Meat and meat products as potential sources of emerging MDR Bacillus cereus: groEL gene sequencing, toxigenic and antimicrobial resistance

Background Bacillus cereus is implicated in severe foodborne infection in humans. This study intended to assess the occurrence, groEL gene sequencing, biofilm production, and resistance profiles of emerged multidrug resistant (MDR) B. cereus in meat and meat product samples. Moreover, this work highlights the virulence and toxigenic genes (hblABCD complex, nheABC complex, cytK, ces, and pc-plc) and antimicrobial resistance genes (bla1, tetA, bla2, tetB, and ermA). Methods Consequently, 200 samples (sausage, minced meat, luncheon, beef meat, and liver; n = 40 for each) were indiscriminately collected from commercial supermarkets in Port Said Province, Egypt, from March to May 2021. Subsequently, food samples were bacteriologically examined. The obtained isolates were tested for groEL gene sequence analysis, antibiotic susceptibility, biofilm production, and PCR screening of toxigenic and resistance genes. Results The overall prevalence of B. cereus among the inspected food samples was 21%, where the highest predominance was detected in minced meat (42.5%), followed by beef meat (30%). The phylogenetic analysis of the groEL gene exposed that the examined B. cereus strain disclosed a notable genetic identity with other strains from the USA and China. Moreover, the obtained B. cereus strains revealed β-hemolytic activity, and 88.1% of the recovered strains tested positive for biofilm production. PCR evidenced that the obtained B. cereus strains usually inherited the nhe complex genes (nheA and nheC: 100%, and nheB: 83.3%), followed by cytK (76.2%), hbl complex (hblC and hblD: 59.5%, hblB: 16.6%, and hblA: 11.9%), ces (54.7%), and pc-plc (30.9%) virulence genes. Likewise, 42.9% of the examined B. cereus strains were MDR to six antimicrobial classes and encoded bla1, bla2, ermA, and tetA genes. Conclusion In summary, this study highlights the presence of MDR B. cereus in meat and meat products, posing a significant public health risk. The contamination by B. cereus is common in minced meat and beef meat. The molecular assay is a reliable fundamental tool for screening emerging MDR B. cereus strains in meat and meat products.


Background
Communicable diarrheal diseases are incriminated in significant morbidity and mortalities all over the world [1].Bacillus cereus (B.cereus) is one the utmost predominant food-borne pathogens, resulting in severe food poisoning.The disease is mainly associated with severe diarrhea, vomiting, liver failure, abdominal pain, and necrotic enteritis [2].Moreover, B. cereus is reported frequently as the most prevailing pathogen found in several food products, including meat and meat products, milk products, and food of plant origin, which is considered a public health threat [3].
B. cereus is a Gram-positive, motile, and spore-forming rod.B. cereus, a ubiquitous pathogen, is widely distributed in food, soil, water, and plants [4].The pathogenicity of B. cereus is endorsed mainly by various virulence factors and toxins encoded by the corresponding genes.The infection takes place as a result of the ingestion of contaminated food with B. cereus.The cells of B. cereus attached to the host intestinal mucosa then undergo colonization, followed by enterotoxin production [5,6].The most common virulence factors associated with B. cereus infection include the heat-labile enterotoxins group: 1-cytotoxin K (regulated by the cytK gene), 2-nonhemolytic enterotoxin (encoded by the nheABC gene complex), and 3-hemolysin BL (encoded by the hblABCD gene complex), the cereulide toxin (regulated by the ces gene), and phospholipase C (regulated by the pc-plc gene) [7][8][9].Several food poisoning outbreaks triggered by B. cereus were reported globally [10].The disease is associated with two clinical forms: diarrheal and emetic forms.Cytotoxin K, a potent enterotoxin, is considered the main virulence determinant incriminated in severe diarrhea.Besides, the heat-labile enterotoxins are responsible for the diarrheal syndrome.Moreover, the emetic syndrome is ascribed mainly to the cereulide toxin [6,11,12].Furthermore, B. cereus is incriminated in severe infections in humans, including pneumonia [13], bacteremia in neonates [5], gas gangrene, bacterial meningitis, and eye infections [14].
The biofilm production is commonly associated with B. cereus to enhance its survival in adverse environmental circumstances.Biofilms produced by B. cereus are considered the principal source of device contamination during food processing [15].Spore production by B. cereus makes biofilm difficult to eliminate due to the resistance of spores to radiation and heat [16].
Antimicrobial resistance is markedly raised all over the world, suggesting a public health hazard [17].B. cereus inherited several antibiotic-resistance genes that enable the bacteria to resist several antimicrobial classes [6].B. cereus is frequently resistant to antibiotics belonging to the β-lactam class [18,19].Moreover, B. cereus could display acquired resistance to some antibiotics such as streptomycin, tetracycline, and erythromycin [20].A previous molecular investigation emphasized that B. cereus harbored resistance genes against macrolides, tetracycline, and β-lactam antibiotics [6].The indiscriminate use of antimicrobial agents consequences the development of multidrug-resistant pathogens [21,22].
Herein, we intended to determine the prevalence, groEL gene sequencing, biofilm production, and resistance profiles of emerged MDR B. cereus in meat and its products.Moreover, this work highlights the virulence and toxigenic genes (hblABCD complex, nheABC complex, cytK, ces, and pc-plc) and antimicrobial resistance genes (bla1, tetA, bla2, tetB, and ermA) inherited and acquired by the obtained isolates.

Sampling
Two hundred samples (sausage, minced meat, luncheon, beef meat, and liver; n = 40 for each) were indiscriminately collected from commercial supermarkets in Port-Said Province, Egypt, from March to May 2021.Consequently, samples were transported as soon as possible to the laboratory.

Isolation, identification, and enumeration of B. cereus
About 25 g of each collected sample was homogenized in 225 mL of tryptic soy broth (BD Difco, USA) using a bagmixer stomacher followed by serial dilution.Afterward, 0.1 ml of each diluted specimen was spread evenly with a spreader onto Mannitol Egg-Yolk Polymyxin (MYP) (BD Difco, USA) agar plates and left incubated at 37 °C for 24 h [23].Typical B. cereus colonies were pink and bounded by a zone of precipitation, specifying the lecithinase activity.Then, colonies were enumerated with a colony counter.B. cereus was identified as consistent with culture and morphological characteristics, endospores, and biochemical tests (catalase, indole, H 2 S production, oxidase, methyl red, sugar fermentation, and Voges-Proskauer tests), according to Maturin and Peeler [24].The identity of isolates was determined genetically using specific primers of groEL gene, as previously described [25].molecular assay is a reliable fundamental tool for screening emerging MDR B. cereus strains in meat and meat products.

groEL gene sequencing
Herein, the retrieved B. cereus isolates revealed similar phenotypic characteristics; therefore, a PCR product of one indiscriminately chosen isolate was used for groEL sequencing in both directions (QIAGEN Sciences Inc., Germantown, MD, USA).The Bigdye Terminator V3.1 cycle sequencing kit was used in gene sequencing (Thermo Fisher Scientific, Waltham, MA, USA).Besides, the phylogenetic tree was assembled in accordance with the neighbor-joining approach using the MEGA 11 software [26].

Hemolytic activity and biofilm production
The retrieved strains were streaked on 5% sheep blood agar (BD Difco, USA) and incubated at 24 °C for 48 h.The occurrence of β-hemolysis specifies the positive result, according to Wiwat and Thiramanas [27].
Using the microtitre plate method, a 200 µl suspension of B. cereus (10 8 CFU/ mL) in tryptone soy broth (Difco, USA) was added to a 96-well polystyrene microtiter plate.The bacterial cell suspension was incubated at 30 °C for 24 h.Subsequently, plates were decanted, washed, dried for 30 min, and stained with crystal violet for 15 min.After staining, the plates were decanted, washed, and dried for 15 min.For each well, 150 µL of ethanol (95%) was added, and plates were left for 30 min.The absorbance was estimated at 595 nm using a microplate reader (un-inoculated wells included as negative controls).Besides, B. cereus ATCC 10876 was involved as a positive control.The tested isolates were classified in consistent their OD value into strong (4 x ODc < ODs), moderate (2 x ODc < ODs ≤ 4 x ODc), weak (ODc < ODs ≤ 2 x ODc), and non (ODs ≤ ODc) biofilm producers [28].

Detection of toxigenic and antibiotic resistance genes
Detection of virulence-related (hblABCD, nheABC, cytK, ces, and pc-plc) and antibiotic resistance genes (tetA, bla1, ermA, tetB, and bla2) was performed using PCR.The extraction of genomic DNA was performed by the QIAamp DNA Mini Kit (QIAGEN Sciences Inc., Germantown, USA).The bacterial pellet was mixed with 180 µL Buffer AL in a 1.5 ml microcentrifuge tube and incubated at 70 °C for 10 min.Subsequently, 200 µL of was added, mixed by pulse-vortex for 15 s, and centrifuged to remove drops from inside the lid of the collecting tube.The mixture was transferred to the spin column and centrifuged at 6500 x g for 1 min.The spin column was then moved to a 2 mL Eppendorf tube and washed multiple times with 500 µL of Buffer AW1 and AW2 at 6,000-20,000 x g for 1-3 min.The extracted DNA was collected in a new collecting tube by adding 200 µL of elution Buffer AE.Moreover, DNA-free reactions were used as negative controls, while reference B. cereus strains (The AHRI, Dokki, Egypt) were used as positive controls.Table 1 illustrates the used primer sequences.

Statistical analyses
G*Power (3.1.9.7) was used to evaluate sample size using Effect size f = 0.25 and power (1-β err prob = 0.80).Categorical variables were presented as frequencies and percentages.Associations between categorical variables were determined using the chi-square test (SAS software, version 9.4, SAS Institute, Cary, NC, USA) (p-value < 0.05 points to a significant variance).The "cor" function in the R-software (version 4.0.2;https://www.r-project.org/) and the "corrplot" package were used to create a correlation matrix for resistance profiles and resistance genes, as well as biofilm production and antimicrobial resistance.The heatmap of antimicrobial resistance patterns was performed using the GraphPad Software (version 8.0.1).
The obtained isolates exhibited the distinctive morphological features of B. cereus, where all the isolates were Gram-positive, motile, short bacilli with non-bulging endospores.On MYP media, colonies were pink with a characteristic precipitation zone (lecithinase positive).Moreover, the obtained isolates tested positive for Voges-Proskauer, catalase, citrate utilization, glucose fermentation, and nitrate reduction.Likewise, the isolates tested negative for oxidase, H 2 S production, methyl red, indole, and mannitol fermentation.Besides, all the obtained isolates inherited the groEL gene.

Phylogenetic analysis of the groEL gene
The phylogenetic analysis confirmed that the examined B. cereus strain (Accession number: MZ424866) revealed a notable genetic similarity with other isolates from various geographical areas.For instance, B. cereus strain Q1 (100%) of China (Accession number: CP000227), strain AH187 (99.79%) of the USA (Accession number: CP001177), and B. cereus strain JEM-2 (99.36%) isolated from the USA (Accession number: CP018935), as illustrated in Fig. 1.
Table 1 Illustrates the oligonucleotide sequences used in PCR assay and their specific amplicons Primers Sequences Amplicons (bp)

Confirmatory gene Chaperonin protein groEL F: T G C A A C T G T A T T A G C A C A A G C T R: T A C C A C G A A G T T T G T T C A C T A C
T 533 [25] hbl complex hblA Resistance genes bla1

T C T A A A A A G C A T G T A A A A G A A R: C T T C G A T A G T T T A T T A A T A T T A G T 645 [38]
Table 2 The prevalence and total count of B. cereus in the inspected food products

The correlation between biofilm production and antimicrobial resistance
In the present study, the strong biofilm producers were resistant to eight antimicrobial agents.Besides, the moderate biofilm producers were resistant to 5-8 antimicrobial agents.Moreover, the weak biofilm producers were resistant to 3-4 antimicrobial agents, while the non-biofilm producers were resistant to 3 antimicrobial agents, as illustrated in Table 6.Statistically, a positive correlation was noticed between biofilm production and antimicrobial resistance (Fig. 7).Fig. 4 The occurrence of toxigenic and antimicrobial resistance genes in the retrieved B. cereus from food samples

Discussion
Bacillus cereus is one the utmost predominant foodborne pathogens, resulting in severe food poisoning in humans.Herein, we intended to investigate the occurrence, groEL gene sequencing, biofilm production, and resistance traits of emerged MDR B. cereus in meat and its products.Moreover, this work highlights the toxigenic and antimicrobial resistance genes.
In this study, all the obtained isolates revealed the characteristic phenotypic traits of B. cereus and disclosed coordination in their biochemical activities, as previously reported by Gdoura-Ben et al. in Tunisia [39].Herein, the total prevalence of B. cereus in the inspected samples was 21%, where the highest predominance was detected in minced meat and beef meat.These findings signify that contaminated ready-to-eat food products could be a probable risk to human consumers.The higher prevalence of minced meat and beef meat, in comparison with luncheon, could be attributed to the heat treatment of luncheon during the processing stages, which reduces the potential B. cereus contamination [40].A higher frequency of B. cereus (35%) was recorded in ready-toeat foods in China; the prevalence was 34% in the cocked meat samples [41].B. cereus is a ubiquitous pathogen, so open-air shops raise the potential for environmental pollution with B. cereus spores.Moreover, poor hygienic procedures during the processing and storage of various food products favor the contamination with B. cereus [42,43].
Alarmingly, in this work, the mean B. cereus counts in the examined food samples exceeded the permissible limits reported by the Health Protection Agency [44] (should be less than 10 3 CFU/g or mL).Moreover, Stenfors et al. [3] confirmed that lower doses of B. cereus in foods could result in severe food poisoning outbreaks in human consumers.Besides, the food is not suitable for human consumers if the B. cereus count is > 10 4 CFU/g or mL [45].The remarkably high counts of B. cereus in food products could be attributed to inadequate hygienic measures during food processing, contamination of equipment, inappropriate handling, bad storage conditions, Fig. 5 The occurrence of MDR among the retrieved B. cereus isolates from food samples and inadequate sterilization of equipment and machines [46].
The findings of this study showed that all the B. cereus isolates carried the groEL gene.Wei et al. [47] reported that groEL is a consistent diagnostic biomarker when compared with the gyrB gene to differentiate B. cereus from other pathogens in food products.The sequence analysis of the groEL gene emphasized that the examined B. cereus strain revealed a notable genetic identity as well as cross-lineage with other strains derived from various geographical areas, such as B. cereus strain Q1 in China [48], B. cereus strain B. cereus strains JEM-2 and AH187 in the USA [49].
Remarkably, all retrieved B. cereus strains exhibited β-hemolysis on sheep blood agar.These findings agreed with those confirmed by Hwang and Park in the Republic of Korea [50].Potent hemolytic activity is usually associated with foodborne B. cereus strains due to inherited factors [51].In the current study, 88.1% of the recovered B. cereus strains tested positive for biofilm production (out of them, 62.2% were strong biofilm producers), suggesting the recovered isolates are highly pathogenic.Our findings agree with Osman et al. [52], who reported biofilm production in 83.3% of isolates (out of them, 33.3%  were strong biofilm producers) obtained from different meat samples in Egypt.Strong biofilm producers are highly pathogenic and frequently resistant to phagocytosis, antimicrobials, and antiseptics [16].The biofilms perform a substantial role in the binding of pathogens to the biotic and non-biotic surfaces that specify the positive correlation between biofilm production and the occurrence of infection.Besides, it reveals the potential public health threat of B. cereus in causing food poisoning to human consumers [53].
In this study, vancomycin and levofloxacin exerted potent antibacterial activity toward the recovered B. cereus strains from various examined food samples.These findings agree with those confirmed by Ikeda et al. in Japan [54], who reported that all the obtained B. cereus strains were highly susceptible to vancomycin, and only 10.3% were resistant to levofloxacin.Moreover, the obtained B. cereus strains were resistant to ampicillin, amoxicillin, tetracycline, cefepime, ceftriaxone, amoxicillin-clavulanic acid, and trimethoprim-sulfamethoxazole.Our findings agree with those highlighted by Savić et al. [55].Also, a previous study in Egypt reported that 40% of the retrieved B. cereus isolates were MDR to eight tested antibiotics [19].Besides, Yu et al. [41] confirmed that the recovered B. cereus isolates from food products in China were MDR to penicillin, cephalothin, ampicillin, cefoxitin, and amoxicillin-clavulanic acid.These outcomes confirmed the occurrence of MDR B. cereus in various food products, suggesting that ready-to-eat foods could be a main source of transmission of foodborne MDR B. cereus to human consumers [56].The haphazard use of antimicrobials in the agriculture and health sectors favors the development of MDR strains [19].
Concerning the dissemination of toxigenic genes, our findings agree with the results confirmed by Tewari et al. in India [43].Moreover, the combination of nheA and nheC genes was noticed in all tested strains, which agrees with the results reported by Fraccalvieri et al. in Italy [57].In this work, all the obtained B. cereus strains inherited two or more enterotoxigenic genes, highlighting their public health significance as a causative agent of severe food poisoning in man, consistent with Owusu-Kwarteng et al. [58].Foodborne B. cereus strains frequently encode one or more hbl complex genes [33].Food poisoning caused by B. cereus is concomitant mainly with these virulence determinants: non-hemolytic enterotoxin (regulated by nheABC complex genes), the hemolysin BL (regulated by hblABCD complex genes), the cytotoxin K (regulated by cytK), and the cereulide toxin (regulated by the ces gene) [9,59].
In this study, the recovered B. cereus strains were MDR to eight tested antimicrobial agents (six different classes) Fig. 7 Illustrates the correlation between biofilm production and antimicrobial resistance and commonly carried bla1, ermA, tetA, and bla2 genes.Our findings are consistent with those confirmed by Fiedler et al. in Germany [6].Moreover, the MARI values were 0.27-0.73(> 0.2), worryingly highlighting that the retrieved strains resulted from high-risk contamination.The bla1 gene is mainly responsible for penicillinase enzymatic activity, whereas the bla2 gene is related to cephalosporins and penicillinase enzymatic activity [60].Furthermore, the combination of bla1 and bla2 resistance genes is mainly responsible for the resistance of the β-Lactam-β-lactamase inhibitor combination [61].Likewise, the resistance of B. cereus strains to tetracycline is mainly accredited to the tetA and or tetB gene, and their resistance to erythromycin is endorsed by the ermA gene.The existence of tetA and tetB genes in B. cereus emphasized the horizontal transfer of antimicrobial resistance genes from resistant pathogens to B. cereus [37].
Herein, a positive correlation was detected between the biofilm production and the antimicrobial resistance.Strong biofilm-producing B. cereus strains are highly virulent and usually resistant to disinfectants and antibiotics [16].Biofilm-producing B. cereus is a highly adapted pathogen that could gain resistance to several antibiotics due to incessant disclosure to the antimicrobials, harboring or acquiring the resistance genes.Moreover, biofilm plays a vital role in drug resistance by delivering a proper environment for resistance determinant transfer [62].
In summary, this study highlights the presence of MDR B. cereus in meat and meat products, posing a significant public health risk.The contamination by B. cereus is common in minced meat and beef meat.MDR B. cereus isolates from food products often exhibit biofilm production and commonly harbor the nhe complex, cytK, hbl complex, ces, and pc-plc virulence genes, and bla1, tetA or tetB, bla2, and ermA antibiotic resistance genes.Vancomycin and levofloxacin demonstrate promising antibacterial activity toward the retrieved isolates.The molecular assay is a reliable fundamental tool for screening emerging MDR B. cereus strains in meat and meat products.

Fig. 2 Fig. 1
Fig.2The rate of biofilm production between the retrieved B. cereus strains

Table 4
Distribution of toxigenic and resistance genes in the obtained B. cereus

Table 5
Resistance patterns and resistance genes of the obtained B. cereus from food samples (n = 42)

Table 6
The correlation between biofilm production and the antimicrobial resistance patterns