multiple formats of cross-genome searches and interactive interfaces have allowed extensive exploration of the database's resources. In this study, we describe web-based data analysis pipelines for genomic analyses and genome-based taxonomy, which could serve as a one-stop platform for the identification of prokaryotic species. The number of type strain genomes that are published will continue to increase as the GCM 10K project increases its collaboration with culture collections worldwide. Data of this project is shared with the International Nucleotide Sequence Database Collaboration. Access to gcType is free at http://gctype.wdcm.org/

First reported genome of an mcr-9-mediated colistin-resistant Salmonella Typhimurium isolate from Brazilian livestock

Objectives: To investigate the genetic context of colistin resistance in anmcr-9-harbouring Salmonella Typhimurium ST19 strain from swine in Brazil. Methods: Minimum inhibitory concentrations (MIC) to colistin were determined by broth microdilution. Whole-genome sequencing was performed on an Illumina MiSeq system, followed by de novo genome assembly using SPAdes 1.13.1. The draft genome sequence was annotated in Prokka using KBase online server. Downstream analyses for resistome and plasmid detection were performed using online tools available at the Center for Genomic Epidemiology. The strain was typed in silico using MLST 2.0. Phylogenetic analysis involving 24 other genomes of Salmonella Typhimurium ST19 and mcr-9-harbouring Salmonella Typhimurium isolated from humans, livestock and foodstuff in different regions was also performed. Results: Assembly of the draft genome resulted in 5245 protein-coding sequences, 14 rRNAs, 83 tRNAs and a GC content of 51.81%. The strain was identified as Salmonella Typhimurium ST19 harbouring a 265.5-kb pN1566-2 plasmid carrying genes encoding resistance to colistin (mcr-9.1), aminoglycosides (aadA1), tetracycline [tet(C)] and sulfonamides (sul1). Our findings indicate that the Salmonella Typhimurium ST19 strain in this study showed low genetic variability compared with Salmonella Typhimurium ST19 isolated from swine and poultry in Brazil, and was less related to those reported in other countries. Conclusions: This is the first reported genome of a phenotypically colistin-resistant Salmonella Typhimurium harbouring the mcr-9 variant in Brazilian livestock. This genome will aid global investigations on epidemiological and evolutionary aspects of plasmid-mediated colistin resistance and the role of colistin-resistant Salmonella Typhimurium ST19 lineage as a zoonotic pathogen

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum β-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway

publishedVersio

Complex Microbiome in Brain Abscess Revealed by Whole-Genome Culture-Independent and Culture-Based Sequencing.

Brain abscess is a severe infectious disease with high mortality and mobility. Although culture-based techniques have been widely used for the investigation of microbial composition of brain abscess, these approaches are inherent biased. Recent studies using 16S ribosomal sequencing approaches revealed high complexity of the bacterial community involved in brain abscess but fail to detect fungal and viral composition. In the study, both culture-independent nanopore metagenomic sequencing and culture-based whole-genome sequencing using both the Illumina and the Nanopore platforms were conducted to investigate the microbial composition and genomic characterization in brain abscess. Culture-independent metagenomic sequencing revealed not only a larger taxonomic diversity of bacteria but also the presence of fungi and virus communities. The culture-based whole-genome sequencing identified a novel species in Prevotella and reconstructs a Streptococcus constellatus with a high GC-skew genome. Antibiotic-resistance genes CfxA and ErmF associated with resistance to penicillin and clindamycin were also identified in culture-based and culture-free sequencing. This study implies current understanding of brain abscess need to consider the broader diversity of microorganisms

Phage Treatment Trial to Eradicate LA-MRSA from Healthy Carrier Pigs

The increase of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) causes a threat to human health. LA-MRSA can be transmitted from animals to animal caretakers, which may further spread MRSA to communities and health care facilities. The objective of this work was to study the efficacy of phage treatment in the eradication of LA-MRSA from healthy carrier pigs. A total of 19 MRSA -positive weanling pigs were assigned to a test (n = 10) and a control group (n = 9). A phage cocktail containing three Staphylococcus phages, or a control buffer was administered to the nares and skin of the pigs three times every two days, after which the phage and MRSA levels in nasal and skin swab samples were monitored for a three-week period. The sensitivity of the strains isolated during the follow-up period to the phage cocktail and each phage individually was analyzed and the pig sera were tested for antibodies against the phages used in the cocktail. The phage treatment did not cause any side effects to the pigs. Phages were found in the skin and nasal samples on the days following the phage applications, but there was no reduction in the MRSA levels in the sampled animals. Phage-resistant strains or phage-specific antibodies were not detected during the experiment. The MRSA load in these healthy carrier animals was only 10–100 CFU/swab or nasal sample, which was likely below the replication threshold of phages. The effectiveness of phage treatment to eradicate MRSA from the pigs could thus not be (reliably) determined

Biochemical and molecular characterization of three serologically different Vibrio harveyi strains isolated from farmed Dicentrarchus labrax from the Adriatic Sea

Vibrio harveyi is recognized as one of the major causes of vibriosis, a disease that threatens the long-term sustainability of aquaculture. Current research shows that the Mediterranean strains of V. harveyi are serologically heterogeneous, though research comparing the traits of different strains is scarce. This study aims to describe the biochemical, physiological and genetic characteristics of three serologically different strains of V. harveyi isolated from farmed European Sea bass (Dicentrarchus labrax) from the Adriatic Sea. A total of 32 morphological and biochemical markers were examined and, the susceptibility to 13 antimicrobials tested, and then compared the results of high-throughput sequencing and in silico analyses. This study also presents the first whole genome sequences of V. harveyi isolated from European sea bass. A large number of nonsynonymous variations were detected among sequences of the three strains. The prediction analysis of resistance genes did not correspond with the in vitro antimicrobial susceptibility tests. Six virulence genes previously unrelated to virulence of vibrios were detected in all three studied strains. The results show that differences were detected at every level of comparison among the three studied strains isolated from the same fish species originating from a small geographic area

Phage Treatment Trial to Eradicate LA-MRSA from Healthy Carrier Pigs

The increase of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) causes a threat to human health. LA-MRSA can be transmitted from animals to animal caretakers, which may further spread MRSA to communities and health care facilities. The objective of this work was to study the efficacy of phage treatment in the eradication of LA-MRSA from healthy carrier pigs. A total of 19 MRSA -positive weanling pigs were assigned to a test (n = 10) and a control group (n = 9). A phage cocktail containing three Staphylococcus phages, or a control buffer was administered to the nares and skin of the pigs three times every two days, after which the phage and MRSA levels in nasal and skin swab samples were monitored for a three-week period. The sensitivity of the strains isolated during the follow-up period to the phage cocktail and each phage individually was analyzed and the pig sera were tested for antibodies against the phages used in the cocktail. The phage treatment did not cause any side effects to the pigs. Phages were found in the skin and nasal samples on the days following the phage applications, but there was no reduction in the MRSA levels in the sampled animals. Phage-resistant strains or phage-specific antibodies were not detected during the experiment. The MRSA load in these healthy carrier animals was only 10–100 CFU/swab or nasal sample, which was likely below the replication threshold of phages. The effectiveness of phage treatment to eradicate MRSA from the pigs could thus not be (reliably) determined