Screening, Isolation, Identification and Antibiogram Study of Enterobacteriaceae in Ready to Cook, Chilled Food Products in Tiruchirappalli, India

The present study was concentrated to screen some members in Enterobacteriaceae family from chilled meat products procured from different retail shops in Tiruchirappalli, Tamilnadu. A total of six varieties of ready to cook chilled food products with five samples in each were randomly purchased from departmental stores, retailer meat shops and local vendors of Tiruchirappalli. Out of 30 ready to cook, chilled food products screened for the presence of Enterobacteriaceae, 28 found to be positive for Enterobacteriaceae. A total of 36 bacterial strains were selected at random and identified. Only 11 isolates were finally confirmed as Enterobacteriaceae and this was shared by Escherichia coli (E. coli), Citrobacter spp., Salmonella spp., Serratia spp. and Proteus spp. Among these Proteus spp. (23.3%) was found predominant in all the samples. Antibiogram study revealed that 54.5% isolates were susceptible to each of Ofloxacin and Ciprofloxacin followed by Ampicillin (45.5%), Chloramphenicol (27.3%) and Gentamycin (18.2%). A high percentage of 54.5% isolates were found to be multidrug resistance (resistant to 3 or more antibiotics). E. coli and Proteus spp. isolated from mixed vegetables and beef respectively, were exhibited 100% resistant to Penicillin, Ciprofloxacin, Chloramphenicol, Ofloxacin, Ampicillin and Gentamycin. The study revealed poor sanitation and cross-contamination in food processing area which resulted in the enhancement of enteropathogenic bacteria which are, known to cause foodborne illnesses. Also, the multidrug resistance noticed in the present study may be linked to the use of antibiotics in cattle rearing which constitute a serious threat to public health.


iNtROduCtiON
The emergence of multidrug resistant bacteria has been reported worldwide and it is a major public health concern 1 . The most important gram-negative bacteria found in the intestinal tract belong to the family Enterobacteriaceae is the challengeable contaminants in raw and cooked food products 2 . Coliforms are indicators of faecal contamination in food 3 . Enterobacteriaceae in ready to cook food products causes foodborne illness associated with mild to severe diarrhoea due to gastrointestinal disorders and clinical manifestations in human 4 . Enterobacteriaceae such as E. coli and Salmonella species are most predominant in some meat products 5,6 . E. coli are considered as a sepathogenic, non-pathogenic and commensal organism found in the intestinal region of both animal and human 7 . Salmonella is the predominant foodborne pathogen causing salmonellosis due to the consumption of undercooked meat and eggs 8 .
Worldwide, 2.2 million adults and children under five years of age die of diarrhoeal disease every year from foodborne and waterborne illnesses. In developing countries, the major cause of foodborne infections is the cross-contamination of meat and raw vegetables by polluted water, soil, air and unhygienic handling. The major factors which affect the quality of perishable foods are microbes present within the animal before slaughtering, enzymatic changes and external sources during processing, microbial accumulation after slaughtering within the tissues, time and temperature of storage, handling at the distribution center 9 . In general, the rate of deterioration is higher in seafoods because of decay organisms present in their gastrointestinal system which rapidly multiply after death 10 . Nowadays, due to lifestyle modernization and raised standard of living, the consumption of ready to cook fresh slaughtered chilled meat, seafoods and vegetable products have increased in India 11,12 . Therefore, the present study was planned to determine the presence of members of Enterobacteriaceae and to study the antimicrobial resistance of isolated gram-negative bacteria in different ready to cook foods obtained in Tiruchirappalli, Tamilnadu.

MAteRiAls ANd MethOds
T

Media preparation
All the media used in the study were prepared according to the manufacturer's instructions.

Sample Collection
A total of six different chilled ready to cook food products with five samples in each such as raw meat samples (chicken, pork and beef meat), seafood samples (fish and prawns) and mixed vegetables were purchased at random from local vendors, departmental stores and local meat retailers located in Tiruchirappalli, Tamilnadu. The samples were transported immediately to the laboratory for microbial evaluation under the packaging condition provided by the retailers and stored under same condition until analysis.

Sample Preparation
Twenty-five grams of all chilled ready to cook samples were finely chopped with a sterile knife and transferred to 225 ml of 10x Phosphate Buffer Solution (PBS) and incubated for 24 h at 37°C for pre-enrichment. The enrichment process was done by transferring 1 ml of pre-enriched broth to 9 ml of Lactose broth, Selenite F broth and Tetrathionate broth (Himedia, Mumbai) and incubated at 37°C for 24 h. A loop full of each enriched broth was streaked on to MacConkey agar plates and Xylose Lysine Deoxycholate (XLD) agar plates (Himedia, Mumbai) in triplicates. The probable morphologically distinct colonies were picked up and streaked on to nutrient agar media (Himedia, Mumbai) and incubated at 37°C for 24 h. Then the pure colonies were subjected to various biochemical analysis for identification.

Identification of Isolated Bacteria
The isolated bacteria were identified using colony morphology and biochemical tests 14,15 . E. coli colonies were further re-streaked on Eosin Methylene Blue agar (EMB) (Himedia, Mumbai) and incubated at 37°C for 24 h for reconfirmation. Colonies with a green metallic sheen were confirmed as E. coli. Black colonies on XLD agar plates were further tested with urease broth. The urease positive was confirmed to be Proteus sp. and Salmonella sp. was confirmed by the H 2 S formation in Triple Sugar Iron (TSI) agar and negative urease tests. The production of red prodigiosin pigment in nutrient agar confirmed the presence of Serratia sp. Citrobacter sp. was confirmed by colony morphology and other biochemical tests such as Indole, Methyl Red, Voges-Proskauer, Citrate (IMViC), Lysin Iron Agar (LIA), Motility Indole Ornithine (MIO), Oxidase, Catalase, Extracellular enzymatic activities test such as Amylase, Protease and Lipase. Oxidative Fermentative test using OF media and carbohydrate fermentation test using Phenol Red Broth (PRB) (Himedia, Mumbai) were performed with glucose and sucrose.

Antibiotic susceptibility test
The antibiotic sensitivity test was performed against isolated colonies by Kirby-Bauer disc diffusion method as described by CLSI standard guidelines 16 . The antibiotics used were Penicillin (P, 10 units), Ciprofloxacin (CIP, 5 mcg), Gentamycin (GEN, 10 mcg), Ofloxacin (OF, 5 mcg), Chloramphenicol (C, 30 mcg) and Ampicillin (AMP, 10 mcg). The 24 h fresh culture of isolated gramnegative bacteria was streaked on to Nutrient Agar (NA) plates and incubated at 37°C for 18-20 h. The isolated single colony of all bacterial cultures were inoculated into the nutrient broth and incubated for 4 h at 37°C until the appearance of moderate The overall Multidrug resistance was found to be 6 out of 11 isolates with 54.5% (S-Susceptible, I-Intermediate, R-Resistant, P-Penicillin, CIP-Ciprofloxacin, GEN-Gentamycin, OF-Ofloxacin, C-Chloramphenicol,

AMP-Ampicillin)
Journal of Pure and Applied Microbiology turbidity. Then the bacterial growth as measured by optical density (OD 600 ) and adjusted. With the sterile wooden swab sticks the bacterial culture was swabbed on 4 mm depth Muller Hinton agar (Himedia, Mumbai) and incubated for 24 h at 37°C with antibiotics discs.

Results
In the present study, 30 ready to cook chilled raw food samples were collected and tested for the presence of members of Enterobacteriaceae bacteria. It was found that only 28 samples showed positive. Among 28 samples, 36 suspected strains were isolated and only 11 (36.6%) bacterial isolates were confirmed to be that of Enterobacteriaceae (Table 1). The biochemical tests revealed the presence of five pathogenic bacterial isolates viz. E. coli, Citrobacter sp., Salmonella sp., Serratia sp. and Proteus sp. Proteus sp. was the most predominant bacteria in all the chilled meat (chicken, beef, pork) and seafood products (fish, prawn). Salmonella sp. and Proteus sp. were recovered from ready to cook chicken sample. From ready to cook chilled beef sample, Citrobacter sp., Serratia sp. and Proteus sp. were isolated. It was found that minimally processed or unprocessed chilled ready to cook food samples were highly contaminated with Enterobacteriaceae. The antimicrobial susceptibility study indicated the resistance of all isolates to penicillin (100%) followed by 72.7% of the isolates to Gentamycin, 54.4% to Ampicillin, 36.4% to Ciprofloxacin, and 27.2% each to Ofloxacin and Chloramphenicol. The gramnegative bacteria of E. coli and Proteus sp. isolated from chilled ready to cook mixed vegetables and beef samples have shown 100% resistance to all the six antibiotics used in this study. 54.5% of bacterial isolates were found to be multidrug resistance (resistant to 3 or more antibiotics) ( Table 2). All the isolated 5 species were found susceptible to Ofloxacin (54.5%), Ciprofloxacin (54.5%), Ampicillin (45.5%), Chloramphenicol (27.3%) and Gentamycin (18.2%). As a result, the isolates were highly susceptible to Ofloxacin and Ciprofloxacin (54.5%) and highly resistant to Penicillin (100%) followed by Ampicillin (54.5%) ( Table 3).

disCussiON
A total of 11 strains were isolated from 28 ready to cook, chilled samples. The occurrence of high Enterobacteriaceae count indicated that there was poor sanitation during slaughtering, storage and hygienic condition inside the butcher's shop which became good sources of crosscontamination from knife, cloth and processing area. Vegetables might get contaminated from manure, water and handling. Hence, this study has clearly shown that the occurrence of different members of Enterobacteriaceae species were common in chilled vegetables and retail meat. In this study, Proteus sp. was present in all the chilled meat and sea food samples with an incidence of 23.3% and is the most predominant strain isolated from chicken, beef, pork, fish and prawn. The predominance of Proteus sp. might be due to cross-contamination by food handlers while removing intestinal content by hands. It is important to wash their hands after processing any food materials to prevent cross-contamination due to the rapid proliferation of the pathogen. In Tiruchirappalli, seafoods are transported from various places of Tamilnadu. The collected sea food samples were observed to be in broken packages. Also, it was suspected to be contaminated by water and the quality of ice used during transportation. Proteus sp. is considered as an indicator of meat contamination during processing. Many researchers have isolated Proteus sp. from different food samples [17][18][19] . In addition, Proteus sp. has been proved to cause urinary tract infection and diarrhoea 20 . E. coli contamination was prevalent among vegetables. The frequency of E. coli in vegetables was found to be high. This study revealed the presence of 3.3% of E. coli in mixed  26 who recorded 89% resistance to Penicillin and 65.8% resistance to Ampicillin. It indicated the indiscriminate use of antibiotics as a growth factor in poultry and cattle. Increasing emergence of horizontal gene transfer could disseminate the virulence factor to other strains of same species or different species, might also be the reason for developing multidrug resistance.

CONClusiON
The study showed the presence of Enterobacteriaceae in all the collected samples of ready to cook, chilled food products. Personal hygiene, storage temperature maintenance and handling during production could have been the reason which led to the microbial contamination.
The knowledge of thawing and storing chilled food should be made aware to the public. Also, improper cooking and storage temperature lead to foodborne illness. The regulated food laws should be followed by both local retailers and consumers. The emergence of multiple antibiotic resistance by the isolated organisms may be occurred due to horizontal gene transfer which has become a global threat to public health.