Retail Market Poultry Meats of North-East India-A Microbiological Survey for Pathogenic Contaminants
A cross-sectional study of different portions of chicken raw meat samples from the local meat markets of North East India was carried out during October 2007 to September 2008 on a total of 110 collected samples using Plate Count Agar, Lactose broth, Violet red bile glucose agar, Brilliant green bile lactose broth, Eosin methylene blue agar, MacConkey agar, Salmonella shigella agar, Bimuth sulphite agar, Xylose lysine deoxycholate agar, Nutrient agar and Potato dextrose agar. Of the 74 different bacteria detected, the bacterial population incidence was highest in chicken wings (83.5%), followed by chicken tails (77%), breasts (70%), thighs (69%) and gizzard (38%). Frequent organisms in the samples were Enterococcus faecalis (100%), Enterobacter aerogenes (100%), Escherichia coli (98%), Klebsiella pneumoniae (98%), Micrococcus sp. (69%) and Candida sp. (80%). The other organisms isolated were Klebsiella oxytoca (35%), Citrobacter sp. (52%), Proteus sp. (49%), Staphylococcus aureus (20%), Staphylococcus epidermidis (20%), Yersinia enterocolitica (23%), Listeria monocytogenes (15%), Shigella dysenteriae (1.8%), Salmonella tyhpi (20%), Bacillus cereus (10%), Aeromonas sp. (5.5%), Alcaligens faecalis (15%), Penicillium sp. (42%), Aspergillus sp. (20%) and Rhodotorula sp. (5.5%). This finding indicates substantial presence of microbial contaminants in retail chicken meat samples in North East India and dearth of proper sanitation in the market places.
Microbial food safety and food-borne infections are important public health
concern worldwide. There have been a number of food-borne illnesses resulting
from the ingestion of contaminated foods such as chicken meats. Most of the
pathogens that play a role in foodborne diseases have a zoonotic origin (Busani
et al., 2006). According to reports of the World Health Organization
(WHO, 2003) and the Centers for Disease Control and Prevention
(CDC, 2000) every year a large number of people are affected
by diseases due to contaminated food consumption. Although, these pathogens
usually cause mild to moderate self-limiting gastroenteritis, invasive diseases
and complications may occur, resulting in more severe cases (Zhao
et al., 2001). Numerous epidemiological reports have implicated foods
of animal origin as the major vehicles associated with illnesses caused by food-borne
pathogens (Petersen and James, 1998; Tood,
1997). Contaminated raw or undercooked poultry and red meats are particularly
important in transmitting these food-borne pathogens (Zhao
et al., 2001). Person-to-person transmission has also been described
Pathogens like Campylobacter, Salmonella and pathogenic E.
coli all colonize the gastrointestinal tracts of a wide range of wild and
domestic animals, especially animals raised for human consumption (Meng
and Doyle, 1998). Studies worldwide have shown that such pathogens are often
present in fresh meat and poultry (Tood, 1997). The
health status of food animals can potentially influence food borne pathogen
levels in three ways. First, diseased animals may shed higher levels of food
borne pathogens. Second, animals that require further handling in the processing
plant to remove affected parts may lead to increased microbial contamination
and cross-contamination. Finally, certain animal illnesses may lead to a higher
probability of mistakes in the processing plant, such as gastrointestinal ruptures,
which would lead to increased microbial contamination and cross-contamination
(Singer et al., 2007).
Raw meat remains an important and probably the major source of human food borne
infection with pathogenic bacteria. In spite of decades of effort it has been
difficult to obtain food animals free of pathogenic bacteria. Meat and poultry
carcasses and their parts are frequently contaminated with pathogens, which
reach the carcasses from the intestinal tract or from faecal material on feet
and feathers. Cross-contamination is a particular problem and several recommendations
have been published to control pathogens throughout the chain from hatcheries
to the preparation in the home (Dinçer and Baysa,
North-East India is inhibited by different ethnic tribes, where chicken meat is one of the highly consumed animal originated food item. With high nutritive value, having both essential macro- and micronutrients, chicken meat makes an important part of a balanced diet for these people. Retail meat and meat products are normally sold in markets in unhygienic conditions. There are no certified meat processing units and the hygiene in retail markets environment is much below the required standards. Therefore, the objective of the present study was to analyze the retailed meat samples of North-East India for the presence of microorganisms, with special emphasis on isolation and identification of pathogenic contaminants.
MATERIALS AND METHODS
Meat samples were collected randomly from October 2007 to September 2008
in sterilized container from different markets spread over seven states of North-East
India and were kept at 4°C until further analysis. Different parts of chicken
meat like thighs, breasts, gizzards, tails, livers, wings and hearts were considered
as source of inoculums for the study.
The General Viable Count (GVC) was done by performing serial dilution of
the sample (10% w/v) in 0.1% peptone water and plated on plate count agar. For
Total Enterobacteriaceae Count (TEC) and Total Coliform Count (TCC), 11 g of
the sample was inoculated in 99 mL of buffered peptone water (0.1% peptone+0.85%
NaCl) and lactose broth (Forward et al., 2004)
containing glass beads, respectively and incubated in shaker for 24 h (Aneja,
2005). TEC was done by plating the serially diluted enriched sample on Violet
Red Bile Glucose Agar (VRBGA) and TCC by MPN technique using Brilliant Green
Bile Lactose Broth (BGLB). Incubation of GVC, TEC and TCC were done aerobically
at 37°C for 24 h. Isolation of E. coli was done by direct inoculation
of the enriched sample from Lactose broth non EMB agar and for other microorganisms
it was done by inoculating the isolates obtained from GVC and TEC on Nutrient
Agar. Lactose fermenters and non lactose fermenters were detected by inoculating
enriched (in buffered peptone water) sample into MacConkey agar plate and incubated
for 24 h at 37°C. For selective isolation of Salmonella enriched
(in buffered peptone water) sample was streaked into Bismuth Sulphite Agar (BSA)
and subcultured into Xylose Lysine Deoxycholate (XLD) agar and Salmonella-Shigella
(SS) agar plates. The inoculated plates were incubated for 48 h at 37°C.
Yeast and mold count were done on Potato Dextrose Agar (PDA) by serial dilution
method and incubated at 25°C for 7 days.
Identification of the Isolates
The bacterial isolates, obtained from the samples were purified by re-streaking
them on the media used for their isolation and were characterized by different
biochemical tests. The biochemically characterized isolates were identified
with the help of Bergeys Manual of Determinative Bacteriology (Holt
et al., 1994). Identification of the mold and yeast were done by
staining and sugar assimilation tests, respectively.
The average microbial load of GVC, TEC and TCC and isolated from various
parts of chicken meat collected from the North Eastern states were listed in
Table 1. Tail and wings revealed higher general viable counts,
which were 1.8x107 cfu g-1 and 4.1x107 cfu
respectively compared to other parts while gizzard showed consistently lower counts (5.0x105 cfu g-1). Total enterobacteriacae count was found to high in tail portion (1.6x105) cfu g-1. Total coliform count was found to be higher in tail and wing portion. It showed MPN index of 0.7.
Yeast and mould was not detected in gizzard portion, whereas high count was
detected again in wing portion. Assam, Manipur and Meghalaya showed high general
viable counts, whereas the other three states showed comparatively low general
population count (Table 2).
Identification of the Isolates
The bacterial isolates were identified based on their morphology and biochemical
characters. Biochemical test results of bacterial isolates were listed in Table
3. However, some isolates need further tests to classify them into species
level. Fungal population were identified based on their morphology and micrograph
and were identified as Penicillium sp. and Aspergillus sp. Yeast
isolates were identified as Candida sp. and Rhodotorula sp. depending
on their morphology and sugar assimilation tests.
||Microbial load of GVC, TEC, TCC and yeast and mould count
of raw meat samples
|ND: Not detected
||Average microbial count detected from various sampling sites
||Biochemical results of the identified isolates from chicken
|+: Positive, -: Negative, A/A,G- H2S-: Acid butt
acid slant gas (-) ve H2S (-)ve, A: Acid production, MR: Methyl
red, A/K G-, H2S-: Acid butt alkaline slant gas (-) ve H2S
(-) ve, AG: acid and gas production, VP: Voges proskauer, A/A G+, H2S+:
Acid butt acid slant Gas (+) ve, H2S (+) ve, ND: Not detected,
TSI: Triple sugar iron
Out of the 110 raw meat samples studies for the microbial load
and Enterobacter aerogenes
showed 100% positive result while
and Klebsiella pneumoniae
showed 98% prevalence.
The frequency of other isolates were Klebsiella oxytoca
sp. (52%), Proteus
sp. (49%) and Micrococcus
sp. (69%). Less
frequently isolated microbes were Staphylococcus aureus
(20%), Yersinia enterocolitica
(23%), Listeria monocytogenes
(15%), Shigella dysenteriae
faecalis (15%) and Bacillus
(10%). Molds and yeast isolated were Penicillium
sp. (20%) and Candida
sp. (80%), Rhodotorula
sp. (5.5%) (Table 4
Escherichia coli is more prevalent in the samples than Salmonella sp. in different portion of the meat samples analyzed (Table 5). Escherichia coli showed 98.14% prevalence, whereas Salmonella sp. was found to be only 12.37%.
||Prevalence of contaminant microbes in the sample
||Prevalence of E. coli and Salmonella sp.
in the chicken meat samples
Lac. fer: Lactose fermenter Non lac. fer: Non-lactose fermenter,
EMB: Eosin Methylene Blue agar, BSA: Bismuth Sulfite agar, SS: Salmonella-Shigella
agar, XLD: Xylose Lysine Deoxycholate agar
Chicken meat is an important source of protein and a valuable commodity in
resource limited tribal communities, such as in North-East India. New problems
of food borne zoonotic diseases are arising in conjunction with the development
of different types of food of animal origin. There are a number of factors responsible
for the spread of zoonotic disease such as pathogenesis related to contaminated
food of animal origin. The present study evaluates that most of the retail shops
do not operate in a safe and sanitized environment. Covering and hanging carcasses
were rarely observed. Further, the chopping blocks of wood were found to be
same everyday without proper cleanliness. This enhanced the chances of cross
contamination of uninfected carcass if any prior carcass happens to be infected.
The processing of carcass surface into parts further spreads contamination by
exposing more carcass surface and susceptible fleshy parts to the contaminants
if the same cutting blocks and knives are used (Satin, 2002).
Wide ranges of microorganisms were found to be associated with the meat samples.
Bacterial count was comparatively higher than yeast and fungal count. It is
expected because meat products are found to be kept in the open environment,
the temperature that allows the bacteria to grow in a good growth rate than
yeast or fungi. The result of the present study indicated the presence of high
microbial load of GVC, TEC, TCC (MPN index) and yeast and mould count isolated
from various parts of chicken meat. Among the various chicken meat parts wings
showed highest bacterial load of GVC and TEC. In this study, Assam, Mizoram
and Nagaland showed high general viable counts, whereas the other three states
showed comparatively low general population count. This may be due to the comparatively
higher ambient temperature prevalent in these areas as high temperature favours
good growth of the microbes. It was found that Enterococcus faecalis
and Enterobacter aerogenes were present in all the samples. Tanimoto
et al. (2005) reported the prevalence of E. faecalis in raw
chicken meat of Japan. Isolation of E. faecalis from raw chicken meat
has also been reported by Simjee et al. (2002).
The results obtained showed that E. coli is more prevalent in the samples
than Salmonella sp. This is in conformity with Zhao
et al. (2001) who also reported high prevalence of E. coli
than Salmonella sp. High prevalence of E. coli in retail meat
market had also been reported by Kumar et al. (2001).
Present findings also revealed the presence of Listeria sp. and
Salmonella sp. which can be supported by the report of Soultos
et al. (2003) and Inoue et al. (2000)
who separately reported contamination levels of these isolates in retail foods.
Prevalence of Salmonella sp. in various raw meat samples of local market
had also reported by Maharjan et al. (2006) which
supports our study. Klebsiella pneumoniae showed 98% prevalence in the
samples. This is in consonance with the finding of Kim et
al. (2005) revealed the prevalence and emergence of multidrug resistant
Klebsiella pneumoniae in chicken.
Yeast and mould population were isolated in low frequency compared to the bacterial
population. Among the yeast isolates, Candida sp. showed 80% prevalence
and among the moulds, Penicillium sp. showed 42% prevalence. There was
no contamination by yeasts and moulds in gizzard. Although, Aspergillus sp.
was found to have 20% incidence, this may be good enough to produce toxins based
on its prevalence and production of food toxins are well reported in species
of Aspergillus. Altalhi and Albashan (2004) had
also reported contamination of chicken meat with Penicillium sp. and
Aspergillus sp. The finding that the presence of Candida sp. and
Rhodotorula sp. in meat is similar to record of Viljoen
et al. (1998) who outlined the presence of yeast population associated
with poultry carcasses.
It can be concluded that the organisms like Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Penicillium sp. Aspergillus sp. Candida sp. and Rhodotorula sp. were the most prevalent microorganisms in the meat samples collected during the study from various market places of North-East India. These contaminants not only possess health hazards to indigenous consumers but also to visitors exposed to consumption of such meats. Though not in the same concentration, these are important microbial contaminants in the retail chicken meats, which need to be taken care of for prevention of health hazards of the consumers by adopting proper sanitation, storage and retail practices.
The authors acknowledge the financial support for procuring chemicals received from UPE-Biosciences Programme of University Grants Commission, India to North-Eastern Hill University for undertaking the present work.
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