Shigella sonnei, an emerging multidrug-resistant sexually transmitted pathogen in Franche-Comté, France

ABSTRACT Shigella sonnei (S. sonnei) is sometimes sexually transmitted. Men, who have sex with men (MSM), may have sexual behaviours different from heterosexual population, and thus may be at risk for S. sonnei infection. We describe three cases of multidrug-resistant S. sonnei in MSM (one HIV-infected patient and two patients receiving pre-exposure prophylaxis against HIV). S. sonnei was isolated from stool specimens and all patients were successfully treated with parenteral third-generation cephalosporins following laboratory confirmation that the isolates were resistant to azithromycin. Two men (patients 2 and 3) were linked epidemiologically. These cases highlight the emergence of this pathogen and its association with some sexual behaviours among MSM in Franche-Comté, France.


Introduction
Shigella, a pathovar of Escherichia coli, is a Gramnegative bacterial pathogen, which causes bacillary dysentery or bloody diarrhoea in humans. This pathovar comprises four groups, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii. Currently, Shigella sonnei (S. sonnei) is an emerging pathogen globally, and a common cause shigellosis predominant in high-income countries [1,2]. Usually, typical risk groups in shigellosis include paediatric population and travellers to developing countries [2]. In the last years, S. sonnei infections have significantly increased among men who have sex with men (MSM) [3][4][5], who are known to have different sexual behaviours and human immunodeficiency virus (HIV)infected patients [3,5] and have been thought to be associated with sexual transmission. Because of widespread resistance to ampicillin (AMP) and trimethoprim-sulphamethoxazole (SMX-TMP), treatment with these drugs is not advised unless isolates are susceptible [6] and azithromycin (AZM) is considered as the first-line or second-line treatment for shigellosis [6].
Nowadays, treatment options for S. sonnei have become more limited due to the emerging multidrugresistant Shigella isolates, particularly those with resistance to AZM and fluoroquinolones (FQ) [3,7].
We report three cases of sexually transmitted S. sonnei infections in MSM diagnosed in Franche-Comté, France: one patient treated with antiretroviral therapy (ART) for HIV infection and two receiving pre-exposure prophylaxis (PrEP) against HIV. The isolates were characterized by the same phenotypic antimicrobial susceptibility testing (AST).

Method
We retrospectively studied three patients with S. sonnei infection in NFC (Nord Franche-Comté) Hospital, France. We collected demographic characteristics, comorbidities, and characteristics of the current infection and outcome, from patients' medical records.
S. sonnei isolates were suspected by studying biochemical characteristics on Hektoen agar (bioMerieux, Marcy-l'Etoile, France) and Kligler-Hajna agar tube (Biorad, Marnes-la-Coquette, France) and identification as Escherichia coli by mass spectrometry MALDI-TOF (Microflex BRUKER, Bremen, Germany) since this technology is unable to distinguish these very similar species. Then, identification was confirmed by performing Api 20E strips (bioMerieux, Marcy-l'Etoile, France) and agglutination (Biorad, Marnes-la-Coquette, France). Antibiotic susceptibility was tested by the disc diffusion assay (disc and Mueller Hinton agar: Biorad, Marnes-la-Coquette, France). Susceptibility to azithromycin was determined by measuring minimum inhibitory concentration (MIC) with E-test® strips (bioMerieux, Marcy-l'Etoile, France). CASFM/ EUCAST breakpoints were used for the interpretation. Strains were sent to the E. coli, Shigella, Salmonella National Reference Center (CNR-ESS) in Paris, France and were identified through multi-locus sequence typing based on the core genome (cgMLST) analysis.

Results
The patients' mean age was 32 years [31-34] and all of them were MSM (one patient was receiving ART for HIV-infection and two patients were PrEP users). In our case series, all patients denied engaging in chemsex or using illicit drugs, and none of them travelled recently. All patients presented with fever and gastro-intestinal (GI) symptoms, especially diarrhoea; however, only one of the three patients was hospitalized. S. sonnei was isolated from stool sample. The complete serotyping by agglutination identified the same serotype of S. sonnei g in all patients, based on biochemical characteristics. In this case series, all strains of S. sonnei were resistant to AMP, AZM, FQ and SMX-TMP and only susceptible to third-generation cephalosporins. They all received antibiotic treatment based on ceftriaxone with favourable outcome.

Case 1, July 2018
A 31-year-old male with no past history sought care for fever and GI symptoms such as nausea, diarrhoea and abdominal pain. He did not report any concurrent diarrhoeal disease in family members or friends, nor recent travels. This patient had multiple sexual contacts with passive anal and oral sex and reported that he had not recently used condoms. He did not smoke tobacco or electronic cigarettes, drink alcohol, nor use illicit drugs. He received PrEP against HIV consisting in tenofovir disoproxil fumarate 200 mg/ emtricitabine 245 mg (TDF/FTC). Physical examination on admission revealed high body temperature of 40°C, tachycardia (110 beats/minute) with a diffuse abdominal tenderness. Laboratory tests showed hypokalaemia at 2.8 mmol/l with normal renal and liver functions and an elevated C-reactive protein (CRP) at 293 mg/l. Peripheral blood cultures were negative. Serology for Hepatitis C, HIV and syphilis was also negative and the patient was vaccinated against hepatitis A and B. Abdominal computed tomography (CT) showed right colitis with mesenteric lymphadenitis. Direct examination of stool sample revealed gram-negative bacilli and the culture isolated S. sonnei. The patient was admitted to the infectious diseases department and started on empiric antibiotics (azithromycin 500 mg daily orally) and support treatment. Susceptibility testing of the organism revealed a MIC > 256 mg/l for azithromycin (R) (Table I). Thus, antibiotics were modified to daily 1 g intravenous ceftriaxone for 7 days with a favourable outcome at discharge.

Case 2, October 2020
A 31-year-old male with Centers for Disease Control and Prevention (CDC) A1 stage of HIV infection presented with a 5-day history of fever, chills, fatigue and abdominal pain. His past medical history revealed several episodes of different sexually transmitted infections (STIs): a successfully treated secondary syphilis and recurrent genital ulcers caused by herpes simples virus type 1. He was currently treated with tenofovir disoproxil fumarate 200 mg/emtricitabine 245 mg/ dolutegravir 50 mg (TDF/FTC/DTG) with a good compliance and undetectable HIV viral load. Physical examination was normal and the patient complained of watery bloody stools. S. sonnei was isolated in stool culture with the same antibiotic resistance profile to AMP, AZM, FQ and SMX-TMP. The complete serotyping by agglutination identified serotype g. The patient received a daily intramuscular (IM) injection of 1 g ceftriaxone for 7 days. All symptoms disappeared after 2 days, with no evidence of relapse afterwards.

Case 3, October 2020
A 34-year-old MSM patient with no significant medical history, except continuous PrEP (TDF/FTC), presented with a 48-hour history of fever followed by mucousy diarrhoea. The patient was treated by his general practitioner with azithromycin and racecadotril for persistence of fluid diarrhoea. The stool culture grew the same pathogen (S. sonnei). The strain was identical (S. sonnei g) with the same susceptibility testing and MICs, as compared to case 2. The patient was treated with IM ceftriaxone (1 g per day for 5 days), which resulted in a rapid resolution of symptoms. The patient 3 reported having had unprotected sex with patient 2.

Discussion
Sexually transmitted bowel and rectal diseases are common in MSM and are caused by a wide variety of infectious agents, such as Campylobacter spp, Yersinia spp, Salmonella spp, Shigella spp, with a recent large increase in the rate of male shigellosis cases associated with antimicrobial resistance.
S. sonnei is one of the bacteria that may be responsible for shigellosis in the general population and particularly in children as one of the major cause of bacillary dysentery in France [8], but is also known to be sexually transmitted with increased levels of antimicrobial resistance. In France, MSM-associated lineages have been isolated and defined within Shigella flexneri serotype 3a, S. flexneri serotype 2a and S. sonnei, with higher-than-normal levels of antimicrobial resistance. Baker et al. showed that four French isolates belonged to the major sub-lineage of S. flexneri 2a, and 13 and 10 French isolates clustered with sublineages 1 and 4 of S. sonnei, respectively, indicating the presence of these three sub-lineages in French MSM, since 2009 [9].
Several outbreaks of S. sonnei have already been reported worldwide in MSM [3][4][5] which may have different sexual behaviours, such as direct/indirect oro-genital intercourse and chemsex practice.
In our cases, all patients denied practising chemsex. Nevertheless, drug use is a known risk factor for STIs, by disinhibiting sexual behaviours, especially in HIV patients [10]. On the other hand, PrEP use for HIV may possibly influence sexual behaviours by reducing condom use, increasing STI prevalence [11] including emergence of Shigella infection in MSM [5].
Regarding antimicrobial resistance, S. sonnei has been found to adapt and equip with antimicrobial resistance genes more profoundly through mobile genetic elements as compared to other Shigella species [12]. With S. sonnei infections in MSM population, a high level of antibiotic resistance has appeared, especially concerning AZM [7] and CIP [3]. In a recent publication, Bourguignon et al. proved that there was a local dissemination of 27 drug-resistant S. sonnei g in Bordeaux, France (regarding serotyping, determination of biotype and cgMLST analysis) with high MICs for AZM (>256), such as our patients [13]. In China, some authors described an significant increase of antimicrobial resistance rate of S. sonnei to third-generation cephalosporin agents in isolates with the extended-spectrum beta-lactamases (ESBLs)-producing genes [14]. In addition to this, oral cephalosporin treatment for shigellosis should be avoided despite in vitro susceptibility, because of a demonstrated clinical failure [15]. Finally, we think that our data are relevant to clinical confirmation and microbiological evidence of S. sonnei infection with the same antimicrobial testing susceptibility and MICs for all three cases in MSM. However, there are a few limits of this study: (i) the number of patients is very limited; (ii) faecal-oral transmission seems to be excluded, based on anamnestic evidence, but cannot be completely ruled out; (iii) without the whole genome sequencing, it cannot be determined with certainty if the strains infecting our patients were from the same cluster.
To conclude, we describe three cases of multidrugresistant S. sonnei infections in MSM in Franche-Comté, France. This case series raises concerns about the ability to manage the spread of resistant Shigellosis in this population, especially with alarming reemergence of STIs. Parenteral third-generation cephalosporins should be an alternative option as an empiric treatment for dysentery in MSM, with possible IM administration in outpatients.