Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics

Authors


Corresponding author and reprint requests: H. M. Holt, Department of Clinical Microbiology, Odense University Hospital, Winsløvparken 21.2, DK 5000 Odense C, Denmark
E-mail: hanne.holt@ouh.fyns-amt.dk

Abstract

The occurrence of the two Shewanella species found in clinical specimens, Shewanella algae and Shewanella putrefaciens, correlates with the temperature and salinity of seawater. This means that Shewanella infections occur in warm climates or during especially warm summers in temperate climates. The infections described most commonly involve ears, skin and soft tissue, with or without bacteraemia. Primary bacteraemia with a fulminant course is also seen in immunocompromised patients. Important differential characteristics between the two species include the ability of S. algae to produce mucoid colonies with β-haemolysis on sheep blood agar, to grow at 42°C and in NaCl 6% w/v, and to reduce nitrite, and an inability to produce acid from maltose, all of which are in contrast to the characteristics of S. putrefaciens. Automated identification systems fail to differentiate between S. algae and S. putrefaciens, as S. algae is not included in the databases of these systems. Presumably for this reason, most Shewanella infections reported during recent years have been attributed to S. putrefaciens. However, when extensive phenotypic characterisation is performed, most human infections are seen to be caused by S. algae. As the two species seem to have different pathogenic potential for humans, correct identification is important, and this is possible in routine clinical microbiology laboratories.

Introduction

Human infections with members of the genus Shewanella are rare, being reported mainly from geographical areas with warm climates, e.g., the USA [1–8], Australia [9–13], Asia [14–22], South Africa [23,24] and southern Europe [25–30]. However, in recent years, cases from countries with temperate climates have also been reported [31–33]. Although the clinical significance of these organisms has often been obscured by their occurrence as part of a mixed bacterial flora, a number of monomicrobial infections have been reported, thereby documenting the pathogenic potential of Shewanella spp. [1,6,12,14,15,24,25,28–31].

Since the publication of Nozue et al.[34], it has become increasingly clear that most reports of human infections attributed to Shewanella putrefaciens have been caused by the species now named Shewanella algae[34–36]. Despite this, most infections caused by Shewanella spp. that have been reported in the literature have been attributed to S. putrefaciens[9,11,12,14,15,17,30]. This review describes the characteristics of S. algae and S. putrefaciens, and the bacteriological and clinical differences between these two species.

History and taxonomy

Shewanella spp. are Gram-negative, motile bacilli, whose most important phenotypic characteristic is the production of hydrogen sulphide. The first description of the species was provided in 1931 by Derby and Hammer [37], who isolated a hitherto unknown bacterial taxon from putrefied butter and water supplies of dairies, and named it Achromobacter putrefaciens. In 1941, Long and Hammer [38] proposed that this species should be transferred to the genus Pseudomonas under the name Pseudomonas putrefaciens. During the next three decades, these organisms were placed in Pseudomonas group IV, according to Shewan et al.[39], and they became known as some of the most important spoilage bacteria of fish [40–43].

In the 1974 edition of Bergey's Manual of Systematic Bacteriology, P. putrefaciens was classified as ‘species incertae sedis’, mainly because the G + C content of 43–55% was below the range for Pseudomonas spp. (58–70%). However, several studies agreed that isolates of P. putrefaciens could be separated into at least two different taxa on the basis of differences in salt tolerance, growth temperatures and saccharolytic activity [44–46]. In 1978, Owen et al.[47] divided isolates into four DNA homology groups, I–IV, a division that supported the results of other investigators [34,36,48]. In 1985, phylogenetic studies resulted in a reclassification of these organisms to the family Vibrionaceae, and the description of a new genus, Shewanella[49], named after James Shewan in honour of his work in marine microbiology. Included in this new genus was S. putrefaciens and the related species Shewanella hanedai and Shewanella benthica.

In the early 1990s, DNA homology group IV of Owen et al.[47] was reclassified as a new species, Shewanella alga (sic), by Simidu et al.[50], emended subsequently by Nozue et al.[34] on the basis of genomic and phenotypic studies. S. alga was found to have a G + C content of 52–54 mol%, compared with 46% for the type strain of S. putrefaciens, while DNA homology between the type strains of S. alga and S. putrefaciens was found to be < 10%. Other studies confirmed the significant genomic differences between the two species by ribotyping, 16S rRNA analysis, gyrB gene sequencing, and DNA–DNA hybridisation [36,51]. In 1997, the name of this new species was corrected to S. algae[52]. On the basis of small-subunit rDNA sequencing, the genus Shewanella was shown to be a robust monophyletic taxon within the gamma subgroup of the phylum Proteobacteria [53]. Recent results of 16S rRNA gene sequence analyses of genera from this group led to a proposal for a new family, Shewanellaceae [54], containing about 30 Shewanella spp., most of which are psychrophilic and therefore of little interest to clinical microbiologists.

Epidemiology and pathogenicity

Shewanella spp. are found throughout the world, mainly in marine environments. They are important in the turnover of organic material, and are capable of dissimilatory reduction of various metals and other substances, such as nitrate, nitrite, thiosulphate and trimethylamine-N-oxide. In Denmark, S. putrefaciens and S. algae can be isolated from seawater with a salinity of 15–20%[32], with the frequency of occurrence being correlated with the temperature of the seawater [55]. These organisms are detected in Denmark only during the months of July–October, when the water temperature is > 13°C [55]. This observation accords with the fact that most Shewanella infections occur in countries with a warm climate, or during especially warm summers in countries with temperate climates.

The only Shewanella spp. found in clinical specimens are S. putrefaciens and S. algae, and it seems likely that > 80% of isolates from humans are S. algae[32,34–36]. Of 164 clinical isolates examined by Holt et al.[56], mostly from ear swabs, only five were identified as S. putrefaciens. As these organisms are frequently isolated together with other bacteria, the pathogenic potential of Shewanella has been controversial. In a study of mice in which 1 mL of seawater was inoculated into surgical wounds, 30% of the mice developed a wound infection from which ‘P. putrefaciens’ isolates were cultured. Three of these isolates were then inoculated intraperitoneally into rats. This procedure resulted in bacteraemia in three of ten rats, with an LD50 of 50%[57]. It is not known whether the isolates used were S. algae or S. putrefaciens. Saxe and Levin [58] found no difference in pathogenicity between strains of S. putrefaciens‘biotype I’ and ‘biotype II’ (equivalent to S. algae). However, another mouse pathogenicity study performed by Khashe and Janda [35] indicated that S. algae was the more virulent species, and it was speculated that the haemolytic activity of S. algae could be an important virulence factor [35]. An ability to form biofilms has been described in detail for Shewanella oneidensis and S. putrefaciens[59,60]. In a Danish study of ear infections involving S. algae, 49% of patients had ear tubes, and biofilm formation could be a pathogenicity factor in such cases [32]. S. algae is tolerant to bile salts, and produces extracellular virulence factors such as siderophores and other exoenzymes [61]. Production of tetrodotoxin, the pufferfish toxin, has been reported [50], but this finding could not be reproduced by other investigators [61,62]. Intraperitoneal inoculation in mice has given rise to specific antibodies against S. putrefaciens[63].

The most obvious source for human infection is exposure to seawater. Such contact has been reported in some of the case reports in the literature [5,8,11,15,16,31,56] and in a Danish study of ear infections, where > 80% of patients had been swimming in the sea shortly before symptoms developed [32]. In Denmark, environmental and clinical isolates of S. algae demonstrate great clonal variability, as determined by ribotyping and random amplified polymorphic DNA profiling, but no systematic differences distinguishing environmental and clinical isolates have been found [64]. Furthermore, a Danish seawater isolate was found to be identical to one of the clinical isolates by all typing methods [64], supporting the theory of a marine source of infection.

Phenotypic characterisation and identification

S. algae and S. putrefaciens are non-fermentative bacilli with a single polar flagellum. They grow well on conventional solid media, including MacConkey agar, with 1–2-mm yellowish-brown colonies after incubation for 18–24 h. Table 1 summarises the main differences between S. algae and S. putrefaciens found by different investigators [32,34–36,50,56,65]. Characteristic traits include production of hydrogen sulphide, decarboxylation of ornithine, and hydrolysis of gelatine. S. putrefaciens is the more saccharolytic of the two species, with S. algae producing acid only from ribose, and sometimes from glucose and fructose, while S. putrefaciens produces acid from maltose and glucose, and sometimes sucrose and arabinose. In contrast to S. putrefaciens, S. algae shows weak β-haemolysis on sheep blood agar after incubation for 48 h, as well as growth at 42°C and in NaCl 6% w/v. Holt et al.[32,56] also found that S. algae was distinguished from S. putrefaciens by a mucoid colony consistency and an ability to reduce nitrite.

Table 1.  Summary of biochemical characteristics of Shewanella algae and Shewanella putrefaciens (type strains and results taken from different studies [32,34–36,50,56,66])
 S. algaeT (IAM 14159)S. algaeS. putrefaciensT (ATCC 8071)S. putrefaciens
  1. Parentheses indicate delayed/weak reactions; d, variable results.

Oxidase++++
Catalase++++
Indole production
Arginine dihydrolase
Lysine decarboxylase
Ornithine decarboxylase++++
H2S production++++
Urea hydrolysis
Gelatine hydrolysis++++
DNA hydrolysis++++
Nitrate reduction++++
Nitrite reduction++
Acid from
 Arabinose+d
 Ribose(+)(+)+d
 Glucose(+)(+)+d
 Fructose(−)(+)d
 Mannitol
 Lactose
 Maltosed
 Sucrosed
Growth
 4°C++
 42°C++
 NaCl 6–6.5% w/v++
Haemolysis (sheep blood, 48 h)++
Mucoid colony consistency++

Automated identification systems are unable to distinguish between S. putrefaciens and S. algae. A large number of clinical isolates identified tentatively as S. putrefaciens were shown to be S. algae by Nozue et al.[34]. A number of reports of human infections allegedly caused by S. putrefaciens do not supply sufficient data to show whether or not the isolates were actually S. algae[9,11,12,14,15,17,30]. This problem is caused by the fact that the semi-automated and automated identification systems used for identification in these reports (API 20E and 20NE; API ID 32 GN and Vitek) include S. putrefaciens, but not S. algae, in their databases. It also appears that these identifications as S. putrefaciens were not confirmed by reference laboratories.

Clinical manifestations

The clinical syndromes reported to be caused by S. algae and S. putrefaciens (hereafter called Shewanella infections unless the species designation is clearly justified) are generally the same as those caused by various species of the other marine halophilic genus Vibrio[11,66,67].

In Denmark, the most common Shewanella infection is infection of the ear with S. algae. This can take the form of acute infection or acute exacerbation in chronic otitis media in predisposed individuals. In a study of 67 ear infections [32], all Shewanella infections were caused by S. algae, with this organism being isolated in pure culture in 50% of cases. Outside Denmark, only a few such ear infections have been reported, and these date from the 1970s [2–4,22,44].

The most common clinical syndrome described in the literature is infection of skin and soft tissue, associated with breaches in the skin such as ulcers or following trauma [10,11,15,16,24,30,31,68,69]. Bacteraemia is often present, but the course is usually benign. In approximately 40 published reports, c. 40% of cases have involved pure cultures of Shewanella. Two cases of S. alga (sic) bacteraemia, one with myonecrosis, involving patients with chronic ulcers have been reported in Denmark [31]. Primary bacteraemia, associated with severe hepatobiliary disease and malignancy with an often fulminant course, has also been described [6,16,24]. A distinction between these two syndromes is not always possible, as cases involving both cellulitis and underlying debilitating diseases have been described.

Another clinical syndrome, hitherto described only in South Africa, is paediatric bacteraemia in underweight babies [24], with 19 cases occurring in two hospitals during 1990–1993. Of 16 neonates, all suffered from respiratory distress at birth, and six subsequently died. In 12 cases, the organism was isolated shortly after birth, thereby indicating the possibility of intrapartum infection.

Respiratory colonisation, and possibly infection, has been seen in a peritoneal dialysis patient with bacteraemia [13], in a patient after near-drowning [8], in patients with tuberculosis [70], and in patients with pleural empyema as a complication of biliary tract infection [16]. There are also anecdotal reports of Shewanella bone and joint infections [2,7,10,28], meningitis [26], cerebellar abscess [14], endocarditis [17], an infected aortic aneurysm [29] and eye infections [1,16].

Among the clinical isolates examined by Holt et al.[56], only 3% were identified as S. putrefaciens. The organism was not detected in blood cultures, and evidence of its pathogenic role in Danish clinical cases is lacking. This is in agreement with the findings of other investigators [34–36].

Antibiotic susceptibility and treatment

S. algae and S. putrefaciens are characteristically susceptible to aminoglycosides, carbapenems, erythromycin and quinolones, but resistant to penicillin [6,16,32,36,45,56]. Susceptibility to ampicillin and cephalosporins is variable, with more isolates being susceptible to third- and fourth- than to first- and second-generation cephalosporins [6,32,45,56]. All Danish S. algae isolates were susceptible to piperacillin, aminoglycosides, ciprofloxacin, erythromycin and tetracycline, while susceptibility to ampicillin and cephalosporins varied [56]. All isolates of S. algae were resistant to colistin, while six clinical isolates of S. putrefaciens were all susceptible. Thus polymyxin susceptibility can be used to differentiate between the two species. The six cases involving S. putrefaciens were regarded as colonisations and were consequently not treated with specific antibiotics. In contrast, many cases with S. algae were treated with local or systemic antibacterial agents [32,56]. According to the literature, most Shewanella infections are treated easily by a combination of surgical therapy/drainage and antibiotics [14,15,29,31,32,56,69]. Poor outcome is associated most often with underlying disease [6,16,24]. Treatment options include β-lactams (provided that the strain is susceptible), aminoglycosides and quinolones.

Conclusions

The increasing number of reports of Shewanella infections in which the organism has been isolated in pure culture indicates the pathogenic potential of this genus. Most human infections are caused by S. algae. Automated identification systems are unable to distinguish between S. putrefaciens and S. algae. Important differences between the two species exist regarding genotypic and phenotypic characteristics, pathogenicity and susceptibility to antimicrobial agents. A prerequisite for further study in this field is correct identification, which is possible in routine clinical microbiological laboratories with careful conventional phenotypic characterisation.

The epidemiology and clinical symptoms of S. algae infections are similar to those of infections involving Aeromonas and halophilic Vibrios. The most common S. algae infections involve ears and soft tissue, but serious infections such as bacteraemia, meningitis and endocarditis have also been described. Marine environments can often be implicated as the source of infection.

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