Chromogenic Agar Assisted Screening of Pathogenic Bacteria from Butcher shops Wastewater Effluent and their Antibiotic Susceptibility Profiling

The research utilized sophisticated technique called chromogenic agar and the Vitek 2 system to examine the bacterial content of fourteen samples of butcher’s shops effluent water, revealing thirty different bacterial species with varying levels of antibiotic resistance, including Staphylococcus aureus, Enterococcus faecalis, Citrobacter freundii, Escherichia coli, and Klebsiella pneumoniae,. The Vitek 2 system verified the initial identification of bacteria with 100% accuracy, providing additional evidence for the reliability of the chromogenic agar method. The Gram staining results revealed the presence of both gram-positive and gram-negative bacteria. These findings emphasize the importance of proper sanitation and hygiene practices to minimize the potential health risks associated with bacterial contamination in effluent water from butcher’s shops.


Introduction
Butchers have a significant role in the food industry as they process and sell meat and meat products to the public.Nonetheless, the refuse generated by these stores might harbour dangerous bacteria which could pose a significant risk to both the environment and public health.Butcher shop wastewater effluent is a potential source of antibiotic-resistant bacteria, which can be hazardous to public health [1].Identifying and isolating pathogenic bacteria from the wastewater effluent of butcher shops, and conducting an antibiotic susceptibility test is vital in preventing the spread of antibiotic resistance.The wastewater effluent from butcher shops contains various types of pathogenic bacteria like Salmonella Escherichia coli, Campylobacter jejuni, Staphylococcus aureus and Listeria monocytogenes, which can cause a range of illnesses from mild gastroenteritis to severe infections like sepsis [2,3].Antibiotic resistance is a worldwide concern, and the misuse and overuse of antibiotics are major contributors to the problem.Identifying and isolating antibiotic-resistant bacteria from wastewater effluent is crucial in identifying the patterns of resistance and finding the most effective antibiotics to treat infections.This knowledge can also help in developing new antibiotics and preventing the spread of antibiotic-resistant bacteria [4][5][6].Chromogenic agar is a type of specialized growth medium that contains chromogenic substrates that can react with specific enzymes produced by bacteria, resulting in the formation of distinct colour colonies.The use of chromogenic agar has revolutionized the screening and identification of pathogenic bacteria, enabling rapid and accurate detection of specific bacterial strains [7,8].Chromogenic agar-assisted screening of pathogenic bacteria is a valuable tool in clinical microbiology and food safety industries.The ability to differentiate between different bacterial species based on colour allows One approach for quickly identifying particular pathogens involves the use of specialized techniques.Chromogenic agar is an illustration of such a technique, which can be utilized for the identification of specific pathogens like Salmonella Escherichia coli, Campylobacter jejuni, Staphylococcus aureus and Listeria monocytogenes, which are common causes of foodborne illnesses [9][10][11].The use of chromogenic agar has several advantages over traditional screening methods.First, it allows for the detection of pathogenic bacteria in complex microbial communities, making it an excellent tool for environmental monitoring.Second, chromogenic agar is easy to use and interpret, requiring minimal training, and producing results in a short amount of time.Finally, the use of chromogenic agar reduces the need for confirmatory tests, saving time and resources [12].In contrast, there are other confirmation platform called Vitek 2, it is a computerized tool commonly used in clinical microbiology laboratories for identifying fungi and bacteria, as well as testing their susceptibility [13,14].This system uses innovative technology to recognize microorganisms by evaluating their biochemical features, delivering rapid outcomes within hours, which is much faster compared to traditional procedures that usually require days to complete.By applying the Vitek 2 system, clinicians can recognize various types of bacteria, both gram-positive and gram-negative, allowing them to provide effective and targeted therapies for infections.Its effectiveness and speed make it a vital instrument in diagnosing and treating infectious ailments, which is why it is increasingly used in clinical settings globally [15,16].Overall, the isolation and identification of pathogenic bacteria in wastewater effluent from butcher shops, and their antibiotic susceptibility profiling, are crucial for preventing the spread of antibiotic resistance and ensuring public health.Chromogenic agar-assisted screening is a valuable tool in detecting and differentiating between potential pathogens, reducing the risk of disease transmission.The use of this technology is a significant advancement in microbial detection and has implications for the future of clinical microbiology and food safety industries.

Material and Methods
Fourteen samples of wastewater were collected from butcher shops located in different areas of Mosul, Iraq between September 1, 2022 and November 1, 2022.The samples were obtained using sterile glass bottles and transported to the laboratory immediately for analysis.Gram staining was performed on each sample to determine the presence of bacteria.

Isolation and Identification of Pathogenic Bacteria
Serial dilution of the collected samples was carried out using sterile saline solution.One milliliter of each dilution was spread onto Chromogenic agar plates including CHROMagar™ Coliform bacteria and CHROMagar™ Staph.After preparing the plates, they were kept in a 37°C incubator for a period of 24 to 48 hours.Any colonies that were suspected to be present were subjected to further purification by sub-culturing on fresh agar plates.The purified colonies were subjected to Gram staining and identified using the Vitek 2 system.The bacterial isolates were then sub-cultured onto blood agar and MacConkey agar plates, which were again incubated for 24 to 48 hours at 37°C.The Vitek 2 system was used to determine the biochemical features and antibiotic susceptibility patterns of the bacterial strains [17].

Antibiotic Susceptibility Testing
The antibiotic susceptibility of the bacterial isolates was confirmed using the Vitek 2 system.The bacterial isolates were tested against a panel of antibiotics including Ceftriaxone, Ciprofloxacin, Clindamycin, Amoxicillin Acid, Cephalexin, Piperacillin, Levofloxacin, Imipenem, Azithromycin, Metronidazole and Trimethoprim (table1Following the recommendations of the Clinical and Laboratory Standards Institute (CLSI), as per the guidelines provided by them [18][19][20].
Table 1.The antibiotics disk used in this study.

Data Analysis
The information obtained from the research was analyzed utilizing Microsoft Excel software.The outcomes of this analysis were subsequently demonstrated in the form of frequencies and percentages.
The results of the antibiotic susceptibility testing were interpreted based on the CLSI guidelines.

Results
The results of using chromogenic agar to identify bacteria from 14 samples of butchers shop effluent water have revealed the presence of 30 different bacteria species.These include Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Enterobacter cloacae , Escherichia coli, Citrobacter freundii and Klebsiella pneumonia (figure1).Further analysis revealed that these bacteria have varying levels of antibiotic resistance against 12 different antibiotics: Ceftriaxone, Ciprofloxacin, Clindamycin, Amoxicillin Acid, Cephalexin, Piperacillin, Levofloxacin, Imipenem, Azithromycin, Metronidazole, and Trimethoprim.This information suggests that butchers shop effluent water may contain a diverse range of potentially harmful bacteria that exhibit resistance to several commonly used antibiotics (figure 2).This underscores the importance of proper sanitation and hygiene practices to prevent the spread of these bacteria to the public (Table2).After the initial identification of bacteria using chromogenic agar, the results were further confirmed using the Vitek 2 system.The identification of bacterial species based on their biochemical properties is a crucial aspect in determining their potential impact on public health.The Vitek 2 system is a dependable and precise method for identifying bacteria using this approach.As depicted in Figure 3, the Vitek 2 system confirmed the initial identification of all 30 bacterial species with 100% accuracy, including Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Enterobacter cloacae , Escherichia coli, Citrobacter freundii and Klebsiella pneumonia, and others present in the samples.This finding is significant as it indicates that the identified bacteria are present in the waste water of butcher's shops and could pose a significant threat to public health if not adequately treated.Moreover, the confirmation of identification through the use of the Vitek 2 system provides additional support for the reliability of the initial identification using chromogenic agar.The Gram staining results confirmed the existence of bacteria that are both gram-positive and gramnegative.in the effluent water samples from butcher's shops.This information is crucial as it provides additional insight into the nature of the bacteria present in the water and can help guide appropriate treatment measures to prevent the spread of bacterial infections (figure4).

Discussion
Our study's findings indicated that there were 30 distinct bacterial species present in the effluent water samples obtained from butcher's shops.These results are in line with previous research that has shown the existence of pathogenic bacteria in wastewater and effluent from food industries.[21,22].
Our study also revealed that the identified bacteria exhibit varying levels of antibiotic resistance against commonly used antibiotics.This is in line with other studies that have reported the presence of antibiotic-resistant bacteria in wastewater from food industries [23][24][25].Moreover, our study confirmed that both gram-positive and gram-negative bacteria were present in the effluent water samples obtained from butcher's shops.This discovery is consistent with prior research that has detected both types of bacteria in wastewater from food industries.[26].
The discovery of multi-antibiotic resistance in certain bacterial strains is especially worrying as it may restrict treatment options and raise the risk of infection transmission.This observation is consistent with prior research that has observed multi-antibiotic resistance in bacteria isolated from wastewater and effluent from food industries [27,28].
Compared to previous studies, our study employed chromogenic agar, Vitek 2 system, and Gram staining to identify and characterize bacteria in the effluent water samples from butchers shops.This allowed for the accurate identification of bacterial species and their resistance to antibiotics, as well as the determination of their gram-positive or gram-negative nature [29].
Our findings underscore the importance of proper sanitation and hygiene practices in food industries to prevent the spread of potentially harmful bacteria to the public.Efforts should be made to develop and implement effective wastewater treatment strategies to reduce the risk of bacterial contamination in effluent water.
Taken together, our study provides valuable insights into the presence and characteristics of bacteria in the effluent water samples from butcher's shops.These results are in line with previous research and emphasize the importance of ongoing surveillance and monitoring of bacterial contamination in effluent water from food industries.[30][31][32].

Figure 1 .
Figure 1.Chromogenic agar plates showing different bacterial species isolated from butchers shop effluent water.

Figure 2 .
Figure 2. The summary of the bacterial strains, their antibiotic resistance or sensitivity, and the results from Muller Hinton agar.Each subplot represents a different antibiotic tested, with the bacterial strains grouped based on resistance or sensitivity and distinguished by color.The subplot includes a legend for the color code and zones of inhibition, represented by symbols or shapes.The figure presents a visual representation of the table data, facilitating comparison and interpretation of results.

Figure 3 .
Figure 3.The identification of bacterial species based on their biochemical properties is crucial in assessing potential risks to public health.The Vitek 2 system is a reliable and precise method for identifying bacteria using this approach.The system confirmed the initial identification of all 30 bacterial species with 100% accuracy, utilizing 64 biochemical tests in total.This finding confirms the presence of various bacteria, including Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Enterobacter cloacae , Escherichia coli, Citrobacter freundii, Klebsiella pneumonia and other species in the samples.

Figure 4 .
Figure 4. Microscopic images of different bacterial strains stained using the Gram staining method, captured using an oil immersion lens.The figure can be used to illustrate the differences in morphology and staining characteristics between strains.

Table 2 .
The information provided on the different bacteria identified and their resistance to various antibiotics.The antibiotics tested include: Ceftriaxone, Ciprofloxacin, Clindamycin, Amoxicillin Acid, Cephalexin, Piperacillin, Levofloxacin, Imipenem, Azithromycin, Metronidazole, and Trimethoprim.