Multilocus Sequence Analysis, a Rapid and Accurate Tool for Taxonomic Classification, Evolutionary Relationship Determination, and Population Biology Studies of the Genus Shewanella.

The genus Shewanella comprises a group of marine-dwelling species with worldwide distribution. Several species are regarded as causative agents of food spoilage and opportunistic pathogens of human diseases. In this study, a standard multilocus sequence analysis (MLSA) based on six protein-coding genes (gyrA, gyrB, infB, recN, rpoA, and topA) was established as a rapid and accurate identification tool in 59 Shewanella type strains. This method yielded sufficient resolving power in regard to enough informative sites, adequate sequence divergences, and distinct interspecies branches. The stability of phylogenetic topology was supported by high bootstrap values and concordance with different methods. The reliability of the MLSA scheme was further validated by identical phylogenies and high correlations of genomes. The MLSA approach provided a robust system to exhibit evolutionary relationships in the Shewanella genus. The split network tree proposed twelve distinct monophyletic clades with identical G+C contents and high genetic similarities. A total of 86 tested strains were investigated to explore the population biology of the Shewanella genus in China. The most prevalent Shewanella species was Shewanella algae, followed by Shewanella xiamenensis, Shewanella chilikensis, Shewanella indica, Shewanella seohaensis, and Shewanella carassii The strains frequently isolated from clinical and food samples highlighted the importance of increasing the surveillance of Shewanella species. Based on the combined genetic, genomic, and phenotypic analyses, Shewanella upenei should be considered a synonym of S. algae, and Shewanella pacifica should be reclassified as a synonym of Shewanella japonicaIMPORTANCE The MLSA scheme based on six housekeeping genes (HKGs) (gyrA, gyrB, infB, recN, rpoA, and topA) is well established as a reliable tool for taxonomic, evolutionary, and population diversity analyses of the genus Shewanella in this study. The standard MLSA method allows researchers to make rapid, economical, and precise identification of Shewanella strains. The robust phylogenetic network of MLSA provides profound insight into the evolutionary structure of the genus Shewanella The population genetics of Shewanella species determined by the MLSA approach plays a pivotal role in clinical diagnosis and routine monitoring. Further studies on remaining species and genomic analysis will enhance a more comprehensive understanding of the microbial systematics, phylogenetic relationships, and ecological status of the genus Shewanella.

Shewanella carassii sp. nov., isolated from surface swabs of crucian carp and faeces of a diarrhoea patient.

Two strains, 08MAS2251T and LZ2016-166, were isolated from diverse samples in China collected from the surface of crucian carp and the faeces of a diarrhoea patient, respectively. Both strains were pink-orange coloured, Gram-negative, oxidase- and catalase-positive, facultative anaerobic and motile bacteria, produced H2S and reduced nitrates to nitrites. Growth occurred in the presence of 0-9 % (w/v) NaCl and at 10-42 °C. The optimum conditions were with 1 % (w/v) NaCl and at 35 °C. The phylogenetic tree of 16S rRNA gene demonstrated that strains 08MAS2251T and LZ2016-166 clustered in a distinctive clade next to the species Shewanella chilikensis JC5T within the genus Shewanella. Meanwhile, gyrB gene sequence analysis indicated that the two strains formed an independent branch that was clearly separate from all the other Shewanella species with sequence similarities from 68.49 to 95.74 %. The DNA G+C content of strain 08MAS2251T was 52.68 mol%. Genomic relatedness of in silico DNA-DNA hybridization between strain 08MAS2251T and phylogenetic neighbours ranged from 50.5-51.8 %, below the cutoff of 70 %. In addition, corresponding average nucleotide identity values were between 93.01 to 93.49%, which were lower than 95 % threshold. The major fatty acids of strain 08MAS2251T were C17 : 1ω8c (27.2 %), iso-C15 : 0 (22.5 %), summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 8.7 %), C16 : 0 (6.2 %), iso-C13 : 0 (5.6 %) and C17 : 0 (4.5 %). Based on phenotypic and genetic analysis, strains 08MAS2251T and LZ2016-166 are identified as a novel species of the genus Shewanella, for which the name Shewanellacarassii sp. nov. is proposed. The type strain is 08MAS2251T (=DSM 104682T=CGMCC 1.16033T).

Genetic and serological characterization of capsular antigen untypeable Vibrio parahaemolyticus strains reveal novel K serotypes and epidemiological characteristics in Shandong, China.

Vibrio parahaemolyticus, which is commonly found in marine and estuarine environments worldwide and isolated from aquatic products, is one of the most important food-borne pathogens. Among the various typing methods, serotyping is widely accepted and utilized by infectious disease specialists and infection control agencies for the detection and epidemiological investigation of this pathogen. Thus far, 13 O serotypes and 71 K serotypes have been defined; however, untypeable strains are frequently isolated during routine detection, and some new O and/or K antigens have been identified and characterized. During a serotyping survey in Shandong province, China from 2016 to 2018, we collected 411 clinical V. parahaemolyticus strains and found that nine of them are untypeable K antigen strains. In this study, we identified three K serotypes of V. parahaemolyticus through in-depth genetic analysis of the K antigen gene cluster, serological tests, and the production of antisera. Among the nine strains, seven possess K untypeable 2 (KUT2) antigens, which have been reported recently by another group. However, two new O and K combinations (O3:KUT2 and O11:KUT2) were first characterized by us, with the remaining two each representing a novel K serotype. Moreover, through comparative genomic analysis, we showed that the Shandong KUT2 strains exhibit different virulence profiles compared to their identical K serotype partners from Zhejiang province, another Chinese coastal province; however, strains from these two regions are clustered into the same linage and may have evolved from a recent common ancestor. Additionally, one isolate, SD2016062, was phylogenetically similar to the strains associated with several local gastroenteritis outbreaks, with similar toxin patterns, suggesting its potential to cause sporadic occurrences of disease or even local pandemics. Finally, we developed a sero-specific PCR assay targeting the three novel K serotypes, which can monitor the V. parahaemolyticus spectrum for clinical and epidemiological purposes. Thus, we identified and characterized novel strains of V. parahaemolyticus and proposed a new technique for tracking the diversity of strains, which can help manage this food-borne pathogen.

Distribution and Genetic Characteristics of SXT/R391 Integrative Conjugative Elements in Shewanella spp. From China.

The genus Shewanella consists of facultatively anaerobic Gram-negative bacteria, which are regarded as potential agents of food contamination and opportunistic human pathogens. Information about the distribution and genetic characteristics of SXT/R391 integrative conjugative elements (ICEs) in Shewanella species is limited. Here, 91 Shewanella strains collected from diverse samples in China were studied for the presence of SXT/R391 ICEs. Three positive strains, classified as Shewanella upenei, were obtained from patients and water from a local mill. In light of their close clonal relationships and high sequence similarity, a representative ICE was selected and designated ICESupCHN110003. The BLASTn searches against GenBank showed that ICEVchBan5 was most closely related to ICESupCHN110003, with the coverage of 76% and identity of 99%. The phylogenetic tree of concatenated core genes demonstrated that ICESupCHN110003 formed a distinct branch outside the cluster comprising ICEValA056-1, ICEPmiCHN2410, and ICEPmiChn1. Comparison of the genetic structures revealed that ICESupCHN110003 encoded uncommon genes in hotspots, such as specific type III restriction-modification system, conferring adaptive functions to the host. Based on the low coverage in the sequence analysis, independent clade in the phylogenetic tree, and unique inserted fragments in hotspots, ICESupCHN110003 represented a novel SXT/R391 element, which widened the list of ICEs. Furthermore, the antibiotic resistance genes floR, strA, strB, and sul2 in ICESupCHN110003 and resistance to multiple drugs of the positive isolates were detected. A cross-species transfer capability of the SXT/R391 ICEs was also discovered. In summary, it is necessary to reinforce continuous surveillance of SXT/R391 ICEs in the genus Shewanella.