Abstract

This study examines bacteria growth in 25 meat samples derived from cattle and sheep, encompassing regional and breed-specific varieties (Iraqi, Indian, Australian, Iranian, calf, and sheep meats). The specimens were cultivated on Nutrient agar, MacConkey agar, and Blood agar to facilitate the proliferation of a diverse array of bacteria. Following initial proliferation, isolates were characterized utilizing the VITEK 2 Compact system for accurate biochemical identification.
Among the 25 samples, several bacterial species were found in just 14 samples. Staphylococcus sciuri was recovered from white Indian, white cow, and yellow Iranian meat, indicating a potential cross-contamination or environmental association. Rothia kristinae was detected in milky calf and white sheep meat, but Staphylococcus warneri was found in both yellow Indian and white Iranian meat. Staphylococcus saprophyticus was detected in dark meat from Iran. A number of samples remained unclassified due to constraints in growth or unusual biochemical characteristics.
The results emphasize the variety of microbial flora in commercially accessible beef and underscore the necessity of regular microbiological monitoring. The utilization of selection and differential media, along with automated identification technologies such as VITEK 2, improves the precision and rapidity of foodborne pathogen detection. This work enhances the comprehension of meat microbiology and underscores the necessity for rigorous cleanliness protocols in meat preparation and handling.

Keywords :Vit D3 ,Periodontitis, Type II diabetic

Introduction

Animal-derived food is perishable and poses potential hazards to living organisms and the environment due to its susceptibility to physical, chemical, and biological contamination, which can jeopardize human, animal, and plant safety, as well as disrupt societal harmony(Argudín et al.,2010).
The elevated presence of microorganisms in meat can diminish its quality and pose health risks to consumers. The supply of beef that is Safe, Healthy, Intact, and Halal is anticipated to comply with the Maximum Microbial Contamination Limit (BMCM), ensuring that the microbial content of the meat does not surpass the established maximum limit, as per SNI 3932, 2008. The maximum total plate count is 1 x 10^6 cfu/g, Escherichia coli must not exceed 1 x 10 cfu/g, and Salmonella must yield negative findings. Additionally, the pH value should range from 5.5 to 5.7 (normal pH) and have a vivid color (Becker et al.,2014 ). The quality of carcasses is significantly affected by environmental conditions, as well as the facilities and infrastructure of slaughterhouses (RPH) (Berends et al.,1997 ). Bacterial contamination originates from the moment the animal is slaughtered until the meat is ingested. Slaughterhouses (RPH & TPH) and traditional marketplaces present the highest risk for bacterial contamination of meat.
Beef available in traditional marketplaces is highly susceptible to bacterial contamination. The market is susceptible to infection by pathogenic bacteria, rendering it a high-risk environment. The sanitation and cleanliness of the sales environment have been neglected by both the merchants and the relevant officials, resulting in elevated levels of microbiological contamination. Establishments offering beef in traditional markets are still interspersed with vendors of other essential goods; furthermore, the meat is merely displayed on tables and suspended at ambient temperature. The sale of meat at traditional markets typically occurs in an uncovered environment. Meat is presented in an environment that lacks cleanliness and maintains elevated temperatures; under these settings, pathogenic microorganisms can proliferate.
In Ramadi City, the absence of centralized animal slaughtering at the Government Slaughterhouse compromises the assurance of meat contamination safety standards, since it lacks oversight from authorized officials and related agencies. Variations in management, infrastructure, staff, and methodologies at government abattoirs and slaughterhouses will undoubtedly influence cattle slaughter outcomes. Numerous slaughterhouses continue to fail to meet regulatory standards and lack oversight concerning livestock health and meat safety. Therefore, it is imperative to conduct research comparing meat quality outcomes based on microbial contamination within the meat supply chain at government abattoirs and slaughterhouses (TPH) in Ramadi City, with the objective of ensuring consumer safety(Brizio et al.,2015;CDC, 2019).

The objective of this research included isolation and identification of bacteria responsible for the contamination of domestic and imported meat.  Utilizing the VITEK2 system methodology for the identification of bacterial species. Also, antibiotic susceptibility testing.

Material and Methods

Collection of Samples

A total of 25 meat samples were acquired from diverse sources to facilitate a representative analysis for the study. The samples comprised both raw and processed meat, sourced from local butcher shops, supermarkets, and open markets across several geographic locations within the study area. The site selection was intended to encompass a variety of meat processing procedures and storage conditions. Each sample was collected aseptically with sterile instruments and containers, appropriately labeled, and transferred to the laboratory under refrigerated conditions to preserve sample integrity and prevent contamination. All samples were processed and evaluated within 24 hours post-collection.
Culture Medium

The medium was created in accordance with the instructions specified on their containers as directed by the manufacturer. The sterilization was conducted using autoclaving at 121°C for 15 minutes under a pressure of 1.5 bars.

Detection of bacteria

The VITEK 2 system (bioMérieux) is utilized for bacterial identification and antimicrobial susceptibility testing (AST).

Antibiotic susceptibility testing via the disc diffusion technique
This type of test is among the common standardized examinations, with its performance consistently refined by the CLSI consensus initiative (CLSI 2012) (Wayne, 2002). The bacterial inoculum was generated by transferring a single colony to a test tube containing nutrient broth, followed by incubation at 37°C for 24 hours. Following the preparation of incubation and inocula. Sterile swabs were collected and placed in a test tube containing inoculum, then equally swabbed across the surface of a Muller-Hinton agar plate. Following inoculation, antibiotics were applied using forceps with firm pressure, resulting in the formation of three discs in each dish. The plates were then inverted and incubated at 37°C for 18 hours. (Mahon & Manuselis, 2000). Subsequently, the zone of inhibition for each antibiotic is evaluated and compared with conventional inhibition diameters.

Bacterial Isolate

A total of 25 raw meat samples, consisting of both beef and sheep meat, were collected from various sources across four countries: India, Iran, Australia, and Iraq. These samples were obtained from local markets and butcher shops, ensuring a wide geographical representation for the study. The collection process was carried out under sterile conditions, with each sample placed in a sterile container and transported to the laboratory under refrigeration to maintain microbial viability. Upon culturing on selective and non-selective media, bacterial growth was observed in only 14 of the 25 samples. This indicates varying levels of microbial presence, potentially influenced by differences in meat handling, hygiene practices, and storage conditions across regions.

 Morphological Diagnosis
On blood agar, colonies of Staphylococcus saprophyticus are smooth, convex, and exhibit a white to creamy yellow coloration. It generally exhibits non-hemolytic or modest hemolytic action. Colonies on nutrient agar have a spherical, opaque, slightly elevated morphology, characterized by a cream hue and a glossy surface. Similar to other Gram-positive cocci, it does not proliferate on MacConkey agar.
When cultivated on blood agar, Staphylococcus sciuri produces spherical, smooth colonies that may demonstrate beta-hemolysis, indicated by clear zones surrounding the colonies resulting from the lysis of red blood cells. On nutrient agar, it forms medium-sized, moist, slightly elevated colonies that are yellow-white in color. It does not proliferate on MacConkey agar, aligning with its Gram-positive characteristics. Rothia kristinae exhibits tiny, round, non-pigmented to slightly yellow colonies on blood agar and is typically non-hemolytic. On nutrient agar, it produces small, spherical colonies that are white to off-white in color and may have a dry or mucoid texture. This species does not proliferate on MacConkey agar due to its Gram-positive nature, which is inhibited by MAC. Refer to figures 1 and 2.

Figure 1: Yellow Colonies on Nutrient Agar

Figure 2: Growth on Blood Agar

Microscopic examination
Figure 3 illustrates Gram-stained bacterial cells observed under a microscope, exhibiting a gram-positive shape. The bacteria manifest as clusters of cocci, indicating their classification within the Staphylococcus genus. The rich violet coloration signifies a robust peptidoglycan coating in their cell wall, typical of Gram-positive bacteria.

Figure 3: Staphylococcus sciuri, Staphylococcus warneri, and Staphylococcus saprophyticus were detected

VITEK2System
Fourteen meat samples from cattle and sheep of diverse origins (Iraq, India, Iran, Australia) and looks (white, yellow, dark, milky) were tested utilizing the VITEK 2 system. Of them, 10 samples produced recognized bacterial species, and 4 samples contained unidentified organisms.
The discovered bacterial species were predominantly Staphylococcus spp. and Rothia kristinae. The bacterium most commonly isolated was Staphylococcus sciuri, identified in three samples: white Indian meat, white calf meat, and yellow Iranian meat. Staphylococcus warneri was identified in both yellow Indian meat and white Iranian meat. Staphylococcus saprophyticus was identified in the dark Iranian meat, whereas Rothia kristinae was isolated from both milky calf meat and white sheep meat, as illustrated in figure 4.
The samples containing unidentified organisms comprised yellow Australian beef, yellow veal, yellow lamb, and white Australian beef, indicating the existence of either uncommon, fastidious, or inadequately plentiful microorganisms that the VITEK 2 system was unable to classify.

Figure 4: Frequency of Identified Organisms in Meat Samples

Table 1: Bacterial Growth Morphology on Culture Media

The prevalence of coagulase-negative Staphylococcus species (CoNS), such as S. sciuri, S. warneri, and S. saprophyticus, corresponds with earlier research indicating common contamination of meat surfaces by skin and environmental bacteria during slaughter and processing (Argudín et al., 2010; Osman et al., 2016).
Staphylococcus sciuri, identified in three samples, is prevalent in animals and the environment and is associated with antibiotic resistance genes, presenting a potential public health threat (Stepanović et al., 2001). S. warneri and S. saprophyticus are opportunistic bacteria frequently associated with illnesses upon introduction into sterile body areas and may infiltrate the food chain through human manipulation (Becker et al., 2014).
The detection of Rothia kristinae, an infrequent bacterium from the human oral cavity, in two samples may suggest contamination from respiratory droplets or inadequate hygiene among handlers (Ramanan et al., 2014). Although generally non-pathogenic, its presence in food necessitates scrutiny as a sign of inadequate sanitary standards.
The failure to detect organisms in four samples may be ascribed to the constraints of the VITEK 2 system, particularly its dependence on a predetermined biochemical profile database (Funke & Funke-Kissling, 2005). This underscores the necessity for molecular identification techniques, such as 16S rRNA sequencing or MALDI-TOF MS, for enhanced detection and classification, particularly of rare or novel organisms.
The findings emphasize the significance of adequate meat hygiene, cold chain preservation, and microbiological quality evaluation. Cross-contamination, especially from personnel and equipment, represents a crucial control point in the meat sector (Jay et al., 2005). The bacterial profiles indicate possible zoonotic and spoilage hazards, underscoring the necessity for regular microbial monitoring and rigorous hygiene standards.

Antibiotic Susceptibility
The antibiotic sensitivity profiles identified in this work offer significant insights into the resistance and susceptibility patterns of bacteria extracted from diverse beef and sheep meat samples. The most abundant Gram-positive cocci discovered were Staphylococcus sciuri, Staphylococcus warneri, and Staphylococcus saprophyticus. These organisms shown uniform susceptibility to Oxacillin, Vancomycin, Ciprofloxacin, and Gentamicin, which are often effective against coagulase-negative Staphylococcus species (Becker et al., 2014). Resistance to Penicillin was universally identified in all Staphylococcus isolates, consistent with global trends demonstrating elevated levels of β-lactam resistance attributed to the development of penicillinase enzymes (Otto, 2013).
Staphylococcus sciuri exhibited resistance to Erythromycin and intermediate sensitivity to Tetracycline in certain samples, indicating the presence of macrolide and tetracycline resistance genes (Hammad et al., 2014). Conversely, Rothia kristinae isolates exhibited complete sensitivity to all evaluated antibiotics, indicating its comparatively low pathogenic potential and minimal exposure to selective antibiotic pressure (CLSI, 2023).
Unidentified organisms were excluded from sensitivity testing owing to identification constraints. Their existence underscores the necessity for enhanced molecular or sophisticated biochemical identification techniques to precisely ascertain their resistance profiles (Kumar et al., 2017).
The findings emphasize the necessity of ongoing monitoring of antimicrobial resistance in foodborne bacteria, especially in meat products, since they may serve as a conduit for the transmission of resistant strains to humans. The findings endorse the judicious application of antibiotics and emphasize the necessity of appropriate hygienic protocols in meat processing and handling to avert the dissemination of resistant bacteria, as illustrated in Table 2 (Kumar et al., 2017).

Table 1: Bacterial Growth Morphology on Culture Media

Conclusions

The predominant bacteria discovered were coagulase-negative Staphylococcus species, specifically S. sciuri, S. warneri, and S. saprophyticus, as well as Rothia kristinae. Their presence suggests contamination, probably from environmental causes or human interaction. Antibiotic resistance was widespread among Staphylococcus isolates, with all exhibiting resistance to Penicillin. S. sciuri demonstrated resistance to Erythromycin and intermediate sensitivity to Tetracycline, raising concerns regarding the infiltration of antibiotic-resistant organisms into the food chain.

Acknowledgment

Non .

Conflicts Interest

Non.

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