Shewanella infection in humans: Epidemiology, clinical features and pathogenicity

ABSTRACT The genus Shewanella consists of Gram-negative proteobacteria that are ubiquitously distributed in environment. As the members of this genus have rapidly increased within the past decade, several species have become emerging pathogens worldwide, attracting the attention of the medical community. These species are also associated with severe community- and hospital-acquired infections. Patients infected with Shewanella spp. had experiences of occupational or recreational exposure; meanwhile, the process of infection is complex and the pathogenicity is influenced by a variety of factors. Here, an exhaustive internet-based literature search was carried out in PUBMED using terms “Achromobacter putrefaciens,” “Pseudomonas putrefaciens,” “Alteromonas putrefaciens” and “Shewanella” to search literatures published between 1978 and June 2022. We provided a comprehensive review on the epidemiology, clinical features and pathogenicity of Shewanella, which will contribute a better understanding of its clinical aetiology, and facilitate the timely diagnosis and effective treatment of Shewanella infection for clinicians and public health professionals.


Introduction
The genus Shewanella comprises Gram-negative, facultative anaerobic, oxidase-positive and motile bacteria [1,2]. Due to the unique physiological and respiratory versatility, Shewanella spp. can survive in a wide range of ecological niches (for example, suboptimal environmental conditions with extreme salinity and high barometric pressure, spoilt foods and clinical specimens) and have been applied in environmental protection and industrial development [2]. Since the first identification of Shewanella putrefaciens in 1931 [3], several Shewanella species have recently emerged as worldwide pathogens, attracting the attention of the medical community [4]. Species like Shewanella algae, Shewanella putrefaciens, and Shewanella xiamenensis [5,6] have been proven to associate with human [4,7] and aquatic livestock diseases [8][9][10][11]. Also, Shewanella spp. can be found in food processing and storage [7]. Nowadays, members of the genus Shewanella are more than 70 (http://www.bacterio.net/shewanella.html). In order to identify the clinical features and to evaluate resistance pattern of Shewanella species, Wincy et al. retrospectively analyzed demographics, antibiotics, microbiology, and outcomes of the 128 patients who has been admitted to a regional hospital in Hong Kong with Shewanella species infection from 1 st April 2010 to 31th December 2020 [4]. In this review, we searched for case reports of Shewanella spp. infections in PUBMED from 1978 to June 2022 and provided an overview of the epidemiological features, clinical manifestations and pathogenicity of Shewanella infection, which may be helpful in guiding treatment strategy determinations and providing responsive therapy.
samples and skin injury swabs, which is consistent with the conclusion drown by Janda [7]. It is worth noting that in Denmark, S. algae has also been isolated in the pure culture of the ear swabs of 33 patients, suggesting its important role in causing ear infections [15]. Moreover, S. putrefaciens has been isolated from patients suffered from peritonitis [20][21][22][23][24] and meningitis [25], and S. xiamenensis has been isolated from intestinal specimen [6].

Infectious pathways and influencing factors
The infection caused by the genus of Shewanella is complex and can be influenced by a variety of factors. Firstly, Shewanella spp. are abundantly distributed in water environments, providing ample opportunities for these bacteria to come into close contact with humans [7]. Recreation (e.g. diving, playing on the beach), occupational exposure (e.g. crabbing, fishing), seafood ingestion, puncture wounds caused by marine organisms (sea urchins, fish), or the direct exposure of a wound to aquatic environments can increase the risk of Shewanella infection. The percentage of exposure to marine environments was reported to be 43.59% among patients with Shewanella infection (Table 1). Secondly, Shewanella infections are commonly found in patients with immunocompromised state, including malignancies, severe heart failure, renal failure, hepato-biliary disease, neutropenia, and chronic ulcerations on the lower extremities [7,26], although infections in healthy individuals with no medical history had also been reported [13,[27][28][29][30]. This kind of microorganisms can also be cultured from the clinical samples of burned or multiply traumatized patients, and patients with diabetes, leukaemia or immunosuppressive therapy. Among the above mentioned, liver diseases appeared to be a strong risk factor [23,[31][32][33][34]. Thirdly, Shewanella spp. have been found to associate with cases of nosocomial infection, leading to the outbreaks of healthcare-associated infections [5,24,[35][36][37]. Invasive procedures like catheterization and intubation are also an important source of infection [22,24,38]. An outbreak of 31 cases of abdominal and biliary tract infections or bacteraemia of Shewanella, caused by the exposure to a shared measuring cup, was reported in a general surgery unit in South Korea [35]. In addition, the differences of development, living styles and environmental pollution conditions result in different incidence rates of Shewanella infection. Brink et al. reported 28 cases of bacteraemia caused by S. putrefaciens in South Africa, and almost all cases were related to poor hygiene [16].

Symptom classification
As opportunistic pathogens, Shewanella spp. can cause a wide spectrum of clinical diseases in human. Documented illnesses by Janda et al. showed that Shewanella-related syndromes can be divided into five categories, including skin and soft-tissue infections (SSTIs), invasive diseases (such as sepsis), hepatobiliary diseases (hepatocirrhosis, liver cancer, cholangitis), otitis media and associated sequelae, and other infection [3]. SSTIs, including cellulitis, abscess, or necrotizing fasciitis, were considered as the most common clinical manifestation of infection. We divided Shewanella-related diseases into eight categories according to the infection site ( Figure 2), including ear, nose, and throat (E.N.T) disorders, central nervous system (CNS) disorders, chest infections, cardiovascular diseases, bloodstream infections (bacteraemia, septicaemia), intra-abdominal infections, bone arthropathy, skin and soft-tissue infections (SSTIs). The first four common clinical manifestations consist of bloodstream infections, SSTIs, E.N.T disorders, and intra-abdominal infections (Table 1). Certain bone or joint diseases such as arthritis [13,38,39], osteomyelitis [40][41][42][43], and discitis [44] can also be caused by Shewanella. SSTIs and bloodstream infections were reported to be predominate in the 16 cases of Shewanella infection in hospitals in Taiwan, followed by biliary tract infections [14]. Consistent with the conclusion of Vignier et al., bloodstream infection was the most common complication caused by Shewanella infection [13]. Bacteraemia was not associated with the ear infection of Shewanella in any case published to date.

Underlying diseases
Among the patients with Shewanella infection, 70.70% of them (n = 193) had underlying diseases (Table 2), some even had multiple diseases. As Wincy et al. concluded, hepatobiliary diseases, malignancy, chronic kidney disease or end-stage renal failure, and diabetes mellitus are important underlying disease [4] ( Table 2). In our study, 31 patients (11.36%) had hepatobiliary, spleen and pancreatic related diseases, like hepatitis, cirrhosis, liver abscess, and gallstones. It needs to be acknowledged that, with the increase in the number of included cases, the status of E.N.T disorders in the underlying diseases cannot be ignored [15]. Cancer infiltration, tumour block compression and other mechanical compression bring about neurological lesions and then result in joint diseases. Shewanella spp. are often found in bile as a part of mixed flora [35,45]. Biliary tract infection and cholelithiasis are mutually causal. Biliary tract obstruction caused by cholelithiasis will lead to cholestasis and bacterial reproduction. Diabetic microvascular lesions can affect the healing of foot ulcers, leaving the wound exposed and prone to bacterial infection [46]. Among the patients with Shewanella infection, 30 (10.99%) were diabetics with the complication of lower limb soft tissue ulcer(  [49], indicating that MALDI-TOF MS is a reliable and powerful tool for the rapid identification of Shewanella strains at the species level. Homology analysis of 16S rRNA gene is the most commonly used molecular identification method in prokaryotic systematics, known as the "molecular clock" of evolution [50]. However, due to the low evolution rate and high sequence conservation of the 16S rRNA gene, it is difficult to distinguish closely related species in the Shewanella genus [51,52]. Although the gene gyrB has higher resolution than 16S rRNA, there are limitations to the accurate identification of Shewanella species at the species level due to the lack of a unified species cutoff value and the uneven quality of sequences submitted in public databases [53][54][55]. Fang et al. established the method of multilocus sequence analysis (MLSA) based on several housekeeping genes [56]. The type strains of fiftynine species were used to describe the phylogenetic relationships and taxonomy of Shewanella, 12 distinct monophyletic clades were defined by using MLSA to clarify the evolutionary relationships of Shewanella spp. Among them, the Shewnaella pathogens isolated from humans are concentrated in two clades, Algae (S. algae, S. indica, S. haliotis) and Putrefaciens (S. putrefaciens, S. xiamenensis). The Shewanella spp. in the same clade are more closely related, showing <4 mol% GC variation and >84% concatenated similarity [56]. Here, after updating the MLSA data of Shewanella type strains, split network tree has been constructed by using SplitsTree 4 program (version: 4.14.4) based on concatenated sequences of 66 Shewanella species type strains ( Figure 3). Thirteen monophyletic clades have been clarified, and the conclusion of GC variation and concatenated similarity within the same clade consistent with that of Fang et al. By the end of 2021, over 300 genome sequences, which belong to 63 Shewanella species, had been collected and collated in GenBank. Genomic data revealed that the average genome size of Shewanella is 4.83Mbp and the average GC content is 46.84 mol%. The genomic sizes of marine strains were significantly correlated with water depth [57]. Typically, deep-sea Shewanella species exhibited larger genomic sizes than their shallow-sea counterparts [57]. As the number of genomic disclosures increases, researchers can achieve accurate identification of strains using conditions of average nucleotide identity (ANI) >95% and digital DNA -DNA hybridization (dDDH) >70% [58]. Thorell et al. quantitatively assessed the genomic diversity within Shewanella using dDDH, and reconstructed the phylogenomic associations among Shewanella strains. The resulting phylogenetic reconstruction revealed two major species cluster show a big difference in G+C content [59]. At the same time, it was emphasized that as new whole genome sequences from Shewanella type strains become available, the complexity of the taxonomic relationships within the genus Shewanella will be clearly realized.

Treatment
Shewanella-related infections can involve multiple parts of the human body. Based on the case reports, we summarized the treatment regimen and the resistance of the strains (Table 3). Most cases recover completely with treatment. However, there was no guideline of the antimicrobial therapy for Shewanella spp. infections in humans. Due to the existence of the patient's underlying disease and the complexity of the disease spectrum, it cannot be clearly summarized what type of diseases were treated with particular antibiotic. It is certain that empirical therapy was initiated with ceftazidime, meropenem, and vancomycin et al. broadspectrum coverage antibiotics, which could effectively control the growth of most pathogens (Table 3) [69]. Piperacillin-tazobactam was used in the initial empiric therapy for sepsis [60]. Usually, targeted treatment is according to the results of in vitro antimicrobial susceptibility testing on isolates. In the majority of cases, β-lactams, aminoglycosides, and quinolones were given to patients, which mostly had effective results. Moreover, there were great differences in drug resistance among different Shewanella strains. The frequent emergence of multidrug resistant (MDR) strains under the selective pressure of antibiotics makes the treatment more difficult, and clinicians need to be more cautious in antibiotic selection.

Morphological, physiological, and biochemical characteristics
There is no uniform biochemical or physiological profile in the genus of Shewanella [2]. As marine microorganisms, Shewanella spp. were originally isolated with marine agar 2216E, forming round, smooth, medium-sized, and raised colonies [56]. On nutrient agar, the colonies are light brown, round and raised with intact margins. Selective media are also used for the initial isolation of Shewanella strains, such as thiosulphate citrate bile salts (TCBS) sucrose agar, MacConkey agar, and blood plates. Colonies on TCBS are mediumsized, smooth, raised, rounded, with colourless edges and black centres. On MacConkey agar, 1-2 mm yellow-brown colonies can be seen, while on sheep's blood agar, some Shewanella species such as S. algae and S. putrefaciens can produce conspicuous β-haemolysis, and some strains can also form mucoid colonies. As researchers studied that β-haemolysis was detected in     all S. algae strains but only in a couple of S. putrefaciens isolates [111,112] (Figure 4). Derby and Hammer described the following general biochemical features of Shewanella spp.: Gramnegative, indole-negative, and gelatinase-, sucrose-, and maltose-positive [113]. This genus is positive for catalase and can reduce nitrate (NO 3 − ) to nitrite (NO 2 − ). Members of this genus are aerobic or facultative anaerobic, and some species can ferment D-glucose and N-acetylglucosamine to produce acid, usually without gas. Most of the Shewanella strains also present H 2 S-producing, urea-, arginine hydrolase-, lysine decarboxylase-negative phenotypes [12,114,115]. This article summarizes physiological and biochemical properties of seven common Shewanella spp. (Table 4) [12,[116][117][118][119][120].

Potential virulence factors
Pathogens must overcome several innate host defenses to attach to and colonize intestinal epithelium. Virulence factors of pathogens include the ability to invade human intestinal epithelial cells through virulence determinates, including flagella, homoserine lactones signal molecules, gelatinases, proteases and biofilm formation, which are essential for the first stage in infectious diseases [121]. Carla et al. characterized the attachment and internalization ability of S. putrefaciens by using human colonic carcinoma (Caco-2) cells, which shared morphological and functional features with normal small intestinal cells in post-confluent stage in vitro. The results showed that S. putrefaciens showed ability to attach and internalized into Caco-2 cells [121]. Members of the genus Shewanella have flagella, which promote motility, adhesion and biofilm formation. The mannose-sensitive haem agglutination (MSHA), and extracellular DNA (eDNA) play important roles in microbial attachment [122,123]. β-haemolysins has been found to involve in haemolytic activity, which is responsible for making S. algae more virulent [111]. Also, quorum sensing (QS), a cell-to-cell communication process in bacteria, has been studied in different Shewanella spp. [124]. In Gram-negative bacteria, the LuxI/R sensing acylhomoserine lactone (AHLs) was the most well-studied QS system [125]. S. putrefaciens were also able to produce AHL molecules, which could coordinate the expression of multiple virulence factors, such as biofilm development [121]. The homologous gene of LuxR has been predicted in the genomes of S. baltica, and the loss of this gene relates to the failure to produce AHLs. However, S. baltica can sense AHLs produced by other bacteria to assist QS-mediated cellular behaviours through LuxR receptor system [126]. Chitinase, lipase, protease, elastin, and alkyl sulphate enzymes produced by certain species of Shewanella may also associate with damage host tissues, being responsible for disruption and depletion of the mucus barrier. The genus Shewanella also has O-side chains or capsules with thickness varying from 20 to 130 nm, depending on  Table 4. Summary of physiological and biochemical characteristics of seven common species of the genus Shewanella.
Characteritics S. algae [12] S. indica [116] S. chilikensis [117] S. xiamenensis [118] S. putrifaciens [12] S. carassii [119] S. seohaensis [120] Type strains  different species [38]. The polysaccharide polymers on the surface of Shewanella are beneficial for its adhesion to solid substances and thus its infectivity [127]. In S. oneidensis, the biofilm facilitates microbial aggregation and then matures from a monolayer state to a three-dimensional structure, mediating by mxdABCD gene cluster and biofilm-promoting factor A (BpfA) [128,129]. Moreover, for tetrodotoxin (TTX), there are evidences that it associates with the pathogenicity of Shewanella [130]. Multiple virulence-specific genes, such as capsular polysaccharide biosynthesis, O-antigen and lasB (vibriolysin related gene) et al. have been identified through genomes scan [131]. In the genomes of S. algae, some putative genes, homologous to hlyA, hlyB, hlyD, and tolC genes encoding haemolysin operon, have been identified. RTX pore-forming toxin α-haemolysin, as the product encoded by hlyA, could change membrane permeability and lead to cell lysis [132]. The genes related to type IV pili, which are regulated by external stimuli for directed movement, and the gene vasF related to VAS T6SS, provide a fresh perspective on the pathogenicity of S. algae. Almost all strains of S. algae contain katA gene, the product of which can decompose hydrogen peroxide, protect cells from the toxic effects of hydrogen peroxide, and enhance the bacterial colonization ability in hosts. Also, the identification of genomic islands (GIs) harbouring a suite of virulence genes and mobile elements implied that the GIs may help cross-species gene transfer and contribute to the independent acquisitions of virulence factors in S. algae [133]. Genes related to the spoilage metabolic pathways (including trimethylamine metabolism, sulphur metabolism, putrescine metabolism, biofilm formation and serine protease production) and to illustrate the specific QS systems were identified, providing additional evidences for its metamorphic potential and pathogenicity to aquatic animals [134]. The study by Tamez et al. proposed new candidate virulence factors including the Fur protein, OmpA, T6SS, type VI secretion effector Phospholipase A1, microbial collagenase, DNase, type IV pili, curli, twinarginine translocation system, and ClpP [135]. Alex A et al. detected genes encoding core components of type III secretion system (T3SS) and type VI secretion system (T6SS) gene cluster, as well as the homologs of T3SS effector molecules, which aid to penetrate host mucous barriers. All structural components of T6SS were detected [136]. Virulence of pathogens depends on the activity of the T3SS injector and the effector proteins delivered to the host cell [137,138]. Previous studies revealed that VPA1328 and VopG are members of a larger family of T3SS2 effector proteins, linking to disruption of host cell survival and suppression of innate immunity in infected cells [139][140][141]. The VopG-like proteins have been found in S. baltica [142].

Antibiotic resistance
The antimicrobial susceptibility testing panel for aerobic Gram-negative bacilli could be used to perform the antimicrobial susceptibility testing of Shewanella by the micro-broth dilution method. Huang [144]. The diameter of the zone of inhibition around each disk was measured and the interpretation of results was acquired according to the guidelines of the CLSI. It should be emphasized that there are no unambiguous criteria for interpretation of antibiotic resistance of Shewanella spp., therefore it is impossible to unequivocally compare the results obtained by different authors. Although the drug susceptibility of the genus Shewanella varies among different species, these bacteria were usually susceptible to the third-and fourth-generation cephalosporins, carbapenems, βlactamase inhibitor combinations, aminoglycosides, chloramphenicol, erythromycin, aztreonam, and quinolones [3,13]. This is consistent with our findings from the summary of case reports (Table 3). Shewanella algae and S. putrefaciens are mostly susceptible to aminoglycosides, carbapenems, erythromycin, and quinolones and usually resistant to penicillin [3]. Their susceptibility to ampicillin and cephalosporins is variable [12]. It has been shown that the strains of S. algae strains harbouring eptA gene were colistin resistant [15,18,145,146], while those of S. putrefaciens were colistin sensitive [111]. Antibiotic resistance is on the rise in the genus of Shewanella. Zhao et al. investigated multidrug resistance in S. xiamenensis isolated from an estuarine water sample in China [147]. The results showed that the strain displayed resistance to ampicillin, aztreonam, cefepime, cefotaxime, chloramphenicol, ciprofloxacin, erythromycin, kanamycin and trimethoprim-sulfamethoxazole. As an important Shewanella pathogenic species, multi-drug resistant S. xiamenensis strains in the aquatic environment may become important reservoirs for resistance genes. Once infected in humans, it may lead to clinical treatment failure.
Bacteria of this genus are possible progenitors of many antibiotic resistance genes. The OXA-54 oxacillinase detected in S. oneidensis MR-1 was the progenitor of OXA-48 identified in Klebsiella pneumoniae [148]. S. xiamenensis was the progenitor of bla OXA-181 found in K. pneumonia [149]. S. algae was considered to be the origin of plasmid-mediated quinolone resistance determinant qnrA [150]. It was also suggested that the genus Shewanella is a reservoir for the MCR-4 mobile colistin resistance genes [151].
In Shewanella spp., the resistant genes were detected on the chromosomal regions surrounding by mobile genetic elements (MGEs) [3,45,152,153]. Numerous multidrug-resistant (MDR) and extensively drugresistant (XDR) Shewanella strains have been reported in recent years [18,147,152]. Most of the bla OXA-55 genes and their variants detected in S. algae are located on the chromosomes, indicating intrinsic resistance to β-lactams. However, the bla OXA-55 gene identified in the MDR S. algae strain MARS 14 is close to a MGEs (transposon) encoding gene, suggesting S. algae may be a possible reservoir of this gene [18]. In the MDR S. xiamenensis strains, MGEs (transposon) or plasmid have been detected, containing a variety of resistance genes and conferring resistance to trimethoprim, aminoglycosides, quaternary ammonium compounds, βlactams, chloramphenicol, sulphonamides, and tetracycline [152]. Not all Shewanella strains carrying β-lactam resistance genes develop a corresponding phenotype [154]. Jianhua Yin et al. discovered that ampicillin with high concentrations (>12.5 µg/ml) could induce the blaA gene, while ampicillin with low concentrations (<5 µg/ml) could not [155].

Outstanding questions
First of all, the diagnostic ability of Shewanella-related infections needs to be strengthened. Shewanella-related infections were easily overlooked in clinical sets. Physicians should take care to diagnose if there exists Shewanella infection when patients had a history of aquatic exposure [7]. Also, clinicians need to be aware of the ability of Shewanella spp. to cause invasive disease, and to understand the clinical and epidemiological characteristics of Shewanella infections, so as to provide an earlier microbiological presumptive diagnosis and to achieve greater controllability and predictability in clinical treatment.
Secondly, methods with more rapidity and accuracy need to be established for Shewanella spp. identification. Considering the complex and variable biochemical characteristics of the existing Shewanella species as well as the discovery of new species, the traditional commercial biochemical identification systems, such as API 20E, API 20 NE, Vitek 2 GN card (bioMérieux, France), cannot correctly identify Shewanella strains at the species level. Therefore, it is necessary to take enough representative strains into account in experimental verification. MALDI-TOF MS, despite the most commonly used instrument in clinical bacterial detection, sometimes can cause inaccurate species-level identification due to its dependence on databases. The development of modern sequencing technology has effectively revised the phylogenetic relationships of bacterial species and improved the accuracy of species identification. However, the 16S rRNA gene or single housekeeping gene lacks discriminatory value for Shewanella identification at the species level. MLSA, which needs PCR of several housekeeping genes, followed by truncation and concatenation of the gene sequences, still cannot meet the requirements of clinical detection timeliness. Researchers can also achieve accurate identification of strains based on whole-genome sequence information. However, it takes a long time to obtain the genome and then to analyze it. Therefore, it is particularly important to establish rapid and accurate detection methods, such as Shewanella speciesspecific gene PCR. Besides, standard procedures for the isolation of Shewanella strains in food and environmental samples should also be established.
Thirdly, considering the increasing antimicrobial resistance, research on effective and judicious use of antimicrobials as well as clarification of the duration of antibiotic use in clinical treatment is needed. As an important vehicle and a reservoir of antibiotic resistance genes, the genus of Shewanella contains a variety of drugresistant genetic elements, showing resistance to many drugs, including β-lactams, quinolones, aminoglycosides, macrolides and carbapenems. The use of broad-spectrum antibiotics provides continuous selection pressure to Shewanella strains, allowing them to evolve and become more resistant. Therefore, it is urgent to conduct drug resistance monitoring for Shewanella spp. and to analyze the resistance characteristics at the species level.

Conclusion
From a clinical and aetiological perspective, this review summarized our present knowledge of the epidemiology features, clinical manifestations and pathogenic characteristics of Shewanella infection, highlighting that some species are associated with a wide spectrum of clinical diseases. Physicians should take care to diagnose Shewanella infection when patients had a history of aquatic exposure. Studies of the clinical and epidemiological characteristics of Shewanella-induced diseases have helped to achieve greater controllability and predictability in clinical treatment. Clinicians need to be aware of the potential of Shewanella spp.
to cause invasive disease and should be able to provide earlier microbiological presumptive diagnosis. With the increasing incidences of Shewanella infection and the emerging drug resistance of Shewanella strains, further research is needed on how to use antibiotics effectively and judiciously, as well as on clarification of the duration of antibiotic use.