Clonal occurrence of <i>Salmonella</i> Weltevreden in cultured shrimp in the Mekong Delta, Vietnam

This study investigated the occurrence, serovar and antimicrobial resistance of Salmonella spp. in shrimp samples from intensive and extensive farms located in three different provinces in the Mekong Delta, Vietnam. Shrimp from 11 of the 48 farms all contained S. Weltevreden, except for one farm yielding S. Agona, with no difference in Salmonella occurrence between the two production systems. Pulsed field gel electrophoresis (PFGE) of S. Weltevreden showed closely related XbaI pulse types, suggesting a clonal relationship despite the farms and shrimp samples being epidemiologically unrelated. S. Weltevreden was susceptible to most antimicrobials tested, with a few strains being resistant to florfenicol, chloramphenicol, sulfamethoxazole or trimethoprim. Future studies of the ecology of S. Weltevreden should establish if this serovar may survive better and even multiply in warm-water shrimp farm environments compared to other Salmonella serovars

Identification and Antimicrobial Resistance of Bacteria Isolated from Probiotic Products Used in Shrimp Culture

Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures

Molecular Characteristics and Zoonotic Potential of<i> Salmonella</i> Weltevreden From Cultured Shrimp and Tilapia in Vietnam and China

Salmonella Weltevreden is increasingly reported from aquatic environments, seafood, and patients in several Southeast Asian countries. Using genome-wide analysis, we characterized S. Weltevreden isolated from cultured shrimp and tilapia from Vietnam and China to study their genetic characteristics and relatedness to clinical isolates of S. Weltevreden ST-365. The phylogenetic analysis revealed up to 312 single-nucleotide polymorphism (SNP) difference between tilapia isolates, whereas isolates from shrimp were genetically more closely related. Epidemiologically unrelated isolates from Vietnam were closely related to isolates from China, e.g., 20 SNPs differences between strains 28V and 75C. In comparison with strains from other parts of the world, our environmental isolates predominantly clustered within the continental South Asia lineage, constituted mostly of strains from human stool with as low as seven SNPs difference, e.g., 30V versus Cont_ERR495254. All sequenced isolates were MLST type ST-365 and contained the major virulence-related genes encoded by the Salmonella Pathogenicity Islands 1-5. Ten of the isolates harbored the IncFII(S) plasmid similar to the virulence genes-mediated plasmid pSPCV of S. Paratyphi C, and one isolate had the IncQ1 plasmid on the same contig with strA/B, sul2, and tetA resistance genes similar to the IncQ1 type, pNUC of S. Typhimurium. A pangenomic analysis yielded 7891 genes including a core genome of 4892 genes, with a closely related accessory genome content between clinical and environmental isolates (Benjamini p > 0.05). In a search for differences that could explain the higher prevalence of S. Weltevreden in aquatic samples, genomes were compared with those of other Salmonella enterica serovars. S. Weltevreden revealed specific regions harboring glpX (Fructose-1;6-bisphosphatase; class II), rfbC (dTDP-4-dehydrorhamnose 3;5-epimerase), and cmtB (PTS Mannitol-specific cryptic phosphotransferase enzyme IIA component) involved in carbohydrate biosynthesis pathways. Our study builds grounds for future experiments to determine genes or pathways that are essential when S. Weltevreden are in aquatic environments and microbial interactions providing survival advantages to S. Weltevreden in such environments.Published versio

Prevalence of <i>Salmonella</i> in extensive and intensive cultured shrimp in the Mekong Delta, Vietnam.

<p>Prevalence of <i>Salmonella</i> in extensive and intensive cultured shrimp in the Mekong Delta, Vietnam.</p

<i>Xba</i>I PFGE patterns and antimicrobial susceptibility of <i>Salmonella</i> isolates from shrimp samples from intensive and extensive production system.

<p>Similarity analysis was performed using the Dice coefficient, and clustering was done by the unweighted pair-group method with arithmetic averages (UPGMA; position tolerance 1.0%). Antimicrobial resistance was determined using the Sensititre Vizion System and MIC values were evaluated in accordance with the protocol of the Clinical and Laboratory Standards Institute. FFC, Florfenicol; CHL, Chloramphenicol; SMX, Sulfamethoxazole and TMP, Trimethoprim; "S" means susceptible to all antimicrobials tested. MIC values (μg/mL) are shown in brackets.</p

Neighbour joining phylogenetic tree of the 16S rRNA gene sequences representing <i>Bacillus</i> spp. isolated from probiotic products.

<p>Type strains of <i>Bacillus</i> species are labeled with accession number and strain number. Strains with identical sequences are included in parenthesis after the strain selected as reference. Strains with sequences shorter than 1 kb were excluded from phylogenetic analysis and their identity only determined by similarity comparison to type strain. The scale bar represents sequence variation considering the model for nucleotide substitution (Jukes & Cantor) and tree-shape used in the neighbour joining analysis. * Strains are with too short sequences to compare.</p

Bacterial species isolated from probiotic products after incubation on blood agar for 24 h at 30°C and subsequent identification by 16S rRNA analysis and their susceptibility to antimicrobials.

<p>^a Accession number for the Ez-Taxon database.</p><p>^b Accession number for the GenBank database.</p><p>^c Unpublished reference.</p><p>^d Nucleotide similarity in percent.</p><p>^e<i>Bacillus cereus</i> identified by <i>gyr</i>B sequencing analysis.</p><p>^f<i>Bacillus cereus</i> identified by MALDI-TOF method.</p><p>^g Strains characterized by whole genome sequencing.</p><p>-^h Not tested.</p><p>ⁱ CLI, clindamycin; CIP, ciprofloxacin; CHL, chloramphenicol; ERY, erythromycin; PEN, penicillin; TET, tetracycline; SXT, trimethoprim/sulfamethoxazole; AMP, ampicillin; GEN, gentamicin; OXA, oxacillin.</p><p>Bacterial species isolated from probiotic products after incubation on blood agar for 24 h at 30°C and subsequent identification by 16S rRNA analysis and their susceptibility to antimicrobials.</p

Information about the seven probiotic products analyzed.

<p>Information about the seven probiotic products analyzed.</p

Oligonucleotide primers used for 16S rRNA analysis and detection of the <i>gyr</i>B gene.

<p>Oligonucleotide primers used for 16S rRNA analysis and detection of the <i>gyr</i>B gene.</p