REVIEW OF LITERATURE
Foodborne listeriosis is mainly caused by L. monocytogenes by the consumption of raw, undercooked and refrigerated foods. The ubiquitous nature of the organism helps it to persist in natural environment and contaminate foods and food-processing environment. The presence of this pathogen in foods not only causes severe health problems but it also causes huge economic loss to the producers.
The literature pertaining to occurrence of the Listeria spp. in vegetables, milk products and meat products, human stool samples, and antibiotic sensitivity has been reviewed under various headings.
2.1. OCCURRENCE OF LISTERIA SPECIES IN VEGETABLES
The vegetables are one of the essential components of human diet due to their nutritional and health benefits and is actively promoted throughout the world. The vegetables are usually consumed raw or in minimally processed forms. The contamination of these vegetables with Listeria spp. can lead to infections in humans with grave consequences in immunosuppressed individuals.
Heisick et al. (1989) analysed 1,000 vegetable samples viz. broccoli, cabbage, carrots, cauliflower, cucumbers, lettuce, mushrooms, potatoes, radishes, and tomatoes, for Listeria spp. L. monocytogenes was found in cabbage, cucumbers, potatoes and radishes. L. innocua was isolated from cucumbers, lettuce, mushrooms, potatoes, and radishes. L. seeligeri was isolated from cabbage and radishes and L. welshimeri was isolated from cucumbers, potatoes, and radishes. Only potatoes (25.8 per cent) and radishes (30.3 per cent) showed significant L. monocytogenes contamination. No Listeria spp. was recovered from broccoli, carrots, cauliflower or tomatoes.
Tang et al. (1994) examined a total of 280 different vegetables samples for the presence of Listeria spp. Listeria spp. found in five samples. L.monocytogenes was detected in lettuce, sengkuang (Pachyrrhizus erosus), Indian pennywort (pegaga) and pegaga (Hydrocotyle asiatica). No Listeria spp. was found in cabbage, cucumbers, celery, tomatoes, spring onions, and carrots.
The frequency of L. monocytogenes contamination in leafy vegetables was found to be 22 per cent. (Arumugaswamy et al.1994).
Pingulkar et al. (2001) described that the vegetables analysed for Listeria spp. from a local market in India. L. monocytogenes was present in 11 per cent of tomatoes, 50 per cent of coriander leaves and spinach, and 25 per cent of cabbage samples.
Dhanshree et al. (2003) screened the vegetable samples for the presence of Listeria spp. L. innocua was isolated from 10 per cent of the palak leaves and 30 per cent of the coriander leaf samples. L. monocytogenes was not detected in any of the cabbage samples.
Seventy vegetable samples comprising of lettuce, cucumber, cabbage, sweet pepper and carrots were analysed for L. monocytogenes. Only one sample of lettuce was found positive for the L. monocytogenes, (Hosein et al. 2008).
Morobe et al. (2008) reported that contamination rate of L. monocytogenes from frozen cabbage was 47.4 per cent and from vegetable salad was 35.1 per cent.
Cordano and Jacquet (2009) reported that contamination of L. monocytogenes from frozen vegetable salads was 25.4 per cent and raw/ cooked RTE vegetable salads were 10.2 per cent.
Ikeh et al. (2010) reported that out of the 20 vegetables (cabbage, lettuce, carrots, green peas) analysed for Listeria spp. 80 per cent were found to be positive.
Schwaiger et al. (2010) reported that out of the 1,001 vegetables screened, 1.1 per cent of samples were contaminated with Listeria spp. L. monocytogenes (4) and L. ivanovii(3) were detected in the samples, L. innocua, L. murrayi and L. seeligeri were observed in one sample each of the total vegetables screened.
Ponniah et al. (2010) reported that occurrence of different raw vegetables for Listeria spp. for cabbages was (30 per cent), cucumber (20 per cent), beans, and carrots (10 per cent). L. monocytogenes was detected in cabbages, beans, and carrots (10 per cent each).
Moreno et al. (2012) analysed 191 samples of vegetables for L. monocytogenes and the isolation rate of 4.19 per cent was obtained by culture technique, with a higher incidence found in frozen vegetables. Multiplex PCR showed a greater number of positive samples (10.47 per cent).
Jamali et al. (2012) reported that the prevalence of Listeria spp.in ready-to-eat (RTE) foods was found to be 17.9 per cent samples of which 11.4 per cent samples were positive for L. monocytogenes
Silva et al. (2012) screened vegetables for the presence of L.monocytogenes. The vegetables (lettuce, cabbage, mukunuwenna, gotukola and tomato) contamination found to be two per cent.
Sunil et al. (2012) analysed Listeria spp. from 120 vegetables comprising of 15 samples each of cabbage, coriander leaves, cucumber, carrot, mint, cauliflower, amaranthus and celery from Thrissur region. The organism was found to be positive 8.3 per cent. The isolates were found to be L. innocua (80 per cent) and L. welshimeri (20 per cent).
Ieren et al. (2013) analysed 250 salad vegetable samples to detect the presence of L. monocytogenes and found that 4.7 per cent of the samples were contaminated with the organism. Cabbage and lettuce showed the highest prevalence of 8.5 and 6.2 per cent respectively.
Wang et al. (2013) analysed 90 vegetables samples comprising of celery, lettuce and tomatoes screened for Listeria spp. and L. monocytogenes and the contamination rate was found to be 73.33 per cent and 13.33 per cent respectively.
Yu and Jiang (2014) studied the Prevalence of L. monocytogenes in vegetables and observed that 1.7 per cent of the samples were positive for the organism.
Ajaykumar (2014) reported that out of 110 vegetables samples comprising up of cabbage (40) and leafy vegetables (70) analysed for the presence of Listeria spp. contamination rate was found to be 9.09 per cent. L. innocua (4.5 per cent), L. grayi and L. welshimeri (1.81 per cent each) and L. monocytogenes (0.9 per cent).
Lucy et al. (2014) analysed 150 raw vegetables for the presence of L. monocytogenes and found to be devoid of L. monocytogenes.
Sangeetha (2014) studied the incidence of Listeria spp. in vegetables marketed in Mysore city, Karnataka. A total of 165 vegetable samples were analysed for the presence of Listeria spp. and samples included amaranthus green, amaranthus red, coriander, dill, spinach, fenugreek, cabbage, curry leaves, cauliflower and radish. L. innocua (1.21 per cent) was detected in spinach and radish samples and L. seeligeri (0.60per cent) from one cabbage sample
Vasu (2014) described that out of 100 vegetables samples comprising of cabbage, celery, coriander from Thrissur and Mannuthy region screened for the presence of Listeria spp. only two samples from cabbage was positive for L.innocua and L. ivanovvi.
2.2. OCCURRENCE OF LISTERIA SPP. IN MILK PRODUCTS
Milk and milk products provides a suitable medium for the growth and survival of Listeria. Moreover, milk and its products have been implicated as a source of foodborne outbreaks due to Listeria monocytogenes.
Piniji and Gilbert (1988) examined retail samples of 222 soft cheeses for Listeria spp. Ten per cent of the soft cheese samples were found to contaminated with L. monocytogenes.
Destro et al. (1991) examined Listeria spp from raw milk and milk products and 12.5 per cent of the samples were found to be contaminated with Listeria spp. L.monocytogenes was detected in five per cent, L. innocua 20 per cent and L. seeligeri and L. welshimeri in 2.5 per cent of the samples.
Cordano and Rocourt (2001) analysed 256 soft cheese and 155 hard cheese samples. L. monocytogenes was detected in 0.8 per cent of soft cheese samples and was not detected from hard cheese samples.
Molla et al. (2004) studied the occurrence and distribution of L. monocytogenes and other Listeria spp in cheese. Out of the 61 samples examined, 1.6 per cent cottage cheese samples were positive for Listeria spp. No L. monocytogenes was detected from the cheese samples.
Vitas et al. (2004) investigated the occurrence of Listeria spp. in a total of 3685 food samples. The samples included natural products derived from poultry, milk and meat, and processed products such as frozen vegetables, smoked salmon, cooked, and cured meats). Listeria spp. was found to be present 8.1 per cent of the soft cheese samples.
Aygun and Pehlivanlar (2005) reported that prevalence of Listeria spp. on unpasteurized milk and white cheese samples was 2.1 per cent and 8.2 per cent, respectively. L. monocytogenes was not detected from unpasteurized milk and was found in 2.3 per cent of cheese samples.
Pintado et al. (2005) examined Listeria spp. in raw ewe soft cheese samples. The contamination rate was found to be 75 per cent with L. monocytogenes detected in 46 per cent of samples and L. innocua in 29 per cent of samples.
The prevalence study carried out by Arslan and Ozdemir (2008) for detecting the presence of Listeria spp. in homemade white cheese samples (142) revealed the presence of Listeria spp. (33.1 per cent) L. monocytogenes, L. innocua, L. seeligeri, L. grayi, L. ivanovii, and L. welshimeri was isolated from 9.1, 9.1, 2.1 and 2.1 per cent respectively.
Fifty milk products viz. pasteurized milk, yoghurt, cheese samples were analysed for the presence of L. monocytogenes. Out of the 30 cheese samples analysed L. monocytogenes was found in 13.33 per cent of the samples. Filiousis et al. (2008)
Jalali and Abedi (2008) screened a total of 88 milk and dairy products for Listeria spp. Only one L. innocua found in ice cream sample and all other dairy products were devoid of Listeria spp.
Morobe et al. (2009) reported that out of 250 cheeese samples obtained from supermarkets and street vendors Gaborone, Botswana, 2.75 per cent of the samples revealed the presence of L. monocytogenes.
Singh and Prakash (2008) reported that out of the 51 bacterial isolates of Listeria spp from curd and cheese samples, 13 isolates were confirmed as L. monocytogenes. Eleven isolates were obtained from curd samples and two from cottage cheese.
Harakeh et al. (2009) analysed 164 samples of baladi cheese (45), kishk (83) and shankleesh (36) for L. monocytogenes. The occurrence of L. monocytogenes was found to be 26.67 per cent, 7.23per cent and 13.89 per cent in baladi cheese, kishk and shankleesh samples respectively.
Jakobsen et al. (2010) investigated the presence of L. monocytogenes in cheese prepared from caprine and bovine raw milk. L. monocytogenes was absent in cheese prepared from caprine milk and only 1.4 per cent of cheese samples prepared from bovine milk revealed the presence of the organism.
Pesavento et al. (2010) studied the presence of Listeria spp. in retail dairy products. The prevalence in fresh soft cheese was found to be 3.5 per cent. L. monocytogenes was found in 20.0 per cent of fresh soft cheeses.
Rahimi et al. (2010) assessed prevalence for Listeria spp. in unpasteurized milk and dairy food stuffs. Out of 594 samples tested revealed that 9.3 per cent of samples contaminated with Listeria spp. unpasteurised sheep milk samples (22.6 per cent) followed by cheese samples (18.9 per cent).
Prevalence of L. monocytogenes in unpasteurized milk and ready to eat (RTE) milk products was studied and the results revealed that 18.1 per cent in raw milk and RTE milk (0.3 per cent) products were positive for the organism L. monocytogenes Kramarenko et al. (2012)
Chen et al. (2013) assesed 65 dairy products for L. monocytogenes from cities of south China but no L. monocytogenes was detected.
A total of 84 dairy products examined for L. monocytogenes by Wu Shi et al. (2014) revealed that 2.4 per cent of the samples were positive for L. monocytogenes.
2.3. OCCURRENCE OF LISTERIA SPP. IN MEAT PRODUCTS
Due to the modernization and changing eating habits of people there has been an increase in intake of ready to eat foods. As ready to eat foods are consumed without further cooking, there are chances of post processing contamination and improper storage which can create a higher health risk for the consumer. So the safety evaluation of the processed foods should be routinely monitored and evaluated.
Destro et al. (1991) screened 60 meat product samples for Listeria spp. and 95 per cent of the samples showed contamination. The Listeria spp found were L. monocytogenes (71.7 per cent), L.innocua (80 per cent), L. welshimeri (3.3 per cent) and L. seeligeri and L. murrayi (1.7 per cent each).
Uyttendaele et al. (1999) reported the contamination rate of L. monocytogenes in different meat products L. monocytogenes was isolated from 13.71 per cent of raw cured meat products, 4.90 per cent cooked meat products, 6.14 per cent cooked minced meat products, 3.96 per cent whole cooked meat products, 14.92 per cent whole cured meat products and 11.69 per cent minced cured meat products.
Cordano and Rockuret (2001) reported that out of the 634 processed meat products screened for L. monocytogenes, 3.6 per cent were found to be positive.
Vitas et al. (2004) investigated the occurrence of Listeria spp. in a total of 3685 food samples. Comprising of raw products (milk, meat, and poultry) and processed food items samples (frozen vegetables, smoked salmon, cooked and cured meats). Listeria spp. was found in 76.3 per cent of raw poultry product samples.
Chao et al. (2006) reported that the prevalence of L. monocytogenes was at 13.06 per cent when two hundred and forty-five samples of delicatessen foods were screened.
Filiousis et al. (2008) studied a total of 210 food samples comprising of milk products, ready-to-eat salads, raw meat and raw meat products for the presence of L. monocytogenes. L. monocytogenes was detected in 14.3 per cent of the samples and the prevalence in raw meat and raw meat products was found to be 27.5 per cent and 18 per cent respectively.
Jalali and Abedi (2008) screened a total of 617 samples comprising of meat, meat products, dairy, vegetables and RTE food samples for Listeria spp. The prevalence of L. monocytogenes was 1.2 per cent. Listeria spp. was present in 6.7 per cent meat and meat products, 1.2 per cent vegetable samples and 12 per cent RTE food samples.
Awaisheh (2010) analysed 240 RTE meat product samples for Listeria spp. comprising of 120 samples each of beef and poultry products. L. monocytogenes, L. innocua and L. welshimeri was detected in 17.1 per cent, 23 per cent and 15 per cent samples respectively. L. monocytogenes and other Listeria spp. was found higher in beef samples. L. innocua was isolated from beef (24.16 per cent), poultry (22.5 per cent), However, L. monocytogenes and L. welshimeri was isolated from 19.16 per cent and 14.16 per cent of beef samples and 15 per cent and 15.83 per cent of poultry samples respectively.
Osaili et al. (2010) analysed total of 280 meat samples of chicken and RTE chicken products (mortadella, chicken-burger, chicken-shawirma and chicken-sausage) for Listeria spp. and reported that 50 per cent were contaminated. The prevalence of L. monocytogenes (18.2per cent), L. ivanovi (26.1 per cent), L. grayi (3.5 per cent), L. seeligeri (1.8 per cent), L. welshimeri (0.7 per cent) was reported. The PCR confirmed all 51 L. monocytogenes isolated from raw dressed broiler chicken (15), chicken-burger (23), chicken sausage (9), and chicken-shawirma (4).
Bouayad and Hamdi (2011) analysed a total of 227 RTE food samples for Listeria spp. The contamination rate was found to be 9.3 per cent. L. monocytogenes, L. innocua, L. ivanovii and L. welshimeri were isolated from 2.6 per cent, 4.8 per cent, 1.3 per cent and 0.4 per cent samples respectively.
Kovacevic et al. (2013) screened a total of 80 samples for Listeria spp comprising of deli meats products and fish products. Ten per cent of the samples were found to be contaminated with Listeria spp. of which L. welshimeri (5 per cent), L. innocua (2.5 per cent), and L. monocytogenes (2.5 per cent) were detected
Alsheikh et al. (2013) collected 250 retail RTE broiler chicken products comprising 50 samples each from chicken burger, frozen chicken sausages, frozen chicken meat balls (kofta), chicken shawerma and chicken mortedella. The samples were examined for Listeria spp. Thirty eight per cent of the samples were contaminated with Listeria spp. where in L. monocytogenes (13.6 per cent), L. ivanovi (20.8 per cent), L. grayi (1.6 per cent), L.seeligeri (0.8 per cent) and L. welshimeri (1.2 per cent) were detected.
Fallah et al. (2012) collected and analysed for Listeria spp. in a total of 402 poultry product samples constituting raw, RTE and ready to cook (RTC) products. The contamination rate was found to be 33.3 per cent in which L.innocua (46.3 per cent), L. monocytogenes (38.8 per cent), L. ivanovii (9.7 per cent) and L. seeligeri (5.22 per cent) were reported. In their study occurrence was more during warm season than in cold season. The percentage of L. monocytogenes detected in raw meat, RTC and RTE poultry products was 14.1, 12.2 and 11.4 respectively.
Jamali et al. (2012) reported the Listeria spp. prevalence in RTE foods was found to be 17.9 per cent samples in which 11.4 per cent of samples were positive for L. monocytogenes. Among the RTE foods, salads and vegetables revealed the highest prevalence (14.7 per cent) followed by chicken and chicken products (13.2 per cent), beverages (10 per cent), eggs and egg products (9.5 per cent), beef and beef products (6.7 per cent), lunch boxes (6.7 per cent), seafood and seafood products (6.7 per cent).
Kramarenko et al. (2012) reported the prevalence of L. monocytogenes from raw meat products and ready-to-eat (RTE) foods. Contamination rate was found to be 18.7 per cent in raw meat products and two per cent in RTE foods.
Marian et al. (2012) analysed L. monocytogenes in 140 raw and RTE food samples sausages (30), burgers (18), canned fish (10), minced meat (8), fish (24), chicken (29) and meat (21). L. monocytogenes was detected from burgers (33.3 per cent), minced meat (25 per cent) and sausages (13.3 per cent).
Chen et al. (2013) screened L. monocytogenes in a total of 223 samples comprising of 158 RTE foods and 65 dairy products. L. monocytogenes was detected in 6.33 per cent samples of RTE samples. However, no L. monocytogenes was isolated from 65 dairy products.
Yu and Jiang (2013) reported that the prevalence of L. monocytogenes in raw meat (pork, beef, mutton and chicken), cooked meat products, seafood, and vegetables was 6.7 per cent, 7.4 per cent, 2.7 per cent and 1.7 per cent respectively.
Gomez et al. (2014) described that out of 336 Listeria isolates obtained from RTE meat products and surrounding of food processing, 206 isolates of L. monocytogenes and 130 isolates of L. innocua were detected
Wang et al. (2014) analysed a total of 628 RTE meat products comprising of 236 sauce pickled products, 260 cured products and 90 smoked and roasted products for L. monocytogenes. The contamination rate found to be at percentage of sauce pickeled product (7.2 per cent), cured products (4.2) and smoked and roasted products (5.6 per cent).
Wu Shi et al. (2014) screened a total of 364 retail RTE foods for L. monocytogenes comprising of dairy products, cold vegetable/noodle dishes in sauce, fried rice/sushi, roast chicken or duck, stewed meat, salt-baked chicken, sausage and ham. Only 6.87 per cent were contaminated with the organism
2.4. OCCURRENCE OF LISTERIA SPECIES IN HUMAN STOOL SAMPLES
Listeriosis has been associated with the consumption of contaminated food and water in humans. The poor hygienic practices and consumption of contaminated foods may be responsible for the occurrence of Listeria spp. in humans. The subclinical infection in humans and dissemination of the organism through stool samples can contaminate environment and food.
Muller (1990) investigated the prevalence of Listeria spp. in 1,000 patients suffering from diarrheal diseases and 2,000 healthy persons. The prevalence of L. innocua and L. monocytogenes in diarrhoea patients was 1.7 per cent and 0.6 per cent respectively. However, the prevalence of L. innocua and L. monocytogenes in healthy patients was two per cent and 0.8 per cent respectively.
Gowan et al, (1994) described that out of the 692 stool samples analyzed for Listeria spp and L. monocytogenes the results revealed that contamination rate was found to be one per cent and 0.6 per cent respectively.
Grif et al. (2003) analysed for L. monocytogenes using PCR and cultural technique for a total of 868 stool samples and revealed PCR (3.5 per cent) and PALCAM selective agar plating method (1.1 per cent).
Sauders et al. (2005) cultured 827 stool samples for detection of L. monocytogenes and found only 0.12 per cent positive stool sample
Kaur et al. (2007) reported that out of 61 faecal swabs screened for L. monocytogenes from the spontaneous abortions cases none of sample were found positive
Malek et al. (2010) reported that out of the 28 stool cultures examined for Listeria spp. from hospitalized children, 7.14 per cent were found positive for Listeria spp. L. grayi and L. seeligeri were the two species commonly found in the samples.
Bhat et al. (2012) studied different methods for detection of L. monocytogenes from stool samples of 505 healthy people. The detection rate of L. monocytogenes was found to be 0.2 per cent by conventional plating, 0.1 per cent positive with enzyme-linked fluorescent immunoassay (ELFA) and ELISA, and 3.6 per cent by PCR.
Dharmendra (2013) reported that out of 300 human clinical samples, 1.7 per cent samples were contaminated with L. monocytogenes and belonged to serogroups 4b, 4d and 4e
Ajaykumar et al. (2014) reported that out of 240 human faecal samples screened for L. monocytogenes none of the isolates were found to be positive.
Hanaa et al. (2014) analysed 72 stool samples analysed for Listeria spp. from immune compromised persons (36), neonates (23) and farm workers (13). Overall occurrence of Listeria spp. was found to be 4.17 per cent. L. monocytogenes was detected in stool samples of neonates (4.35 per cent) and L. ivanovii was detected in the immunocompromised patients (5.56 per cent).
Negi et al. (2014) described that out of the 165 human infants stools screened for L. monocytogenes two samples were positive for the organism. Contamination rate found to be (1.2 per cent).
2.5. METHODS OF DETECTION AND MOLECULAR CHARACTERISATION
2.5.1. Conventional techniques for detection
The isolation of fastidious Listeria spp. in general purpose bacteriological media such as trypticase soya agar, nutrient agar, and blood agar is usually difficult. However, enrichment protocols have proved to be useful in the isolation of the organism and has been reviewed in this section.
Capita et al. (2000) described that for detection of Listeria spp. higher sensitivity (100 per cent) and specificity (85.07 per cent) was obtained on Modified Oxford agar (MOA) when compared to PALCAM agar where the sensitivity and specificity was 94.29 per cent and 80 per cent respectively.
Pinto et al. (2001) reported that comparison of Listeria monocytogenes blood agar (LMBA) with conventional selective agar medium (PALCAM/OXFORD), higher recovery of the Listeria monocytogenes organism was obtained from LMBA agar then conventional selective agar.
Mahmood et al. (2003) adopted a method for detection of L. monocytogenes from poultry meats and its products from 320 samples by directly plating onto blood agar plates comprising 0.05 per cent potassium tellurite. The plates were incubated for a period of 37′ C for 24 h and found that 23.75 per cent of samples were contaminated with the Listeria spp.
Jalali and Abedi (2008) analysed food products for isolation of Listeria spp. in vegetables, dairy, meat products and ready to eat foods. Enrichment was done using Listeria enrichment broth (UVM). After primary enrichment and secondary enrichment with UVM, selective plating was done on Oxford agar and PALCAM agar and incubated at 37′ C for 48h. The contamination rate of the organism in these food products was found to be 6.33 per cent.
Malek et al. (2010) reported that detection of Listeria spp. from meat, chicken products and human stool samples was done by one step enrichment with the LSEB (Listeria Selective Enrichment Broth) comprising of 0.6 per cent yeast extract with Listeria selective supplement. Selective plating was done onto Listeria selective agar incubated at 35”C for 24-48 h 33.59 per cent was found to contaminated with Listeria spp.
Moreno et al. (2012) analysed the vegetables for the presence of L. monocytogenes in vegetables by using double enrichment with Frazer broth and streaked on to selective PALCAM and ALOA agar and recovered 4.18 per cent positive samples.
Jamali et al. (2012) reported that sensitivity and specificity for isolation of L. monocytogenes was low in Listeria selective agar and PALCAM agar when compared with CHROM agar. The sensitivity and specificity in CHROM agar was found to be 96.9 and 99.1 per cent respectively.
Ieren et al. (2013) employed the protocol (ISO 11920-1) method for detection of L. monocytogenes from vegetables. Enrichment was done using a Fraser broth and selective plating was carried out on Oxford Listeria agar at an incubation of 37”C for 24-48 h. The contamination level of organism in vegetables was 3.9 per cent.
Yu and Jiang et al. (2013) studied isolation of L. monocytogenes by using primary and secondary Listeria enrichment broth and streaked onto PALCAM agar. The contamination rate found to be 6.2 per cent in retail food samples
Ajaykumar et al. (2014) compared two different enrichment broths by using Buffered Listerial broth (BLEB) and University of Vermount broth (UVM) and plated on to (PALCAM and Oxford agars). Combination of UVM and PALCAM (4.4 per cent) was more effective than the combination of UVM and oxford (3.35 per cent).
Wang et al. (2014) analysed L. monocytogens in ready to eat meat products according to the protocol of China national food safety standard methods. Enrichment of samples protocol carried out by using Listeria enrichment broth (LB) by incubating at 37′ C for 24 h to 48 h. The enriched samples was streaked onto selective PALCAM agar cultural method 5.3 per cent was found to be positive for L. monocytogenes.
2.5.2. Molecular characterisation
The recommended and approved microbiological isolation protocol for Listeria spp. are time consuming and identification of species is difficult due to biochemical variation. Molecular characterisation by PCR procedure has proved to be highly sensitive, substantially faster, and more cost effective for identification of species and virulence of the isolates.
Thomas et al. (2002) reported that out of 282 L. monocytogenes isolates obtained from the meat and fish products specific serotype was found to be 1/2a, 1/2b and 1/2c.
Lawrence and Gilmour (1994) investigated 154 samples comprising of raw (91) cooked (96) poultry products for the presence of Listeria spp. by using primer LI1 and Ul for amplification of 16S rRNA gene at 938 bp hlyA gene for confirmation of L. monocytogenes by using primer LM1 and LM2 they found that contamination rate of Listeria spp. and L. monocytogenes in the samples was 26 and 15 per cent respectively.
Bansal et al. (1996) analysed 350 food samples comprising variety of products including seafood, vegetables, dairy products, processed meats and raw poultry by multiplex PCR. Initial enrichment with the enrichment broth for detection of Listeria was used followed by detection of 16S rDNA gene for Listeria. Virulence gene hlyA, Listeria internal postive control (LIPC) and yielded a product size at 938, 750 and 525 bp respectively. 28 per cent contained Listeria spp. and 16 per cent was positive for L. monocytogenes
Borucki and Call, (2003) studied the serogrouping of L. monocytogenes isolates (122) by) utilising three Division III primer sets. The results from PCR found to be similar with those of the conventional slide agglutination method for 97, 100, 94, and 91 per cent of strains belonging to serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively.
By utilising a multiplex PCR Doumith et al. (2004) differentiated serovars by PCR for 222 isolates of L. monocytogenes was carried out by utilising a primer target gene for imo0737, imo1118, ORF 2819, ORF2110, prs and showed the amplification at 691, 906, 471, 597 and 370 bp respectively.
Gouws and Liedemann (2005) analysed L. monocytogenes in 21 sorts of foods products comprising of cheese (7), raw meat and fish product (3), ready to eat chicken products(5) by PCR by extracting the DNA from the colonies obtained on RAPID L mono agar. The hly gene (730bp) was detected in the isolates and contamination rate was found to be 37 per cent through PCR.
Hong et al. (2007 ) molecular serotyping was carried out for the food (270) and human (179) L. monocytogenes isolates and found that 58 per cent of the food isolates belong to 1/2a and 3a and the isolates of human 48 per cent was found positive for 4b, 4d and 4e.
Rawool et al. (2007) studied multiplex PCR to identify the virulence genes plcA, hlyA, actA and iap genes for confirmation of L. monocytogenes. in milk. All the genes were detected in the isolates.
Jalali and Abedi (2008) analysed L. monocytogenes in 373 food samples comprising of meat, meat products, poultry, sea food and chilled ready to eat foods by PCR. The blyA gene was detected in 8.5 per cent of the samples.
Mauro et al. (2008) describes that out of 120 isolates obtained from food and food processing environments serotypes was found to be 53. 8 per cent (1/2a), 23.1 per cent (1/2b), 13.2 per cent (1/2c) and 99 per cent (4b).
Cordano and Jacquet (2009) out of 110 L. monocytogenes isolates obtained from the frozen vegetable salads of different sero group was found to be IIb (1/2b) 45.5 per cent and 4b (4b) 37.3 per cent and IIa (1/2a) 15.5 per cent and IIc (1.8 per cent).
Malek et al. (2010) reported that out of the 100 samples comprising of fifty samples each of meat (Minced frozen meats and Luncheon) and chicken products (Frozen chicken fillets and chicken frozen legs) was examined for the presence of L. monocytogenes by PCR. The prfA gene (271 bp) was detected in five per cent of the samples analysed.
Ponniah et al. (2010) screened a total of 306 vegetables by PCR utilising by the amplification of 16S rDNA gene (938 bp) to detect Listeria spp. and hly gene (701-bp) for detection of L. monocytogenes. The contamination rate was found to be 33.3 per cent for Listeria spp. and 22.5 per cent for L. monocytogenes respectively.
Moreno et al. (2012) analysed for L. monocytogenes in a total of 191 vegetables by multiplex PCR method. By utilising species specific primer for amplification at 750bp for hlyA gene they found 10.47 per cent of the samples to be contaminated with the organism
Chen et al. (2013) analysed 37 L. monocytogenes isolates obtained from the ready to eat foods for the presence of seven virulence genes viz., actA, iap, plcA, and inlA, prfA, plcB, mpl genes by duplex PCR. All the isolates found positive for inlA, plcA, actA, mpl, iap, and prfA genes.
Ryu et al. (2013) reported that by utilising multiplex PCR for detection of Listeria spp. from meat-processed foods. By utilising primers for Listeria genus specific and Listeria spp specific primers for amplification of prs, oxidoreductase, lin0464, namA, imo 1030, imo0333 and scrA genes results found that Listeria genus, L.grayi, L.innocua, L.ivanovii, L.monocytogenes, L.seeligeri and L.welshimeri at product size of 370 bp, 201 3bp, 749bp, 463bp,509 bp,673bp, 281bp respectively
Ajaykumar (2014) reported that out of 76 Listeria spp. isolated from cattle (16), goat (20), human (5), soil (16), water (9) and vegetables (10), screened for the presence of six virulence genes iap, hlyA, actA, prfA, plcA and inlA. Only four isolates of L. monocytogenes obtained from cattle, goat, soil and cabbage showed the presence of all six virulence genes. Out of the 54 isolates of L. innocua four isolates showed the presence of hlyA gene.
Sunil et al. (2013) described that out of 18 isolates of Listeria spp. obtained from fish, leafy vegetables and soil samples, all the isolates were positive for carrying virulence genes iap, hlyA, actA, prfA, plcA and inlA and showed amplification at 131, 456, 839, 1060, 1484 and 820 bp.
Wang et al. (2014) analysed 628 ready to eat products for L. monocytogenes through PCR and 5.3 per cent of the samples positive for the organism. All the isolates were found to be positive for five-virulence genes viz., iap, inlA, inlC, inlJ and lmo2672
2.6. ANTIBIOTIC SENSITIVITY OF LISTERIA SPP.
The emergence of antibiotic resistant microorganism can pose a public health hazard which can lead to clinical complications. The development of antibiotic resistance is mainly due to the acquisition of antibiotic resistance genes. So identification of antibiotic sensitivity profile is important for effective treatment of the disease
Dhanashree et al. (2003) studied the antibiotic susceptibility pattern of food isolates of L. monocytogenes from food and clinical samples. L. monocytogenes was sensitive to ampicillin, chloramphenicol, ciprofloxacin, cotrimoxazole, erythromycin, gentamycin and penicillin. Few of the L. innocua isolates were resistant to erythromycin and cotrimoxazole.
Stonsaovapak and Boonyaratanakornkit (2010) carried out the antibiotic sensitivity of 64 isolates of Listeria spp. Listeria spp. were resistant to penicillin (6.3 per cent) chloramphenicol (3.1 per cent) and tetracycline (1.6 per cent) but sensitive to amoxicillin, vancomycin, ampicillin, rifampicin and sulfamethoxazole. The level of resistence to L. monocytogenes was low (5.6 per cent) as compared with L. innocua (16 per cent), L. ivanovii (33.3 per cent) and L. seeligeri (50 per cent). None of antibiotics were resistant to L. grayi and L. welshimeri. However L. innocua showed multidrug resistance.
The antimicrobial assay of L. monocytogenes isolates from raw chicken and RTE chicken products was conducted by Osaili et al, (2010). The results revealed that 18.2 per cent of the samples were found positive for L. monocytogenes L. monocytogenes isolates were resistant to tilimicosin (17.6 per cent) and tetracycline (11.8 per cent). Only one isolate was resistant to both tilimicosin and tetracycline.
Bouayad and Hamdi, (2011) studied the antimicrobial sensitivity pattern of L. monocytogenes isolates obtained from food samples and reported that the isolates were sensitive gentamicin (10mg), piperacillin (30mg), tetracycline (30mg) trimethoprim/sulfamethoxazole (1.25/23.75 mg), amoxicilin/Clavulanic acid (20/10mg) and chloramphenicol (30mg).
Lotfollahi et al. (2011) found that out of the nine L. monocytogenes isolates obtained from the 100 human clinical samples, 77.77 per cent of the isolates were showed resistance to multiple antibiotics. The isolates showed resistance to penicillin G (77.77 per cent), cephotaxim (77.77 per cent), chloramphenicol (11.11 per cent) and streptomycin (11.11 per cent). L. monocytogenes was sensitive to tetracyclin, trimethoprim, ciprofloxacin, norfloxacin, ampicillin and erythromycin.
Fallah et al. (2012) reported that out of the 25 isolates of Listeria spp isolated from poultry products, 10.5 per cent were susceptible to the common antimicrobials. The level of multidrug resistance was highest in L. monocytogenes isolates (60.2 per cent) followed by L. innocua (50.9 per cent) and L. ivanovii (23.8 per cent). More isolates were resistant to ampicillin and penicillin was observed for L. monocytogenes.
Korsak et al. (2012) carried out the antibiotic sensitivity for L. monocytogenes isolates from foods. Out of 200 isolates, phenotypically resistance for two L. monocytogenes strains (0.42 per cent). Resistant for tetracycline and minocycline was observed in each isolate and second isolate to ciprofloxacin and norfloxacin
Marian et al. (2012) described the antimicrobial susceptibility profile of 23 isolates of L. monocytogenes obtained from raw and ready to eat food samples. All the isolates were resistance towards ampicillin and penicillin G and sensitive to streptomycin. Moderate sensitive to erythromycin (56.5 per cent) and chloramphenicol (52.2 per cent) was also observed. A total of 82.6 per cent isolates were resistant against rifampicin
Chen et al. (2013) conducted the antimicrobial susceptibility of 37 isolates of L. monocytogenes and found that 83.8 per cent isolates were susceptible to chloramphenicol, ciprofloxacin, erythromycin, levofloxacin, vancomycin and sulbactam/ampicillin. Two isolates were penicillin-resistant and one isolate was multidrug-resistant to kanamycin, tetracycline, sulfamethoxazole, rifampacin, gentamycin, penicillin, and ampicillin.
Grouping of L. monocytogenes isolates into different lineages and serotyping was done using specific primers (Gamboa et al. 2013). Restriction enzyme analysis also used in conjunction with the PCR in serotyping. Out of the 259 L. monocytogenes isolates, maximum belonged to 1/2a serotype (24.3 per cent) and the least was 4a/4c (0.4 per cent).
Ieren et al. (2013) conducted a study to determine the antimicrobial resistance of L. monocytogenes isolated from salad vegetables and ready to eat vegetable salads the isolates were resistant to ampicillin (92.9 per cent) followed by oxacillin (85.7 per cent) gentamicin (21.4 per cent )and ciprofloxacin (14.3 per cent).
Ajaykumar (2014) depicted the antibiotic resistance pattern of various Listeria spp. L. ivanovii was resistant to ampicillin, L. monocytogenes to ampicillin, amoxicillin and rifampicin, L. grayi to ampicillin, azithromycin and streptomycin and L. welshimerri to amoxacillin .
Gomez et al. (2014) studied the antibiotic sensitivity pattern for 336 Listeria isolates obtained from RTE meat products and surrounding area of meat-process comprising of 206 L. monocytogenes, and 130 L. innocua isolates. Oxacillin resistance was observed in all Listeria isolates. An intermediate resistance observed to clindamycin (39.3 per cent) and tetracycline (3.9 per cent). higher antimicrobial resistance (31.3 per cent) was observed in RTE meat products than those from the environment (13.4 per cent)
Vasu (2014) described that antibiotic profile of Listeria isolates obtained from food samples. They reported that give resistance and sensitivity according to type of sample cloxacillin and ampicillin showed resistance. cefotaxime, chloramphenicol, cotrimoxazole, doxycycline, erythromycin, gentamicin, streptomycin and vancomycin showed sensitive.
Wang et al. (2014) investigated the antimicrobial susceptibility of 33 isolates of L. monoctogens obtained from RTE meat products. All the isolates were susceptible to penicillin, ampicillin, amikacin, tobramycin, gentamycin, and streptomycin. All isolates were resistant to trimethoprime sulfamethoxazole. Eleven, ten, and four isolates were resistant to chloramphenicol, ciprofloxacin, and tetracycline respectively.
Yu and Jiang, (2014) analysed the antibiotic susceptibility of L. monocytogenes isolated from raw meat, cooked meat products, sea food and vegetables All isolates were susceptible to moxifloxacin, levofloxacin, gentamycin, and chloramphenicol. Resistance to cefotaxime (30.5 per cent) and ciprofloxacin (13.5 per cent) and intermediate susceptibility was observed for cefotaxime (11.9 per cent), ciprofloxacin (3.4 per cent), erythromycin (3.4 per cent), and streptomycin (1.7 per cent)
Wu Shi et al. (2015) reported that out of 80 L. monocytogens isolates obtained from ready to eat meat products analysed for serovars the results revealed belongs to 1/2a and 3a (46.25 per cent), 1/2c and 3c (6.25 per cent), 4b, 4d and 4e (26.25 per cent) and 1/2b, 3b and 7 (21.25 per cent).