Bacteriological characteristics and resistance profiles of Vibrio cholerae O1 strains isolated in the Republic of Benin in 2020

This study aims to carry out a bacteriological characterization and determine the resistance profile of Vibrio cholerae O1 strains isolated during the epidemiological season of 2020 in Benin. To achieve this goal, 43 diarrheal stool samples were analyzed. The samples were taken during the epidemic period of 2020. Bacteriological analyses consisted of enrichment of the samples in buffered peptone water followed by culture on SBCT agar. Then the characteristic colonies were subjected to microscopy, biochemical identification (oxidase, seeding and reading of TSI agar and API 20 E gallery), serotyping, and antibiotic sensitivity tests using the diffusion technique in agar medium according Kirby-Bauer method. The median age of the patients included in this study was 25 years (IQR: 15-40) with predominantly female patients. Individuals aged 11 to 25 were the most represented. Of the 43 stool samples analyzed, 22 were culture positive for V. cholerae and belonged to serogroup O1. The clinical manifestations observed in patients with cholera were watery diarrhea, vomiting and severe dehydration before admission to hospital. It should be noted that all of V. cholerae O1 strains isolated were multidrug resistant with a strong resistance to erythromycin (81.13%), ampicillin (79.96%), chloramphenicol (79.06%), and cotrimoxazole (78.12%).


INTRODUCTION
Cholera is a very virulent and fatal diarrheal disease caused by ingestion of water or food contaminated with the bacteria Vibrio cholerae (Levade et al., 2017). Vibrio strains can grow and remain for a long time in coastal waters polluted by human feces (Gidado et al., 2018). Seven cholera pandemics have been recorded worldwide *Corresponding author. E-mail: denric2000@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License and continue to cause local and regional outbreaks on the African continent (Danso et al., 2020). Cholera is a major public health problem in many sub-Saharan African countries. According to World Health Organization (WHO), in 2014, 190,549 cases and 2,231 deaths were reported. Out of 2231 deaths, 1882 (84.3%) were from Africa (Bwire et al., 2017). Cholera remains endemic in many countries, in Southeast Asia, Africa, Central and South America. Transmission is closely linked to a lack of access to safe drinking water and sanitation facilities (Abana et al., 2019). Strains of vibrios produce an enterotoxin which causes profuse, painless, watery diarrhea associated with vomiting, leading to severe and fatal dehydration if treatment is not administered promptly (Ngandjio et al., 2009). The main treatment for cholera is based on rehydration. Antimicrobials are a useful therapy for decreasing the duration of diarrhea and bacterial stool and therefore reducing the volume of fluid replacement needed for treatment (Ohene et al., 2016). Widely used antimicrobials include doxycycline or tetracycline for adults, cotrimoxazole (trimethoprim = sulfamethoxazole) for children, and furazolidone for pregnant women (Ngandjio et al., 2009). Since the first pandemic that reached West Africa, some outbreaks have been frequently reported across the region . However, only a few small-scale studies have been performed to investigate the dynamics of recent cholera outbreaks in West Africa. Overall, the cholera epidemics studied in the region exhibited different characteristics in different countries (Moore et al., 2017). In some countries such as Benin, Ghana, Nigeria and Togo, cases of cholera have been reported every year, although their incidence is relatively low (Landoh et al., 2013;Sule et al., 2017;Moore et al., 2018;Danso et al., 2020). However, in Benin there is a glaring absence of epidemiological data on annual cholera epidemics. It is to remedy this situation and to provide recent epidemiological information on cholera that this study was initiated. It aims to carry out a bacteriological characterization and to determine the resistance profile of the strains of V. cholerae O1 isolated in 2020.

Study framework
This study was conducted in the Republic of Benin. It is a coastal country in West Africa, with an area of 114,763 km 2 . The annual average temperatures vary from 26 to 28°C. The climate is subequatorial in the south, with a humid tropical transition in the center and dry tropical in the north (Houehanou et al., 2015). The population was estimated at 9,983,884 inhabitants in 2013 (INSAE, 2016) of which 51.2% were women. The population is elderly with a majority of individuals over 15 years of age. The health system is structured in three levels (central, intermediate and peripheral) in a pyramidal manner (Houehanou et al., 2015). Specifically, the bacteriological diagnosis of the samples was carried out in the bacteriology section of the National Public Health Laboratory, Ministry of Public Health, Benin.

Sampling
Stool samples (43) were systematically taken during the study period (May 8 to October 20, 2020). These samples came from all suspected cases of cholera, notified by health centers and departmental health services. Thus, stool samples from each of these cases were taken into sterile jars and sent to the laboratory immediately for analysis. Samples not sent directly to the laboratory for transport were put on Cary Blair medium and sent to the laboratory within 24 h. Each sample was identified and accompanied by a completed notification form. The information on the sheet concerns the socio-demographic and epidemiological information of the patient.

Isolation and identification of strains of V. cholerae
Isolation of V. cholerae began by enriching samples in buffer peptone water (BPW) (pH 8.4) at 37°C for 6 h, and the resulting solution was streaked onto agar with sucrose bile citrate thiosulfate (SBCT). After 24 h of incubation at 37°C, yellow colonies (sucrose fermenting, 2-3 mm in diameter) suspected of being V. cholerae were purified on Müeller Hinton agar for 24 h at 37°C. The purified colonies were characterized by Gram stain (Gram negative comma shaped rods), positive oxidase reaction and reaction in triple sugar iron (TSI) agar. Confirmation of the identity of the putative isolates of V. cholerae was achieved by seeding and reading the API 20E gallery.

Serogrouping
The previously identified serogroups of V. cholerae strains were serologically confirmed by a slide agglutination test using polyvalent antisera specific for V. cholerae O1 and O139, followed by a serotype specific monoclonal antibody.

RESULTS
The sex ratio in the sampled patients was 1.39 in favor of the female sex. The median age of this study population was 25 years (IQR: 15-40). Individuals aged 11 to 15 and 21 to 25 were the most represented with a common percentage of 13.94%, followed by individuals aged 16 to 20, 26 to 30 and 36 to 40 with the same percentage of 9.30% (Table 1). Of the 43 stool samples analyzed, 22 were culture positive for V. cholerae and serogrouping resulted in V. cholerae O1, that is to say, a prevalence of 51%.
Of the 22 strains of V. cholerae, 12 were from samples from female patients and 10 were from samples from male patients: 48% of the samples from female patients and 55.56% of samples from male patients, respectively.  Analysis of the data showed an absence of statistical significance (p ˃ 0.05) between the positive proportions of the samples in the culture of V. cholerae and the sex of the patients from whom these samples were taken.
Of the 22 culture-positive samples for V. cholerae, samples from patients aged 11 to 15 and 26 to 30 years old represented the highest proportion with 13.64% each (n = 3) (Figure 2).
Of the 8 symptoms or groups of symptoms observed in patients during sampling, only watery diarrhea + vomiting + dehydration was observed in patients whose specimens tested positive for V. cholerae culture (Table  2). Among the culture negative samples of V. cholerae, the most prominent symptoms were vomiting + dehydration (23.82%), followed by watery diarrhea + dehydration (19.05%) and severe diarrhea (09.52%). But there was no statistically significant difference (p ˃ 0.05) between symptoms and positive culture.

DISCUSSION
The suspected cholera patients included in the study are predominantly female, with a median age of 25 years. The most represented age groups were [10][11][12][13][14][15] and [20][21][22][23][24][25]. However, statistical analysis of the data has shown that the occurrence of cholera is not related to age or gender. Blacklock et al. (2015) showed that there were more women than men admitted to the Referral Hospital during the cholera epidemic in 2012 in Sierra Leone. As for Danso et al., (2020), they showed that the patients involved in the cholera epidemics in southern Ghana from 2012 to 2015 were mostly men (54.9%), which justify moreover the conclusion according to which the occurrence of cholera in an individual did not depend on sex. However, the fact that in the present study women are more represented than men, could be explained by the exclusive involvement of women in the performance of household chores and the associated risks of cholera transmission. Of the 43 samples analyzed, 22 were declared positive in the culture of V. cholera and confirmed to be all strains of V. cholera O1, that is, a prevalence of 51%. This prevalence is lower than that found by Koley et al. (2014) 65.6% and that found by Chhotray et al., (2002) in India. All strains of V. cholerae isolated in this study are serogroup O1. Both V. cholerae serogroups O1 and O139 are involved in cholera epidemics in Africa and globally (Chhotray et al., 2002;Rashed et al., 2012;Lessler et al., 2018). Several studies have shown that in West Africa the V. cholerae serogroup most involved in annual cholera epidemics was serogroup O1 (Dalsgaard et al., 1996;Landoh et al., 2013;Moore et al., 2018;Danso et al., 2020). This justifies the results we have obtained. All patients diagnosed positive for V. cholerae O1 in the present study were doing watery diarrhea, vomiting and dehydration. The main symptoms of cholera are profuse diarrhea and vomiting, after an incubation period of about 2 h to 5 days (Mengel et al., 2014). Many authors have shown in their studies that the clinical manifestations of patients diagnosed with cholera are diarrhea and vomiting and that on admission to hospital they are severely dehydrated (Ndour et al., 2006;Sule et al., 2017;Elimian et al., 2019). All 22 strains of V. cholerae O1 isolated were multidrug resistant with high resistance of the strains to erythromycin, ampicillin, chloramphenicol, cotrimoxazole (trimethoprim/sulfamethoxazole), flucloxacillin and nalidixic acid. Rashed et al. (2012) also showed that the strains of V. cholerae O1 responsible for the 2008 to 2010 cholera epidemic in Bangladesh had high resistance to erythromycin and trimethoprim/ sulfamethoxazole. As for the resistance of the strains to ampicillin and nalidixic acid, Eibach et al., (2016) made the same observations on the strains of V. cholerae O1 isolated during the cholera epidemics that occurred in Ghana in 2014. However, the results of the present study showed that all the strains of V. cholerae O1 obtained were sensitive to gentamicin, ciprofloxacin, doxyclyne and cefotaxime. The sensitivity of the strains of V. cholerae responsible for cholera epidemics to gentamicin, ciprofloxacin and docycline have been reported by several authors (Akoachere et al., 2013;Abana et al., 2019;Danso et al., 2020). Cotrimoxazole (trimethoprim = sulfamethoxazole), formerly first-line treatment in children, was ineffective in 78.12% of isolates. Tetracycline, recommended by the WHO for the treatment of cholera in adults, was found to be effective against 51.8% of the strains tested and ineffective against 48.2% of the latter. This situation then poses a serious problem of therapeutic failure in the treatment of cholera with antibiotics. However, it should be noted that in the treatment of cholera, rehydration remains the main treatment.

Conclusion
This study confirms that the annual cholera epidemics in Benin are the cause of the same V. cholerae serogroup, which is serogroup O1 and has shown that the disease can affect all ages, and 90% of them manifest as watery diarrhea, vomiting, followed by severe dehydration. The results showed an increasing trend of multi-resistant V. cholerae O1 strains in Benin. Ciprofloxacin, tetracycline and doxycycline remain effective against clinical strains. But a strong resistance of these same strains to erythromycin, tetracycline, ampicillin and trimethoprim/sulfamethoxazole were noted. The results indicate an urgent need for the appropriate use of antibiotics, hygiene and sanitation of coastal villages in Benin in general to prevent the cholera epidemic.