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Multi-drug Resistant and Extended Spectrum β-lactamase Producing Salmonella Species Isolated from Fresh Chicken Liver Samples

Out of 200 samples analyzed, 61 (30.5%) samples harbored Salmonella species out of which 15 (7.5%) samples showed the presence of Salmonella Typhi. A significant association was noted in the incidence of Salmonella with various factors pertaining to the butchers, such as age, sex, literacy rate, practices of washing knives and chopping board, wearing aprons and gloves and type of water used (p < 0.05). Salmonella isolates were highly sensitive to amikacin (82.0%) and least sensitive to tetracycline (3.3%). All the isolates were resistant to colistin. Sixty (98.4%) isolates were identified as multi-drug resistant (MDR). The total number of extended spectrum betalactamase producers reported among Salmonella isolates was 29 (47.5%).


INTRODUCTION
Avian Salmonella infections are significant causes of clinical disease in poultry and a potential source of foodborne transmission of Salmonella in humans. 1 About 95.0% of salmonellosis cases were estimated to originate from food materials and the colonization of Salmonella covers humans and animals including livestock, poultry, rodents and birds. [2][3][4] 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 many developing as well as developed countries. [7][8][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. 10,11 Salmonella serotypes differ significantly in their pathogenic potentials and a study suggested the confirmed cases of Salmonella sp. in the surveillance network Food Net from the period 1996-2006. 12 Chicken liver is an important low-cost source of animal protein, phosphorus, others minerals, and B-complex vitamins; 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. [13][14][15][16] The frequency of isolation of Salmonella strains resistant to several antimicrobial agents has increased in several countries worldwide including Nepal. [17][18][19] 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 slaughterhouses in Bharatpur.

METHODS
A descriptive cross-sectional study was conducted for 5 months from February to June 2018 in Bharatpur Metropolitan City and laboratory analyses were performed at the Microbiology laboratory of Birendra Multiple Campus, Nepal. Random sampling was done to collect 200 non-repeated single whole liver samples from different slaughterhouses located at different places of Bharatpur (Baseni, Dipendra Chowk, Hope Chowk, Junhal Road, Bel Chowk, Malpot Chowk and Gitanagar). An approval was initially sought from the Research Committee of Birendra Multiple Campus to conduct this work. Animal samples were processed according to the animal research ethical guidelines. The sample size was determined in accordance with the incidence rate based on the previous study. 8 Properly labeled fresh chicken liver samples were included in this study whereas refrigerated and improperly labeled samples were excluded. 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 a brief interview using semi-structured questionnaires and through observations as well.
Whole 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 o C for 24 h to obtain a 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 o C 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. Blackcentered red colonies on XLD agar were sub-cultured on NA plates at 37˚C for 24 h to obtain a pure culture of the isolates. 19 For further identification of Salmonella sp., 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 to detect S. Typhi O9, poly O and H antigens.
All the isolates were tested for susceptibility to antimicrobial agents on MHA by Modified Kirby-Bauer disc diffusion method as recommended by the Clinical Laboratory Standards Institute. 20 The antibiotic discs used were amikacin (30 µg), cotrimoxazole (25 µg), ciprofloxacin (5 µg), colistin (10 µg), tetracycline (30 µg), gentamicin (10 µg) and azithromycin (15 µg). The turbidity of inoculums from a pure culture of Salmonella isolates on nutrient agar plates incubated at 37°C for 24 h were adjusted to the equivalent turbidity of 0.5 McFarland standards before spreading uniformly over the surface of Mueller-Hinton agar (MHA) (Titan Biotech Ltd. Bhiwadi-301019, Rajasthan, India) plates. Using sterile tweezers, the antibiotic discs were placed widely spaced aseptically on the surface of the MHA plate. The organism was classified as resistant, intermediate or sensitive according to CLSI performance standards. 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 chi-square test (χ 2 ) was performed using SPSS V. 20 software. P value ≤ 0.05 was assigned to have a significant association. 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).

RESULTS
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%). Moreover, the highest incidence of S. Typhi (20.0%) was reported from samples collected from Junhal Road ( Table 2).
Amikacin was found to be the most effective antibiotic inhibiting the growth of 82.0% of the bacterial isolates followed by gentamicin. 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 multidrugresistant. 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).

DISCUSSION
In the present study, out of 200 samples, 61 (30.5%) were Salmonella positive and 139 (69.5%) were negative. Within the positive samples, 15 (7.5%) were identified as S. Typhi and the remaining 46 (23.0%) were other Salmonella species. This result showed a higher incidence of Salmonella than a study by Gupta which reported that the presence of Salmonella in layer chicken was 9.3%. 23 Similarly, in another study in Yangzhou city, China, between April 2011 and March 2012, a total of 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 which is lesser than the prevalence of Salmonella reported in the current study. 25 These differences might be due to differences in geography, time and season of study.
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). Female butchers usually involve in household activities, children caring and cleanliness which increase their exposures to microbes as compared to the male. This 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 as the people of this age group are mostly involved in children caring 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 The highest number of the sample (37) was collected from Bel Chowk area in which 11 (29.7%) samples showed the presence of Salmonella including 3 S. Typhi. The lowest number of samples (15) was collected from Junhal Road area in which 8 (53.3%) were positive to Salmonella including 3 S. Typhi. Most of the sample collected areas were densely populated and the butchers weren't aware of good hygienic practices while handling the poultry. In every location, most of them used bare dirty hands to slaughter the chicken which might be the reason for the contamination. A significant association was noted between the contamination of liver samples with water sources, practices of washing knives and chopping board and wearing aprons and gloves (p < 0.05). Therefore, the use of municipal water, gloves, aprons, good hygienic environment of the slaughterhouse as well as proper personal hygiene of the butchers should be prioritized.
Amikacin was found to be the most effective antibiotic inhibiting the growth of 50 (82.0%) bacteria followed by gentamicin which was able to inhibit 46 (75.4%) isolates. Thus, amikacin and gentamicin can be used for the treatment against Salmonella species causing various diseases in humans due to the consumption of contaminated poultry products. Colistin was found to be 100.0% resisted followed by tetracycline (96.7%) and azithromycin (77.0%) which means they are not appropriate antibiotics for Salmonella. Moreover, the use of colistin has been banned due to their side effects of nephrotoxicity and neurotoxicity and has been replaced by other antibiotics. 29  of Salmonella sp. as ESBL producer which is very low compared to our study. 31 Extreme and haphazard use of broad-spectrum antibiotics might be associated with a higher rate of ESBL production in Salmonella.
This study only determines the prevalence of Salmonella species and their MDR pattern with special focus on ESBL production. The source of contamination of meat was not assessed though. We were unable to perform the molecular characterization of bacteria due to the financial and laboratory scarcity. Future studies should address these limitations.

CONCLUSION
The present study shows that chicken meat sold at Bharatpur Metropolis are contaminated with MDR and ESBL producing Salmonella sp. This suggests a dire need of taking initiatives to control the dissemination of such pathogens. Haphazard use of antibiotics, poor personal hygiene, illiteracy rate of the butchers, improper handling and storage practices are some of the factors concerned authorities should address.