Salmonella in Indian ready-to-cook poultry: antibiotic resistance and molecular characterization

The availability and popularity of processed, ready-to-cook (RTC) poultry products are increasing in India. Though fresh poultry is known to be contaminated with Salmonella, the prevalence of this foodborne pathogen in RTC poultry products is not reported. Eighty-seven chilled and frozen RTC poultry samples of 4 different brands obtained from supermarkets and departmental stores in Mumbai were analyzed for the presence of Salmonella. The prevalence of Salmonella was higher (51%) in chilled RTC samples as compared to the frozen RTC samples (5%). The frozen RTC samples of one brand were free from Salmonella. S. Typhimurium (75.2%) was the most prevalent serovar, followed by S. Enteritidis (23%) and S. Weltevreden (1.7%). A high percentage (81.4%) of the isolates were found to be resistant to 5 or more antibiotics and class 1 integron, which has been shown to confer multi-drug resistance, was detected in 69.9% of the isolates. Multiple antibiotic resistance index of isolates was high (0.6) indicating the indiscriminate use of antibiotics during poultry farming. High genetic diversity was observed among the Salmonella serovars based on Pulsed Field Gel Electrophoresis profiles. Results showed the presence of multi-drug resistant Salmonella serovars in processed, chilled RTC poultry products marketed in Mumbai, India.


Introduction
Salmonella is one of the most important foodborne pathogens.Poultry meat, eggs and foods of animal origin are important sources of human Salmonella infections. 1,27][8] A multistate outbreak of Salmonella Heidelberg infections linked to foster farm brand chicken has been reported. 9mergence of multi drug resistant Salmonella has been reported worldwide and it is a major public health concern. 6,10- 12The antimicrobial resistance is frequently associated with integrons, transposons, and plasmids, which are involved in horizontal transfer of antibiotic resistance genes among bacteria and increase in the overall resistance gene pool. 11,13,14Integrons are genetic elements able to capture, integrate and rearrange open reading frames (ORFs) embedded in variable regions of genes cassette units and convert them to functional genes by ensuring their correct expression. 15Integrons do not transfer themselves; instead facilitate transmission of antibiotic resistance genes via transposons or conjugative plasmids. 16Class 1 integron, the most common integron located on Salmonella genomic island 1 (SGI 1), has been detected in different Salmonella serovars such as S. Typhimurium, S. Bareily, S. Oslo, and S. Newport in several countries. 4,10,13There are reports on multidrug resistant Salmonella isolated from India. 4,13,17However, incidence of Salmonella in RTC food samples in India is not well documented.Moreover there are very few studies on molecular characterization of these Salmonella isolates. 4,18resh poultry slaughtered in local shops is generally preferred by consumers in India.However, due to changes in life style and modernization, fresh chilled and frozen RTC poultry products has become readily available in retail high end shops and supermarkets in major cities.But, there are few studies on the presence of Salmonella in these RTC products from India. 18The aim of present study was to i) screen branded RTC poultry products from Mumbai for the presence of Salmonella, ii) study the antimicrobial resistance profile of Salmonella isolates and presence of class 1 integron in MDR strains and iii) characterize these isolates by Pulsed Field Gel Electrophoresis (PFGE).

Sampling
Forty eight chilled and thirty nine frozen RTC poultry samples of four differ-ent brands were obtained from various supermarkets and departmental stores in Mumbai (Supplementary Table S1).The chilled RTC samples included mixed boneless chicken, leg cut, precut, soup pieces, kheema, assorted cut pieces, lollipops or drumsticks.The frozen RTC samples were comprised of sausages, kheema, cutlet, nuggets, tandoori chicken nuggets, tandoori chicken tikka, chicken samosa, salami slices, sheekh kebab, burger patty, lollipops, and spring roll.The frozen RTC samples contained ingredients such as flour, onion, water, spices and condiments (coriander leaves, garlic, ginger, red chili powder, coriander powder, curry powder, turmeric powder, green chili, kasoori methi, edible vegetable oils, soya bean granules, iodized salt, and acidity regulators-INS 330, 452).The samples were brought in ice and analyzed immediately.

Isolation of Salmonella
The isolation of Salmonella was carried out as per US-FDA Bacteriological analytical manual 8 th edition. 19Microbiological media and antibiotic disc were from HiMedia Laboratories, Mumbai, India.Twenty five grams of poultry meat were homogenized in 225 mL lactose broth and incubated at 37°C for 24 h.After the initial pre-enrichment step, the samples were further enriched in Tetrathionate broth and Rappaport-Vassiliadis medium at 43°C for 24 h.A loopful culture from each of these media was streaked on Bismuth Sulfite Agar (BSA), Xylose lysine Deoxycholate Agar (XLDA), and Hektoen Enteric Agar (HEA) and plates were incubated at 35°C for 24 h.After pre-enrichment, enrichment and plating on selective agar plates, typical Salmonella were isolated, and identified by biochemical tests like glucose test by Triple sugar iron agar (TSI), lysine decarboxylase test by Lysine Iron Agar (LIA), Urease test (Urea broth), IMViC test. 19Isolates were serotyped at the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, India.

Antimicrobial susceptibility test
Salmonella isolates were screened for antibiotic sensitivity using 15 different antibiotics by agar diffusion method as described by Clinical and Laboratory Standards Institute (CLSI). 20Salmonella isolates were grown in Mueller-Hinton broth (HiMedia) overnight to prepare inoculum in order to achieve colony suspension to match with McFarland standard 0.5.The culture suspensions were evenly spread on Mueller-Hinton Agar (HiMedia) and antibiotic discs were placed on agar surface followed by further incubation at 37°C for 24 h.Antibiotic resistance profiles were assigned according to CLSI as resistant (R), intermediate (I), or sensitive (S) after measuring average zone diameter. 5The type and concentration of antibiotics in disc were as follows, Ampicillin (AMP) 10 µg; Chloramphenicol (CHL)

Molecular characterization of Salmonella
Salmonella isolates were tested for the presence of invA gene by PCR amplification using the primers as previously described by Chiu et al. 21The Integron region was PCR amplified from MDR isolates using class 1 integron specific primers (CSL1 and CSR1) as previously described by Khan et al. 13 PFGE was performed as per the Pulse Net USA protocol with 50 U of XbaI (Bangalore Genei, Bangalore, India) at 37°C. 22PFGE was carried out with Gene Navigator System (Amersham Biosciences, Sweden) in 1% agarose gel [Seakem ® Gold Agar (Lonza, Rockland, USA)] in 0.5 X Tris-Borate EDTA buffer at 9°C.Pulse times ramped from 5 to 120 s during a 25 h run at 160 V. Lambda ladder PFGE marker (New England Biolabs, Ipswich, MS, USA) was used as molecular weight standard.The gels were stained with ethidium bromide.The bands were analyzed visually and the 0 and 1 matrix (binary matrix) was developed based on the presence or absence of particular size band on the gel in all the samples.The matrix was analyzed using FREETREE software (Version 0.9.1.50,Folia Biologica, 2001).Strains differing by one band were considered as different pulsed field profiles (PFPs).The relatedness of the isolates was analyzed using Nei and Li/Dice distance similarity calculations and neighbor joining as the tree building algorithm.The output tree was visualized using the Tree View software (Version 1.5.2,Roderic D. M. 2005).

Prevalence of Salmonella
High percentage of chilled RTC poultry samples (53% of brand 1 and 50% of brand 2) were positive for Salmonella as compared to frozen RTC samples (5% samples of brand 4).The samples from brand 3 were free from Salmonella (Table 1).Present investigation shows both fresh chilled and processed frozen RTC poultry products are contaminated with Salmonella.Poultry meat and egg are established as a major source of contamination by Salmonella. 6,22resence of Salmonella in fresh poultry has been well reported worldwide. 6,8In the present study, samples were taken from very diverse products.The comparison between these products with respect to Salmonella incidence is difficult, but irrespective of the products, high incidence of Salmonella was found in all the samples except one.It was found that minimally processed, fresh RTC poultry samples (mixed boneless, soup pieces, mixed boneless, precut, drumstick and leg cut ) and also processed fresh/frozen RTC poultry samples (from brand 1 lollipops and kheema) were contaminated with S. Typhimurium and S. Enteritidis.The fresh RTC poultry (brand 1 and 2) comprised of both processed and un-processed poultry products.Therefore, there is a high level of the Salmonella incidence in these products (Table 1 and Supplementary Table S1).Out of 113 Salmonella isolates, 75.2% were S. Typhimurium, 23% were S. Enteritidis and 1.7% were S. Weltevreden (Table 1).Thirty four percent of Salmonella positive samples were contaminated with at least 2 serovars (Table 1 and Supplementary Table S1).S. Typhimurium and S. Enteritidis were the most frequently reported serovars associated with human foodborne illnesses and poultry industry in India. 6,8Also, S. Typhimurium is one of the most commonly detected serovars from animals used for food and retail meat in the USA. 10

Antibiotic resistance
More than 80% of Salmonella isolates   2).Multidrug resistance has been reported in a number of serovars of Salmonella from different foods. 13,17Salmonella isolates from brand 1 and brand 2 were resistant to 9 different antibiotics; whereas, Salmonella isolates of brand 4 were resistant to 4 antibiotics (Table 2).Ninety percent of S. Typhimurium isolates were resistant to NAL, CTC, KAN, OTC and TET.All S.

Article
Enteritidis isolates were resistant to KAN and OTC, while more than 80% of isolates were resistant to NAL and CTC.Both the S.
A total of 19 antibiotic resistance pat-terns were observed.The most predominant antibiotic pattern was NAL, SMZ, CTC, KAN, OTC, TMP, TET (29 isolates) followed by AMP, CEP, NAL, SMZ, CTC, KAN, OTC, TMP, TET (16 isolates) (Table 2).Multiple antibiotic resistance (MAR) index of both S. Typhimurium and S. Enteritidis was 0.6 while that of S. Weltevreden was 0.53 (Table 2).The high MAR index indicates indiscriminate use of antibiotics in poultry farming for growth promotion, prophylaxis as well as therapeutic purposes.The injudicious use of antibiotics in poultry which has increased the emergence and maintenance of MAR bacteria in the environment has been reported. 1,6

Molecular characterization
All the Salmonella serovars isolated from poultry were invA positive.The invA gene is located on Salmonella Pathogenicity Island 1 (SPI-1), which is essential for the invasion of host's epithelial cells by Salmonella.This gene is highly conserved in Salmonella serotypes and has been used as a potential target for Salmonella detec-tion. 23In a study of 630 different strains of Salmonella enterica, 99.4% of strains were found to harbour the invA gene. 24Whereas another study reported the presence of invA gene in Salmonella is not universal, as during their study out of 35 tested strains of Salmonella, 33 harboured sseL as well as invA virulence genes, however two strains (Salmonella Molade and Salmonella München), did not harbour the invA virulence gene. 25eventy percent of the Salmonella isolates carried the class 1 integron.Class 1 integron specific PCR detected double amplicons (1 kb and 1.2 kb) in 53.9% isolates (Supplementary Figure S1).The rest of the class 1 integron positive isolates showed multiple bands.][28] Integrons may carry antibiotic resistance gene cassettes, which confer resistance to antimicrobials. 13In the present study, 82% of the integron positive isolates were MDR.But interestingly, 18% of the integron positive isolates were non-MDR (resistant to less than 5 antibiotics).Ampicillin and 30 µg; Streptomycin (STR) 25 µg; Cephalothin (CEP) 30 µg; Nalidixic acid (NAL) 30 µg; Ciprofloxacin (CIP) 10 µg; Ceftriaxone (CTR) 30 µg; Sulfamethizole (SMZ) 300 µg; Enrofloxacin (EFX) 10 µg; Chlortetracycline (CTC) 30 µg; Kanamycin (KAN) 30 µg; Oxytetracycline (OTC) 30 µg; Ofloxacin (OFX) 2 µg; Trimethoprim (TMP) 30 µg; and Tetracycline (TET) 30 µg (HiMedia).Multiple antibiotic resistance (MAR) index is calculated as the ratio of number of resistant antibiotics to which organism is resistant to total number of antibiotics to which organism is exposed.6 S. Typhimurium (4) S. Enteritidis (2) a Chilled RTC poultry samples comprises of raw meat only.b Frozen RTC poultry samples contained ingredients such as flour, onion, water, spices and condiments.c ND stands for Not detected.weremulti-drug resistant (MDR) with resistance to 5 or more different antibiotics.S. Typhimurium (17 isolates), S. Enteritidis (11 isolates) and S. Weltevreden (2 isolates) with MAR index from 0.5333 to 0.6 were resistant to more than 8 antibiotics belonging to different class of antibiotics (Table