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Multi-drug Resistant (MDR) and Extended Spectrum β-lactamase (ESBL) Producing Salmonella species isolated from fresh chicken liver samples

Objective: Emergence of antibiotic resistance among microbes contaminating the fresh meat and meat products is a global public health concern as they can be easily transmitted to humans through their consumption and contact. The current study aimed to isolate and identify Salmonella sp. from fresh chicken liver samples and determine their antibiotic susceptibility patterns with special emphasis on multidrug resistance (MDR) and extended spectrum beta-lactamase (ESBL) production. Results: Out of 200 samples analyzed, 61 (30.5%) samples harbored Salmonella species among which 15 (7.5%) samples showed the presence of Salmonella Typhi isolates. A significant association was noted in the incidence of Salmonella with various factors pertaining to the butchers such as age, sex and literacy rate. Salmonella isolates were highly sensitive to amikacin (82.0%) and least sensitive to tetracycline (3.3%). All the isolates were resistant to colistin. Moreover, 56 isolates were identified as multidrug-resistant. The total number of ESBL producers reported among Salmonella isolates was 29 (47.5%). The study reported the presence of antibiotic-resistant Salmonella species in fresh chicken liver samples sold in Bharatpur metropolis suggesting a need of serious attention by the concerned authorities.


Introduction
Avian Salmonella infections are important causes of clinical disease in poultry and a potential source of foodborne transmission of Salmonella in human [1]. About 95.0% of salmonellosis cases were estimated to originate from food materials [2] and the colonization of Salmonella covers humans and animals including livestock, poultry, 3 rodents and birds [3] [4]. The adaptive ability of pathogen itself, the changing characteristics of the population, the increasing globalization of the food trade, and the changes in industrial structure, poor hygienic environment, improper storage or cooking, cross-contamination, infected stocks contribute to the development of Salmonella in poultry and poultry products leading to the major source of human foodborne illness and loss of product shelf-life [5] [6].
Poultry products have always topped the incidence of salmonellosis in India, Egypt, Brazil, Zimbabwe, Nepal and other developing countries [7] [8] and are the most seriously perceived food risks in chicken meat, even in the developed countries [9]. The incidence of human salmonellosis has increased greatly over the past 20 years and this can mostly be attributed to epidemics of S. enteritidis in poultry in numerous countries [10] [11].
Salmonella serotypes differ significantly in their pathogenic potentials and a study suggested the confirmed cases of Salmonella sp. in the surveillance network FoodNet from the period 1996-2006 [12]. Chicken liver is an important low-cost source of animal protein, rich in nutrients, phosphorus, others minerals, and B-complex vitamins [13]; however, the presence of MDR resistant Salmonella sp. in chicken livers have become the solemn concern of food safety and one of the major public health problems [14][15] [16]. Different food items have been documented as a reservoir of ESBL producing bacteria and such food items are probable sources for the acquirement of beta-lactamase-producing bacteria. The frequency of isolation of Salmonella strains resistant to several antimicrobial agents has increased in several countries worldwide including Nepal [17][18] [8]. Thus, the purpose of the present study was to determine the prevalence of MDR and ESBL producing Salmonella sp. from chicken livers sold at different slaughter houses in Bharatpur.

Main Text
A cross-sectional study was carried out among the slaughter houses of Bharatpur  [8]. Each butcher was briefed of the purpose of sample collection and verbal informed consent was taken assuring them of total confidentiality. Slaughterhouse's sanitary and salubrious status was studied by brief interview using semi-structured questionnaire and through observations as well.

Methodology
Fresh chicken liver samples were collected separately in sterile zip-locked plastic bags with the help of sterile forceps and scissors, stored in cold box and transported aseptically to the laboratory for further processing within an hour. The samples were ground in sterile mortar and pestle to make fine particles and 1 g of them was inoculated into 9 ml of distilled water and dilutions up to 10 -5 were made. From each of 10 -3 , 10 -4 and 10 -5 dilutions, 0.1 ml of inoculum was spread in nutrient agar plates in triplicate and incubated at 37℃ for 24 h to obtain viable count of the bacteria. For the isolation of Salmonella, 1 ml of the inoculum was enriched in Selenite F-broth (Himedia, M025S) and incubated at 37℃ for 24 h. A loopful of culture in Selenite F-broth was directly streaked on XLD agar (Himedia, MH031) and incubated at 37˚C for 24 h. Black-centered red colonies on XLD agar were sub-cultured on NA plates at 37˚C for 24 h to obtain pure culture of the isolates [19]. For further identification of Salmonella species, Gram staining and various biochemical tests (SIM, MR-VP, citrate, catalase, oxidase, urease and TSI) were performed.
A slide agglutination test using antisera (Statens serum institute, Copenhagen) was used 5 to detect S. Typhi O9, poly O and H antigens.  [20]. Resistance to more than three structural classes of the antimicrobials tested was considered as MDR [21]. Salmonella Typhimurium ATCC 14028 was used as a reference strain for quality control purposes.
Primary screening test for ESBL production was done by using ceftazidime and cefotaxime discs against which the organisms showing the zone of inhibition ≤22 mm for ceftazidime (CAZ) (30 µg) and ≤27 mm for cefotaxime (CTX) (30 µg) were considered to be probable ESBL producers. The phenotypic confirmatory test was done for suspected ESBL producing isolates for which antibiotics combinations of ceftazidime + clavulanic acid (CAZ/CAC) (30/10 µg) and cefotaxime + clavulanic acid (CTX/CTC) (30/10 µg) were used according to the protocols recommended by CLSI [22]. An increase in the zone of inhibition by ≥5 mm around the discs containing cephalosporin with clavulanate over the discs containing cephalosporin alone were ESBL producers [22].
The data obtained from laboratory investigation were tabulated and presented in defined tables and p-value of the obtained results was calculated using SPSSv20 software. P-value 6 ≤ 0.05 was considered to have a significant association.

Results
Total viable counts for the collected samples ranged from 7.8×10 4 -1. and from the butchers without apron and gloves (52.8%). A significant association was noted between the contamination of liver samples with the age, gender and literacy rate of butchers, type of water used, practices of washing knives and chopping board and wearing aprons and gloves (p ≤ 0.05) ( Table 1).

Area-wise variation in the isolation of Salmonella
Out of seven different locations the samples were collected from, the highest proportion of Salmonella sp. was recovered from Junhal Road (53.3%) followed by Baseni (40.9%) and Malpot Chowk (37.0%). Samples collected from Hope Chowk showed the lowest prevalence of Salmonella (12.9%) ( Table 2). 7 Antibiotic susceptibility pattern, MDR and ESBL Producers Amikacin was found to be the most effective antibiotic inhibiting the growth of 82.0% of the bacterial isolates followed by gentamicin which was sensitive to 75.4% of the isolates.
All of the isolates were resistant to colistin. A large proportion of the isolates showed resistance to tetracycline (96.7%) and azithromycin (77.0%). Out of 61 isolates, 60 (98.8%) isolates were found to be multidrug-resistant. The frequency of Salmonella isolates that gave ESBL screening test positive was found to be 33 (54.1%). The confirmed ESBL production was reported in 29 (47.5%) isolates (Table 3).   between April 2011 and March 2012, total 240 chicken carcasses were tested, and the overall contamination rate for Salmonella was 33.8% [24]. However, the incidence of Salmonella in the present study is higher than a study by Shrestha et al who isolated 26.2% Salmonella in poultry meat in Chitwan district of Nepal [8]. In another study in the same district, 26.1% occurrence of Salmonella was reported from the poultry meat samples [25] which is lesser than the presence of Salmonella reported in the current study. These differences might be due to differences in geography, time and season of study among the researchers.
A large number of Salmonella sp. (52.6%) was isolated from the meat collected from female butchers compared to male butchers (28.2%). There was a significant association between the gender of the butchers with the number of Salmonella isolates (p ≤ 0.05).
Females usually involve in household activities, children caring and cleanliness and mainly for various physiological reasons chances of microbes present in female might be comparatively more as compared to the male which may possibly lead them to be the carrier of bacteria and cause more contamination in the food products they handle [26] [27]. The occurrence of Salmonella in the fresh chicken liver sample was significantly affected by age of butchers (p ≤ 0.05). In the current study, maximum contamination was found in the age group 36-45 years probably due to the lack of sanitation and personal hygiene because the people of this age group are mostly involved in children caring, raring and cleaning which might make them more likely to be contaminated with bacteria.
The presence of Salmonella in liver samples was significantly affected by the literacy rate of butchers (p ≤ 0.05). This might be due to the lack of knowledge about the importance of sanitation in illiterate ones. In contrast, the literate butchers might know the importance of sanitation and so they use clean water and clean the slaughter area frequently [28].   [31].
Extreme and haphazard use of broad-spectrum antibiotics might be associated with a higher rate of ESBL production in Salmonella.

Conclusion 12
The present study shows that chicken meat sold at Bharatpur Metropolis are contaminated