Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade

Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade Maria de Oliveira Firmino , Mykyta Forofontov , View ORCID Profile Ricardo O. Louro , Alberto J. Martín-Rodríguez , Mário Ramirez , View ORCID Profile Catarina M. Paquete doi: https://doi.org/10.1101/2025.02.24.639908 Maria de Oliveira Firmino a Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Av. da República, 2780 – 157 Oeiras, Portugal b Instituto de Microbiologia da Faculdade de Medicina da Universidade de Lisboa , Avenida Prof. Egas Moniz, P-1649-028 Lisboa, Portugal Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mykyta Forofontov b Instituto de Microbiologia da Faculdade de Medicina da Universidade de Lisboa , Avenida Prof. Egas Moniz, P-1649-028 Lisboa, Portugal Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ricardo O. Louro a Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Av. da República, 2780 – 157 Oeiras, Portugal Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ricardo O. Louro Alberto J. Martín-Rodríguez c Department of Clinical Sciences, University of Las Palmas de Gran Canaria , Paseo Blas Cabrera Felipe, s/n, 35016, Las Palmas de Gran Canaria, Spain d Department of Microbiology , Tumor and Cell Biology, Karolinska Institutet , Solnavägen 9, 171 65 Solna, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mário Ramirez b Instituto de Microbiologia da Faculdade de Medicina da Universidade de Lisboa , Avenida Prof. Egas Moniz, P-1649-028 Lisboa, Portugal Find this author on Google Scholar Find this author on PubMed Search for this author on this site Catarina M. Paquete a Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Av. da República, 2780 – 157 Oeiras, Portugal Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Catarina M. Paquete For correspondence: cpaquete{at}itqb.unl.pt Abstract Full Text Info/History Metrics Preview PDF Abstract Shewanella is an environmentally ubiquitous genus with significant roles in bioelectrochemical applications and human infections. However, identification problems involving Shewanella putrefaciens , Shewanella xiamenensis, and Shewanella seohaensis , have been reported, potentially hindering research progress in these areas. In this study, we explored how to discriminate between these species. By comparing the genomes of Shewanella spp. available in public databases with that of the newly sequenced strain DSM9451, we showed that this strain is a member of the species S. seohaensis . Of the eight public genomes associated with this species, only two were correctly identified in public databases. Phenotypic analysis revealed distinct features of S. seohaensis with respect to S. putrefaciens and Shewanella decolorationis. However, only differences in the intensity of biochemical reactions were observed between S. xiamenensis and S. seohaensis. To discriminate between these closely related species and explore their diversity, a whole-genome multilocus sequence typing scheme was developed. The scheme distinguished related species and revealed significant diversity among S. seohaensis and S. xiamenensis isolates, suggesting that both species may harbor isolates with significantly different metabolic properties. These differences are so wide within the clade that may explain why these two species are difficult to distinguish. The identification of exclusive genes of each species allowed the design of a simple molecular method to differentiate S. seohaensis from closely related species, which will help in clarifying its role in human infections and environmental processes. Importance Misidentification within the Shewanella genus, particularly between S. putrefaciens , S. seohaensis, and S. xiamenensis , has been reported and misperceives scientific research on Shewanella spp . in diverse fields, including both biotechnological applications and human infections. Near-complete 16sRNA gene sequencing fails to correctly classify many Shewanella species, and MALDI-TOF systems used in clinical microbiology laboratories are suboptimal species-level identification. In this study, we identified phenotypic characteristics that can guide differentiation and classification, and building upon the identification of species-specific genes, we suggest an accurate and cost-effective molecular test as an alternative to genome sequencing. The proposed wgMLST scheme allows the exploration of species and strain diversity, highlighting the limitations of generalizing results from studies of a single strain. As an emergent pathogen and biotechnological candidate, the proper identification by a single molecular test will enhance the insights about these species towards biotechnology development and public health safety. Introduction The genus Shewanella, proposed by MacDonell and Cowell in 1985 ( 1 ), is a ubiquitous and diversified genus that belongs to the class of Gammaproteobacteria, order Alteromonadales, and family Shewanellaceae ( 2 ). The first isolate reported from this genus had several classifications, including Achromobacter putrefaciens , Pseudomonas putrefaciens, and Alteromonas putrefaciens, until its final classification as Shewanella putrefaciens ( 3 ). Shewanella spp. can be found in fresh and seawater and human samples ( 4 ). In recent years, the interest in this genus has increased due to its importance in bioremediation, microbial electrochemical technologies, and biosensing ( 5 ) and by being identified as a potential emerging pathogen ( 6 – 8 ). All human isolates reported until 1995 were mistakenly identified as either Pseudomonas putrefaciens or Alteromonas putrefaciens ( 9 ). Shewanella algae was recognized as a pathogen in the early 1990s, being easily distinguished due to its distinctive phenotypic differences, such as the ability to grow at higher temperatures and salt concentrations ( 3 , 10 ). It was only after the first DNA sequencing of several isolates of the Shewanella genus in 2002 ( 11 ) that other Shewanella species, including Shewanella xiamenensis , were identified as pathogenic ( 12 ). The identification of other Shewanella species in samples from human patients, such as Shewanella carassii ( 13 ), suggests that the breadth of potentially pathogenic species within this genus might be wider than initially thought. Improved identification systems and changing environmental conditions (such as climate change and socio-geographic distribution) ( 8 , 14 ) are contributing to a rise in documented cases of Shewanella infections ( 15 ). Shewanella spp. are frequently present in polymicrobial infections ( 3 , 8 ) and are considered important environmental reservoirs of antimicrobial resistance genes ( 16 ). Genome sequencing stands out as the most suitable method to achieve accurate identification within the Shewanella genus. To resolve the taxonomy of the genus Shewanella, Martín-Rodríguez and Meier-Kolthoff sequenced all Shewanella type strains that lacked information in public repositories and reconstructed a genus-wide phylogeny with all sequenced Shewanella strains at that time ( 17 ), demonstrating that relying solely on 16S RNA sequence-based identification has limitations within this genus. Given that sequencing of new isolates is a laborious and expensive identification method, the issue of misidentification of Shewanella species persists. This problem is particularly evident in infection-associated Shewanella isolates ( 16 , 18 , 19 ), as standard clinical microbiology identification methods, frequently carried out through automated or semi-automated methods, often rely on incomplete or inaccurate databases ( 20 ). A notable example is the systematic difficulty in correctly identifying Shewanella seohaensis, S. xiamenensis, and S. putrefaciens ( 21 ). Thorell and colleagues demonstrated the existence of misidentified isolates, including S. putrefaciens SA70 and S. putrefaciens NCTC12093 ( 18 ), while the work of Martín-Rodríguez and Meier-Kolthoff determined that these isolates belong to the species S. seohaensis ( 17 ). In this study, we focus on differentiating S. seohaensis , S. xiamenensis, and S. putrefaciens by phenotypic characteristics and molecular methods. Towards this, the phylogenetic classification and phenotypic characteristics of two pathogenic Shewanella strains, S. putrefaciens DSM9451 and S. xiamenensis (HI32665) isolated from human clinical samples, were determined. This research enhances our current understanding of Shewanella taxonomy and also investigates the differences between S. seohaensis and closely related species. It provides the tools for their differentiation in laboratories lacking genome sequencing capabilities, which potentially helps to clarify the role of this species in human infections and environmental processes. Additionally, this study proposes a whole-genome multilocus sequence typing (wgMLST) scheme to help differentiate these species and better understand their population structure. Results and Discussion Shewanella putrefaciens DSM9451 is a member of the Shewanella seohaensis species The type strain of S. xiamenensis was first isolated from a sediment sample of the coastal area of Xiamen, China ( 22 ). This species was also isolated from human infections ( 13 , 23 ), establishing it as one of the pathogenic members of the Shewanella genus. The S. xiamenensis strain (HI32665) used in this work was isolated from a coproculture at a Portuguese Hospital and was initially identified as Shewanella xiamenensis by MALDI-TOF (MALDI Biotyper MSP Identification). The species assignment for this strain was conclusively achieved by analyzing its genome sequence relatedness with respect to the reference genome of the type strain of S. xiamenensis . The values of d DDH (74.9 % [confidence interval 71.9 – 77.7]) and ANIb (97.0%) were concordant with the identification of HI32665 as S. xiamenensis . Shewanella putrefaciens DSM9451, initially designated as CL 256/73 and first described by Holmes et al. ( 24 ), was isolated from a mixed culture from the cerebrospinal fluid of a 1-year-old infant, with a suspicion of meningitis. This strain was acquired for this study from the DSMZ repository, where it is identified as S. putrefaciens . However, MALDI-TOF (MALDI Biotyper MSP Identification) classified this strain as S. xiamenensis . Distinction between these species often eludes such systems ( 21 ), since their accuracy depends on the databases supporting the analysis. To evaluate the capacity of MALDI-TOF MS to discriminate between the species S. seohaensis , S. xiamenensis, and S. putrefaciens , the strains S. seohaensis SA70, S. putrefaciens 95 T and S. seohaensis CCUG60900 T were analyzed by the MALDI Biotyper MSP Identification system. The results revealed that while the system could differentiate S. putrefaciens, it was unable to distinguish between S. seohaensis and S. xiamenensis , identifying strains of both species as S. xiamenensis . Analysis of genomic data using Kraken suggested that S. putrefaciens DSM9451 was indeed Shewanella bicestrii . However, this taxon is a non-validated species, also referred to as Shewanella sp. JAB-1 ( 15 , 16 ). According to Martín-Rodríguez and Meier-Kolthoff ( 17 ), the affiliation of Shewanella sp. JAB-1 with the species S. seohaensis suggested a different classification for S. putrefaciens DSM9451, which we will further refer simply as DSM9451. For a deeper understanding of the phylogenetic classification of DSM9451, a comprehensive phylogenetic analysis was undertaken. This analysis involved comparing the genome of DSM9451 with all 822 genomes of Shewanella available on the National Center for Biotechnology Information (NCBI) on the 17 th of June 2024 by GBDP. The results revealed that DSM9451 and S. seohaensis NCTC12093 have nearly identical genomes ( d DDH = 99.9 % [99.9 - 100]) (see Figure 1 ). Furthermore, the comparison between DSM9451 and S. seohaensis CCUG6090 ( d DDH = 70.7 % [67.7 - 73.6]), S. seohaensis SA70 ( d DDH = 76.2 % [73.2 - 79]), S. seohaensis JAB-1 ( d DDH = 71.9% [68.9 - 74.8]), and S. seohaensis BC20 ( d DDH = 69.4% [66.4 - 72.3]) confirmed that they all belong to the same species. DSM9451 also had a d DDH value >70% with Shewanella sp. GD03713 (74.8 % [71.8 – 77.6]), indicating that Shewanella sp. GD03713 is of the same species as DSM9451. When compared with S. putrefaciens 95, the type strain of S. putrefaciens , the value of d DDH is 22.70 % [20.4 - 25.1] ( Table 1 ). Comparing these strains with the type strain of S. seohaensis, S. seohaensis CCUG60900, we obtained similar results, except for Shewanella sp. GD03713, where the dDDH values are on the limit to consider this strain as not belonging to this species. However, the ANI results support the identification of GD03713 as S. seohaensis ( Table 1 ). The ANI values presented in Table 1 , reinforce that DSM9451 does not fall within the S. putrefaciens species. Download figure Open in new tab Figure 1. Whole-Genome-based GBDP tree. Blue shades represent the G+C percentage with darker corresponding to a higher percentage. User-provided genomes are marked by a red cross. Branch lengths are scaled in terms of GBDP distance formula d6. The numbers above branches are GBDP pseudo-bootstrap support values from 100 replicates. View this table: View inline View popup Download powerpoint Table 1. Matrix of dDDH and ANIb results. dDDH values cut-off is at 70% and ANI values cut-off is at 95 %. The grey shadow boxes are those that belong to the same species (the confidence interval for the estimate includes 70%). A - Shewanella sp. DSM9451; B - S. seohaensis NCTC12093; C - S. seohaensis JAB-1; D - S. seohaensis SA70; E - S. seohaensis CCUG60900; F - S. seohaensis BC20; G - Shewanella sp. GD03713; H - S. putrefaciens 95. View this table: View inline View popup Download powerpoint Table 2. Bacterial strains used in this study. The TYGS online service, which employs the same technique (i.e., GBDP) coupled with G+C content, provides a whole-genome-based GBPD tree phylogenetic analysis ( 25 ) ( Figure 1 ). The tree was inferred with FastME from GBDP distances calculated from genome sequences obtained from the TYGS online service. The S. seohaensis strains, S. xiamenensis HI32665, and the type strain of S. putrefaciens and S. decolorationis were introduced to guarantee the representation of the genomes of interest. A duplicated entry means an automatic addition to the analysis by the TYGS service database. In the generated tree, DSM9451 clusters within S. seohaensis strains. These results further strengthen the suggestion that the correct classification of DSM9451 is S. seohaensis . Although genome sequencing could identify DSM9451 as S. seohaensis , this method is not available in routine clinical microbiology laboratories as well as in many research laboratories. To explore alternative reliable methods of identification, a study focused on the phenotypic differentiation between S. seohaensis, S. xiamenensis, and S. putrefaciens was conducted. Phenotypic analysis provides incomplete discrimination between Shewanella species To determine phenotypic differences between Shewanella species, the growth capacity of strains S. seohaensis DSM9451, S. seohaensis SA70, S. seohaensis CCUG60900, S. putrefaciens 95, and S. xiamenensis HI32665 at different temperatures, and their biochemical characterization were performed. The results were compared between the different strains and with those described in the literature for S. decolorationis, which has been used as a comparison species in previous studies ( 22 , 26 ). All strains of S. seohaensis exhibited growth at 37ᵒC, although they did not grow at temperatures of 4ᵒC or 42ᵒC (Supplementary Table 1). S. putrefaciens 95 showed poor growth at 37ᵒC but could grow at 4ᵒC. In contrast to what is described in the literature, all S. seohaensis strains and S. xiamenensis strains tested grew at 40ᵒC, indistinguishably from growth at the optimum temperature, as also described for S. decolorationis ( 22 ). Biochemical analysis revealed differences between strains of the same species as well as from phenotypes previously described in the literature for those strains. The KIA test revealed that CCUG60900 could produce hydrogen sulfide, contrary to what was described ( 26 ). In the API panels, divergences were observed within the S. seohaensis species (Supplementary Table 1). In the API ZYM panel and among the tested enzymes, alkaline phosphatases, butyrate esterase, caprylate esterase lipase, leucine aminopeptidase, α- chymotrypsin, acid phosphatase, and trypsin exhibited activity in all tested S. seohaensis strains. The trypsin activity observed on CCUG60900 contrasts with what was described previously ( 26 ). Notably, only DSM9451 presented enzymatic activity for N-acetyl-β-glucosaminidase. In the API 20NE panel, all strains tested positive for gelatine hydrolysis but displayed diverse responses regarding nitrate reduction: DSM9451 and CCUG 60900 tested positive, while SA70 tested negative (Supplementary Table 1). As for assimilation tests, all strains were able to use arabinose, N-acetyl-glucosamine, maltose, and malate as carbon sources. An exception occurred with the glucose utilization test, which was only positive for CCUG60900. Overall, it is possible to phenotypically distinguish S. seohaensis from S. putrefaciens since the latter demonstrated the ability to grow at 4ᵒC, exhibit glucose fermentation in the KIA test, and does not hydrolyze gelatine or utilize arabinose and maltose as the carbon source in the API 20NE panel (Supplementary Table 1). In comparison to the phenotype described for S. decolorationis , these two species exhibit variations in leucine aminopeptidase activity and the assimilation of arabinose. Interestingly, the phenotype of S. xiamenensis closely resembled that of S. seohaensis . Our results revealed only minor discrepancies among these species, notably in the intensity of the signal for trypsin activity (negative) and esculin hydrolysis (positive). While other inconsistencies have been documented in the literature (Supplementary Table 1), we were unable to definitively confirm them in this study ( 22 , 26 ). Whole-genome multilocus sequence typing scheme reveals species segregation and large strain diversity within species For a more comprehensive understanding of the relationship between the isolates and to identify specific genes associated with each species, a wgMLST scheme was developed for all the strains analyzed in this study. For comparative analysis, closely related species including representatives of S. decolorationis and S. xiamenensis , were also incorporated. This scheme evaluates the diversity of the strains under study and identifies genes that are either present or absent in the various species. An exclusive gene was defined as being present in all isolates of a species and absent in isolates of all other species represented in the scheme. The scheme and the alleles identified are available in the chewie-NS ( 27 ) platform ( https://chewbbaca.online/ ). Using the PHYLOViZ online software, it is possible to observe a minimum-spanning tree for the core-genome MLST (cgMLST) ( Figure 2 ), i.e., of the allelic profiles of the genes present in all strains (cgMLST 100 = 922 loci). Download figure Open in new tab Figure 2. PHYLOViZ. The numbers represent the number of allelic differences between the two nodes. The green round box highlights the strains identified as S. oneidensis but in the S. xiamenensis cluster. The yellow rectangular box highlights the S. seohaensis strains. In Figure 2 it is possible to observe sixteen different species, whose strains mostly segregate together in the tree. However, there are also several cases in which strains identified as a given species group with others of a different species. This is the case of the strains identified as S. oneidensis S2_009_000_R2_72 (GCA_003241225.1) and S. oneidensis SRR9109399 (GCA_945952185.1) that can be found among the S. xiamenensis cluster. Phylogenetic studies confirm they indeed belong to S. xiamenensis species ( d DDH values are 76.50% [73.5 – 79.3%] and 71.20% [68.2 – 74%], respectively). The same situation could be observed with strains identified as Shewanella putrefaciens . In the S. seohaensis cluster (yellow rectangular box), S. seohaensis SA70 and S. seohaensis NCTC12093 have been misidentified as S. putrefaciens while S. seohaensis JAB-1 has been misidentified as Shewanella bicestrii , visible by the three circles with two colors in Figure 2 . A comparison of the genes of S. seohaensis with those of closely related species reveals a list of 8 exclusive genes (Supplementary Table 2). Interestingly, cgMLST analysis demonstrates extensive diversity within species, with the number of allelic differences between pairs of closest isolates of the same species being similar to pairs of closest isolates of different species (numbers on Figure 2 ). This phenomenon could explain the phenotypic differences observed within the same species. Additionally, without distinct phenotypic features for each species, phenotypic discrimination among them is not possible. Given the absence of distinctive phenotypic characteristics between S. seohaensis and S. xiamenensis, this study allowed to identify exclusive genes that could function as targets for differentiating S. seohaensis from S. xiamenensis and other closely related species (see below). Novel method for the identification of S. seohaensis by detecting an exclusive gene Among the 8 exclusive genes to S. seohaensis compared to closely related species in the previous section, the gene that encodes for a phosphatase PhoE (ID: VEE60752.1) was selected and corresponding primers were designed ( Table 3 in Materials and Methods). A PCR was performed using genomic DNA from different strains, and the resulting agarose gel (Supplementary Figure 1) showed that amplification occurred only for the S. seohaensis strains. This confirmed the utility of this assay in identifying this species. View this table: View inline View popup Download powerpoint Table 3. Primers used to confirm the presence of the exclusive genes. These results provide a novel tool for distinguishing S. seohaensis from closely related species, including S. xiamenensis and S. putrefaciens , effectively resolving the issue of misidentification among these species. Conclusion The misidentification of species within the Shewanella genus is well-documented ( 17 , 18 ), especially between S. seohaensis , S. putrefaciens, and S. xiamenensis ( 21 ). S. putrefaciens often emerged as the initial taxonomic choice after preliminary tests ( 19 , 24 ), which led to lumping together a highly diverse group of isolates. Moreover, the overall similarity between related species and the biochemical diversity within the same species increases the complexity of arriving at an accurate identification. Despite advances afforded by using MALDI-TOF for routine species identification in clinical microbiology laboratories, our results show that it still fails to accurately identify some species within the Shewanella genus, similar to what happens with other genera ( 28 ), possibly due to insufficient representation in existing databases. Although we identified several characteristics that can be used to distinguish S. putrefaciens from S. seohaensis, and S. xiamenensis, including the ability of these strains to grow at 4 ᵒC and ferment glucose in the KIA test, no phenotypic tests could reliably distinguish the latter two species. Distinguishing S. seohaensis from closely related species can be achieved by the PCR reaction proposed here, targeting a gene exclusive of S. seohaensis . Although we did not test strains representing all Shewanella spp. of the clade, in silico analysis indicates that this PCR reaction will also distinguish S. seohaensis from all other Shewanella genomes used to construct the wgMLST scheme proposed here. Our work illustrates a way to overcome the limitations of several techniques for the identification of species within the Shewanella genus. While phenotypic traits may offer initial insights, complementing this information with the presence of species-specific genes, is an effective strategy for accurately identifying Shewanella isolates, serving as a practical and cost-effective alternative to genome sequencing. The wgMLST scheme proposed constitutes a framework allowing researchers to explore the diversity of each of these closely related species with potential consequences for our understanding of their pathogenic potential, environmental role, and gene flow between them. Several studies report in vitro experiments to obtain insights into the potential role of different Shewanella species in bioremediation processes (see for instance ( 29 )). However, the large diversity observed within each of these species suggests that generalizing the results obtained from a single strain to the whole species may be particularly questionable in Shewanella spp. The wgMLST scheme proposed can help in providing a population context and help guide the choice of strains for further study. The growing recognition of the importance of Shewanella as potential infectious agents, in bioremediation, and in the environment at large highlights the relevance of accurate species identification to understand their interactions and specific roles in these processes. Materials and Methods Bacterial strains The bacterial strains used in this work are described in Table 1 . All strains were routinely cultivated on Luria-Bertani (LB) agar or in LB broth ( 30 ) at 37 ᵒC with aeration (at 150 rpm), except S. putrefaciens 95, which was cultured at 30 ᵒC. The genomes of S. putrefaciens DSM9451 and S. xiamenensis HI32665 were sequenced to validate their identification, while the genomes of all other isolates are accessible on public databases. Genome sequencing Genomic DNA from S. putrefaciens DSM9451 and S. xiamenensis HI32665 was extracted using the Invitrogen PureLink Genomic DNA mini kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions and using cultures grown in LB broth at 37 ᵒC overnight. The genomes were sequenced using an Illumina Nextseq® 500 system (2 X 150bp PE) (Illumina ©, San Diego, CA, USA), following the low-volume Nextera protocol ( 32 ). The reads were assembled with the INNuca pipeline v3.1 ( 33 ). For taxonomic classification, Kraken software 2.0.9 ( 34 ) was used. Phylogenetic analysis The intergenomic distances were evaluated by digital DNA-DNA hybridization ( d DDH) ( 35 ) and Average Nucleotide Identity (ANI) ( 36 ) methods. The technique used was Genome-BLAST Distance Phylogeny (GBDP), through the GGDC 3.0 online service ( 37 ), where the d DDH was calculated between the target genome and all the Shewanella genomes available in the database on 17 th June 2024. Strains of the same species are expected to exhibit dDDH values >70%, while isolates corresponding to the same subspecies should present values >79% ( 35 ). The values of d DDH were determined according to formula d6 , which preserves most information ( 35 ). The TYGS online service, which employs the same technique (GBDP) combined with the G+C content, creates a whole-genome-based GBPD tree ( 25 ). ANI is a similar method to d DDH ( 36 ). An ANI value >95% indicates that the genomes belong to the same species ( 36 , 38 ). Calculation of ANI values using the BLAST algorithm (ANIb) was achieved using JSpeciesWS v.4.1.1 ( 38 ). Phenotypic analysis Temperatures permissive for growth were assessed by cultivating the different strains on LB agar at 4ᵒC, 30ᵒC, 37ᵒC, 40ᵒC, and 42ᵒC. The biochemical characterization of the different strains was performed following the protocols described in the “Clinical Microbiology Procedures Handbook” ( 39 )and in line with the established standards for the Shewanella genus ( 4 ). Hydrogen sulfide production, fermentation of glucose and lactose, and gas production were tested in Kligler’s Iron Agar (KIA) ( 39 ). Enzymatic activities and carbon assimilation were tested using Analytical Profile Index (API) kits: API 20 NE and API ZYM ( 40 – 42 ). All phenotypic tests were performed in triplicate. Creation of a whole-genome multilocus sequence typing (wgMLST) scheme To establish a finer relationship between isolates, a whole-genome multilocus sequence typing (wgMLST) scheme was developed. This was used to characterize the isolates sequenced in this study, as well as all genomes of closely related species of the Shewanella genus, including S. baltica, S. putrefaciens, S. oneidensis, S. glacialipiscicola, S. morhuae, S. oncorhynchi, S. mangrovisoli, S. hafniensis, S. shenzhenensis, S. acanthi, S. profunda, S. septentrionalis, S. decolorationis and S. xiamenensis , available on NCBI GenBank on the 17 th of June 2024. Supplementary Table 3 presents the list of all the assembly identifiers used in this study. Based on 202 draft or complete genomes, the wgMLST scheme was generated with chewBBACA v3.2.0 CreateScheme module ( 43 ), resulting in a scheme with 21046 loci representing the genetic diversity of the selected genomes. Upon verifying the quality of the allelic profiles, it was decided to remove 11 genomes due to poor quality (low representation of core genome loci) or unverified origin resulting in a final data set of 191 genomes. This data was visualized in PHYLOViZ online ( 44 ). Polymerase chain reaction for the identification of S. seohaensis To differentiate S. seohaensis from closely related species, particularly the S. putrefaciens type strain and S. xiamenensis , one gene exclusive to S. seohaensis was selected, and specific primers were designed (VEE60752.1_F and _R – see Table 3 ). This gene ( phoE ), with a size of 609 bp, encodes for a phosphatase PhoE (ID: VEE60752.1). In silico PCR was performed to validate the amplification of a single product exclusively in the genomes of S. seohaensis. A PCR assay was performed for each strain using genomic DNA, the NZYTaq II 2x Green Master Mix (NZYTech, Lda, Lisboa, Portugal), and the primers indicated in Table 3 . The 16sRNA gene, amplified with primers suitable for the Shewanella genus (She211f and She1259r) ( 45 ), was used as a control for identification as Shewanella . Data availability To promote open science and reproducibility, all resources associated with this schema have been made publicly available. These include: the wgMLST schema and its annotations; the GenBank files used that can be used for functional annotation of loci; AlleleCall reports and AlleleCallEvaluator output; the wgMLST matrix of genetic profiles; and the metadata for all genomes used for schema creation and allele calling. The full dataset is available at: https://doi.org/10.5281/zenodo.13735156 . The Schema and its annotations is available at: https://chewbbaca.online/species/ . Acknowledgement Financial support was provided by the following institutes and funding agencies with the respective projects specified: MOSTMICRO-ITQB base funding with references UIDB/04612/2020 and UIDP/04612/2020, LS4FUTURE Associated Laboratory (LA/P/0087/2020), Portuguese Foundation for Science and Technology (FCT) with the project grant PTDC/BIA-BQM/4143/2021 and the PhD grant 2022.12339.BD for Maria de Oliveira Firmino. References 1. ↵ MacDonell MT , Colwell RR . 1985 . Phylogeny of the Vibrionaceae, and Recommendation for Two New Genera, Listonella and Shewanella . Syst Appl Microbiol 6 : 171 – 182 . OpenUrl CrossRef Web of Science 2. ↵ Satomi M . 2014 . Shewanella . Encyclopedia of Food Microbiology 3 : 397 – 407 . OpenUrl 3. ↵ Hau HH , Gralnick JA . 2007 . Ecology and Biotechnology of the Genus Shewanella . Annu Rev Microbiol 61 : 237 – 58 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Bowman JP . 2015 . Shewanella . Bergey’s Manual of Systematics of Archaea and Bacteria 1 – 22 . 5. ↵ Fonseca BM , Soares RM , Paquete CM , Louro RO . 2021 . Bacterial Power: An Alternative Energy Source , p. 215 – 246 . In Moura , JJG ; MI; ML (ed.), Enzymes for Solving Humankind’s Problems . Springer International Publishing , Cham . 6. ↵ Sharma KK , Kalawat U . 2010 . Emerging infections: Shewanella - A series of five cases . J Lab Physicians 2 : 61 . OpenUrl CrossRef PubMed 7. Hong J , Steen C , Wong E , Keong B . 2020 . Shewanella : an important, emerging and lethal pathogen in a patient with recurrent presentations of cholangitis . BMJ Case Rep 13 : e237655 . OpenUrl CrossRef PubMed 8. ↵ Yu K , Huang Z , Xiao Y , Wang D . 2022 . Shewanella infection in humans: Epidemiology, clinical features and pathogenicity . Virulence 13 : 1515 – 1532 . OpenUrl CrossRef PubMed 9. ↵ Gilardi GL . 1972 . Infrequently Encountered Pseudomonas Species Causing Infection in Humans . Ann Intern Med 77 : 211 – 215 . OpenUrl CrossRef PubMed Web of Science 10. ↵ Nozue H , Hayashi T , Hashimoto Y , Ezaki T , Hamasaki K , Ohwada K , Terawaki Y . 2008 . Isolation and Characterization of Shewanella alga from Human Clinical Specimens and Emendation of the Description of S. alga Simidu et al., 1990, 335 . Int J Syst Bacteriol 42 : 628 – 634 . OpenUrl 11. ↵ Heidelberg JF , Paulsen IT , Nelson KE , Gaidos EJ , Nelson WC , Read TD , Eisen JA , Seshadri R , Ward N , Methe B , Clayton RA , Meyer T , Tsapin A , Scott J , Beanan M , Brinkac L , Daugherty S , DeBoy RT , Dodson RJ , Durkin AS , Haft DH , Kolonay JF , Madupu R , Peterson JD , Umayam LA , White O , Wolf AM , Vamathevan J , Weidman J , Impraim M , Lee K , Berry K , Lee C , Mueller J , Khouri H , Gill J , Utterback TR , McDonald LA , Feldblyum T V. , Smith HO , Venter JC , Nealson KH , Fraser CM . 2002 . Genome sequence of the dissimilatory metal ion– reducing bacterium Shewanella oneidensis . Nat Biotechnol 20 : 1118 – 1123 . OpenUrl CrossRef PubMed Web of Science 12. ↵ Antonelli A , Di Palo DM , Galano A , Becciani S , Montagnani C , Pecile P , Galli L , Rossolini GM . 2015 . Intestinal carriage of Shewanella xiamenensis simulating carriage of OXA-48–producing Enterobacteriaceae . Diagn Microbiol Infect Dis 82 : 1 – 3 . OpenUrl CrossRef PubMed 13. ↵ Fang Y , Wang Y , Liu Z , Lu B , Dai H , Kan B , Wang D . 2017 . Shewanella carassii sp. nov., isolated from surface swabs of crucian carp and faeces of a diarrhoea patient . Int J Syst Evol Microbiol 67 : 5284 – 5289 . OpenUrl CrossRef PubMed 14. ↵ Janda JM . 2014 . Shewanella : A Marine Pathogen as an Emerging Cause of Human Disease . Clin Microbiol Newsl 36 : 25 – 29 . OpenUrl CrossRef 15. ↵ Firmino MO . 2022 . The Shewanella genus – understanding an emerging human pathogen . Master thesis. Master thesis - Universidade de Lisboa . 16. ↵ Jousset AB , Dabos L , Bonnin RA , Girlich D , Potron A , Cabanel N , Dortet L , Glaser P , Naas T . 2018 . CTX-M-15-Producing Shewanella Species Clinical Isolate Expressing OXA-535, a Chromosome-Encoded OXA-48 Variant, Putative Progenitor of the Plasmid-Encoded OXA-436 . Antimicrob Agents Chemother 62 . 17. ↵ Martin-Rodríguez AJ , Meier-Koltho JP . 2022 . Whole genome-based taxonomy of Shewanella and Parashewanella . Front Microbiol 10 : 1 – 14 . OpenUrl 18. ↵ Thorell K , Meier-Kolthoff JP , Sjöling Å , Martín-Rodríguez AJ , Bowman JP . 2019 . Whole-Genome Sequencing Redefines Shewanella Taxonomy . Front Microbiol 10 : 1861 . OpenUrl CrossRef PubMed 19. ↵ Potter RF , D’Souza AW , Wallace MA , Shupe A , Patel S , Gul D , Kwon JH , Andleeb S , Burnham C-AD , Dantas G . 2017 . Draft Genome Sequence of the bla OXA-436 - and bla NDM-1 -Harboring Shewanella putrefaciens SA70 Isolate . Genome Announc 5 : 12 . OpenUrl 20. ↵ Janda JM , Abbott SL . 2014 . The genus Shewanella : from the briny depths below to human pathogen . Crit Rev Microbiol 40 : 293 – 312 . OpenUrl CrossRef PubMed 21. ↵ Yu K , Huang Z , Li Y , Fu Q , Lin L , Wu S , Dai H , Cai H , Xiao Y , Lan R , Wang D . 2021 . Establishment and Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Detection of Shewanella Genus . Front Microbiol 12 : 1 – 10 . OpenUrl CrossRef 22. ↵ Huang J , Sun B , Zhang X . 2010 . Shewanella xiamenensis sp. nov., isolated from coastal sea sediment . Int J Syst Evol Microbiol 60 : 1585 – 1589 . OpenUrl CrossRef PubMed 23. ↵ Zong Z . 2011 . Nosocomial peripancreatic infection associated with Shewanella xiamenensis . J Med Microbiol 60 : 1387 – 1390 . OpenUrl CrossRef PubMed 24. ↵ Holmes B , Lapage SP , Malnick H . 1975 . Strains of Pseudomonas putrefaciens from clinical material . J clin Path 149 – 155 . 25. ↵ Meier-Kolthoff JP , Göker M . 2019 . TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy . Nat Commun 10 : 2182 . OpenUrl CrossRef PubMed 26. ↵ Yoon J-H , Park S , Jung Y-T , Lee J-S . 2012 . Shewanella seohaensis sp. nov., isolated from a tidal flat sediment . Antonie Van Leeuwenhoek 102 : 149 – 156 . OpenUrl CrossRef PubMed 27. ↵ Mamede R , Vila-Cerqueira P , Silva M , Carriço JA , Ramirez M . 2021 . Chewie Nomenclature Server (chewie-NS): a deployable nomenclature server for easy sharing of core and whole genome MLST schemas . Nucleic Acids Res 49 : 660 – D666 . OpenUrl CrossRef 28. ↵ Arinto-Garcia R , Pinho MD , Carriço JA , Melo-Cristino J , Ramirez M . 2015 . Comparing Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and Phenotypic and Molecular Methods for Identification of Species within the Streptococcus anginosus Group . J Clin Microbiol 53 : 3580 – 3588 . OpenUrl Abstract / FREE Full Text 29. ↵ Xia L , Cheng L , Xi W , Zhang X , Shi X . 2024 . Distinct influence of model electron shuttles on anaerobic mononitrophenols reduction in aquatic environments by Shewanella oneidensis MR-1 . J Hazard Mater 477 : 135348 . OpenUrl CrossRef PubMed 30. ↵ Bertani G . 2004 . Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems . J Bacteriol 186 : 595 – 600 . OpenUrl FREE Full Text 31. Derby H , Hammer B . 1931 . Bacteriology of Butter - Bacteriological Studies on Surface Taint Butter . Iowa Agric Exp Stn Res Bull 11 : 387 – 416 . OpenUrl 32. ↵ Baym M , Kryazhimskiy S , Lieberman TD , Chung H , Desai MM , Kishony R . 2015 . Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes . PLoS One 10 : e0128036 . OpenUrl CrossRef PubMed 33. ↵ Machado M , Halkilahti J , Jaakkonen A , Silva D , Mendes I , Nalbantoglu Y , Borges V , Ramirez M , Rossi M , Carriço J . 2021 . INNUca GitHub , https://github.com/B-UMMI/INNUca . Retrieved 4 August 2021. 34. ↵ Wood DE , Salzberg SL . 2014 . Kraken: ultrafast metagenomic sequence classification using exact alignments . Genome Biol 15 : R46 . OpenUrl CrossRef PubMed 35. ↵ Meier-Kolthoff JP , Auch AF , Klenk H-P , Göker M . 2013 . Genome sequence-based species delimitation with confidence intervals and improved distance functions . BMC Bioinformatics 14 : 60 . OpenUrl CrossRef PubMed 36. ↵ Richter M , Rosselló-Móra R . 2009 . Shifting the genomic gold standard for the prokaryotic species definition . Proceedings of the National Academy of Sciences 106 : 19126 – 19131 . OpenUrl Abstract / FREE Full Text 37. ↵ Meier-Kolthoff JP , Carbasse JS , Peinado-Olarte RL , Göker M . 2022 . TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes . Nucleic Acids Res 50 : 801 – D807 . OpenUrl CrossRef 38. ↵ Richter M , Rosselló-Móra R , Oliver Glöckner F , Peplies J . 2016 . JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison . Bioinformatics 32 : 929 – 931 . OpenUrl CrossRef PubMed 39. ↵ Isenberg HD . 1992 . Clinical Microbiology Procedures Handbook . American Society for Microbiology . 40. ↵ Humble MW , King A , Phillips I . 1977 . API ZYM: a simple rapid system for the detection of bacterial enzymes . J Clin Pathol 30 : 275 – 277 . OpenUrl Abstract / FREE Full Text 41. BioMerieux . 2019 . API Reference Guide .. 42. ↵ Geiss HK , Piotrowski HD , Hingst V . 1985 . Evaluation of API 20 NE in routine diagnostics of nonfermenting gram-negative rod-shaped bacteria . Zentralbl Bakteriol Mikrobiol Hyg A 259 : 35 – 42 . OpenUrl PubMed 43. ↵ Silva M , Machado MP , Silva DN , Rossi M , Moran-Gilad J , Santos S , Ramirez M , Carriço JA . 2018 . chewBBACA: A complete suite for gene-by-gene schema creation and strain identification . Microb Genom 4 : 1 – 7 . OpenUrl 44. ↵ Ribeiro-Gonçalves B , Francisco AP , Vaz C , Ramirez M , Carriço JA . 2016 . PHYLOViZ Online: web-based tool for visualization, phylogenetic inference, analysis and sharing of minimum spanning trees . Nucleic Acids Res 44 : 246 – 251 . OpenUrl CrossRef 45. ↵ Todorova SG , Costello AM . 2006 . Design of Shewanella -specific 16S rRNA primers and application to analysis of Shewanella in a minerotrophic wetland . Environ Microbiol 8 : 426 – 432 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted February 26, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade Maria de Oliveira Firmino , Mykyta Forofontov , Ricardo O. Louro , Alberto J. Martín-Rodríguez , Mário Ramirez , Catarina M. Paquete bioRxiv 2025.02.24.639908; doi: https://doi.org/10.1101/2025.02.24.639908 Share This Article: Copy Citation Tools Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade Maria de Oliveira Firmino , Mykyta Forofontov , Ricardo O. Louro , Alberto J. Martín-Rodríguez , Mário Ramirez , Catarina M. Paquete bioRxiv 2025.02.24.639908; doi: https://doi.org/10.1101/2025.02.24.639908 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7624) Biochemistry (17651) Bioengineering (13871) Bioinformatics (41882) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19867) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17714) Microbiology (40331) Molecular Biology (17148) Neuroscience (88483) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)