DETERMINATION OF ANTIBIOTIC RESISTANCE OF SALMONELLA SPP. ISOLATED FROM ICE CREAM

The current study sought to determine the extent of antibiotic resistance and prevalence among Salmonella spp. The Salmonella spp. Antibiotic resistance threat and bacteriological safety of food goods like ice cream present therapeutic management problems within the public health system. Salmonella bacteria are Gram-negative, facultative anaerobic, flagellated bacilli containing antigens like V, Hi, and O. Nearly 1800 serovars have been found. Each of them is categorized as a different species under the current classification system. To obtain the concentration of food borne pathogens, Ice cream was gathered from various locations and put on SS and MacConkey agar to test for the presence of Salmonella spp. in ice cream. Azithromycin, gentamycin, cefotaxime, and ciprofloxacin were among the many classes of antibiotics present in the SS agar on which it was also dispersed. To lessen the burden of Salmonella spp. prevalence in milk, the study advises farmers and raw milk vendors to adopt substantial interventions in animal husbandry and milk marketing, respectively. Additionally, the enforcement and animal health divisions’ active involvement in ensuring prudent antibiotic usage at the farm level may aid in limiting Salmonella spp. antimicrobial resistance. Antibiotic resistance might be reduced by concentrating on alternative remedies for treatment of GIT infections including the use of probiotics, organic approaches, and preventing the horizontal gene transfer of resistant genes.


Introduction
Food borne disease has great impact on health of the society. The only method to recognize the cause and source of the food borne diseases and overcome this issue is controlling the cause of spread. The identification of the cause of food borne disease is very difficult and log process including bundle of various steps. This also includes eating pattern and transferring mode of the contaminated foods (Jacobs's et al., 2020). Salmonella infection causes fever, diarrhea, and stomach cramps 1 to 3 days after the onset of first infection. Symptoms usually last 4 to 6 days, although in certain cases, the patient may have needed to admit to the hospital. Despite the fact that there are a lot of possible routes, the intake of raw meat is a source of Salmonella transmission, or undercooked or raw eggs or poultry are known to be of concern of great significance. Salmonella species have wide variety with number in thousands. Different serotypes have been identified, each with its own set of characteristics and their transmission channels. Salmonella enteritidis, for example, is frequently connected with eggs, but Salmonella typhimurium is related with a broader range of foods. Infections caused by Salmonella are more common in the summer because it is climate sensitive. Studies demonstrate that Salmonella infections are higher in warm temperature. Furthermore, since Salmonella may thrive in food maintained at room temperature, there is a strong biological knowledge of the processes involved. As a result, Salmonella infections may rise as the planet warms (Akil et al., 2014). The number of infections is currently reducing across Europe as a result of successful intervention, such as better biosecurity, animal immunization, and slaughtering. Salmonella has a close relationship with the environment, particularly the ambient temperature. Salmonella reproduction is aided by higher ambient temperatures. Despite this biological process, UK infectious disease specialists believe Salmonella is not one of the illnesses most likely to be impacted by change in climate (Lake, 2017). Salmonellosis is regarded as one of the most serious zoonotic diseases caused by bacteria that may affect animals, humans, and fowl. There are around 2500 Salmonella serovars in the world (Dróżdż et al., 2021). Salmonella enterica typhi causes typhoid fever and Salmonella enterica Paratyphi cause paratyphoid fever in humans. Whereas, salmonellosis is an umbrella term that encompasses invasive infection with all Salmonella serovars as well as the normally gut-confined infections of food poisoning (Alphons et al, 2014). Several additional Salmonella serovars, rather than paratyphi and typhi, cause non-typhoidal salmonellosis. Salmonella typhimurium and S. enteritidis, which may be transferred to humans, are widely found in poultry and animals. Human salmonellosis epidemics have been linked to animal products such as milk, chicken meat, eggs, wild, domestic and aquatic animals, pets, and rodents (Teklemariam et al., 2023). It has been shown that the chicken industry may be responsible for up to 50% outbreaks caused by Salmonella. Serovars of Salmonella have identified as a significant and dangerous food-borne zoonosis with public health implications. Typhoid fever outbreaks and occasional cases in humans are widespread throughout the world (Ferrari et al., 2019). Although gastrointestinal symptoms are the most common sign of typhoid fever, other clinical syndromes such as pulmonary disease and disseminated intravascular coagulation can also occur. The clinical illness manifests as diarrhea with vomiting, fever, abdominal discomfort, nausea, abdominal, bradycardia, headache, and cough between 12-72 hours of infection. The disease normally lasts 4-7 days without antibiotic therapy before fully recovering. The intensity of the sickness worsens among the immunocompromised people, elderly, very young, and the diarrhea might be severe enough to require hospitalization. In such individuals, infection can spread and infiltrate the hepatobiliary system, leading to mortality if antibiotics are not administered. Cirrhosis of the liver, malignancies, acute pancreatitis, renal failure, and liver abscess are all seen in complicated instances because of the numerous risks linked with the use of various antibiotics, particularly in zoonotic illnesses, bacterial resistance in humaninfecting strains has become quite prevalent (Abudabos et al, 2020). Human-to-human transmission happens by direct in-contact with animals, through environment, or through consumption of eggs, meat and milk, or items containing eggs. It has been reported that Salmonella that is resistant to cephalosporin has emerged in Egypt. The incidence of multidrug-resistant (MDR) isolates has been documented, and this has had an impact on typhoid fever in impoverished nations. Salmonella typhi AMR genes isolated from 29 percent of patients demonstrated resistance to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole in a research conducted in Lower Egypt. In diarrheic Egyptian individuals with Salmonella typhoid illness, multiple quinolone resistance has been found (Marchello et al., 2020). Ampicillin, trimethoprim-sulphamethoxazole tetracycline and chloramphenicol resistance was found in high numbers of individuals with non-typhoid Salmonella isolates (Chong et al, 2017). In addition, non-typhoid Salmonella isolates had a significant incidence of AMR genes to chloramphenicol and tetracycline. In Egypt and Algeria, a total of 76 nontyphoid Salmonellae were recovered from poultry and humans, with a significant rate of cephalosporin resistance among the isolates. Eissa et al. recently revealed that individuals with typhoid fever were resistant to combination cefotaxime and ciprofloxacin therapy. Typhoid Salmonella is found in only humans and could be spread by oral and fecal contact or by an in direct contact with an infected people. It is characterized by a high temperature that lasts for a long time hepatosplenomegaly, rose patches, neutropenia leukopenia, as well as relative bradycardia (Abd El-Ghany, 2020). The condition is essentially cured by in an undiagnosed patient at the conclusion of the fourth week. The symptoms include chills, a high temperature, stomach discomfort, and a headache. Typhoid is a bacterial infection caused by S. typhi. ecause of the existence of the resistome, Salmonella develops resistance to most antibiotics and multidrug resistance (MDR) develops. A resistome is a collection of bacterial genes that are resistant to drugs. here are a few therapeutic options available for salmonellosis, however they are ineffective. The parenteral nutrition commercially available Vɩ vaccine and liveattenuated Ty21a vaccination are only marginally effective in preventing this illness. These vaccinations also have certain drawbacks, including short-term immunity in adults and limited efficacy in children under the age of two (Kumar, 2022). arge number of bacteria are responsible for many food borne diseases. Most of them also develop resistance against antibiotics. Salmonella spp. is leading cause of gastrointestinal infections. With increasing use of antibiotics, antibiotic resistance has become the global issue. The aim of this study is to check prevalence, and antibiotic resistance of Salmonella spp. isolated from ice-cream. Objective of this study are analysis of ice cream to determine the prevalence of Salmonella spp. and determination of antibiotic resistance in isolated Salmonella spp.

Materials and methods Sample collection and transportation:
Since the sample being tested is ice cream, it was collected in an ice-chest to prevent melting and facilitate handling. 10 samples were collected from nearby ice cream point and bakeries that located such as jay bees, gourmet, and the cafeteria of Lahore College for Women University Punjab, Pakistan.
The prevalence of Salmonella is the most common in dairy products. So, all collected ice creams samples were dairy ice creams. For the collection of the sample, first containers were sterilized by autoclaving them then used them for the sample collection purpose. In the table 1 sample name were given just for convince and prevent them from sample mixing. Here ICD is for dairy ice cream and ICND is for non-dairy ice cream  (Salman et al., 2021). Sterilization of materials: Micro tips have been autoclaved at 180 to 220C for 20 minutes with prepared MacConkey media and petri plates. The UV light was turned on for ten to twelve minutes. After that, the laminar flow was held on for a little while, followed by sterilizing it with an ethanol spray and drying it with a cotton plug. Subsequently, the spirit lamp was ignited. Gloves were worn as a precaution, and hands had been sterilized (Sastry and Bhat, 2018).

Media pouring:
The temperature of the media was reduced to a certain extent, following which it was introduced into the laminar airflow. Each plate was filled with 20ml of the media and allowed to dry for a period of time. Subsequently, the plates were covered with their respective lids and appropriately labeled. (Sastry and Bhat, 2018).

Spreading of sample:
The work was conducted within the laminar airflow. For each sample, a volume of 200-300µl was extracted using a micro pipette. The sample was then poured onto the media, while the spreader was sterilized with alcohol. Subsequently, the spreader was heated on the spirit lamp and sterilized again using the alcohol present in the petri plate within the laminar airflow area. The spreader was rotated in both clockwise and counterclockwise directions to ensure complete absorption of the sample onto the media. The petri plates were covered with lids after the procedure was repeated for the remaining six plates. Any remaining sample was discarded. (Siddique et al., 2021). Incubation: The set of plates were placed in incubator for overnight at 27C (Leng et al., 2019). Data analysis: After 24 hours, the plates were removed from incubator and count CFU of bacterial species for data analysis.

Results
The results are analyzed by counting the colonyforming units on the petri plates. These species of bacteria could be differentiated by color and morphology of bacteria. Salmonella and Shigella gives pink or sometimes reddish brown (medium-colored) colonies on the media. On SS Agar Salmonella produced black-centered colored colonies. Shigella produced colorless colonies on the SS Agar. On the other hand, E. coli gives pink to red color on the SS Agar. Table 2 indicates that Salmonella is prevalent in the dairy ice creams. This tabular data shows that there is not only Salmonella but also the prevalence of Shigella and E. coli also observed . Fig 1shows the number of Shigella is in excess but Salmonella is in very small quantity. Figure 2 shows that on MacConkey agar the number of E. coli is in excess. There are some resistant colonies that grow on SS Agar containing a different class of antibiotics such as gentamycin, ciprofloxacin, cefotaxime, and azithromycin as shown in table no 2 and 3. These results indicate bacterial colonies are less resistant to ciprofloxacin and cefotaxime while gentamycin and azithromycin are strong antibiotics.

Discussion
The prevalence of bacteria such as Salmonella, Shigella, and E. coli, which are responsible for food poisoning and foodborne diseases, is alarmingly high. Antibiotics are commonly employed as therapeutic agents to combat these bacterial infections. Regrettably, our bodies are progressively developing resistance to these medications. It is important to note that foodborne illnesses are not solely limited to typhoid; Salmonella poses a significant global public health concern. In the United States, laboratoryconfirmed cases of salmonellosis among infants reached 121.6 infections per 100,000 infants, which is approximately eight times higher than the incidence observed in other age groups. It is worth mentioning that there is a scarcity of data on this topic in numerous other countries, particularly in developing nations (Yang et al., 2014).
Third-generation antibiotics such as gentamycin, azithromycin, cefotaxime, and ciprofloxacin are commonly utilized in clinical practice. SS Agar, a culture medium, consists of various constituents including pancreatic casein digestion, beef extract, sodium thiosulphate, bile salts, brilliant green, agar, peptic digestion of animal tissues, lactose, sodium citrate, ferric ammonium citrate, and neutral red. The medium maintains a pH of approximately 7. Its selectivity is primarily based on its ability to inhibit the growth of Gram-positive bacteria. The inclusion of lactose in the medium allows for the detection of acid production by lactose-fermenting microorganisms, which results in the formation of red colonies when reacting with the neutral red indicator. On the other hand, lactose non-fermenters produce colorless colonies. Salmonella Shigella (SS) Agar, serving as a moderately selective and differential medium, is employed for the isolation, cultivation, and differentiation of Salmonella spp. In contrast to lactose fermentation, Salmonella bacteria produce hydrogen sulphide (H2S) gas. Consequently, the resulting bacterial colonies exhibit a white color with black centers (Meteab and Abed, 2018).
Dairy-based ice cream predominantly comprises milk, eggs, and egg products, which provide favorable conditions for the growth of Salmonella bacteria. These ingredients serve as nutrient-rich substrates, facilitating the proliferation of Salmonella within the ice cream. Analysis of the tabular data presented in Tables 1 and 2 suggests that local vendors and their handling practices of ice cream, as well as the storage compartments or containers utilized by them, contribute to the creation of an environment conducive to the flourishing and propagation of these bacteria.
We do not consume food in microliters, despite the fact that all of the bacteria in the sample were put into microliters and their prevalence in food items was close to 200 CFU. We often calculate our food intake in milliliters and liters. So the incidence of the bacteria will rise, reaching a maximum of 200 x 5 = 1000 CFU, which will undoubtedly be the cause of food poisoning. As shown by the data analysis in the mentioned tables 1 and 2 which shows the concentration of bacteria, particularly Salmonella, in each serving, our consumption of bacteria present in food is excessive, which is what causes food related illness. Salmonella CFU concentrations are frequently excessive in ice cream purchased from local vendors or produced in unsanitary circumstances. Under conditions where stringent hygiene standards are maintained and samples are collected in a controlled environment, the presence of bacteria is minimal, and no colonies are observed on antibiotic-containing media. The study area under investigation represents one of the most socioeconomically disadvantaged regions in Pakistan, characterized by prevailing poverty and a higher burden of illness. In this context, the current study demonstrated elevated prevalence rates of Salmonella spp. across various areas in Southern Punjab, indicating a heightened frequency of salmonellosis. Notably, in the Multan area, the overall incidence of Salmonella spp. was found to be 25.89%, while comparable findings revealed 21.89% prevalence in other sampled locations. Previous research also reported prevalence levels ranging from 7.61% to 11.9% for Salmonella spp., attributing these variations to factors such as sanitation practices, training of food handling personnel, and overall hygiene conditions (Qamar et al., 2020).