Prevalence of Staphylococcus aureus and Antimicrobial Drug Residues in Chicken Liver its Antibiogram Analysis and Implications on Public Health

Abstract

Chicken is a primary source of food worldwide. Ensuring food safety and preserving public health is crucial, and chicken meat inspection plays a critical role in achieving this goal. The transfer of pathogenic bacteria from animal intestines to carcasses during processing, and subsequently from meat products to consumers, is a significant contributor to the growing incidence of resistance and foodborne illnesses. This study aimed to examine the antimicrobial resistance of Staphylococcus aureus present in chicken liver. Fifty samples of chicken liver were collected from various areas of Peshawar and screened for resistant Staphylococcus aureus. The bacteria were tested against different antibiotics, including Moxifloxacin, Vancomycin, Linezolid, Gentamycin, and Clindamycin. Gentamycin demonstrated the highest susceptibility against the maximum bacterial isolates, with 100% susceptibility as its P-value was less than 0.05. Other antibiotics showed effective results. Linezolid was 66.7% susceptible. Both vancomycin and Clindamycin were 41.6% susceptible, while Moxifloxacin was 33.3% susceptible. This study concludes that Staphylococcus aureus isolated from chicken liver has the potential to cause multiple diseases. The screened pathogens were not only resistant to single antibiotics but also showed resistance to multiple antibiotics. The cut-off values of the isolates were two folds higher than those reported in CLSI 2023. The findings highlight the need for effective measures to control the spread of antimicrobial-resistant bacteria and to ensure food safety.

Introduction

Chicken is one of the biggest source of foods all around the globe, Chicken meat is not being used only in Pakistan but all around the globe. Worldwide production of the chicken is exceeded from 74 million metric tons as compared to beef meat that is 65 million [1]. China is one of the biggest chicken meat producer in the world while USA is prominent for the highest consumption of chicken meat [2]. In Pakistan poultry source is playing important role in bridging the gap of protein demand, not only this, poultry sector of Pakistan is helping in GDP (growth domestic product) about (1.3%) [3]. It has proved that chicken meat is the significant source of daily protein. As chicken is inexpensive than other meat forms. According to the World Health Organization (WHO) annual report, on average a person must have 27 g of protein daily [4]. Unfortunately, in Pakistan, it is only 17 g, whereas the percentage of poultry meat is only 29.4% out of 17 g. This leaves a deficiency of 10 g (71%) of the daily protein requirement per person [5]. On the other hand, there are many problems related with poultry, due to poor quality and poor hygienic condition there are many things that lack in chicken meat [6, 7]. There are a number of challenges related to poultry meat, such as; disease cult breaks, like Newcastle, Marek’s disease, infectious bronchitis, Gambaro disease, botulism, fowl cholera, thrush, air sac disease, mushy chick, and bumble foot [8]. Most of the enteric bacteria reside in chicken liver [9–11]. Due to undercooked food, such pathogenic bacteria can easily transfer to humans and can cause diarrhea, dysentery and many other diseases.

Staphylococcus aureus plays a major role. This bacterium causes food poisoning, boils, cellulitis and toxic shock [12–14]. Staphylococcus aureus is Multi Drug Resistance (MDR) micro-organism making a single bacterium resistant to three or more than three drugs at the same time, Thus, this bacterium is said to be the multiple drug resistance [15]. Not all strains of S. aureus isolated from various matrices are inherently multi-resistant to antibiotics. However, there is a concerning trend of increasing multi-resistance among S. aureus strains over time. This trend is largely attributed to the widespread and often indiscriminate use of antibiotics, which exerts selective pressure on bacterial populations, favoring the survival and proliferation of resistant strains [16]. Additionally, the ability of S. aureus to acquire resistance genes through horizontal gene transfer further exacerbates the problem. Several factors contribute to the development of multi-resistance in S. aureus. These include the overuse and misuse of antibiotics in healthcare settings, agriculture, and community settings, as well as inadequate infection control measures. Furthermore, the genetic plasticity of S. aureus allows for the rapid evolution and dissemination of the resistance mechanisms, such as the acquisition of mobile genetic elements carrying resistance genes [17]. The major source of contamination or spreading of such microorganism in chicken is the feed they consume [18]. Broilers are kept in dirty places and directed towards hormonal injections / artificial hormones so that they can grow in a matter of weeks rather than their natural time period, this artificial infusion of hormones has reportedly resulted in baldness and homosexuality of chicken and consumers. Other factors include slaughter facility, poor handling during feather removal, and the container where they are put after slaughter. Due to bad sanitation practices and poor health conditions of animals, pathogenic microorganisms can easily invade their bodies as their immune system [19]. The pathogenic microorganisms are transferred from chicken meat to humans if the meat or liver is not cooked properly. Over 90% of the cases of food poisoning is due to undercooked food in weddings or other catering venues [20]. Poultry livers carry a particularly high risk of Campylobacter, Salmonella typhi, Salmonella enteritis’s, Staphylococcus aureus, Campylobacter jejuni, Aeromonas sp, Clostridium perfringes and E. coli as these bacteria can be present throughout the liver, and may remain a source of disease if they are not cooked properly. People with immunocompromised system can readily get infected by pathogenic microbes via food [20]. Furthermore, the excessive use of antibiotic with no check and balance leads to the resistance against a number of drugs [21]. Many studies reveal that these pathogenic microorganisms are resistant towards antibiotics such as; streptomycin (100%), ampicillin (60%), nalidixic acid (50%) ciprofloxacin (47%) and tetracycline (45%), (% shows the amount of resistance) [22]. The aim of the current study was to isolation and screening of Staphylococcus aureus from chicken liver and its antibiogram analysis.

Materials and Methods

Study Area

The current study is associated with antimicrobial resistivity of Staphylococcus aureus isolated from chicken liver sold in retail market in Peshawar and was conducted at Department of Biotechnology, Institute of Integrative Biosciences (IIB), CECOS University of IT and Emerging Sciences, Peshawar, Pakistan.

Sample size

For this case study a total of 50 samples of chicken liver were isolated from different vendors’ shops in Zipper bags to avoid contamination, these samples were then transferred directly to laboratory refrigerator using an ice box.

Sample Collection & Sites

In Peshawar, samples were obtained from various regions. Samples 1–10 were gathered from Zaryab Colony, while samples 11–20 were obtained from Dalazak Road. Additionally, samples 21–30 were collected from Chicken Chock, while samples 31–40 were collected from Azhar Khani. Finally, samples 41–50 were obtained from Nowshehra Road. Each of the 50 samples was acquired from a retailer shop, placed in a zipper bag, and kept refrigerated at 4 °C.

Isolation of Microbes

For isolation of microorganisms from chicken liver, nutrient agar media was used. For this purpose, 0.5 g peptone, 0.3 g yeast extract, 0.5 g Nacl and 1.5–2 g technical agar was dissolved in 100 ml (mL) of distilled water. The media was autoclaved (Hirayama HV-110 made in Japan) for 15 min at 121 °C and 15psi (Pounds per square inch). After autoclaving, the media was poured in petri plates under sterile conditions using a biosafety cabinet. Media was allowed to solidify and petri plates were shifted to incubator (MCO-18ACL-PA made in USA) to ensure sterility for 24 h at 37 °C. Sterile saline was used for serial dilutions of each liver sample made up to10⁻³. Culturing was done with the help of sterile cotton swabs so that inoculum could be spread evenly on media plate via spread plate technique. After culturing, media plates were incubated at 37 °C for 24 h for growth. Afterwards the petri plates were transferred to refrigerator at 4 °C. From the first swab of non-selective medium on the plates 188 colonies were observed [23].

Purification

And then the 50 colonies were again isolated from non-selective medium. 50 microbial colonies were picked randomly from previously cultured petri plates and then to fresh the culture we were poured previous cultured on freshly prepared nutrient agar plates using inoculation loop. Cultured Petri plates were shifted to incubator for 24 h at 37 °C [24].

Screening

Screening for Staphylococcus aureus was done using Mannitol Salt Agar (MHA) medium. A total of 200 ml (mL) medium was prepared using distilled water (Nacl 75 g, D-Mannitol 10 g, phenol Red 0.025 g, agar 15.0 g) 200 ml (mL) solution was autoclaved for121 °C, 15 min at 15psi (Pounds per square inch). After autoclaving pouring was done in petri plates under sterile condition. These plates were then shifted to incubator for 24 h at 37 °C. After 24 h sterility check, discrete and pure colonies from the previous petri plates were cultured using streaks plate technique. All the 50 colonies were positive for S. aureus. These plates were than incubated for 24 h, cultured plates were transferred to refrigerator at 4 °C. For long term storage we have cryopreserved the colonies in broth and glycerol and place it at − 80 ºC [25].

Disc Diffusion Method/Kirby Bauer Test

Disc diffusion or Kirby Bauer test was done to check zone of inhibition, that to which extent antibiotic is working. After pouring MHA (Mannitol Salt Agar) media in petri plates, it was allowed to cool, and was kept for sterility check for 24 h in incubator. Afterward, the bacterial suspension 20 µL of bacterial culture, typically grown overnight in a broth medium, into a sterile container containing 20 mL of sterile saline solution, the bacterial culture and diluent are mixed thoroughly to achieve a homogeneous suspension. The concentration of the bacterial suspension was compared with the McFarland standard. And the inoculum was prepared from pre-culture in broth. After lawn preparation, five antibiotics of different concentrations such as Clindamycin (20 μg), Moxifloxacin (10 μg), Vancomycin (15 μg), Gentamycin (30 μg) and Linezolid (10 μg), were placed in equal distance on petri plates and was incubated for 24 h. Zone of inhibition was measured for these antibiotics. And to validate the diameter of the zone of inhibition we measure the zone according to the latest CLSI report [26, 27].

MIC (Minimal Inhibitory Concentration)

MIC is the lowest concentration of drug which prevents visible growth of microorganism. For MIC determination, previous cultures were freshed using nutrient agar media, and were incubated for 24 h. For inoculum preparation, nutrient broth was prepared using bacteriological peptone 0.75 g and yeast extract 0.45 g in 150 mL of water. Broth inoculum was compared with McFarland standard Dilutions for various antibiotics were prepared including Clindamycin, Moxifloxacin, Vancomycin, Gentamycin and Linezolid. For this purpose, predetermined cutoff values of each antibiotic was checked using CLSI 2023. The highest concentration for dilutions were 2 folds greater and 2 folds lesser than predetermined cutoff value so a total of 5 concentrations were prepared for each antibiotic. Both antibiotic solutions and inoculum were added to 96 well plate (195 µl antibiotic dilution and 5 µl bacterial culture). After pouring, OD (Optical Density) was checked via spectrophotometer (VT-UV1900 made in Japan) for initial reading at 600 nm wavelength. Micro titer plate was then incubated for 24 h at 37 °C and OD (Optical Density) was again measured via spectrophotometer [28].

Statistical Analysis

Data analysis in this study involved the utilization of statistical software, including SPSS, Graph Pad, and Origin. T-test was conducted, with a significance level (P value) of ≤ 0.05. Both statistical analysis and data reconfiguration were performed. The t-test was specifically for MIC was employed to compare the initial and final readings of bacterial growth in the presence of antibiotics.

Results

Antibiotics are commonly used in the poultry industry to prevent and treat bacterial infections in broiler chickens. However, their overuse can lead to the development of antibiotic-resistant bacteria that can spread to humans through the food chain and the environment. When broiler chickens are given antibiotics, residues can be found in their meat, eggs, and other poultry products. These residues can then enter the food chain and be consumed by humans, potentially leading to the development of antibiotic resistance in bacteria that can cause infections in people. Additionally, the use of antibiotics in poultry farming can lead to the contamination of water and soil, as well as the development of antibiotic-resistant bacteria in the environment. Ultimately, the overuse of antibiotics in the poultry industry can have significant impacts on human health, including an increased risk of antibiotic-resistant infections, reduced effectiveness of antibiotic treatments, and potential long-term harm to the environment which is also shown in Fig. 1.

Fig. 1.

Graphical representation of the study which shows the use of antibiotics in broiler chickens contributes to the development and spread of antibiotic-resistant bacteria in the environment, which can eventually reach humans and cause illnesses. This cycle highlights the importance of responsible antibiotic use in agriculture and the need for monitoring and managing environmental contamination

Antibiotics List

Staphylococcus is a genus of bacteria that can cause a range of infections in humans, from mild skin infections to life-threatening conditions such as sepsis. Over time, many strains of Staphylococcus have developed resistance to various antibiotics, making them difficult to treat. Some of the antibiotics to which Staphylococcus species can be resistant include Cefoxitin, Chloramphenicol, Clavulanic acid, and Streptomycin. The increasing prevalence of antibiotic-resistant Staphylococcus strains highlights the need for responsible antibiotic use and the development of new treatments to combat these infections list of some antibiotics are shown in Fig. 2 [29].

Fig. 2.

Antibiotic resistance profile of Staphylococcus species: The figure illustrates the spectrum of resistance exhibited by various Staphylococcus species against a range of commonly used antibiotics. The antibiotics tested Cefoxitin, Ceftiofur, Chloramphenicol, Gentamicin), Clavulanic Acid, ciprofloxacin, Ampicillin, Kanamycin and Streptomycin. The data highlight the prevalence of multidrug-resistant strains within the Staphylococcus genus, emphasizing the clinical challenge posed by these pathogens in the treatment of infections

Isolation of Microbes

Different kinds of microbes were present in chicken liver sample, sample collected from different area had various numbers of microorganism. Isolation was done from serial dilution number 2 and 3 via spread plate method result are also shown in Fig. 3 [23].

Fig. 3.

Isolation of microbial species on nutrient agar: The figure shows the isolation of diverse microbes from environmental samples using nutrient agar, which supports the growth of various bacterial species. Distinct colony morphologies indicate different species, essential for obtaining pure cultures and further microbiological analyses

Purification of Bacteria

Random colonies were picked from isolated microbes on base of size, shape and color, these colonies were than streaked on Nutrient Agar plate to get pure isolates. For this purpose, 4 Quadrant streak plate technique was used which is also shown in Fig. 4 [24].

Fig. 4.

Purification of microbial isolates using the streak plate method with the quadrant technique: The figure depicts the process of isolating and purifying microbial colonies on agar plates. This method facilitates the isolation of pure colonies from a mixed culture, which are essential for accurate identification and further microbial analysis

Screening of Bacteria

Isolated bacterial colonies were screened on Mannitol Salt Agar media. Only positive isolates showed growth as MSA is selective for Staphylococcus spp. Moreover, those bacterial isolates that turn the media color from red to yellow were identified to be Staphylococcus aureus. They produce an acidic as a byproduct that converts phenol red of MSA (Mannitol Salt Agar) media in to yellow. MSA (Mannitol Salt Agar) is also differential for other Staphylococcus spp. such as Staphylococcus epidermidis gives pale yellow colonies on this media also shown in Fig. 5 [25].

Fig. 5.

Screening of Staphylococcus aureus on Mannitol Salt Agar (MSA): The figure illustrates the selective and differential screening of Staphylococcus aureus using Mannitol Salt Agar. MSA, a high-salt medium, selectively promotes the growth of Staphylococcus while inhibiting non-halotolerant organisms

Kirby Bauer Method

Disc diffusion method was performed foe selected Antibiotics, zones of inhibition were observed and measured. The test results are reported as sensitive, intermediate and resistant. Figure below shows zone of inhibition of five antibiotics results are also shown in Fig. 6a, b [26, 27].

Fig. 6.

a Antimicrobial susceptibility testing of bacterial isolates using the disc diffusion method: The figure displays the results of the disc diffusion assay, where bacterial cultures were exposed to various antibiotic-impregnated discs on agar plates. The zones of inhibition around each disc indicate the effectiveness of the antibiotics against the tested bacterial strain. Larger zones of inhibition reflect higher sensitivity of the bacteria to the corresponding antibiotic, while smaller or absent zones suggest resistance. This method provides a visual and quantitative measure of the antibiotic susceptibility profile of the bacterial isolates. b Antimicrobial susceptibility testing using the disc diffusion method: The figure demonstrates the zones of inhibition produced by the antibiotics Vancomycin, Moxifloxacin, Gentamycin, Linezolid, and Clindamycin against different bacterial strains, including strain S3-1. The zones of inhibition reflect the effectiveness of each antibiotic, with larger zones indicating greater bacterial sensitivity. Notably, strain S3-1 exhibits smaller zones of inhibition for several of these antibiotics, particularly Gentamycin and Moxifloxacin, indicating a higher level of resistance compared to the other tested strains. This underscores the multidrug-resistant nature of the S3-1 strain, highlighting the challenges it poses for treatment

Minimal Inhibitory Concentration

MIC (Minimal Inhibitory Concentration) was performed for each Antibiotic ( Moxifloxacin, Vancomycin, Clindamycin, Linezolid, Gentamycin) were test against Staphylococcus aureus, For this purpose, 96 well plate (Corning 96 well plate) was used, in which various concentrations of antibiotic solutions were tested against isolated Staphylococcus aureus spp. OD (Optical Density) results were obtained before and after 24 h. OD (Optical Density) results showed that Moxifloxacin showed 33.3% susceptibility, Vancomycin and Clindamycin showed 41.6% susceptibility, Linezolid showed 66.7% susceptibility whereas Gentamycin gave the highest susceptibility against all isolates which was 100% which is also shown in Fig. 7 and Table 1 [28].

Fig. 7.

MIC results for antibiotics Moxifloxacin (Mxf, 10 μg), Vancomycin (VA, 15 μg), Clindamycin (CN, 20 μg), Linezolid (LZD, 10 μg), and Clindamycin (DA, 30 μg) against various strains. Clindamycin shows 100% resistance the highest, while Linezolid exhibits 33.3% resistance the lowest. The data highlight the varying levels of antibiotic susceptibility across different strains

Table 1.

MIC results of Antibiotics against various strains. This table contains the percentage of bacteria for each antibiotic calculated on the P > 0.05 bacteria is said to be resistant and P < 0.05 or equal to, antibiotic is susceptible

Staphylococcus aureus
	Mxf (10 μg)	VA(15 μg)	CN (20 μg)	LZD (10 μg)	DA(30 μg)
Susceptible	4 (33.3%)	5 (41.6%)	0 (0%)	8 (66.7%)	5 (41.6%)
Resistance	8 (66.7%)	7 (58.4%)	12 (100%)	4 (33.3%)	7 (58.4%)

Discussion

Fifty samples of chicken liver were taken from different vendor’s shops of Peshawar city and were checked for Staphylococcus aureus, these bacteria are involved in skin infections, meningitis, pneumonia etc., and was said to be antibiotics resistant. Liver samples were immediately brought to the lab for the screening of Staphylococcus aureus spp. First of all microbes were isolated from chicken liver, liver is primary source of many bacteria as it is exposed to gut-derived factor [30], so many bacteria’s including staph attacks it, as chicken liver is undercooked most of time due to this reason it causes Staphylococcal infections., After isolation of microbes purification was done discrete random colonies were taken from isolated plates and were cultured on nutrient agar media, colonies were selected keeping Staphylococcus aureus morphology in mind so round yellow colonies were picked and recultured. After purification screening was done using MSA (Mannitol salt agar), It encourages the growth of a group of certain bacteria while inhibiting the growth of others. It is a selective medium prepared for the isolation of pathogenic Staphylococci, specifically, this agar inhibits growth of gram positive/ negative and Streptococcus and allow Staphylococcus and micrococcus to grow. Staphylococcus aureus produces yellow zone in the agar. After screening process [31]. Disc Diffusion Method was performed main function of Disc diffusion method was to check either to which extent antibiotics is working, Antibiotics were placed on surface of agar where bacterial lawn was made and is incubated for 24 h. After incubation period zone of growth inhibition around each antibiotics disc were measured in millimeter. The purpose of this method is using this measured reference table, the size of zone can be related to MIC to check whether organism is susceptible, intermediately susceptible, or resistant. After disc diffusion method MIC was performed to determine the lowest concentration at which a particular antibiotic works [32]. Five different antibiotics were used including Vancomycin, Moxifloxacin, Linezolid, Gentamycin and Clindamycin. P value of Vancomycin shows five strains on which antibiotic works (out of 12) and seven strains that are completely resistance to Vancomycin, similarly in Moxifloxacin four strains shows that antibiotic is susceptible while other eight strain shows significant growth of bacteria, Linezolid has eight such strain on which antibiotics worked but four strains show massive growth. Gentamycin shows no growth of bacteria as all of its P values are less than 0.05 which means that this antibiotic did not allow any bacterial strain to grow. Clindamycin shows five strain on which antibiotic work while other seven shows significant growth of bacteria, the reason of bacterial growth against antibiotics may be due to excessive use of antibiotics due to which bacteria have developed tolerance, or may be because of mode of action, these five antibiotics have different mode of action, Clindamycin works by inhibiting ribosomal translocation, Vancomycin inhibits cell wall synthesis resulting in inhibition of trans peptidase, Moxifloxacin work by inhibiting DNA gyrase, linezolid works by inhibiting the initiation process of protein synthesis and Gentamycin works by binding 30S subunit of bacterial ribosome may be due to this reason gentamycin is more effective than other antibiotics [33].

Conclusion and Future Prospects

The current study highlights the alarming potential of Staphylococcus aureus isolated from chicken liver to cause various diseases. The pathogens identified exhibited not only resistance to individual antibiotics but also multi-drug resistance, with resistance cut-off values twice as high as those reported in CLSI 2023. This underscores a significant public health concern, emphasizing the need for further research. Future investigations should focus on the molecular identification of these resistant Staphylococcus aureus strains and explore the pathogenic genes responsible for their antibiotic resistance. Additionally, precise breakpoint values for resistant antibiotics need to be established to guide effective treatment and control measures. Addressing these issues is crucial for enhancing food safety and mitigating the risks associated with antibiotic-resistant bacteria.

Authors Contribution

Dr. Sulaiman Faisal supervised the project. Muhammad Ijaz, Gulzar Ahmad, Malik Muhammad Rizwan, Faryal Azam, Ainee Urooj Khan, Saqib Rauf, Hafsa Shah, Noor Muhammad, Zia Ur Rahman, Numan Fazal, Sajjad Ullah, Sidra Ahmad, Shamsher Khan, Muhammad Khan and Dr. Shahid Ullah conducted and analyzed the experiments. All authors contributed to writing and reviewing the manuscript and have agreed for submission.

Funding

This work don’t receive any funding.

Data availability

All relevant data are within the paper.

Declarations

Conflict of Interest

All authors of this work have declared that there is no conflict of interest.