After half a century of antibiotic use, the emergence and spread of bacterial resistance are a critical public health issue. Bacterial resistance is growing in importance worldwide, especially in hospitals, and has now reached dangerous proportions in all regions of the world . The extensive use of broad-spectrum antibiotics in human medicine to treat infections without diagnosis of the specific pathogen involved and in animal husbandry is considered to be a major factor in bacterial resistance to antimicrobials. Resistance can also be coupled with poor environmental conditions . Indeed, infections caused by antibiotics resistant bacteria lead to high morbidity or mortality . Fish is one of the most popular foods in the world population. It is a source of protein and essential oligonucleotides and is very valuable for nutritional balance  . The diversity of cultures in the world has given rise to a wealth of recipes and methods for preparing fish. In the Republic of Congo in rural areas as well as in urban areas including Brazzaville, fresh fish is eaten in stews, sauces, barbeques, fried in oil, grilled and stewed. Fish in stews are very popular and eaten a lot. In addition to households, fish stews are prepared and then sold in markets, on public roads, in restaurants and drinking establishments, and at fish auctions by itinerant women. Due to the high consumption of these products, it is therefore necessary to ensure their hygienic quality. To date, no studies have been carried out on the antibiotic resistance of bacteria isolated from fish to smothering. Therefore, we proposed to establish the antibiotic resistance profile of pathogenic bacteria isolated in fish stewed sold and consumed in the markets of Brazzaville in order to assess the health risks related to the consumption of these products.
2. Materials and Methods
2.1. Sampling of Smothered Fish
Smothered fishes sold in Brazzaville were taken from Moukondo market (Site 1), Total market (site 2) and Moungali market (site 3). Three samples were purchased per site. At the time of purchase, the samples were placed in ice box and sent to the laboratory for analysis.
2.2. Isolation and Identification of Bacteria from Smothered Fish
In a test tube containing 9 mL of distilled water, 1 mL of the smothered fish soup was added as the stock solution from which the decimal dilutions were made. The inoculations were made on agar plate of four different culture media: 1) Mannitol egg Agar (Mannitol egg Yolk Polymixin Agar, GranuCult) for isolation of bacteria of the genus Bacillus, in particular Bacillus cereus; 2) Mannitol Salt Agar (Bio RAD) for isolation of bacteria of the genus Staphylococcus including mannitol positive Staphylococcus (Staphylococcus aureus); 3) Salmonella-Shigella Agar (Bio RAD) for isolation of bacteria of the genus Salmonella whose colonies are black-centered (H2S+) and lactose-positive and the genus Shigella whose colonies are colorless (H2S−) and lactose-negative; and 4) Methylene Blue Eosin Agar (Bio RAD) for isolation of enterobacteria in which the major species is Escherichia coli which is characterized by a metallic sheen.
The plates were incubated aerobically at 37˚C for 24 hours. Each colony was purified by separate streaking until distinct and homogeneous colonies were obtained. To ensure the purity of the bacterial isolates; microscopic observation was carried out. The isolates were identified based on cellular morphology by Gram stain and color of colonies and biochemical reaction on the growth media . Isolates were stored at 4˚C in cryotubes containing 20% glycerol in LB broth.
2.3. Antibiotics Sensitivity Test
The antibiotics tested on S. aureus and B. cereus were: penicillin G (P., 10IU), oxacillin (OXA., 1 μg), cefoxitin (CX., 30 μg), vancomycin (VA., 30 μg), Kanamycin (K., 5IU), gentamicin (CN., 10IU), tobramycin (TOB., 10 μg), fosfomycin (FF., 200 μg), ciprofloxacin (CIP., 5 μg), norfloxacin (NOR., 10 μg), erythromycin (E., 15 μg), clindamycin (DA., 2 μg), pristinamycin (PT., 15 µg), fusidic acid (FA., 10 µg), rifampicin (RA., 5 µg). For Salmonella spp, Shigella spp and E. coli, the antibiotics tested were: ticarcillin (TIC., 75 μg), ticarcillin + clavulanic acid (TIM., 75 + 10 μg), piperacillin (PRL., 75 μg), piperacillin + tazobactam (TPZ., 110 μg), cefepime (CEF., 30 μg), ceftazidime (CAZ, 30 μg), aztreonam (ATM., 30 μg), Meropenem (MEM., 10 μg), Kanamycin (K., 5IU), gentamicin (CN., 10IU), tobramycin (TOB., 10 μg), fosfomycin (FF., 200 μg), ciprofloxacin (CIP., 5 μg), norfloxacin (NOR., 10 μg).
The antibiotic resistance profile of the bacterial strains was evaluated by the standard Kirby-Bauer disc diffusion method  . The inoculum was prepared by suspending of a well-isolated colony of a young; pure bacterial culture (24 hours on agar medium) in 5 ml normal saline and the turbidity of the suspension was adjusted using spectrophotometer to 0.1 at 625 nm. The optical density of 0.1 at a wavelength of 625 nm is equivalent to 0.5 Mac Farland . The culture medium, Mueller-Hinton agar, was inoculated using the swab as recommended by CLSI (Clinical and Laboratory Standard Institute) . The antibiotic discs were then applied to the inoculated Mueller Hinton agar medium. The plates were incubated at 37˚C for 18 - 24 h. The diameter of bacterial growth inhibition area around the disc after incubation were measured and the antibiotics susceptibility was interpreted based on the breakpoint values published by the Antibiogram Committee of the French Society of Microbiology . The strains were categorized as either: sensitive, intermediate or resistant against the antibiotics.
3.1. Isolation and Identification of Bacterial Isolates
A total of 25 pathogenic bacteria of which 15 (60%) were gram-negative and 10 (40%) were gram-positive were isolated from the smothering fish sold in the markets of Brazzaville. Figure 1 shows the number of bacteria isolated per site. Bacteria were mostly isolated from the samples from site 3 (Moungali market).
Figure 2 shows the repartition of the identified strains. Of these strains, 5
Figure 1. Number of bacteria isolated per site (Site 1: Moukondo market, Site 2: Total market, Site 3: Moungali market).
Figure 2. Frequency of identified bacteria.
(20%) were Shigella spp, 5 (20%) Salmonella spp, 5 (20%) E. coli, 5 (20%) S. aureus and 5 (20%) B. cereus.
3.2. Antibiotic Sensitivity Test
The results of the antibiotic sensitivity test are shown in Table 1 and Table 2. Table 1 represents the antibiotics resistance rate in S. aureus and B. cereus. These strains were resistant to beta-lactam antibiotics, which were sensitive to aminoglycosides and quinolones. Table 2 shows that Shigella spp, Salmonella spp and E. coli were resistant to beta-lactam antibiotics with the exception of meropenem. These strains were sensitive to tobramycin. Salmonella spp and E. coli were susceptible to quinolones.
Figure 3 shows the resistance profile of S. aureus and B. cereus. This figure shows that S. aureus and B. cereus were susceptible to kanamycin, tobramycin, ciprofloxacin and norfloxacine. High levels of resistance were observed to beta-
Table 1. Antibiotic resistance rate of S. aureus and B. cereus.
ATB tested = Antibiotics tested; R = Number of resistant strains; I = Number of intermediate resistant strains; S = Number of susceptible strains; (%) = Percentage of sensitivity and resistance; MLSB = Macrolides-lincosamides-Streptogramines; P = Penicillin G; OXA = oxacillin; CX = cefoxitin; VA = vancomycin; FF = Fosfomycin; K = Kanamycin; TOB = tobramycin; CN = Gentamycin; CIP = ciprofloxacin; NOR = Norfloxacin; E = Erythromycin; DA = Clindamycin; PT = pristinamycin; FA = fusidic acid; RA = Rifampicin.
Table 2. Antibiotic resistance rates of Shigella spp, Salmonella spp and E. coli.
ATB tested = Antibiotics tested; R = Number of resistant strains; I = Number of intermediate resistant strains; S = Number of susceptible strains; (%) = Percentage of sensitivity and resistance; TIC = ticarcillin; TIM = ticarcillin + Clavulanic acid; PRL = piperacillin; TPZ = piperacillin - tazobatam; CAZ = ceftazidime; FEP = cefepime; MEM = meropenem; ATM = aztreonam; FF = fosfomycin; TOB = tobramycin; CN = gentamycin; K = kanamycin; NOR = norfloxacin; CIP = ciprofloxacin.
Figure 3. Antibiotic resistance profile of S. aureus and B. cereus.
lactam antibiotics. The resistance of these bacteria (S. aureus and B. cereus) to beta-lactam antibiotics was characterized by P OXA CX. The most apparent MLSB resistance phenotype was EDAPT (20%) in S. aureus and the least apparent in B. cereus. The DA PT phenotype was widely encountered in B. cereus (40%).
Of all the antibiotics tested, Shigella spp, Salmonella spp and E. coli were beta-lactam resistant apart from meropenem, which retained its activity. Tobramycin retained activity on all strains. Salmonella spp were susceptible to ciprofloxacin and norfloxacin (Figure 4). In Shigella spp, Salmonella spp and E. coli, only one type of beta-lactam resistance phenotype was observed: TIC TIM PRL TPZ CAZ FEP ATM. In Salmonella spp, 2 strains showed the CN K phenotype for aminoglycosides. The CIP NOR phenotype was widely observed in E. coli and Shigella spp.
In our study, we investigated the antibiotic resistance of strains of S. aureus, B. cereus, Shigella spp, Salmonella spp and E. coli. The susceptibility test in S. aureus showed the following results: penicillin G (80%), oxacillin (100%), cefoxitin (60%), vancomycin (20%), kanamycin (0%), gentamicin (0%), tobramycin (0%), fosfomycin (80%), ciprofloxacin (0%), norfloxacin (0%), erythromycin (60%), clindamycin (40%), pristinamycin (20%), fusidic acid (40%), and rifampicin (60%). In the Republic of Congo, a study conducted by  in S. aureus isolated at the Brazzaville University Hospital Centre (CHUB) showed resistance rates for penicillin G 55.10%, oxacillin 53.06%, kanamycin 14.26%, tobramycin 12.24%, and gentamycin 42.55%. These results differ from those reported in this study. This difference could be explained by the fact that acquired resistance is evolutionary, it evolves with time and location.  in Morocco, reported for vancomycin, gentamicin, ciprofloxacin, erythromycin, pristinamycin and fusidic acid whose respective resistance rates are: 0%; 66.6%; 72.2%; 50%; 16.6% and 61.1%.
Figure 4. Antibiotic resistance profile of Shigella spp, Salmonella spp and E. coli.
These rates are quite close to those reported we found. A study conducted by  in Congo Brazzaville on community and clinical settings showed similar rates on community strains to those found in this study. Beta-lactam resistance was dominated by the POXACX phenotype, with similar results reported by . This could be due to the mecA gene encoding a PLP2a. Resistance to MLSB was marked by the EDAPT phenotype. This could be due to ErmC-type methylases.
Bacillus cereus is ubiquitous in nature and is easily spread through food production systems. Contamination by this species is almost inevitable. B. cereus, was reported to be resistant to penicillin G and sensitive to aminoglycosides, fluoroquinolones, glycopeptides, lincosamides and rifampicin . These are explained by the fact that B. cereus produce beta-lactamases and are therefore considered resistant to beta-lactam antimicrobial agents . In our study, the resistance rates for penicillin G, oxacillin, erythromycin, gentamycin, clindamycin and rifampicin were 80%, 100%, 40%, 20%, 80% and 80% respectively. These results differ from those reported by  in Egypt on the prevalence of Bacillus cereus resistance, where the respective rates were 100% for penicillin G and 0% for the rest of the above antibiotics. The work carried out by  in Ghana on the prevalence of virulence factors and antibiotic resistance genes in Bacillus cereus shows resistance rates of 88% for oxacillin and 0% for the same antibiotics mentioned previously.
All E. coli strain isolates were resistant to Cefrazidime. This differs from rate of resistant of 77.3% reported by  on E. coli isolated at the CHUB.  in the Republic of Congo working on E. coli isolated from CHUB inpatients and outpatients reported resistance rates of 46.51%, 37.20%, 34.88% 13.95% and 46.51% respectively for piperacillin-tazobactam, cefepime, gentamicin, ciprofloxacin and fosfomycin in the extended-spectrum beta-lactamase producing strains. These levels differ from those reported in our study.  in Morocco reported resistance rates of 60.2% for gentamycin, 75.5% for ciprofloxacin and 75.5% for norfloxacin. These differ from those we found for norfloxacin, ciprofloxacin and gentamycin in E. coli where the resistant rates were 100%, 100% and 40% respectively.
The genus Salmonella is one of the leading causes of collective foodborne illness. Although some cases may be directly transmitted from pets, reptiles or contaminated water, the percentage of transmission through food is estimated at 95% . The results of our study show that these strains presented resistance rates of 100% to kanamycin, piperacillin-tazobactam, cefepime and 0% to ciprofloxacin and tobramycin. In Chad,  reported resistance rates of 10.5%; 0%; 11%; 0%; 31.8% for kanamycin, piperacillin-tazobactam, cefepime, ciprofloxacin and tobramycin respectively in Salmonella strains isolated from semi-traditional farmers. On the other hand, traditional farmers reported resistance rates of 14.29%; 0%; 0%; 0% and 19.05% respectively for the same antibiotics.
Antibiotic sensitivity testing showed that Shigella spp isolates were 100% resistance to ciprofloxacin and ceftazidime. In a study conducted by  in America, resistance rates of 50% to ciprofloxacin and 0% to ceftazidime were reported. The work of   revealed a total sensitivity of the strains to ciprofloxacin and gentamycin. Our results on kanamycin (80%) differed from those found by  (0%). In Asia, 81.8% resistance to norfloxacin was demonstrated  while it was 100% in our results. With piperacillin, our results are consistent with those of , where 100% resistance was observed. The presence of the TIC TIM PRL TPZ CAZ FEP ATM beta-lactam resistance phenotype in E. coli, Salmonella spp and Shigella spp could be explained by inactivation of these antibiotics by extended-spectrum beta-lactamases.
The presence of these pathogens in smothered fish could be explained by human activity (hands, kitchen utensils, water, ingredients, packaging sheets) on the one hand and by the microbial flora of the fish on the other hand. The resistance of the bacteria to the cooking temperature would be explained either by sporulation as is the case for Bacillus cereus or by the short cooking time. The high resistance rates observed could be explained by the dissemination of resistance genes between bacteria by horizontal and vertical transfers.
The study showed that the pathogenic bacteria S. aureus and B. cereus contained in the smothered fish are highly resistant to beta-lactam antibiotics and MLSB, while the enterobacteria Shigella spp and E. coli were resistant to beta-lactam and quinolones. Beta-lactam and MLSB resistance in Gram-positive bacteria have been characterized by the presence of resistance phenotypes highlighting the involvement of penicillinase (PLP2a) and methylasses (ErmC) respectively. In contrast, in Gram-negative bacteria, the resistance phenotype reporting the production of extended-spectrum beta-lactamases was observed to beta-lactam. However, vancomycin, kanamycin, tobramycin, gentamycin, ciprofloxacin, norfloxacin can be used for the treatment of community-acquired infections caused by Gram-positive bacteria; meropenem and tobramycin for infections of Shigella spp, Salmonella spp and E. coli.
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