Identification of blaVIM , blaIMP and blaSPM-1 Metallo-β-Lactamase Genes in Bacillus subtilis Strains Isolated From Raw Milk and Cheese Samples

Introduction: Studies on bacterial resistance to common antibiotics are often based on bacteria isolated from patients, with less attention towards foodborne bacteria. This study aimed to investigate the antibiotic resistance pattern and the presence of metallo-β-lactamase (MBL) genes viz blaVIM, blaIMP, and blaSPM-1, in the Bacillus subtilis strains isolated from raw milk

method and renders lower validity. 5,6BL is resistant to β-lactamase inhibitors such as sulbactam, tazobactam, and clavulanic acid. 3his resistance poses a major clinical problem since MBL can potentially influence various antibiotics including penicillin, broad-spectrum cephalosporins, and carbapenems (except for monobactam such as aztreonam).Carbapenems such as imipenem and meropenem are the most important antibiotics which can be used as antibacterial agents for treating infections caused by pathogens. 6Unfortunately, resistance towards these antibiotics is increasing.For example, Centers for Disease Control and Prevention (CDC) in the United States has announced that the resistance rate of 9% among isolated bacterial samples in 1995 has increased considerably to 40% in 2004. 7IMP-1 in Serratia marcescens was identified as the first MBL enzyme in 1991. 8Over the past decade, MBLs have been categorized based on their molecular structures into 6 groups of GIM (German imipenemase), IMP (Imipenemase), SPM (São Paulo metallo-β-lactamase), SIM (Seoul imipenemase), VIM (Verona integron-encoded metallo-β-lactamase), and AIM (Adelaide imipenemase). 9Recently, a new type, KHM (Kyorin Health Science MBL), has been reported in Citrobacter freundii from Japan. 10 The genes encoding MBLs are present on both chromosomes as well as plasmids.The MBL-encoding genes including blaVIM, blaIMP, and blaSPM-1 are found as a genetic cassette in class I integrins and have the potential for being transferred to other bacteria. 4acillus subtilis is a gram-positive and cylindricalshaped bacterium which is mostly found in soil and usually does not cause disease in humans.Characteristically, it forms large, flat, cloudy pale, yellow or brown colonies. 11,12n comparison with gram-negative bacteria, such as Escherichia coli, B. subtilis is considered GRAS (generally recognized as safe) according to a classification of the Food and Drug Administration (FDA), since it is generally found to be harmless towards mammals.For this reason, even today this bacterium is considered as a good host for the production of secretory heterologous proteins. 13With the active and efficient secretion system, this bacterium is capable of secreting various proteins into the peripheral environment, and is a suitable bacterium for cell growth and differentiation studies.These properties render the facilitation of production of recombinant proteins. 14o provide a generalized overview of MBL producers, Pseudomonas aeruginosa could be proposed as another interesting example along with B. subtilis.In a study by Doosti et al 15 in Zanjan in 2011, out of 70 isolates of P. aeruginosa obtained from intensive care units (ICUs), 78% were MBL producers. 15Based on molecular epidemiology studies on P. aeruginosa strains as MBL producers around the world, they were mostly prevalent in Netherlands, Greece, Italy, Japan, and Canada, and the most prevalent type was VIM. 4 In another study by Yousefi et al in 2010, 39 out of 104 isolates of P. aeruginosa were reported as MBL positive strains, though in none of them, blaSPM-1 gene was detected in polymerase chain reaction (PCR) analysis. 16icrobial resistance has been extensively studied in bacteria isolated from disease cases.This is while few studies have been conducted on antibiotic resistance genes in foodborne bacteria.Therefore, our study was designed to survey the antibiotic resistance profile and genotype of B. subtilis strains isolated from samples of raw milk and cheese, along with detection of MBL encoding genes, blaVIM, blaIMP, and blaSPM-1 in these strains.Further effort should be directed to understand the probability of transferring resistance from these foodborne strains to other strains.The current study also provided an overview of the current state-of-art of the topic.

Sample Collection and Processing
At first a total of 100 samples of raw milk and cheese (50 samples of each) were collected in aseptic conditions from the Dairy Products Preparation and Distribution centers in Ardabil city, northwest province of Iran.First of all, the samples were treated with thermal enrichment for 10 minutes at 80°C to maintain spores and remove vegetative cells.Then, using serial dilution method, successive dilutions of the samples were prepared in sterile distilled water.Afterwards, 1 mL of each suspension was dispensed in sterile plates.Nutrient agar culture medium was then prepared and added to the petri dish under sterile conditions and incubated at 37°C for 24 hours.White and creamy colonies with rough and marginal surfaces were selectively purified through four-way streaking technique on nutrient agar-coated plate.

Isolation and Identification of Bacillus subtilis
To isolate B. subtilis strains, gram and malachite green stains were used in addition to biochemical tests (i.e.hydrolysis of lecithin, catalase test, indole test, tests with methyl red, citrate, and cell wall hydrolysates were used for glucose, arabinose, mannitol and xylose test. 17o investigate the proteolytic activity, bacteria were cultured on 50% skim milk agar medium and incubated at 37°C for 24-48 hours.The presence of a bright area around the colony in the culture medium indicated bacterial proteolysis.The lipolytic activity of the strains was investigated by growing the bacteria in tributyrin agar culture medium at 37°C for 3 days.Finally, the growth inhibition zone around the colony was investigated. 18
For each strain, the turbidity equivalent to 0.5 mg MacFarland tube (1.5×10 8 bacteria) of the bacterial suspension was prepared and streaked using a sterile swab on a solid culture medium (Mueller-Hinton agar).Using sterile forceps, the antibiotic discs were placed on the culture medium.The plates were incubated at 35°C for 18 to 24 hours.Inhibition zones were measured in millimeters and the bacterial susceptibility to antibiotic was reported.The measurements of the diameter of inhibition zone for antibiotics and their comparison were made according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, and the strains were considered as either susceptible (S) or resistant (R).
Detection of MBLs Using Double-Sided E-Test Strip MBL enzymes were characterized by the phenotypic method using double-sided E-test strip (Imipenem and Imipenem + EDTA) purchased from Bio Merieux (SA, France). 20One side of the strip contained varying concentrations of imipenem (0.125 to 8 μg/mL) and the other side contained varying concentrations of imipenem + EDTA (0.032 to 2 μg/mL).In the next step, a turbidity equivalent to 0.5 McFarland tube of the bacterial suspension was inoculated in Muller-Hinton agar medium, on which the E-test strip was placed.The plates were then incubated at 35°C for 24 hours.To determine the presence of MBL enzymes, the intersection point of the halo formed on both sides of the strip (imipenem and imipenem + EDTA) was considered as an inhibitory concentration (IC) and was quantitatively reported as microgram per milliliter.According to the manufacturer's instructions, if imipenem + EDTA inhibitory dilution was reduced by more than three dilutions in comparison to imipenem, or the proportion of imipenem inhibitory dilution was greater than or equal to 8, the strain was considered to be an MBL producer. 21tudies have shown that the accuracy of the E-test in evaluating MBL activity is higher than that of other conventional methods. 5In the E-test method, one side of a plastic strip is used to increase the concentration of an antimicrobial agent while the other side has a continuous minimum inhibitory concentration (MIC) grading.Thus, an effective antibiotic gradient is warranted against the bacteria.If the isolate is susceptible towards the antibiotic, growth inhibition zone is seen as an oval area on the agar plate.The peak of the strip that breaks the oval growth inhibition zone denotes the MIC.In the strips, imipenem is placed on one half of the gradient strip, while imipenem + EDTA is placed on the other half of the gradient (Figure 1).In cases where the imipenem MIC is greater than 8, or is equal to the imipenem + EDTA, the production of MBL enzyme is detected. 13,22lecular Confirmation of MBLs The bacterial DNA was extracted using boiling method for PCR.Firstly, 3 to 5 fresh bacterial colonies were removed from the culture medium and dissolved in 200 µL sterile distilled water and boiled for 10 minutes at 100°C.This was followed by centrifugation at 12 000 rpm for about 10 minutes.The supernatant containing DNA was used for PCR analysis. 23The PCR reaction was performed in thermocycler as follows: initial denaturation at 94°C for 5 minutes; 30 cycles of amplification including denaturation at 94°C for 1 minute, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds; and final extension at 72°C for 5 minutes. 24The PCR test was performed to identify blaVIM (382 bp), blaIMP (587 bp), and blaSPM-1 (650 bp) genes using VIM, IMP and SPM-1 primers, respectively (Table 1).

Results
Our results showed that from a total of 100 isolates obtained from raw milk samples (non-pasteurized: 50 isolates) and cheese samples (50 isolates), 42 (84%) isolates out of raw milk and 37 (74%) isolates out of cheese samples contained B. subtilis.Upon biochemical analysis of MBL enzymes using phenotypic double-sided E-test strip, 25 (59.52%)isolates of raw milk and 16 (43.24%)isolates of cheese were able to produce MBL enzyme.
Totally, all MBL-producing isolates (100%) exhibited  antibiotic resistance towards cotrimoxazole and cloxacillin, while the number of isolates showing resistance to the cefixime and cefotaxime were 67.40% and 64.60%, respectively (Figure 2).
The resistance rate of MBL negative isolates to the antibiotics has been graphically represented in Figure 3.The highest antibiotic resistance was observed to cotrimoxazole (68.29%), while the lowest value was recorded for cefixime (46.34%).
The results of PCR reaction for blaVIM, blaIMP, and blaSPM-1 genes from B. subtilis strains isolated from raw milk and cheese samples are shown in Figure 4, Panel A. While all the strains revealed the presence of blaVIM gene, expressing MBL enzyme, none showed the presence of blaIMP and blaSPM-1 genes (Figure 4, Panel B).

Discussion
Milk and dairy products play a significant role in human nutrition because of their high nutritional value.However, due to the presence of most of the elements and nutrients, it is a very good environment for the growth and activity of many pathogenic microorganisms.Thus, non-compliance with the principles of hygiene in the preparation and maintenance of dairy products poses many health problems and dangers to consumers of such foodstuffs.The consumption of raw milk, unpasteurized or uncooked

A B
contaminated dairy products or even pasteurized dairy products is one of the most common routes of transmitting diseases, along with several antibiotic-resistant bacteria.Production of beta-lactamases is the most common cause of antibiotic resistance in bacteria. 25Food contamination with antibiotic-resistant strains increases the risk of passing antibiotic resistance to the intestinal bacterial flora; hence, it should be considered as a significant risk factor. 25,26Researches have shown that a high percentage of raw milk samples are contaminated with bacillus, and warrants application of effective methods to eliminate such pathogens.
Based on the results of this study, B. subtilis isolates, producing the MBL enzyme, exhibited the highest (100%) resistance towards cotrimoxazole and cloxacillin, while the resistance to cefotaxime (64.60%) and cefixime (67.4%) antibiotics was the lowest.MBL-driven resistance to carbapenems, as the most effective antibiotics against bacterial infections, is an issue of utmost concern. 27arbapenems such as imipenem, meropenem, biapenem, and ertapenem are an important class of β-lactam drugs to which beta-lactamases are resistant. 28In a study by Magalhães et al, 29 48 samples of P. aeruginosa were collected, of which 24 (50%) were imipenem-resistant, and 15 (31.25%) samples were found to be producers of MBL enzymes. 29In a study by Franco et al 24 on blood bacterial isolates, the prevalence of imipenem-resistant P. aeruginosa strains was reported to be 34%, of which 77% were MBL producers. 23A similar and consistent observation was found in our study where 75% of milk samples and 75.42% of cheese samples were MBL producers.
In their study in 2008, Khosravi and Mihani showed that in all the strains they studied, the expression of blaVIM gene was observed. 30In the present study, PCR was used to confirm the presence of blaVIM, blaIMP, and blaSPM-1 MBL genes in B. subtilis strains isolated from raw milk and cheese samples.The results confirmed the presence of blaVIM gene in the same strains that produced the enzyme in the phenotypic test.While all the MBL-producing strains harbored blaVIM gene, no strains were found to carry either blaIMP or blaSPM-1 genes.
In a study by Varaiya et al, 31 they reported the expression of blaIMP in neither of 20% imipenem-resistant strains.They also showed that 12.4% of the strains expressed blaVIM gene.The findings are consistent with our results in only showing the presence of blaVIM gene and the absence of blaIMP and blaSPM-1 genes in MBL producer B. subtilis.A study carried out by Franco et al on MBL producer blood isolates of P. aeruginosa, 34% of the strains were imipenem-resistant while 77% of the samples were MBL producers from which 81% contained the blaSPM-1 gene. 24Likewise, in a study performed by Sadeghi et al 32 on 108 isolates of P. aeruginosa, the absence of blaSPM-1 gene was observed in all the strains. 32Torkar and Bedenic in their study on Bacillus cereus isolates (closely related strain to B. subtilis) obtained from clinical specimens and foods (raw and pasteurized milk) confirmed that 27.3% of strains expressed BlaVIM gene. 33However, 37% of strains that were positive in double disc diffusion method showed negative results in PCR analysis of blaSPM gene.They also detected MBL sequences encoding blaII (BCII) in all tested strains. 33In another study on 28 clinical isolates of P. aeruginosa, 15 isolates were found positive for blaVIM gene. 34Overall, the blaVIM gene is the most frequent gene in MBL producer bacterial strains and therefore the importance of this gene should not be neglected in nosocomial infections and even outbreaks.Moreover, Jovcic et al showed that 3% of imipenem-resistant isolates of P. aeruginosa expressed MBL genes. 35In their study, Faghri et al found no expression of blaVIM-1 and blaSPM-1 in P. aeruginosa strains, highlighting the importance of other potential MBL-related genes than blaVIM-1 and blaSPM-1. 36In 2015, Mehdi et al isolated MBL-producing VIM-1, SPM-1, and IMP-1 genes from clinical Pseudomonas species.A total of 5.7% of the isolates showed resistance to imipenem.Furthermore, 9 (4.7%) of the isolates were MBL producers, of which VIM-1 was found by PCR test in 7 isolates. 37A study in 2017, carried out by Chika et al, was done for the molecular identification of MBL genes blaIMP-1 and blaSPM-1. 38n E. coli isolates from cows, 31% were MBL producers with 8% expressing blaIMP-1 gene. 38This is while all of the isolates were negative for blaSPM-1 gene in the recent report. 38

Conclusion
In this study, MBL producer B. subtilis strains were assessed in raw milk and cheese samples as key dairy products.It was shown that 100% of the MBL producer strains were resistant to the antibiotics cotrimoxazole and cloxacillin.The high occurrence of the β-lactamaseproducing microorganisms is a matter of concern for the medical and food industries.They are still known for the rapid infection and spread of nosocomial diseases.
Furthermore, their resistant nature makes them very difficult to eradicate.Thus, proper infection control measures and hygiene programs need to be implemented.According to our results, although it may not render a very sensitive approach, PCR can be used as a complementary method for detecting MBL genes in bacterial strains.

Figure 1 .
Figure 1.Representation of E-test Method With Imipenem on One Half of the Gradient Strip, and Imipenem + EDTA on the Other Half.

Figure 2 .
Figure 2. The Susceptibility of MBL Positive Isolates of Bacillus subtilis Towards Antibiotics by Disc Diffusion Method (The result has been expressed as % of antibiotic resistance).

Figure 3 .
Figure 3.The Susceptibility of MBL Negative Isolates of Bacillus subtilis Towards Antibiotics by Disc Diffusion Method (The result has been expressed as % of antibiotic resistance).