Shewanella putrefaciens: a rare cause of purulent otorrhoea

Shewanella putrefaciens is a Gram-negative, non-fermenting, motile and oxidase-positive bacillus. Its incrimination in human pathology is very rare, although there has been a resurgence in Shewanella infections in recent years. We report the first case in Morocco of a purulent otorrhoea caused by S. putrefaciens , resistant to conventional treatment, occurring in a 25-year-old female, afebrile, without deterioration of the general state and possibly acquired during sea bathing. We also describe the bacteriological characteristics of and antibiotic susceptibility results for the isolate.


INTRODUCTION
The genus Shewanella is in the family Shewanellaceae, which includes Gram-negative, non-fermentative, motile and oxidasepositive bacilli. Shewanella putrefaciens was formerly known as Pseudomonas putrefaciens; it was classified in the family Vibrionacae until the 1990s, when it was reclassified into the genus Shewanella [1]. The majority of these bacteria were originally found in aquatic environments [2].
Human infections are rare, but have been increasingly reported in recent years, including bacteraemia and skin and soft tissue infections [3]. However, purulent otorrhoea due to S. putrefaciens is very rare. We report the first case in Morocco of a purulent otorrhoea caused by S. putrefaciens, resistant to conventional treatment, occurring in a 25-year-old female, afebrile, without deterioration of the general state and possibly acquired during sea bathing. We also describe the bacteriological characteristics of and antibiotic susceptibility results for the isolate.

CASE REPORT
The patient is a 25-year-old woman, without any medical history, treated in our ENT department for chronic otitis media with open eardrum. Her ear had been dry for more than 6 months after treatment including amoxicillin-clavulanic acid 3 g/day associated with ofloxacin ear drops. Currently, she has been admitted for purulent foetid otorrhoea following a swim at the beach. The illness is 1 month old. The symptomatology started 4 days after her swim, motivating her consultation with a general physician who prescribed the same probabilistic treatment as before, based on amoxicillin-clavulanic acid 3 g/day associated with ofloxacin ear drops for 15 days without improvement. Clinical examination on admission was normal. Blood tests showed a CRP of 150 mg l −1 and hyperleukocytosis of 12 000 GB ml −1 .
As part of the aetiological exploration, an auricular pus sample was taken in order to identify the germ responsible for the infection and adapt the treatment. Direct microscopic examination of the pus showed numerous neutrophils, a few epithelial cells and OPEN ACCESS numerous Gram-negative bacilli. Pus was cultured in Columbia agar, polyvitex chocolate agar, Columbia agar with nalidixic acid, nystatin and colistin, and Sabouraud agar with chloramphenicol. Cultures were placed in an incubator supplemented with 5-10 % CO 2 for 24 h at 37 °C. All cultures were pure with pigmented colonies, showing no haemolysis on Columbia agar after 2 days of incubation. Colonies were oxidase-positive. Identification was performed using an API 20NE gallery (bioMérieux, Marcy l'étoile, France), resulting in an excellent identification (code 3051354) of the S. putrefaciens group. However, the API 20NE gallery cannot distinguish between Shewanella algae and S. putrefaciens. Therefore, we required additional tests based on the criteria of Nozue et al. [4] for better species identification (see Table 1).
Antibiotic susceptibility testing was performed using the microdilution method with Sensititre Gram-negative MIC plates (Thermo Scientific, France) from a young culture. Results were interpreted in accordance with the Comité De l'Antibiogramme de la Société Française de MicrobiologieCASFM (CASFM)/European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2021 recommendations.
Our isolate was sensitive to all antibiotics tested except amoxicillin and amoxicillin-clavulanic acid ( Table 2).
The patient was placed on levofloxacin 500 mg twice a day for 15 days with a good clinical evolution marked by the interruption of purulent discharge after 10 days of treatment.

DISCUSSION
Shewanella spp. are Gram-negative bacilli found in seawater [5]. They allow a renewal of organic matter and also a reduction of various metals and substances [1]. The Shewanella spp existing in clinical samples are S. putrefaciens and S. algae, and currently more than 80 % of human isolates are S. algae [4,[6][7][8]. At present, there are molecular methods based on 16S rRNA and gyrB to differentiate S. algae from S. putrefaciens, but these tests are used more in the research laboratory than in the routine laboratory. However, these two species can easily be distinguished by phenotypic properties such as the capacity of S. algae to develop at 42 °C and a high concentration of NaCl (e.g. 6 %), unlike S. putrefaciens. In a study reported by Holt et al., of 164 clinical isolates from ear samples, 5 isolates were identified as S. putrefaciens [9]. Another case of S. putrefaciens isolated from an ear swab was reported by Martín-Rodríguez et al. [10]. Our case represented the seventh case of S. putrefaciens otitis in the literature and the second in our Mediterranean region. Using molecular methods, other species have been reported in the literature, such as Shewanella chilikensis, Shewanella carasii and Shewanella xiamenensis [11][12][13] .
These Shewanella infections have been reported in coastal areas and hot climates. In recent years, cases have begun to occur in temperate regions [6,14,15]. These infections are frequent in July, August and September, with a few cases in October [1]. Our case occurred inSeptember, which is in accordance with the literature. Contact with seawater remains the most common source of human infection. Cases with contact with seawater have been reported in numerous studies [9,14,[16][17][18][19][20] and in another Danish study on ear infections, where more than 80 % of the patients had some exposure [6]. Our case supports the data in the literature. Shewanella infections predominantly occur among males, although this predominance may be due to genetic or sociocultural factors [10]. Skin and tissue infections following skin tears or trauma are the clinical syndrome most described in the literature [14,[18][19][20][21][22][23][24][25]. According to a Danish study, most patients present with symptoms of acute or chronic otitis or non-specific ear discharge and are between 3 and 15 years of age [6] . On the other hand, our patient was 25 years old and had a chronic otitis with purulent discharge.
Holt et al. reported that the time to onset of symptoms varies between 1 to 5 days after exposure to seawater [6]. Our patient reported that she had a discharge 4 days after swimming in the beach. According to the same Danish study cited above, 49 % of S. algae isolates were isolated in pure cultures, documenting the pathogenic potential of this emerging germ [6]. Our strain has been identified in pure culture.
S. algae and S. putrefaciens retain good sensitivity to aminoglycosides, carbapenems, erythromycin and quinolones except penicillin [6,8,9,20,26,27]. These isolates have variable susceptibility to amoxicillin and cephalosporins, with more isolates susceptible to third-and fourth-generation cephalosporins than to first-and second-generation cephalosporins [6,9,26,27]. In a Danish study, all S. algae isolates were susceptible to piperacillin, aminoglycosides, ciprofloxacin, erythromycin and tetracycline, with variable susceptibility to ampicillin and cephalosporins [9]. Holt et al. reported that all isolated S. algae showed resistance to colistin, and six S. putrefaciens isolates were susceptible [6]. Therefore, polymyxin sensitivity can be used to differentiate between the two species. Our S. putrefaciens isolate was susceptible to all antibiotics tested except amoxicillin and amoxcillin-clavulanic acid. According to the literature, the treatment of Shewanella infections is easy and includes surgical and medical treatment [6,9,14,19,25,28,29]. Medical treatment is based on β-lactams, aminoglycosides and quinolones, provided that the strain is sensitive to these molecules [1].

CONCLUSION
The emergence of Shewanella infections in our region requires us to consider them in patients with a history of ear disease and recent contact with seawater.
Peer review history 5-Shewanella infections can be severe when they involve other organ systems (e.g. skin and soft tissue infections, hepatobiliary infections, bactereaemia) and patients may have underlying medical co-morbidities. In cases with ear infections, severe infections are rare and most of them occur in young and immunocompetent patients, according to a large case series on Shewanella ear infections. Please consider rephrasing your lines 100-102, as Shewanella ear infections are already known to occur in immunocompetent subjects.
Answer: thank you for this comment.It has been deleted.
6-There are already a number of case reports and case series on Shewanella ear infections in existing literature. What is the innovation of your case report among the existing case reports and case series?
Answer: thank you for this comment.The innovation of our report lies : -The rarity in our Mediterranean region -To enrich the literature.
7-Consider including the references of existing case series and case reports of Shewanella ear infections and compare your case report with these references.
Answer: thank you for this comment.They were included. Line 141,156.
8-You have mentioned in line 111 that a lack of treatment guidelines contributes to emergence of antibiotic resistance. You then stated in the conclusion that the increasing incidence of Shewanella infections requires us to consider empirical coverage of Shewanella infections with marine exposure. What is your recommendation of antibiotic choice to empirically cover for Shewanella infections without contributing to antibiotic resistance?
Comments: 1. What is the season in which the described case occurred in? Is this in alignment with the usual climate as stated in existing literature? 2. What is the time frame between the exposure to seawater to the development of symptoms? 3. Any documentation on the history of "chronic otitis media" -could it be a recurrence of the same species in the same patient? 4. Co-infection has been commonly described in infections due to Shewanella species. Were any other organisms isolated besides Shewanella putrefaciens? 5. Shewanella infections can be severe when they involve other organ systems (e.g. skin and soft tissue infections, hepatobiliary infections, bactereaemia) and patients may have underlying medical co-morbidities. In cases with ear infections, severe infections are rare and most of them occur in young and immunocompetent patients, according to a large case series on Shewanella ear infections. You have mentioned in line 111 that a lack of treatment guidelines contributes to emergence of antibiotic resistance. You then stated in the conclusion that the increasing incidence of Shewanella infections requires us to consider empirical coverage of Shewanella infections with marine exposure. What is your recommendation of antibiotic choice to empirically cover for Shewanella infections without contributing to antibiotic resistance?