Meat is widely acknowledged as one of the most perishable food items (Mohebi & Marquez, 2015). These things happen because of the chemicals that are in it. These chemicals encourage the growth of germs to levels that are not acceptable. This makes the meat go bad much faster. When raw meat contains a high concentration of bacteria, it goes through changes that make it unattractive and unfit for human eating (Doulgeraki et al., 2012).  The consumption of meats is a common nutritional practice that provides a vital supply of essential micronutrients and macronutrients necessary for human growth and development. Meat is a very perishable product due to its high water content (0.99 water activity) and abundance of proteins, minerals, and other elements conducive to microbial growth, making raw meat one of the vehicles of food-borne infections in humans (Tsehayneh et al., 2021). Meat may be contaminated by bacteria either endogenously or by subsequent contamination from blood, gastrointestinal contents, feet, hide or skin, water, knives, instruments used in slaughterhouse vehicles, or working personnel. Most bacteria are found to cause serious food-borne diseases and are also involved in spoilage of foods, eventually imposing a significant threat to human health and the countrys economy (Diyantoro & Wardhana, 2019; Rahman et al., 2019).

Antibiotics are frequently employed in the treatment of infectious diseases in both humans and animals. One of the primary outcomes of antibiotic residues in foods of animal origin is the proliferation of antibiotic-resistant bacteria (Khatibi et al., 2021). Hence, the presence of antibiotic-resistant infectious agents is currently compromising the safety of meat on a global scale (Baah et al., 2022). Foods contaminated with antibiotic-resistant bacteria could be a significant threat to public health via the transmission of antibiotic-resistance determinants to other bacteria in humans (Javadi & Khatibi, 2017) (González-Gutiérrez et al., 2020). Drug-resistant bacteria can and do travel on meat (Tshipamba et al., 2018). Unfortunately, there is an increasing concern about global public health due to the widespread presence of antibiotic-resistant bacteria in food animals and the uncontrolled use of antibiotics. It can also lead to more prolonged illness and hospitalizations with more extended stays. The presence of such bacteria in food items and their related environment could play a role in spreading antimicrobial resistance amongst food-borne pathogens (Mancuso et al., 2021).  It is a common practice to administer antibiotics to food animals for therapeutic and non-therapeutic objectives. Typically, microorganisms contaminate meat and meat products during the slaughtering and processing of food (Messele et al., 2017). The primary concern worldwide is the rise of antibiotic-resistant bacteria, especially bacteria that live in animals, which could affect people.

In this study, we assessed the bacteriological profile and analyzed the drug resistance pattern of the bacteria. The purpose was to develop effective strategies for treating, controlling, and preventing food-borne diseases. We collected a sample of beef meat from a local butchers shop and determined the presence of bacteria using standard microbiology protocols. Once the bacteria were separated, we used gram staining, looked at their drug resistance profile, and performed biochemical tests to confirm their identity. We found both Staphylococcus aureus and Escherichia coli, mostly S. aureus.

Area of Study

A cross-sectional study was conducted in the Gopalganj district, situated in the southern region of the Dhaka division in Bangladesh. This region spans an area of approximately 1490 square kilometers and is located at latitude of 23.470° N and a longitude of 89.490° E. The population of this region exceeds 1.2 million (Rahman et al., 2018).

Determination of Sample Size and Procedures

Two raw meat samples were obtained from the butchers shop in the local market in Gopalganj. Both of the samples were beef specimens. A simple random sampling technique was utilized to collect meat samples from randomly selected butcher shops in an aseptic manner. 1 gram of each beef sample was immersed in a 0.9% phosphate-buffered saline (PBS) solution inside falcon tubes. The tubes were then packed in sterile plastic zipper bags and stored in a cold box containing ice below 4 degrees Celsius. The samples were transported within 2 hours to the Microbiology Laboratory of the Department of BGE at Bangabandhu Sheikh Mujibur Rahman Science and Technology University for further bacteriological analysis. 

Bacteriological Analysis

The samples obtained were prepared for analysis using standard methods. A homogenate uniform suspension was prepared by combining 1 gram of meat samples with 9 ml of PBS solution. Subsequently, the tenfold serial dilutions (10-1 to 10-6) were performed following the method described by Michael et al. (DoÄŸruer et al., 2015). An aliquot of 0.1 ml from each serial dilution was inoculated onto nutrient agar, Mannitol salt agar (MSA), MacConkey agar, and Eosin methylene blue (EMB) agar plates using the spread plate method. Following the injection of each dilution onto nutrient agar, the plates were incubated at 37 °C for 24 hours. Subsequently, the cultures were transferred to MSA, MacConkey, and EMB agar media. The enumeration of colony-forming units (CFU) was conducted on nutrient agar. Dilutions that yielded 30–300 colonies were deemed countable and included in the calculations. To identify the bacteria, we obtained 25 well-isolated colonies from each sample by subculturing and checking for purity onto MSA agar, MacConkey agar (Oxoid, UK), and EMB agar. The identification of pure culture colonies was conducted through the analysis of colonial morphology, Gram staining, and the assessment of biochemical reactions such as the Methyl Red (MR) test, Motility test, Indole test, and Urease test (Messele et al., 2017).

Isolation of Staphylococcus aureus

The colonies obtained from the nutrient agar media were streaked onto mannitol salt agar plates and incubated at 37°C for 48 hours to assess the abundance of S. aureus.

Isolation and Identification of E. coli

To isolate E. coli, the isolates on nutritional agar were streaked onto sterile Petri dishes containing MacConkey agar medium. The dishes were then incubated at a temperature of 37˚C for 24 hours. Next, the sample was put onto Eosin Methylene Blue (EMB) agar, and the presence of E. coli was confirmed by its distinct translucent green metallic sheen color. A representative colony from a culture grown on EMB agar was transferred to a tube containing Tryptone water and incubated at 44°C for 24 hours. Indole can be detected by adding approximately 0.1 ml of Kovacs reagent to tryptone water and gently mixing the solution. If indole is present, a red hue will appear in the Kovacs reagent, forming a film on the surface of the aqueous phase of the medium. The confirmatory assays indicate the indole is present, and the metallic sheen observed on EMB agar confirms the presence of E. coli (Kwiri et al., 2014).

Isolation of Klebsiella pneumonia

To separate K. pneumoniae, the isolates on nutritional agar were streaked onto sterile Petri dishes with Mac Conkey agar medium. The plates were placed in an incubator and maintained at 37 °C for 24 hours. Subsequently, the specimen was applied to eosin methylene blue agar (EMB), and its existence was verified by its distinctive pink mucoid hue (Zhang et al., 2018).

Biochemical Tests

In addition to Grams staining, the isolates were subjected to biochemical characterization using conventional techniques as outlined by Mooizman et al. (Mooijman et al., n.d.). Urease tests were conducted. The indole-methyl red test was employed to identify E. coli (Preethirani et al., 2015).

Antibiotic Susceptibility Tests

The Kirby-Bauer disk diffusion method (CLSI & Antimicrobial Susceptibility Testing (AST), n.d.) was used to conduct the antimicrobial susceptibility test, following the Clinical Laboratory Standards Institute (CLSI) criteria. We measured the diameter of the inhibitory zone and classified it as resistant, intermediate, or susceptible using the interpretive categories and zone diameter breakpoints provided by CLSI. The samples were added to the nutrient broth and incubated at 37 C for 24 hours. A Muller Hinton media was prepared for the antibiotic susceptibility test. A total of 8 different antibiotics were used, including Ampicillin (10 mcg), Amoxicillin (30 mcg), Ciprofloxacin (5 mcg), Azithromycin (15 mcg), Cefotaxime (30 mcg), Erythromycin (15 mcg), Kanamycin (5 mcg), and Streptomycin (25 mcg). The antibiotic discs were positioned on the agar at 3 cm intervals and incubated at 37 °C for 24 hours. The measurement of the zone of inhibition around each disc allowed for categorizing the isolates as resistant, intermediate, or sensitive, following the guidelines set by CLSI. 

Calculating CFU/ml of bacteria

The colony-forming unit (CFU) quantifies the number of viable cells capable of forming colonies per millilitre (CFU/mL). These measurements quantify the number of cells that retain the ability to undergo cell division and generate small clusters (Messele et al., 2017). The CFU/ml can be calculated using the formula: CFU/ml = (No. of colonies x Dilution factor) / Volume of the culture plate.

Bacteriological profile of the raw beef meats

Cultural, staining, and biochemical characteristics

The sample preparation process was carefully designed to ensure the utmost accuracy and reliability of the studys results. The primary microbial load was substantially decreased through tenfold dilutions (10-1-10-6) and then spread over nutrient agar media. Serial dilution significantly reduced the concentrations of microbial load in the sample, making it much easier to isolate and identify any potential bacterial pathogens (Ben-David & Davidson, 2014). Fig. 1 depicts the undiluted and tenfold dilution (10–3) of raw beef meat samples inoculated in nutrient agar media.

Isolation of S. aureus, E. coli, and K. pneumoniae

Staphylococcus aureus is the predominant and economically impactful bacterium responsible for causing infections in meat-producing ruminant animals. It is a pathogenic bacterium that causes multiple human diseases. The capacity to induce various illnesses may be linked to its secretion of a broad spectrum of extracellular toxic substances and other variables contributing to its virulence, such as toxic shock syndrome, exotoxins, and enterotoxins (I-OE et al., 2010). The colonies in the yellow and pink zones on the mannitol salt agar were separated and transferred to nutrient agar as presumptive Staphylococcus aureus (Fig. 2A) (Dixit & Luqman, 2015). 

E. coli is a well-established pathogen that causes diarrhea and other foodborne illnesses when contaminated food is consumed (COLLINS, 1995). Typically, E. coli is among the bacteria that constitute the beneficial microorganisms found in the intestines of humans and warm-blooded animals. Insufficient hygiene practices such as touching meat without gloves, not using an apron, failing to cover ones hair, and handling money while working can all contribute to unclean conditions during sample collection. This technology could create a conductive environment for the proliferation of E. coli and other disease-causing microorganisms (science & 2010, 2010) (Mensah et al., n.d.). The current investigation established the appearance of E. coli by observing its distinct, clear green metallic sheen color (Fig. 2B) upon transfer to Eosin methylene blue agar (EMB). The isolation of K. pneumoniae was conducted using the usual technique. Following incubation at 37°C overnight, the development of K. pneumoniae was distinguished based on their colony features, pigment generation (ranging from pink to colorless flat or mucoid colonies), motility (Fig. 2C), and Gram-staining techniques (Gram-negative rods, non-sporing, and non-capsulated) (Diriba et al., 2020).

Gram staining

The Gram stain is a technique that enables the differentiation of bacteria into Gram-positive and Gram-negative based on their staining properties with the crystal violet-iodine complex and safranin counterstain. Gram-positive organisms retain the intact complex in their cell walls after alcohol treatment, giving them a purple color. Besides, gram-negative organisms lose their color and appear pink after the same treatment (Beveridge, 2001). Of 25 samples, ten were gram-negative, and the remaining showed gram-positive bacteria. Fig. 3 illustrates the results of Grams staining where the gram-positive bacteria retain the violet color while others keep the counterstain safranin color.

Fig. 1: Serial dilution of Beef meat samples for isolation of Bacteria. The undiluted beef meat samples were spread onto nutrient agar media for total bacterial growth. (A) Representing bacterial growth of the first sample (Sample T-1) and (B) Representing bacterial growth of the second sample (Sample T-2). The Fig. C shows tenfold dilutions (10-3) of first sample T-1 and (D) second sample T-2 that were inoculated in nutrient agar media. 

Fig. 2: Isolation of different bacteria on selective culture media. (A) Bacterial isolates on MSA media with characteristic yellow and pink colonies of S. aureus; (B) Bacterial isolates on EMB media with characteristic metallic green sheen colonies of E. coli; and (C) Bacterial isolates on EMB media with characteristic colorless to pink mucoid colonies.

Fig. 3: Gram staining of isolated bacteria and photograph were taken at 100X magnification using immersion oil. Fig. A-B shows gram-positive bacteria and Fig. C- D shows the gram-negative bacteria.

Fig. 4: Results of Biochemical tests of bacterial isolates obtained from beef meat samples. In Fig. A, 1, 2, and 3 showed motility whereas 4 showed the non-motility with indicated control of 5. In the Methyl Red (MR) test Fig. B, 1, 2, and 3 significantly showed positive results by changing the color sample from no color to red-purple with one negative and one control sample here.

Identification of bacteria using various biochemical tests

Three biochemical tests, Indole, Urease, and Methyl Red, were used to identify bacterial species.  The results of these tests are summarized in Table 1 and Fig. 4. The MR test determines an organisms capability to carry out mixed acid fermentation of glucose. The experiment requires the addition of methyl red indicator to a culture that has been incubated with glucose. The appearance of a red color (Chauhan & Jindal, 2020) indicates the presence of acidic end-products. The indole test is utilized to identify whether an organism can produce tryptophanase, an enzyme that breaks down tryptophan into indole, pyruvic acid, and ammonia. The experiment requires the addition of Kovacs reagent to a culture that has been incubated with tryptophan. The presence of indole is indicated by the appearance of a red color in the organic layer (Tille, n.d.).

Table 1: The biochemical tests of isolated bacterial species (S. aureus, E. coli, and K. pneumonia).

Antibiotic Susceptibility Test

The antibiotic susceptibility patterns of bacterial strains found in beef samples were determined using an antibiotic susceptibility test. The study aimed to evaluate the effectiveness of several antibiotics against bacterial strains in livestock production. The antibiotics that were examined include Streptomycin (S), Erythromycin (E), Kanamycin (K), Ciprofloxacin (CIP), Cefotaxime (CTX), Ampicillin (AMP), Amoxicillin (AMX), and Azithromycin. The susceptibility of the samples to each antibiotic was measured, along with the diameter of the zone of inhibition in centimeters (cm). The study involved an antibiotic susceptibility test on beef meat samples (T2-4, T2-5, T2-9, and T2-12) cultivated on Nutrient agar media. The bacterial isolates obtained from T2-4 exhibited resistance to ciprofloxacin, but both T2-5 and T2-12 displayed resistance to ampicillin and amoxicillin. Conversely, T2-9 exhibited sensitivity to all of the antibiotics. 

Table 2: Antibiotic susceptibility test for the bacterial isolates from beef meat sample against 8 different antibiotics using disc diffusion method.

The study emphasizes the significance of ongoing surveillance of antibiotic resistance patterns in bacterial strains discovered in livestock. Providing information is essential for choosing the right antibiotics to treat bacterial illnesses effectively. The results of the antibiotic susceptibility tests performed on bacterial isolates from beef meat samples are displayed in Fig. 5 and Table 2. It provides crucial information about the prevalence of antibiotic-resistant microorganisms in food animals. 

Fig. 5: Antibiotic susceptibility test for the bacterial isolates from beef meat sample against 8 different antibiotics. In this test, there has been shown a maximum zone of inhibition (3.2cm) from the ciprofloxacin with a minimum inhibiting zone of (1cm) erythromycin. There were five antibiotics which had shown insensitivity with these samples.

Fig. 6: Bar chart demonstrating the antibiotic susceptibility test for the bacterial isolates from beef meat samples against the 8 different antibiotics. This reflecting graph shows some resistance by five samples with the measurement of centimeters proportionally.

Research has indicated that humans can obtain essential nutrients by including meat. The food item discussed is a source of protein, lipids, and necessary vitamins critical for maintaining good health (Baghbaderani et al., 2020). Based on the antibiotic susceptibility testing outcomes, our research findings suggest that the bacterial strains obtained from beef samples displayed resistance to specific antibiotics while showing sensitivity to other antibiotics. The T2-4 isolates resisted Ciprofloxacin, while Cefotaxime and ampicillin showed intermediate sensitivity. Furthermore, our research revealed a significant sensitivity to the remaining antibiotics. Our research findings indicate that azithromycin, cefotaxime, kanamycin, and streptomycin demonstrated considerable efficacy against the T2-5 and T2-12 isolates.

On the other hand, erythromycin exhibited only intermediate sensitivity. However, its important to note that these isolates resisted ampicillin and amoxicillin. Regarding beef T2-9, our research findings indicate no resistance was detected against any of the 8 antibiotics tested. The study emphasizes the importance of addressing potential public health risks associated with consuming contaminated meat and the need for implementing improved food safety measures, including the responsible use of antibiotics in animal farming. The study shows that germs found in beef meat samples from Gopalganj, Bangladesh, are not easily killed by many drugs. It also stresses the importance of monitoring drug resistance in pathogens that are spread through food. It is necessary to note that the data in this study come from experiments done in vitro on different types of bacteria. There are other ways these bacteria may react to medicines when alive. Because of this, you must be careful about how you interpret the data. It is also suggested that more research be done to support the results.

This study aims to find out how standard and how they spread multidrug-resistant bacteria in beef meat samples from different markets in Bangladeshs Gopalganj district. The studys results suggest that many of the germs isolated from the meat samples were not sensitive to more than one antibiotic. This is called multidrug resistance. The antibiotics for which resistance was observed are Ciprofloxacin, Ampicillin, and Amoxicillin. The study identified Escherichia coli and Staphylococcus aureus as the most common antibiotic-resistant bacteria. The studys findings indicate that the prevalence of antibiotic-resistant bacteria in the food supply chain in the Gopalganj district is a cause for concern, and urgent measures must be taken to address this issue. Enhancing awareness among the general public and healthcare professionals about the proper utilization of antibiotics and the potential repercussions of excessive antibiotic use is of utmost importance. The study can also be used as a starting point for more research on how antibiotic-resistant bugs in food can be found, which could lead to new ways to fight microbes. The study data can help make public health policies and plans to lower the risk of infections and inhibit the spread of antibiotic-resistant bacteria in Gopalganj and others.

Conceptualization; S.K.B.: Data curation and figure; S.K.B.; T.T.T.; A.H.: Data curation and table; S.K.B.; T.T.T.: Formal analysis; S.K.B.; A.H.; T.T.T.; Methodology; S.K.B.; T.T.T.; A.H.: Writing manuscript; T.T.T.; S.K.B.; A.H.: review & editing: S.K.B.; A.H.

The authors received funding from the Bangabandhu Sheikh Mujibur Rahman Science and Technology University Research Cell (BSMRSTU-RC).

We want to thank the BSMRSTUs Department of BGE for all the technology help theyve given us.

This article is original work and the author ensures that there is no conflict of interest relevant to this article.