Methods for identification and differentiation of different Shewanella spp. isolates for diagnostic use

Abstract

Shewanella spp. are Gram-negative, rod-shaped, motile bacteria that are widely distributed in marine and freshwater environments. The bacteria are present in the physiological microflora of fish from temperate waters and are known as fish spoilage species. From clinically healthy fish and from fish with skin ulcerations, Shewanella spp. is regularly isolated, indicating a possible role as fish pathogen. In this study, 74 isolates of Shewanella spp. were analysed. For species identification, biochemical techniques, 16S rRNA sequencing, MALDI-TOF MS and the Sherlock Microbial Identification System (MIS) based on the composition of fatty acid ethyl esters were compared. The phylogenetic relationship, cytotoxicity in vitro and resistance against antibiotics were tested. The most reliable method for species identification was 16S rRNA sequencing. From diseased fish, clinically healthy fish and the aquatic environment, different Shewanella species were isolated. This indicates that Shewanella spp. is widespread in the aquatic milieu and acts as a secondary pathogen. The virulence of Shewanella spp. is probably not depending on the species but on the isolate itself. Many isolates of Shewanella spp. were showing multiresistances against antibiotic substances, especially in samples derived from retailers and in routine diagnostics, all Shewanella spp. should therefore be tested for resistances against antibiotic agents.

Genotypic diversity among Shewanella spp. collected from freshwater fish

“…Our study showed that Shewanella isolates collected from freshwater fish are a diverse group of microorganisms belonging to previously identified species S. putrefaciens , S. xiamenensis and S. oneidensis (Supporting Information Table S1; Figure ). S. putrefaciens was dominant in contrast to study by Jung‐Schroers et al () who described mainly S. oneidensis in freshwater fish. Both studies indicate S. xiamenensis as isolated mainly from ornamental fish and less frequently from farmed fish.…”

“…Shewanella spp ., gammaproteobacteria belonging to the Alteromonadaceae family, are widespread in marine and freshwater environments (Buller, ). Different Shewanella species were isolated from healthy fish as well as from fish exhibiting clinical signs of a disease (Al‐Harbi & Uddin, ; Cocchi et al, ; Esteve, Merchán, & Alcaide, ; Jung‐Schroers et al, ; Kim et al, ; Korun, Akgun‐Dar, & Yazici, ; Pakingking, Palma, & Usero, ; Pękala, Kozińska, Paździor, & Głowacka, ; Qin, Zhu, & Xu, ; Rusev, Rusenova, Simeonov, & Stratev, ; Saeed, Alamoudi, & Al‐Harbi, ). However, contemporary typing methods were not sufficient to determine genotypic diversity of Shewanella fish isolates.…”

“…Furthermore, S. oneidensis and S. xiamenensis were included in group III (Huang, Sun, & Zhang, ; Venkateswaran et al, ). The most recent studies focused on analysis of 16S rRNA or gyrB genes (Beaz‐Hidalgo, Agüeria, Latif‐Eugenín, Yeannes, & Figueras, ; Dailey et al, ; Jung‐Schroers et al, ; Pękala et al, ). The other methods such as random amplified polymorphic DNA (RAPD‐PCR), enterobacterial repetitive intergenic consensus (ERIC‐PCR) and pulsed‐field gel electrophoresis (PFGE) were also used to characterize Shewanella (Beaz‐Hidalgo et al, ; Kim et al, ; Oh et al, ; Roh et al, ; Ziemke, Brettar, & Hofle, ).…”

Diagnostic methods for identifying different Aeromonas species and examining their pathogenicity factors, their correlation to cytotoxicity and adherence to fish mucus

“…In former studies, this product could be detected more often in clinical isolates than in environmental isolates of Aeromonas spp. and it has to be proposed as an important virulence factor (Krovacek et al., ). Aerolysins and Haemolysin were detected in different numbers of samples.…”

“…that include virulence genes are potentially suspected to cause severe infections, like gastrointestinal signs in humans, especially in children (Soltan Dallal, Mazaheri Nezhad Fard, Kavan Talkhabi, Aghaiyan, & Salehipour, ). As it was shown that clinical isolates as well as isolates from the aquatic environment can be tested positive for virulence factors (Krovacek et al., ), the significance for fish health and also for public health has to be determined in further epidemiological studies.…”