Antibiotics Resistance Profile of Bacillus cereus Strains Isolated from Soil and Pepper in Brazzaville ()
1. Introduction
Infectious diseases are nowadays responsible for high morbidity. These diseases are mostly linked to food contamination of microbial origin and result in gastrointestinal symptoms occurring after the consumption of a meal [1]. Many studies have reported a high level of contamination of food products by microorganisms and several consummated foods around the world have been identified as vectors of microorganism transmission [2] [3].
B. cereus is one of the bacteria with the highest rate of food contamination. It forms an independent group belonging to the Bacillus genus, currently comprising eight species: B. cereus (sensu stricto), B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. cytotoxicus and B. toyonensis [4]. More recently, 13 species have been proposed as new: B. gaemokensis, B. manliponensis, B. bingmayongensis, B. wiedmanni, B. paranthracis, B. pacificus, B. tropicus, B. albus, B. mobilis, B. luti, B. proteolyticus, and B. nitirireducens [5].
Mainly known to cause mild or severe food poisoning, sometimes leading to death, Bacillus cereus is a germ with many characteristics such as the omnipresence in different environments, the formation of spores, the adaptation in variable conditions, the production of virulence factors and harmful toxins in food, making it an opportunistic human pathogen even at very low loads [6]. Most of the studies affirmed that the emetic and diarrheal syndrome caused by Bacillus cereus could occur whenever bacterial load in a food reaches 5 to 8 × 104 CFU/g [7]. A recent study showed its presence in pepper (Capsicum), a food widely consummated around the world during meals, in sauce or in powder form, inside condiment dishes [8].
However, some studies have shown that B. cereus group strains could serve as a transferable source of antibiotic resistance genes in the food chain. Moreover, the emergence of antibiotic resistance in B. cereus currently poses a real problem in the treatment of patients. Most B. cereus strains produce β-lactamase-like enzymes and are considered resistant to β-lactam antimicrobial agents. The treatment of other Bacillus cereus infections is complicated because those Bacillus have rapid and progressive evolution and a high incidence of multidrug resistance [9].
Consequently, the presence of transposons and plasmids in B. cereus helps them in the acquisition and transmission of resistance genes characterized by an increasing level of resistance, particularly against several drugs [9]. Antibiotic resistance in B. cereus currently poses a real public health problem and remains a serious problem in searching for resolutions. Thus, the emergence and spread of antibiotic resistance is the subject of several studies and is reported by the WHO as one of the major health issues of the 21st century [1]. This work is initiated in order to determine the antimicrobial resistance profile of B. cereus group strains isolated from soil used and pepper consummated in Brazzaville.
2. Material and Methods
2.1. Study Design
The study was conducted in the bacteriology department of the national public health laboratory in Brazzaville and in the cellular and molecular biology laboratory of the Faculty of Sciences and Technics of Marien NGOUABI University between March and April 2021.
2.2. Sampling
The Bacillus cereus strains were isolated from two types of samples: (a) an environmental dry soil taken at 30 cm depth at the Faculty of Sciences and Technics, using a sterile tube. (b) The second sample was a fermented pepper, close to the pepper’s characteristics of Capscicum annum species, purchased at a market in Brazzaville.
2.3. Isolation and Identification of Bacterial Strains
Using each sample, decimal dilutions have been performed for Bacillus cereus isolation on Mossel agar medium. Petri dishes were incubated at 37˚C for 24 hour. A total of 16 strains were obtained, notably 10 and 6 from pepper and soil, respectively. These different strains were phenotypically characterized using conventional methods. After a morphological reading, a typical pink colony purification was realized by successive culture on agar medium. Strains were kept from a culture contained in LB broth supplemented with 40% of glycerol and stored at 4˚C.
2.4. Bacterial Strains and Culture Conditions
Sixteen (16) strains of Bacillus cereus pre-isolated from soil and pepper samples according to the ISO method are subcultured on specific Mossel agar. Of these 16 strains, six were isolated from soil, namely: two strains of Bacillus cereus (sensu stricto: Ri7 and Ri9) and each one strain of Bacillus weidmani (Ri8), Bacillus thuringiensis (Ri10), Bacillus anthracis (Ri4), and Bacillus albus (Ri1). In addition, 10 strains were isolated from pepper: 6 strains of Bacillus cereus (sensu stricto: Ri14, Ri19, Ri20, Ri21, Ri25a, and Ri11), 1 Bacillus sp. (strain Ri25b), 1 Bacillus albus (strain Ri22), and 2 Bacillus thuringiensis (strains Ri23, Ri17).
2.5. Antibiotic Sensitivity Test
A sensitivity test of strains to different antibiotics was conducted using the Kirby and Bauer’s standard disc diffusion (susceptibility) method [10] [11]. Antibiotics tested are penicillin G (10 U), Amoxicillin AML (10 µg), Ceftazidime CAZ (10 µg), Doripenem DOR (10 µg), Ciprofloxacin CIP (5 µg), chloramphenicol C (30 µg), Tigercycline TGC (15 µg), Rifampicin RD (5 µg), Vancomycin VAN (30 IU), Colistin CT (50 µg) and Linezolids LNZ (10 µg). Reading of inhibition diameters and their interpretations were made according to the guidelines of the Clinical Laboratory Standards Institute [12].
The results obtained were notified on a form and recorded on a computer file. These values were transcribed into sensitive (S), resistant (R), and intermediate (I) categories after comparison with the critical diameters according to the recommendations [12].
The presence or absence of resistance phenotypes was indicated by the signs (+) and (−) respectively, and a numeric coding was used to indicate the number of multidrug resistant (MDR) phenotypes.
2.6. Statistical Analyzes
A Microsoft Excel program was used for statistical analysis. The diameters have been analyzed, and percentages calculated statically. Experimental values were represented as the mean and standard deviation.
3. Results
3.1. Sensitivity Study
B. cereus strains studied showed different levels of resistance to antibiotics tested (Figure 1). All strains tested showed resistance against β-lactams (penicillin, amoxicillin, and ceftazidime), Ansamycins and polypeptides with a resistance rate of 100% (Table 1). Doripenem was the most active antibiotic of the β-lactam family with a rate of 18.75%. This same resistance was observed with chloramphenicol and tigecycline (Table 1). However, linezolid remained the most active antibiotic on all strains tested with a rate of 100%.
3.2. Antibiotic Resistance Profile
Bacillus cereus strains showed varying levels of resistance depending on the antibiotic tested (Table 2). Strain Ri10 showed resistance against 5 antibiotics of different families. 6 strains showed resistance to 4 antibiotics from different families while all other strains were resistant to at least 3 antibiotics (Figure 2).
A multiresistance profile has been observed in a variable proportion of strains depending on antibiotics classes. (16/16) 100% of strains were resistant to 3 antibiotic classes (betalactams, polypeptides and ansamycins), 43.75% (7/16) of strains resistant to 4 antibiotics classes and only one strain (Ri10) was resistant to 5 antibiotic classes (betalactam, polypeptide, ansamycins, cyclins and glycopeptides) (Figure 3). Table 2 describes the multiresistant (MDR), ultraresistant
![]()
Figure 1. Antibiotics susceptibility profile of B. cereus strains (Ri9, Ri20, and Ri11).
![]()
Table 1. Antibiotics sensitivity of 16 Bacillus cereus strains isolated from soil and pepper samples consummated in Brazzaville.
![]()
Figure 2. Antibiotics resistance profile of strains by class. Multidrug resistance (MDR), ultra resistance (XDR) and Broad insensitivity (PDR) proportions of B. cereus strains according to antibiotic classes.
(XDR), and largely insensitive (PDR) frequency of each strain according to antibiotics tested. (+) and (−) sign marks presence and absence of resistance respectively.
![]()
Table 2. Resistance level of variable strains depending on the antibiotic tested.
Legend: Penicillin G (P), Amoxicillin (AML), Ceftazidime (CAZ), Doripenem (DOR), Vancomycin (VA), Colistin (CT), Rifampicin (RD), Linezolid (LNZ), Tigercycline (TGC), Chloramphenicol (C), (+): presence of resistance and (−): absence of resistance.
![]()
Figure 3. Multi resistance (MDR), Ultra resistance (XDR) and broad insensitivity (PDR) frequency of 16 B. cereus strains according to the number of antibiotic classes (multi resistant phenotype to 3, 4 and 5 classes of antibiotics).
3.3. Resistance Phenotypes
Resistance phenotype analysis of strains to antibiotics has mostly presented resistance phenotypes. Figure 3 shows the multidrug resistance (MDR) phenotype, with a predominance of strains resistant to at least three different families of antibiotics (MDR 3).
4. Discussion
The increasing frequency of multiple antibiotic resistance is currently a concern for both food processors and public health. This study aimed to determine the resistance profile of B. cereus isolated from soil and pepper samples consummated in Brazzaville. The great adaptation of B. cereus in unfavourable growing conditions leads to increase its ability to resist antibiotic pressure. [13] reported that food contamination rates due to B. cereus in Switzerland were 78%, while, [14], reported only 8.2% contamination of samples of aquatic products sold in China.
Consequently, results obtained in our study showed that most B. cereus strains are resistant to the betalactam classes (penicillin G, amoxicillin, and ceftazidime). It confirms the hypothesis of the natural resistance of B. cereus to the majority of betalactam antibiotics, supporting the results obtained by [11] on the resistance of B. cereus isolated from ready fast foods in China. In fact, B. cereus strains are known as intrinsic producers of Metallo-β-Lactamases with chromosomal resistance to penicillin and cephalosporins. [15] reported strong betalactam resistance of B. cereus in studies focused on raw cow’s milk sold in Ethiopia.
A strain resistance rate of 18.75% (3/16) to doripenem has been observed. Doripenem is degraded by dehydropeptidase I, a renal tubular dipeptidase enzyme. Thus, this resistance could be explained by the production of betalactamases capable of carbapenem hydrolysis. In fact, these enzymes inactivate the majority of betalactams and are encoded by genes carried in transposons, plasmids, or other mobile genetic elements that could be horizontally transferred towards other bacterial species [16]. Regarding the class of cyclins, 18.75% of the resistance rate has been observed. This rate is different to results reported by [17] on the resistance of B. cereus isolated from rice in the United States, which showed a resistance rate of 98% to tetracycline [17]. This difference could be explained by using tigecycline as the cyclin instead of tetracycline.
We report a resistance percentage of 11.76% (5/16) to vancomycin, lower than that obtained by [18], with a resistance rate of 87% for B. cereus isolated from pasteurised milk in China. Additionally, B. cereus can acquire resistance to commonly used antibiotics such as ciprofloxacin, cloxacillin, erythromycin, tetracycline, and streptomycin [19]. In contrast to the findings of [20] on the resistance of B. cereus sensu lato isolated in Ghanaian dairy farms and traditional products, these results have shown that the B. cereus strains show resistance to chloramphenicol, which may be the consequence of the acquisition and presence of chloramphenicol resistance genes.
Our results are similar to those obtained by [21] on the resistance of B. cereus isolated from environmental samples in the United States. Variation in antibiotic resistance extent and pattern of B. cereus could be impacted by factors such as sources of strain isolation, geographic areas, or frequency of antibiotic applications.
[22] reported that B. cereus strains from stool, food, and environmental samples in Serbia have higher resistance proportions than those from food and the environment. Results in our study shows that the 16 B. cereus strains are sensitive to linezolid, tigecycline, ciprofloxacin in significant proportions, similar results obtained by [23] during their work reported in dairy local products in China, [24] in Cameroon obtained the same in raw cows and processed milk.
All B. cereus strains tested here proved to be resistant to at least 3 different families of antibiotics. This result indicates that B. cereus strains tested during this study are multiresistant (MDR) as reported by [25]. As a result, the rate of increasing antibiotic resistance of B. cereus could result from the occurrence of high severity infections which could be fatal.
The prevalence of pepper consumption among the Congolese population makes pepper an unavoidable support for Bacillus cereus growth, owing to endospore production, which provides resistance despite adverse environmental conditions. B. cereus spores could potentially contaminate raw food material and be found in all stages of manufacture, particularly in the production as well as in the sale of pepper jars, with a high probability of favouring its deterioration and causing food poisoning [8].
In our study, antibiotic resistance proportion and multidrug resistance emergence attributed to Bacillus cereus strains therefore constitute a public health problem. In particular, the emergence of multi-resistant strains is a medical situation to be feared because antibiotic resistant genes could be transferred to other important pathogen agents on horizontal transfer and facilitate their dissemination within the bacterial population from the presence of mobile genetic elements such as plasmids and transposonson present in B. cereus [26].
In addition, these strains could survive in the gastrointestinal tract and complicate treatment for younger or older users or for people with weakened immune function after infection [27]. All these complications set up a problem for infectious disease management caused by increased infection risk and treatment failures.
Thus, it remains to highlight the presence of genes responsible for multiresistance, as well as production of Metallo-β-Lactamases, in knowing Bacillus cereus strains’ impacts on mechanisms of resistance.
5. Conclusion
The study shows the characteristic resistance to antibiotics of B. cereus strains. A strong resistance (100%) of the strains has been observed against antibiotics of the polypeptide classes and ansamycins. Linezolid and ciprofloxacin were the most active antibiotics against the strains tested. The strains isolated in this work are multiresistant. The multiresistance phenotype is observed in 100% of cases against 3 classes of antibiotics (betalactams, polypeptides, and ansamycins). These results indicate that antibiotic resistance such as tigecycline, ciprofloxacin, and chloramphenicols could occur. These data could lead to other in depth studies focused on resistance mechanisms of B. cereus strains isolated in the Republic of Congo. B. cereus therefore represents a potential source of transferable resistance genes in the food chain and to other medically important pathogens.