Virulence and Antibiotics Susceptibility Genes in Bacillus species Isolated from Freshly Expressed Breast Milk

Breast milk can sometimes spread acute infections from bacterial and viral agents to babies. There are several recognised instances of infections caused by several microorganisms contracted through breast milk. Bacilli are Gram-positive, aerophilic, spore-producing bacteria that are ubiquitous in the environment. They are found not only in the ground, dirt, and water but also in vegetation, animals, and people. The genus Bacillus encompasses a very significant number of species as it has experienced massive taxonomic growth with the application of 16SrRNA sequencing. This research work was focused on the isolation and molecular identification, determining the presence of virulence genes (hblA, hblD, nheA, nheB and cesB), antibiotics resistance genes (blaVIM, vanA and vanB), and antimicrobial susceptibility of eighteen Bacillus species isolates obtained from freshly expressed breast milk (these isolates were predominantly isolated from mothers within the first 6 weeks of lactation). The result obtained indicated that all the Bacillus isolates were motile, spore formers and could not form biofilm. They were all susceptible to ciprofloxacin (100%) and resistant to ceftazidime and lincomycin (100%). All the isolates showed a distinctive band of suitable size of around 300bp for the molecular identification except isolate 12. The antibiotics resistant genes (blaVIM, vanA and vanB) were absent. Bacillus cereus possessed most of the virulence genes. nheB gene had the highest occurrence 50.0 % followed by hblA gene 33.3 %, hblD gene 16.6 %, cesB gene 5.56% while none of the isolates screened had the nheA gene. Based on these findings, we conclude that breast milk, when not properly handled or when there is little or no observation of strict hygiene practices by the breast-feeding mother, can harbor pathogenic microorganisms such as Bacillus species which possess virulence genes as well as increase the rate of spread of antibiotics resistance from mothers to infants.

Keywords: Breast milk, Bacillus species, Virulence genes, Antibiotics susceptibility, Antibiotics resistance genes.

Milk from humans is generally recognized to be the finest diet for babies; it is a highly nutritious liquid that comprises energetic nutrients, including various bioactive complexes, carbohydrates, proteins, immune cells, and immune globulin, which supplies satisfactory dietary and protecting necessities for toddlers [1]. It may also include several commensal or lactic acid bacteria strains [2]. These bacteria when present in milk can be taken in by infants, confirming the information that these commensal bacteria when present in milk may act as major contributors in establishing the microbiota of the infant gastrointestinal tract (GIT). Numerous current reports have presented that commensal or food-borne bacteria can function as storage of antibiotic resistance genes, that are alike to those observed in bacterial strains that are pathogenic [3].

Amongst the microbes that can be found in milk during processing on farms or processing lines in dairy, bacteria that produce endospores belong to the Gram-positive (comprising about two hundred species) group and are of major concern because they can survive severe environmental conditions such as pressure, extreme temperatures, desiccation, ultraviolet radiation, drought, biocides, and lack of nutrients [4]. Sadly, bacteria that produce spores are everywhere; usually isolated from the soil and seen as microflora in gastrointestinal tract of mammals and insects [5].

Genera Bacillus are made up of Gram-positive rods that can produce endospores and can be differentiated from other endospore-producers due to their aerophilic character (strict or facultative), cells that are rod-shaped and ability to produce the catalase enzyme [6]. Spore production, generally observed in this group, is believed to be an approach for existence in the soil, where these bacteria dominate.

Most times, species of Bacillus are identified and separated by approaches based on the endurance of their endospores to heat or ethanol. Nevertheless, straightforward isolation of species needs a discriminating medium or other discriminating conditions that are obtainable only for a limited number. Conventional classification has been centered on Gram staining, colony morphology, motility, and biochemical tests, which are timewasting, slightly biased, and exhaustive techniques. Both conventional and automatic classification methods have problems recognizing some Bacillus species and do not distinguish amongst strains.

Approach through phylogenetic studies to Bacillus classification has been achieved essentially by exploration of 16S rRNA gene by oligonucleotide sequencing. This technique, of course, also reveals phylogenetic relationships. Sequences of the 16S rRNA gene have played an important role in studies of phylogenetic at the level of genus, its usefulness has been interrogated in instances of closely connected species collections including Bacillus, where inadequate separation in 16S rDNA prohibited the resoluteness of species and strains relations. Successive usage of genes that are housekeeping are fundamental thus not absent from genomes, therefore evolving faster than 16S rDNA, has attested to be beneficial for taxonomical categorization [7].

The pathogenic potential of Bacillus species that do not belong to the anthrax group has not been investigated lengthily, aside from B. cereus. The pathogenicity of this bacterium has been linked to the exudation of numerous virulence proteins [8] and to factors that contribute to motility i.e., swarming and swimming [9]. The virulence proteins comprise numerous hemolysins i.e., trimeric toxins (hemolysin BL, (HBL) complex, the non-hemolytic enterotoxin (NHE) complex), cereulide, proteases, phospholipases, and cytotoxin K (CytK) [10]. These virulence proteins can act as damaging/ highly reactive proteins detrimental to the integrity of the plasma membrane of numerous cells [8]. Cereulide has been shown to be the highly dangerous virulent protein produced by B. cereus. This is seen in an unusual situation where a boy who was seventeen years old and his father suffered from acute gastroenteritis after consuming pesto and spaghetti which was cooked four days ago. Within two days, the boy died because of failure of the liver (fulminant) and rhabdomyolysis. His father suffered rhabdomyolysis and hyperbilirubinemia and got well [11]. Additionally, B. cereus can form biofilm, which can play a key part in attaching to catheters [12]. Certain other Bacillus spp. was indicated to have genetic sequences coding hemolysin BL, cytotoxin K or non-hemolysin complexes [13] or have the capability of forming biofilms.

Acting as an opportunistic microorganism, B. cereus can trigger two kinds of food poisoning in people, categorized by both queasiness and throwing up or pain in the abdomen and diarrhea [14]. The pathogenicity of B. cereus sensu lato is ascribed to several causes. Diarrheal sickness is linked with the release of enterotoxins such as enterotoxin FM, HBL, NHE, CytK [15], whereas the pathogenicity of emetic strains is ascribed to the release of cereulide which is heat-stable, produced on the ces genes by a non-ribosomal peptide synthetase [16] (Table 1). The emetic toxicant, commonly performed in diet, is not deactivated throughout processing of food or movement in the GIT because it is extremely unaffected by application of heat, extremes in pH, and action by protease. Cereulide has been shown to be toxic to mitochondria by acting as a potassium ionophore and has been reported to inhibit human natural killer cells. Thus, consumption of live B. cereus is not required for sickness of this form to happen.

Table 1. Most tested virulence (associated) genes in Bacillus species [17]

Food poisoning (causing diarrhea) does not result from preformed toxicants in food, instead it is caused by live B. cereus cells releasing enterotoxins into the small intestine. Presently, there are three known types of enterotoxins implicated in food poisoning occurrences: HBL and NHE complexes, CytK which is a single protein [18], HBL is a three-constituent hemolysin that comprises two lytic segments (L1 and L2, encoded by hblC and hblD genes) and a protein B that binds (encoded by hblA). NHE is also a three-component; non-hemolytic toxin that is encoded by three genes nheA, nheB and nheC. These two toxin complexes are arranged in operons and the genes of the enterotoxin complex NHE which are corresponding have been shown to be transcribed simultaneously [19].

Bacillus licheniformis, along with Bacillus cereus, has been confirmed to be amongst the extremely common Bacillus species identified in unprocessed milk and in the processing field of dairy [20]. Though B. licheniformis has not been classified as pathogenic to humans, the endospores produced are acknowledged to bring about putrefaction of milk and milk products, increase problems that prevent compliance and have antagonistic effects on milk organoleptic and useful qualities [21].

Bacillus circulans, which also produces endospores, can be in the ground, dirt, food, and baby bile. Spores allow these organisms to become inactive for an unlimited period when environmental surroundings are hostile, nevertheless when circumstances become good again the endospore can revive itself into its vegetative form [22].

Bacillus subtilis, known also as grass or hay bacillus, can be isolated from soil and GIT of humans and ruminants. It can produce a hard, protecting spore, enabling it to survive severe environmental situation [23]. It has proven to be greatly responsive to genetic technology, and has been commonly accepted as a prototype for research, specifically of sporulation, which is a straightforward case of cell specialization [24]. Though not conventionally considered as pathogenic to humans, this organism has been stated to be implicated in food poisoning incidents, e.g., there was an occurrence in 2005 in a kindergarten triggered by milk powder, with throwing up as the major indication but is frequently associated with loose stool [25].

An overlooked aspect of research is the effect of antibiotic intake amid women who are breastfeeding as it relates to increase in the rate of resistance to antibiotic [26]. Women who are nursing and who suffer mastitis can be given amoxicillin/clavulanic acid, ciprofloxacin, or an alternative antibiotic for which the impacts on babies have been investigated thoroughly [27]. Selective action of bacteria that are resistant can arise at strengths of antibiotics lower than the minimal inhibitory concentration [26]. Use of antibiotics by nursing mothers can be a contributing factor to the development of resistance to antibiotics when babies are exposed to less therapeutic levels.

Evidence based on scientific findings regarding Bacillus species that are resistant to antibiotics is inadequate, and unease concerning the capability of these species to transmit genes that are antibiotic resistance is ever-increasing [28]. Almost all Bacillus strains are resistive to ticarcillin-clavulanate and broad spectrum cephalosporins; Bacillus species such as B. cereus, antibiotic treatment is still the main therapy approach given for the illnesses [29]. Nonetheless, the rise of B. cereus strains that are antibiotic resistant, mostly owing to antibiotic misuse or acquiring genes that are resistance via horizontal gene transport, leads to antibiotic therapy failing. Therefore, acquiring the antibiotics resistance profile of B. cereus is greatly significant to public health.

This research work is carried out to study the occurrence of Bacillus species. and its various enterotoxigenic and antibiotic susceptibility genes from freshly expressed human breast milk given to infants as food.

Collection of Breast milk Specimens

Breast milk specimens were collected from breastfeeding mothers at six selected Government hospitals in Lagos. Hundred (100) samples consisting of 50 pre-cleaned and 50 post-cleaned manually expressed milk were collected from the breastfeeding mothers.

Bacterial isolates

A total of eighteen Bacillus species isolates comprising seven B. cereus, six B. subtilis, three B. licheniformis, one B. brevis and one B. circulans strains were investigated. The isolates which were previously isolated on Tryptic Soy Agar (TSA) and identified using Gram staining technique and biochemical testing used for clinical diagnosis amongst the isolates. Bacillus isolates were recognised based on the morphology of their colony, catalase production, indole production, nitrate reduction, urease activity, citrate production and fermentation of sugars.

Motility test

Semi solid nutrient agar was prepared in a sterile test-tube, a sterile inoculating needle was used to select a good-isolated colony and stab the agar to about 1 cm of the base of the test-tube. It was ensured that the needle was drawn-out from the agar on the same line as it was introduced. It was incubated at 37ºC for 18 to 24 hours till growth was obvious. A motility test that is positive is seen having a dispersed cloud of growth away from the area of inoculation. Growth along the line of stab inoculation means the test is negative for motility [30].

Endospore staining

A thin smear of a well-isolated 2-week colony was made on a grease free microscope slide. It was allowed to air dry, heat-fixed, and then placed a blotting paper on it. The paper was soaked in malachite green (a stain mixture) and heated for a little time without boiling, ensuring the paper is wet while pouring additional dye as needed. Then the slide was gently rinsed under slow running tap water followed by counterstaining for thirty seconds with safranin. The slide was then washed and placed at an angle of 45º to air dry. The stained slides were observed under oil immersion (X100) using a binocular compound microscope for the appearance of spores. On observation, spores appeared sharp green and cells that are vegetative were pink [31].

Screening for biofilm using congo red method

All isolates were cultured on Congo red agar prepared by adding Congo red dye (0.8gm/l), to Brain heart infusion broth (37gm/l), agar number 1 (10gm/l), and sucrose (5gm/l). Congo red dye was made as a concentrated aqueous mixture and sterilized at 121ºC for 15 minutes. It was afterwards introduced to sterilized Brain heart infusion agar plus sucrose at 55ºC [32]. Petri-dishes were inoculated with the isolates and incubated at 37ºC for 24 – 48 hours aerobically. Black colonies with a dry crystalline consistency indicated slime-forming strains; red coloured colonies indicated non-slime forming strains.

Antibiotics susceptibility test

Antibiotics sensitivity testing of the isolates was performed according to 0.5 Mac Farlands standard using Kirby-Bauer disk diffusion method on Mueller-Hinton agar. The isolated bacterium was suspended in normal saline and incubated for 30 minutes or more to ensure the turbidness is standardized to be equivalent to that of a 0.5 MacFarland standard (consistent to around 1.5 X 10⁸ CFU/ml). Beginning from the top, the Mueller Hinton agar plate surface was streaked with the inoculum via a sterile swab stick.

The whole plate was covered by swabbing forward and backward from one edge to another. Then was turned to about 60º and the swabbing process was done again. The plate was rotated approximately 60º over and the whole plate was swabbed for the third time to guarantee that the inoculum is uniformly spread. Antimicrobial discs were added around fifteen minutes of inoculating the plate containing Mueller-Hinton agar. The discs were positioned on the plate one by one using sterile forceps, guaranteeing enough space between all discs. Each disc was pressed down firmly using forceps to guarantee total level placement with the agar. The plates were turned upside down and incubated at 37ºC for 24 hours. After incubation, plates were held a little distance over a black non-reflecting top and zone of inhibition was determined to the closest millimeter with a ruler.

The antibiotics used were Amoxicillin Clavulanic acid, Ceftazidime, Vancomycin, Streptomycin, Ciprofloxacin, Lincomycin and Tetracycline (Table 2).

Table 2. Antibiotics used in this research and their mode of action.

The millimeter of zones of inhibition were translated as Resistance (R), Intermediate (I) and Susceptible (S) corresponding to the criteria advised by the National Committee for Clinical Laboratory Standards [33] (S1 Table).

Deoxyribonucleic acid (DNA) extraction

The boiling method of bacterial DNA extraction was executed accordingly [34] with a slight modification. Pure isolates were subcultured overnight; the cells were collected into 1000µL of distilled water (sterile). Vortex until they were entirely dispersed. It was then centrifuged at 10,000rpm for 5 minutes, the supernatant was thrown away, then the pellet suspended in 200µl of sterile distilled water and vortexed until thoroughly mixed. The sample was then boiled for 10 minutes at 100ºC in a heat block and immediately cooled on ice. It was then centrifugated at 10,000rpm for five minutes. The supernatant having the genomic DNA was moved to a pristine Eppendorf tube and stored at 4ºC and used for subsequent PCR amplification.

Polymerase chain reaction (PCR)

PCR screening for antibiotics resistance genes and virulence genes (Table 3) was performed on bacterial DNA from breast milk. PCR reaction was achieved by utilizing the Solis Biodyne 5X HOT FIRE Pol Blend Mater mixture. PCR reaction was executed in 20 µl of the reaction mix, and then the concentration of the mix was reduced from 5X to 1X concentration which contained the 1X Blend Master Mix buffer (Solis Biodyne), 2.0mM MgCL₂, 200µM of the deoxyribonucleotides each (Solis Biodyne), 20pMol of the primer each (Stabvida, Portugal), 2 unit of Hot FIREPol DNA polymerase (Solis Biodyne), 5µl of extracted DNA, enzyme for proofreading, and sterile distilled water needed to make up the reaction mixture.

Thermal cycling was carried out in a Pielter thermal cycler 100 (MJ Research) for the first denaturation at 95ºC for five minutes accompanied by thirty-five cycles of denaturation at 95ºC for thirty seconds; followed by annealing at 72ºC required by each primer for one minute and one minute thirty seconds. Finally, it ended with an extension period at 72⁰C lasting for ten minutes. The amplified product was resolved on a 1.5% agarose gel followed by electrophoresis carried out at 80V for one hour thirty minutes and the DNA molecular weight standard used was 100bp DNA ladder. After electrophoresis, ethidium bromide staining was used to visualize DNA bands.

Table 3. Primers used in PCR amplification of virulence genes and resistance genes.

Biochemical identification of virulence factors from isolates

A total of eighteen Bacillus species isolates comprising seven B. cereus, six B. subtilis, three B. licheniformis, one B. brevis and one B. circulans were investigated. Their ability to produce endospores, motility and form biofilm was investigated in this research work and is presented in Table 4. It was noted that all the tested isolates produced endospores when subjected to harsh conditions, were motile but lacked the ability to form biofilm.

Table 4. Biochemical Characterization of selected Gram Positive (rods) isolates from breast milk

KEYS: R= Rod; + = positive; – = negative

Antibiotics susceptibility for isolated Bacillus spp

Resistance and susceptibility to different antimicrobial agents by the Bacillus isolates can be seen in Table 5. It was noted that all tested isolates were susceptible to Ciprofloxacin and resistant to the antibiotics Lincomycin and Ceftazidime.

Table 5. Antibiotics susceptibility for Bacillus spp isolated from breast milk.

AMC; Amoxicillin clavulanic acid 20/10µg, TE; Tetracycline 30µg, CIP; Ciprofloxacin 10 µg, MY; Lincomycin 15µg, S; Streptomycin 10µg, CAC; Ceftazidime clavulanic acid 30/10µg, VA; Vancomycin 30µg, R; Resistance, I; Intermediate, S; Susceptible [33].

Molecular identification of Bacillus spp

The Bacillus spp. isolates were identified molecularly based on the arrangement of the 5’ hypervariable areas of the 16S rRNA gene which was intensified using primers of the 16S-HV. All the isolates showed a distinctive band of suitable size of around 300bp for the molecular identification except isolate 12 (Fig 1). Other strains used as negative controls gave no PCR product or product with size 300 bp.

Fig 1.  Agarose gel electrophoresis for amplification of 16S rRNA in Bacillus species isolated from breast milk.

M= Marker; -V= Negative control; 1= B. circulans; 2= B. cereus; 3= B. cereus; 4= B. cereus; 5= B. cereus; 6= B. licheniformis; 7= B. subtilis; 8= B. brevis; 9= B. cereus; 10= B. cereus; 11= B. subtilis; 12= B. cereus; 13= B. licheniformis; 14= B. cereus; 15= B. subtilis; 16= B. licheniformis; 17= B. subtilis; 18= B. subtilis

During this research, five B. cereus enterotoxin genes were determined by PCR amplification.  Bacillus species isolated from freshly expressed breast milk were investigated for different enterotoxin genes such as hblA, hblD, nheA, nheB and cesB (Figs 2 – 4).

Fig 2. Agarose gel electrophoresis for amplification of hblA and hblD in Bacillus species     isolated from breast milk.

M= Marker; -V= Negative control; 1= B. circulans; 2= B. cereus; 3= B. cereus; 4= B. cereus; 5= B. cereus; 6= B. licheniformis; 7= B. subtilis; 8= B. brevis; 9= B. cereus; 10= B. cereus; 11= B. subtilis; 12= B. cereus; 13= B. licheniformis; 14= B. cereus; 15= B. subtilis; 16= B. licheniformis; 17= B. subtilis; 18= B. subtilis

Fig 3. Agarose gel electrophoresis for amplification of nheA and nheB in Bacillus species isolated from breast milk.

M= Marker; -V= Negative control; 1= B. circulans; 2= B. cereus; 3= B. cereus; 4= B. cereus; 5= B. cereus; 6= B. licheniformis; 7= B. subtilis; 8= B. brevis; 9= B. cereus; 10= B. cereus; 11= B. subtilis; 12= B. cereus; 13= B. licheniformis; 14= B. cereus; 15= B. subtilis; 16= B. licheniformis; 17= B. subtilis; 18= B. subtilis

Fig 4: Agarose gel electrophoresis for amplification of cesB in Bacillus species isolated from breast milk.

M= Marker; -V= Negative control; 1= B. circulans; 2= B. cereus; 3= B. cereus; 4= B. cereus; 5= B. cereus; 6= B. licheniformis; 7= B. subtilis; 8= B. brevis; 9= B. cereus; 10= B. cereus; 11= B. subtilis; 12= B. cereus; 13= B. licheniformis; 14= B. cereus; 15= B. subtilis; 16= B. licheniformis; 17= B. subtilis; 18= B. subtilis

Three antibiotics resistance genes were tested in this study which are the blaVIM, vanA and vanB genes (Figs 5 and 6). The results obtained showed that none of these genes were existent in any of the isolates.

Fig 5: Agarose gel electrophoresis for amplification of blaVIM in Bacillus species isolated from breast milk.

M= Marker; -V= Negative control; 1= B. circulans; 2= B. cereus; 3= B. cereus; 4= B. cereus; 5= B. cereus; 6= B. licheniformis; 7= B. subtilis; 8= B. brevis; 9= B. cereus; 10= B. cereus; 11= B. subtilis; 12= B. cereus; 13= B. licheniformis; 14= B. cereus; 15= B. subtilis; 16= B. licheniformis; 17= B. subtilis; 18= B. subtilis

Fig 6: Agarose gel electrophoresis for amplification of vanA and vanB in Bacillus species isolated from breast milk.

M= Marker; -V= Negative control; 1= B. circulans; 2= B. cereus; 3= B. cereus; 4= B. cereus; 5= B. cereus; 6= B. licheniformis; 7= B. subtilis; 8= B. brevis; 9= B. cereus; 10= B. cereus; 11= B. subtilis; 12= B. cereus; 13= B. licheniformis; 14= B. cereus; 15= B. subtilis; 16= B. licheniformis; 17= B. subtilis; 18= B. Subtilis

Supplementary files

Table S1. Antibiotics susceptibility interpretation table

Table S2. Primers used in PCR amplification of virulence genes and resistance genes

The microbiota of the gastrointestinal tract of human is an indisputably essential part of the body, necessary for the appropriate working of numerous activities which include digestion, drug response, functioning of the immune-system and brain [39]. It is yet to be agreed upon whether colonization of the gut begins before or after birth. Nevertheless, the generally dynamic period of bacterial colonization in the gut begins from the time of birth and the microbiota structure of the gut varies significantly throughout the initial weeks of birth [40]. Once commencement of nursing, the baby is continuously provided with breast milk-related bacteria [41].

The eighteen Bacillus spp. isolates were identified molecularly based on the arrangement of the 5’ hypervariable areas of the 16S rRNA gene which was intensified using primers of the 16S-HV. All the isolates showed a distinctive band of suitable size of around 300bp for the molecular identification except isolate 12 (Fig 1). An investigation was performed previously by applying the restricted 16S rDNA sequence to explore the identification approach of Bacillus species [7]. Primarily, to explore the major information section of the 16S rDNA, sixty-nine Bacillus species strains were exposed to a comparableness of the 16S rDNA sequences. It was shown that the hypervariable area was the 5’ end region which was extremely particular for every strain. Likewise, studies on the sequence of the hypervariant area from fifty-one strains being part of 4 clusters hinted that the hypervariable area was greatly preserved among species. These outcomes established that the hypervariant area is a very effective marker for the prompt recognition or classifying of Bacillus species. Other type of bacteria specie used as negative controls gave no PCR product or product with size 300 bp.

It was observed that all the Bacillus isolates produced endospores when subjected to harsh conditions, were motile but lacked the ability to form biofilm (Table 4). Endospores of Bacillus species can persist in their inactive and resistive conditions for a long period of time, but subjection to conditions like certain nutrients can initiate endospores’ reverting to existence within a short period of time [42].  The result also showed that none of the isolates had the ability to create biofilm. According to a study, 10% of Bacillus species that were non-psychrotolerant were enumerated from lettuce leaf (green) gathered during the low-temperature process having frail biofilm-development capability, however; about ninety percent of the isolates were unable to create a biofilm at 7ºC [43]. Motility of bacterial motility driven by flagellum, i.e., swarming or swimming, may ease the penetration of host cellular blockades in humans [44].

Antimicrobial sensitivity test of the isolates was performed utilizing seven of the standard antibiotics. The antibiotic susceptibility of Bacillus spp. gotten from milk of humans has not been well studied. The antimicrobial sensitivity of Bacillus species is presented in Table 5. All the isolates showed differences in their sensitivity and resistance to the seven antibiotics. The highly efficient antibiotics were ciprofloxacin, tetracycline, and streptomycin.

Although 33.3% of the Bacillus isolates were susceptible to the penicillin Amoxicillin Clavulanic acid, all the isolates were resistive to the cephalosporin Ceftazidime. Three of the isolates studied were resistive, while nine were intermediate to the penicillin Amoxicillin Clavulanic acid. Resistance to vancomycin was strain dependent. All Bacillus subtilis (isolates) were resistant to the antibiotic Vancomycin, alongside two strains of Bacillus cereus and Bacillus brevis. 22.2% and 27.8% of the Bacillus strains were susceptible and intermediate to the antibiotic Vancomycin respectively.  A study carried out showed that B. cereus, B. clausil, B. circulans, B. thurungiensis, B. weihenstephanesis have identification with VanA, VanD and VanM and VanY genes in the findings [45].

From Table 5, 83.3% of the Bacillus isolates were susceptible to the antibiotic Tetracycline while one strain of B. subtilis was intermediate and one strain of B. cereus and B. brevis were resistant to Tetracycline. A study was stated for a group of isolated B. cereus (29 strains), the result showed that 89.7% of the strains were susceptible to tetracycline [21]. None of the Bacillus isolates were susceptible or intermediate to Lincomycin; they were all resistant to it.

Many of the Bacillus isolates were susceptible to the antibiotics Streptomycin while 22.2% and 5.6% were intermediate and resistant to Streptomycin respectively. This result is alike to the research carried out where erythromycin, vancomycin and streptomycin were the most effective antibiotics and resistances were strain dependent [46].

All the Bacillus isolates were sensitive to Ciprofloxacin. Good susceptibility to ciprofloxacin, gentamicin and tetracycline has also been described in France [47]. Similarly, another report showed sensitivity of one hundred per cent to ciprofloxacin from patients’ samples [48]. Hence, this information proved that ciprofloxacin is quite efficient against strains of B. cereus from several sources as one agent.

Polymerase chain reaction (PCR) test has been utilized for quick recognition and differentiation of enterotoxins genes found in Bacillus cereus and other Bacillus species [35]; and for fast recognition of food contaminated with enterotoxigenic B. cereus also [49]. Some of these virulence genes were investigated in the isolates. B. cereus possessed most of the virulence genes (Fig 2 – 4). nheB gene had the highest occurrence 50.0 % followed by hblA gene 33.3 %, hblD gene 16.6 %, cesB gene 5.56% while none of the isolates screened had the nheA gene (Fig 3). One research carried out, showed that the nheB and nheC gene, which are frequently identified in isolates linked with food poisoning in Japan was regularly observed [50].

The results obtained were nearly alike to the study performed by [35], who discovered that B. cereus isolates gotten from food lacked one or two of the HBL and NHE genes 36% and 63% out of the 88 strains of B. cereus, respectively by PCR amplification. One study found that NHE genes were present in higher percentage in almost all the B. cereus strains than HBL genes [51]. Another study reported that NHE complex genetic sequence was observed in about 80 % of all the B. cereus identified from baby food [52]. They also stated that HBLA protein sequence was discovered from B. cereus obtained from milk, dairy goods, and baby food in the proportions of 9.09, 20 and 20%, correspondingly. Difference in the acquired outcomes is received based on several considerations which include time of year, type of sample, gathering requirements, it was carefully similar with quantitative and qualitative outcomes.

All the strains of B. cereus (100%) had a minimum of one genetic material of the hemolysin BL and non-hemolysin complexes. Similarly, [53] informed that the strains of B. cereus they studied were confirmed to have at least one of the hemolysin BL (55.2%) and non-hemolysin (89.7%) genes. These findings disagree with a study who stated that out of the four hundred and eleven strains of B. cereus they examined none displayed the occurrence of just a specific or dual genes in both the NHE or HBL complexes [54].

The discovery of a minimum of a gene3from the NHE and/or HBL complex implies the occurrence of both complex operons [55]. The polymorphism amongst HBL and NHE complexes is the possible reason for the inability to detect all genes present in most B. cereus isolates by PCR method [35]. Therefore, when strains of B. cereus harbor a minimum of one virulence gene, this afterwards might be a point indicator for checking for B. cereus strains that are toxigenic in food.

As a result of complications in refining cereulide, few information is available about this peptide. Only one of the isolates (isolate 9) in Fig 4 had the emetic specific fragment EM 1 (cesB gene). Cereulide is an exceptionally lasting toxin, which can withstand treatments with pepsin and trypsin, pH 2 to11 and temperature of 121ºC for ninety minutes [11]. Production of cereulide is majorly constrained to a one evolutionary descent amongst the group of B. cereus including B. anthracis [16, 45], and is linked to a specific serovars and certain biotype of B. cereus.

All the eighteen Bacillus isolates assayed for the presence of resistant genes (blaVIM, vanA and vanB) in Figs 5 and 6 showed no amplification which means they lack resistant genes. However, in previous studies carried out, the blaVIM gene was detected in all the B. subtilis obtained from unprocessed milk and cheese products [56] while the vanA and vanB genes have been identified to be present in B. cereus and B. circulans isolated from a catheter related infection [45].

Feeding babies with human milk is undoubtedly nourishing, and ought to be promoted. However, breast milk can sometimes lead to newborn septicemia and mortality because of contamination by pathogenic microorganisms. The results obtained from this research show that breast milk can harbor pathogenic microorganisms such as Bacillus species which possess virulence factors and genes as well as increase the rate of spread of antibiotics resistance in infants. The precise dosage of antibiotics a child being breast-fed takes in is determined by specific factors, such as the dosage the mother takes in, how soon the baby feeds after maternal dosing and the quantity milk the baby drinks. The likelihood that the use of antibiotics by mothers while nursing might influence the spread of antibiotic resistance in babies should always be considered.

Ethics and consent

Ethical approval was sought from Health Research Ethics Committee (HREC) of Nigerian Institute for Medical Research, Yaba; Lagos with Project number IRB/18/046. The patients were assured that research outcomes will be made available through the hospital management and no third party will be involved except the physician in charge of the individual concerned.

Funding information

This work received no special grant from any funding agency.

Competing interests

The authors have no competing interests to declare.

Author contributions

Margaret Toluwalayo Arowolo- Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Resources, Writing- Original draft.

Aminat Oluwatoyin Adelowotan- Conceptualization, Project administration, Supervision, Validation, Writing- review and editing.

Morohunranti Sekinat Sanusi- Conceptualization, Data curation, Visualization, Writing- review and editing.