Phenotypic diagnosis and genotypic identification of Bacillus cereus causing subclinical mastitis in cows

Background and Aims: Bovine mastitis is a disease that affects dairy cows and impacts the global dairy industry. Bacillus spp. can infect the mammary gland during lactation, intramammary treatment, or dry cow therapy. This study aimed to isolate and identify Bacillus spp. in raw milk samples from cows with subclinical mastitis from dairy farms in Beheira, Giza, Alexandria, and Menoufia Governorate, Egypt. We also investigated their antibiotic sensitivity and detected the enterotoxigenic and antibiotic resistance genes. Materials and Methods: A total of 262 milk samples (15-20 ml each) were examined microscopically, biochemically, and phenotypically. A polymerase chain reaction was used for genotypic identification and detecting antibiotic-resistance and enterotoxigenic genes. Antibiotic sensitivity was tested using the agar well diffusion test. Results: Bacillus cereus was identified in 47.7% of samples. Nhe and hblD enterotoxin genes were found in 93.64% (103/110) and 91.82% (101/110) of the samples, respectively. Tetracycline and β-lactam antibiotic-resistance genes were present in 0% (0/110) and 98.18% (108/110), respectively, of the samples. All isolates were resistant to cefepime, cefixime, and oxacillin, while they were susceptible to amoxicillin-clavulanic, chloramphenicol, ampicillin/sulbactam, and levofloxacin. Conclusion: These results highlight the need to promote awareness regarding B. cereus, the most common pathogen causing mastitis in Egyptian dairy cows. We also emphasized that antibiotic misuse during mastitis is a potential public health threat.


Introduction
Bovine mastitis is a disease affecting dairy cows characterized by pathological, chemical, and physical changes in the milk-producing glandular tissues [1]. It is a pernicious disease of great concern for the global dairy industry, leading to decreased milk production and rejected milk [2]. Bacillus cereus is a rodshaped, Gram-positive, facultative-anaerobic, and endospore-forming pathogen that causes mastitis in cows and severe food poisoning in humans [3]. The spores can survive in dry and hot conditions and stay dormant for several years. They are resistant to heat and chemicals [4]. Soil, straw, and other fodders are the most common contaminants in dairy farms. The bedding used is also a potential contaminant when the cows are housed indoors during winter. Contaminated udders eventually result in the presence of B. cereus in raw milk [5]. Bacillus spp. can also infect the mammary gland during lactation, intramammary treatment, or dry cow therapy. Moreover, it can be introduced into the mammary gland through unsterilized injections. Some Bacillus spp. can cause fatal gangrenous mastitis. Bacillus cereus is the most common foodborne bacteria in raw milk and dairy farm environments [6]. Bacillus cereus is a species complex with high phenotypic and genotypic similarity [7]. The proteins encoded by the groEL and sodA genes are essential for bacterial cell viability, and hence, these genes can be used for phylogenetic analysis to identify B. cereus. The groEL gene has been investigated as a phylogenetic marker [8]. However, genomic studies are required to assess the genetic mechanisms and factors enabling toxin production to differentiate between Bacillus spp. [9].
Bacillus cereus causes several diseases in humans and animals [10]. They are the most frequently isolated foodborne bacterial pathogens and can produce several powerful toxins [11]. Consequently, they endanger public health by forming spoilage enzymes and toxins in dairy products, resulting in enormous economic losses [9]. Bacillus cereus causes two types of food poisoning: diarrheal and emetic, which negatively affect human health. The diarrheal type is linked to the production of enterotoxins such as hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) [12]. Further, tetracycline-resistant genes tetA and tetB have been reported for the 1 st time in B. cereus [13]. Most B. cereus strains are resistant to β-lactam antibiotics as they produce the lactamase enzyme [14]. Bacillus cereus infections are still primarily treated using antibiotics. However, the emergence of antibiotic-resistant B. cereus strains due to antibiotic misuse [15] and transmission of resistance genes through horizontal gene transfer [16] has resulted in the failure of antibiotic treatments.
Therefore, understanding the antibiotic resistance profile is crucial before treating B. cereus. Further, the importance of B. cereus as a major cause of mastitis among Egyptian dairy farms should be elucidated. This study aimed to detect antibiotic-resistance and toxigenic genes from B. cereus found in raw milk of sub-mastitic cows from different governorates in Egypt.

Ethical approval
Ethical approval was not required for this study; however, samples were collected as per the standard sample collection procedure.

Study period and location
The study was conducted from January 2018 to January 2020 at the National Research Centre in Dokki, Egypt and Animal Reproduction Research Institute Agriculture Research Center (ARC), Giza, Egypt.

Sample collection
A total of 262 milk samples were collected aseptically using sterile vials from cows with subclinical mastitis from dairy farms in Beheira, Giza, Alexandria, and Menoufia governorates, which were suffering from decreased milk yield, recurrent mastitis, and failure of antibiotic treatment. The milk samples were placed immediately in an ice container and transported to the microbiology laboratory. The samples were collected in compliance with the rules of the local Commission for Ethics in Animal Experimentation and Investigation

Bacterial culture
The milk samples were cultured on Bacillus selective agar (HiMedia, India), and after 24 h-48 h of incubation at 37°C, the plates were examined for bacterial growth. The B. cereus colonies displayed a distinct turquoise-peacock blue color and were surrounded with egg yolk-like precipitate of the same diameter. The color of the indicator dye around the colony remained unchanged as B. cereus does not ferment mannitol. We performed morphological and biochemical tests on all suspected B. cereus colonies. The Gram-stained smears were microscopically examined to identify their cell shape, motility, and hemolysis. We also evaluated nitrate reduction and the production of enzymes, including catalase, oxidase, urease, and lecithinase [17].

Identification of B. cereus using HiCrome ™ Bacillus agar (HiMedia)
We observed one or more blue colonies on each Bacillus selective agar media plate. The lecithin-positive colonies appeared as light-blue colored, large, flat colonies with blue centers, and pink edges on chromogenic B. cereus agar after adding Bacillus Selective Supplement (FD324) and incubating at 30°C for 24-48 h [18].

Detection of the groEL gene
A polymerase chain reaction (PCR) analysis was performed on all 125 chromogenic-positive isolates. A single typical colony was inoculated on brain heart infusion broth and incubated overnight at 37°C. We investigated the potential of the groEL gene as a phylogenetic marker by extracting deoxyribonucleic acid (DNA) from the broth culture using a positive reference strain (B. cereus ATCC 14579).

Detection of the virulence genes (enterotoxigenic and antibiotic resistance genes)
A polymerase chain reaction was performed to detect the virulence genes, including hblD and nhe, tetA, and beta lactam-resistant (bla) genes in the positive isolates identified using groEL.

Deoxyribonucleic acid extraction
The DNA was extracted from the samples using the QIA amp DNA Mini kit (Qiagen, Germany, GmbH) based on the manufacturer's recommendations with slight modifications.

Polymerase chain reaction amplification
The PCR reaction was performed using a reaction mixture containing 12.5 µL Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µL of each primer (20 pmoL), 5.5 µL water, and 5 µL DNA template in a final volume of 25 µL using an Applied Biosystems thermal cycler type 2720.

Analysis of PCR products
The PCR products were separated by running a 1.5% agarose gel (Applichem, Germany) at a 5 V/cm gradient in 1× Tris borate ethylenediaminetetraacetic acid buffer at room temperature. Each lane was loaded with 15 µL of the product, and the fragment sizes were determined using the Generuler 100 bp ladder (Fermentas, Germany). The gel was photographed using a gel documentation system (Alpha Innotech, Biometra, Germany), and the data were analyzed using computer software.

Statistical analysis
Data presented in tables as percentages were subjected to an exact test using IBM-SPSS 20.0 software (IBM Corp., NY, USA). In addition to Pearson Chisquare, 'Fisher's Exact, Linear-by-Linear Association, and McNemar tests were also performed.

Results and Discussion
Bacillus cereus is a Gram-positive bacteria found in nature [25]. When present in milk, B. cereus causes milk spoiling, which results in food poisoning in humans [26]. It is considered one of the major causes of mastitis in cows on dairy farms [27].
In the present study, we isolated 125 (93.2%) B. cereus strains from cows with subclinical mastitis based on colony morphology and biochemical tests (Table-2). These isolates were confirmed by culturing on chromogenic B. cereus agar media (Table-3), consistent with the study by Hammad et al. [28] reporting that B. cereus is 85% prevalent in raw milk in Egypt. However, this prevalence rate is higher than others reported by Meng et al. [6], Hassan et al. [29], Haughton et al. [30], Rezende-Lago et al. [31], who found that B. cereus were 46.6%, 59%, 50%, and 61.1% prevalent, respectively. Conversely, other studies by Alemneh [32], Seblewongel [33], and Gilles et al. [34] found lower isolation rates of 15.4%, 15.86%, and 15.4%, respectively. Furthermore, Hayat et al. [35] determined that B. cereus is associated with subclinical mastitis in buffaloes in swats, with a (3.27%) prevalence. In addition, Ghazali et al. [36] identified that 23 of 78 milk samples from subclinical mastitic goats contained B. cereus. These variations in results can be attributed to weather variations or the hygiene conditions in the farms that differ from those observed in this study.
Polymerase chain reaction analysis is a simple, fast, and reliable tool for effectively identifying microorganisms from numerous sources [37]. The groEL gene was used to detect B. cereus [38], as its efficacy has been demonstrated in previous phylogenetic research [39]. In this study, the PCR results revealed that 110 isolates (88%) harbored the groEL gene while 15 isolates (22%) did not (Table-4). The toxin hemolysin is a virulence factor that can potentially cause diarrhea    (Figure-3) and necrosis [40]. Species containing the enterotoxin genes nhe and hbl primarily cause food deterioration, resulting in food poisoning [41]. Bacteria produce diarrheal toxins when they multiply in the intestines. At least three bacterial toxins are known to be involved in diarrheal syndrome: hbl, nhe [42], and the genes hblA, hblC, and hblD that encode the three-component hemolysin BL enterotoxin [43]. In this study, the toxigenic genes (nhe and hblD) were detected in 110 B. cereus isolates, of which 103 were nhe-positive (93.64%) and 101 were hblD-positive (91.82%) ( Table-5 and Figures-1 and 2) [44]. Remarkably, nhe was identified in all isolates, while only 50.7% had hbl genes. However, Owusu-Kwarteng et al. [7] found that 13% (12/96) of the isolates found in the raw milk and other dairy products of farm-raised cattle had all three hemolytic hbl complex enterotoxin genes (hblA, hblC, and hblD), whereas 25% had no hbl gene, and 63% had one or more of the three hbl genes. Moreover, they showed that 14% (13/96) had only one nhe gene, 60% (57/96) had all three nhe genes (nheA, nheB, and nheC), and 8% had no nhe genes. In addition, Meng et al. [6] showed that 12.77% and 8.51% of B. cereus isolates obtained from farm environments and raw milk harbored the hblACD and nheABC genes, respectively. The high percentage of toxigenic genes indicates the importance of detecting virulence factors to understand the involvement of the production of various toxins and enzymes. Bacillus cereus is a global health threat as they are extremely resistant and have genetic mechanisms for responding to various environmental conditions. The antibiogram pattern against several commonly used antibiotics showed 100% resistance to FEP and cefixime (CFM). However, they are 100% sensitive to amoxicillin, C, ampicillin, and levofloxacin (LE), followed by CIP (93.6%), azithromycin (AZM) (90.9%), getamicin (88.2%), CXM (79.1%), VA and cefaclor (CF) (68.2%), tetracycline (TE) (54.5%), and amikacin (AK) (20.9%) ( Table-6 and Figure-3). Our results were consistent with Owusu-Kwarteng et al. [7], who reported that B. cereus was susceptible to C (99%) and CIP (100%). The results by Sadashiv and Kaliwal et al. [45] showed that B. cereus was resistant to ampicillin (50.67%), C (6.33%), and AZM (5.42%). Furthermore, B. cereus showed 54.75%, 51.13%, 12.21%, 17.64%, and 7.69% resistance to CFM, CF, gentamicin (GEN), AK, and TE, respectively, which contradicted our findings. Moreover, similar results were detected regarding CIP (4.07%) and VA. According to Rosovitz et al. [46], B. cereus is susceptible to VA, and most strains are sensitive to C, CIP, erythromycin, and GEN. Few B. cereus strains are moderately sensitive to clindamycin and TE [47]. Tetracycline resistance was observed in 45.5% (50/110) of B. cereus isolates, significantly higher than that reported by Whong and Kwaga [48], who showed that 6.7% of B. cereus isolates were TE-resistant. These results indicate the importance of effectively selecting specific antibiotics to treat antibiotic-resistant B. cereus strains in dairy farms.
Pearson Chi-square, Likelihood Ratio, Fisher's Exact Test, and Linear-by-Linear Association (p < 0.0001), Goodman and Kruskal tau, and  showed that 98.18% (108/110) of the identified carried the bla gene (Table-5      Somers'd (p < 0.0001) indicated a significant difference between resistance and sensitivity to different antibiotics (Table-6). All symmetric measures of the exact test (Phi, Cramer's V, Contingency Coefficient, Kendall's tau-b, Kendall's tau-c, Gamma, Spearman Correlation, and Pearson's R) showed significant (p < 0.0001) with fair (k = −0.153) and significant (p = 0.0001) measure of agreement (Kappa). Based on these findings, suspected B. cereus infections should be clinically treated with VA or LE rather than broad-spectrum cephalosporins and penicillin. Furthermore, we found that several B. cereus isolates were multidrug-resistant, implying that raw milk infected with B. cereus is a major concern [49]. We agree with Chen et al. [50], who discovered that VA should be the drug of choice for B. cereus infections.
The molecular examination of the antibiotic-resistant genes bla and tetA revealed that despite the absence of the tetA gene (Table-5 and Figure-4), 45.5% (50/110) of B. cereus isolates displayed TE resistance phenotypically. Our results agree with Agers et al. [51], who found that phenotypically three isolates showed TE resistance despite the lack of tetA, tetB, or tetC. This might be due to the presence of other TE resistance genes, for example, tetM and tetL, or other gene mutations. When a bacterial cell becomes resistant, it can swiftly transmit the antibiotic resistance genes to numerous species [52], transferring TE resistance genes [53].

Conclusion
The B. cereus strains isolated from subclinical bovine mastitis cases showed high rates of resistance to most tested antibiotics due to the presence of several antibiotic-resistant and virulence genes (hblD and nhe). This suggested the emergence of multidrug resistance among these isolates in Egypt, which makes it necessary for milk producers and conventional dairy processors to follow strict sanitary and manufacturing practices to avoid contamination and subsequent disease outbreaks caused by B. cereus. Furthermore, it is crucial to determine the antibiotic resistance profile of B. cereus to identify treatment regimens and raise awareness for B. cereus as one of the most important causes of mastitis.