Prevalence of Extended Spectrum Beta-Lactamase Producing Enterobacteriaceae in Commercial Broilers and Backyard Chickens

| The present study demonstrated the prevalence of extended spectrum beta-lactamase (ESBL) -producing enterobacteriaceae in liver samples of commercial broilers (commercial broilers) and backyard chickens (backyard chickens). Results demonstrated that Escherichia coli (E. coli) was the most common isolate recovered from both commercial broilers (44.58%) and backyard chickens (57.03%), that followed by salmonella (commercial broilers: 35.06%; backyard chickens: 21.09%), klebsiella (commercial broilers: 12.98%; backyard chickens: 13.28%), proteus (commercial broilers: 04.76%; backyard chickens: 06.25%), and enterobacter (commercial broilers: 02.59%; backyard chickens: 02.34%). The prevalence of E. coli and salmonella was found higher (P < 0.05) in commercial broilers as compared to backyard chickens. However the prevalence differences of klebsiella between commercial broilers and backyard chickens were found statistically non-significant (P > 0.05). Among these isolates, 7.76% and 10.95% E. coli isolates were recorded as ESBL -producing from commercial broilers and backyard chickens respectively. While 12.34% and 7.40% salmonella isolates were found positive for ESBL production from commercial broilers and backyard chickens respectively. However, 13.33% klebsiella isolates of commercial broilers were declared as ESBL -producers; whereas klebsiella isolates of backyard chickens and proteus and enterobacter of both commercial broilers and backyard chickens were found negative for ESBL production. These results indicates that microbiota of commercial broilers established a higher number of enterobacteriaceae as compared to backyard chickens, moreover, the prevalence of ESBL -producing enterobacteriaceae in liver of commercial broilers was also higher than backyard chickens.


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used as indicator organism by the water and food industry.Currently, both coliforms and enterobacteriaceae are isolated from foodstuffs for showing evidence of poor sanitation or inadequate processing (especially heat-treatment), post-process contamination of foods and process failure.Improper handling during evisceration and removal of intestinal tract may cause the rupture of intestine that results the contamination of meat and other visceral organs being contaminated (Khan et al., 2016;ILSI, 2011).
Meat-type poultry chickens (broilers) have a complex population of bacteria, however, the digestive tract microflora of backyard chickens suggested a greater diversity (Saliu et al., 2012).Among the isolates, Escherichia coli, lactobacillus spp., Enterobacter aerogenes, Bacillus cereus and Bacillus subtilis were found colonized in both, backyard chickens and broilers.However, Staphylococcus epidermis, Proteus vulgaris, Staphylococcus aureus, Enterococcus facium, Streptococcus pyogenes, and Bacillus megaterium were habitating the free range (backyard) chickens only.Many isolates found with the capability of hydrolyzing cellulose and starch.So these microflora have important roles in the carbohydrates digestion especially cellulose (Saliu et al., 2012).Furthermore, the backyard chickens may be critical environmental indicators of multidrug resistance and they might take part as spreaders and long-term reservoirs of medically threatening pathogens with resistance genes, more actively than previously thought (Badrul et al., 2012).
There is a global rise in infections caused by Gram-negative bacteria of enterobacteriaceae family, producing extended spectrum beta-lactamases (ESBL).The incidence of ESBL-producing pathogens in the poultry gastrointestinal tract increased from 3% in 2003 to 15% in 2008.The poultry industry now has been considered a likely reservoir of ESBL-producing Gram-negative bacteria (Leverstein et al., 2011).The diseases caused by these pathogenic microbes (ESBL producers) are producing high economic losses all over the world, in terms of high morbidity, stress, mortality, decreased hatchability and egg production (Numan et al., 2005).The ESBL show resistance to most of the beta-lactam antibiotics, including third and fourth cephalosporin generation, causing major problem of treatment promises for infections produced by these pathogens (Serefhanoglu et al., 2009).Keeping in view the above facts, the present study was planned to investigate the prevalence of enterobacteriaceae in liver (an edible part) of commercial broilers and backyard chickens.Moreover, the results will also provide the first insights on prevalence of ESBL -producing enterobacteriaceae in both chicken types.

SaMple ColleCtion
For present study apparently sick commercial broilers (n = 150) and backyard chickens (n = 150) of adult age (body weight, commercial broilers: 1.6-2.0kg, backyard chickens: 1.5-2.1 kg) were obtained from different farms of district Peshawar and brought to the post mortem room of Veterinary Research Institute (VRI) Peshawar.The birds were sacrificed, eviscerated and the fresh liver samples (about 25 g) were collected aseptically in sterilized sample bottles and stored immediately at -20˚C until analyzed (Nzouankeu et al., 2010).All the experimental protocols were approved by the institutional Animal Care and Use Committee.

iSolation of Enterobacteriaceae
All the samples were processed for isolation of major enterobacteriaceae viz, E. coli, salmonella, klebsiella, proteus and enterobacter following the procedure adopted by Roy et al., (2012).The samples were aseptically treated with 225 ml of Buffered Peptone Water (Oxoid UK, CM0509) as per procedure of Mossel et al., (1963) and appropriate decimal dilutions were prepared that were streaked on to MacConkey agar, tryptose agar and Nutrient agar of (Hi-Media Laboratories Pvt. Ltd.India) and incubated at 37 o C for 24 hours to get the primary bacterial growth.After 24 hours, the colony morphology was examined and each type of colony was picked and sub-cultured onto separate media plates (Roy et al., 2012).Pure cultures were achieved as per procedures described by OIE, (2000).The obtained pure bacterial growth was transferred to sterilize tryptose and nutrient slants, which were then incubated for 24 hours at 37 o C.After 24 hours period, the growth were examined and the stock cultures of different purified organisms were kept in the refrigerator at 4 o C for further investigation.The organisms were characterized as Gram-positive or Gram-negative by Gram's staining method according to the technique described by Merchant and Packer, (1967).Further confirmation of the isolates was made by API (Analytical profile Index) RapID-One strips (Remel Co, Lenexa, USA), which is a rapid detection method for the identification of important enterobacteriaceae members.

eSbl deteCtion
For determining the ESBLs -producing species of family enterobacteriaceae, the Double Disk Synergy method was used as adopted by Sirot (1996).In brief, nutrient agar plates were inoculated with test organisms and after inoculation, the combination disc of amoxicillin (20μg) and clavulanic acid (10μg) were placed at centre of nutrient agar plate while discs of cefotaxime (30μg), ceftazidime (30μg) and ceftriaxone (30μg) of Liofilchem Pvt. Ltd.Company, Italy were placed 15mm apart from central disc.The plates were incubated at 37°C for 18 to 24 hrs.An expansion zone of inhibition between any of the cephalosporin and combination disc ≥5mm indicated as ESBL positive according to the guidelines recommended by CLSI (2006).

StatiStiCal analySiS
On completion of the study, the data obtained were stated in absolute values and percentages.For the determination of percentages and calculations, the Microsoft Office 2013 software package was used.The incidence of different enterobacteriaceae isolates and their extended spectrum beta-lactamase production difference between commercial broilers and backyard chickens were compared by the Chi-square test at a P < 0.05 probability level using Instat GraphPad software (San Diego, California).

prevalenCe of eSbl -produCing enterobaCt-eriaCeae in CoMMerCial broilerS and baCKyard ChiCKenS
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DISCUSSION
Enterobacteriaceae are a major part of gut microbiota that play a critical role in enteric diseases and competitive exclusion (ILSI, 2011).For present study we targeted five genera/species (viz., E.coli, salmonella, proteus, enterobacter and klebsiella) of enterobacteriaceae which is considered as the key part of gut ecosystem, and also play major role in host health.Our results revealed a significant difference in gut microbiota of both chicken types and showed a higher population of enterobacteriaceae in commercial broilers as compare to backyard chickens, however, there was no difference in quality/diversity of isolated organisms from the liver of both chicken types.The same findings have been reported by Saliu et al. (2012), who had found commercial broilers with high prevalence of enterobacteriaceae as compared to backyard chickens.The variation in prevalence and diversity of enterobacteriaceae of commercial broilers and backyard chickens could be associated with mood of nutrition Saliu et al. (2012).The high prevalence of bacteria in broiler's intestine might be due to high nutrients ratio found there because commercial broilers are supplied with diet enriched with nutrients for the improved productivity and FCR.The gut microflora take advantage of these nutrients and thus multiply there rapidly as much as they found suitable conditions (Salah et al., 2015;Saliu et al., 2012).On the other hand, backyard chickens are omnivores, generally, they roam freely around the farmers' house at day time and pick food including wheat/maize grains, plant parts/leaves, and vegetable waste from the soil and/ or in the area around the cooking place.These botanicals contains phytochemicals including flavonoids, stilbenes and polyphenols, which are known as strong anti-inflammatory and antimicrobial agents (Kamboh et al., 2015).The low microflora level (enterobacteriaceae) in backyard chickens could be associated with the antimicrobial activities of these phytochemicals.Ojo et al. (2012) reported Escherichia coli, klebsiella, salmonella and enterobacter in free range chickens, which is somehow in line with our findings.Our results are also supported by the findings of Naldo et al. (1998), who had reported Escherichia coli, klebsiella, proteus and enterobacter in free ranging kori bustard chickens.Current finding of E. coli isolates is somewhat consistent with that of Roy et al. (2012) and Awad-Alla et al. (2010), who reported the prevalence of E. coli as 52% in commercial broilers.The high prevalence of E. coli in chicken's digestive tract are not unexpected as the coliforms are the main flora of farm animals as well as human beings (2, 7).Our further investigations showed that the overall prevalence of salmonella spp.was 30.08% in chicken, which is supported by Roy et al. (2012).However, a significant difference may be found in other findings like, Takehisa et al. (2013) who had examined 1,472 faecal samples of commercial broilers in Japan and found 93 isolates of salmonella spp.with 6.31%, which might be due to differences in geographical locations, feed ingredients and management conditions particularly those provided during embryonic life or early days of life (Ahmed et al., 2015;Nghonjuyi et al., 2015).

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The prevalence of ESBL -producing gram negative enterobacteriaceae in poultry and poultry products and their subsequent transmission to human beings have been proposed by several studies.Some workers have reported the similar prevalence percentages of ESBL -producers in poultry as declared in our study.Like, Smet et al. (2008), reported 27.2% ESBLs positive samples out of 489 samples.Besides this, Blanc et al. (2006) examined 192 enterobacteriaceae positive samples for the screening of extended spectrum βeta-lactamases and found 51 (26.57%) isolates with ES-BLs -producing enzymes.
In Pakistan, the frequent use of antibiotics in commercial poultry farming may also play a major role in the occurrence of resistance to the bacterial organisms by producing the enzyme ESBLs.Many bacteria attain resistance by exposure to antibiotics.The exposure may in shape of use of growth promoting antibiotics in poultry feed and/or non-judicial use of antibiotics for treatment/prevention of bacterial diseases (Ansari et al., 2014).There are two groups of resistance.In the first group: the bacteria have natural ability to resist against antibiotics by enzymatic inactivation while, in the 2 nd group, the bacteria have the ability to survive in the antibiotic environment by gene action without the interaction/alteration of antibiotics (Apata, 2009).
Our study have reported first time the prevalence of ES-BL-producing enterobacteriaceae in backyard (free rangers) and commercial chickens.It is generally hypothesized that backyard chicken farming system results the less or no dissemination of antibiotic resistance (Mirandaa et al., 2008).Because, in rural areas there is no high scale of farming of backyard chickens, usually the birds are kept in small cages in houses where they have less chances of getting antibiotics as they pay no significant attention for vaccines or treatment purposes.Our present findings were also in agreement with the above theory, as we have find less distribution of ESBL -producing enterobacteriaceae isolates in backyard chickens.Moreover, our results have also indicated the difference in diversity of ESBL-producers between both chicken types (as no klebsiella isolate was recorded as ESBL-producer in backyard chickens).This could be associated with the host-response mechanisms for stimulation of ESBL and non-ESBL strains (Demirel et al., 2013).Furthermore, all the isolates of proteus and enterobacter were declared as non-ESBL -producers regardless of chicken type.proteus are usually dispersed in nature everywhere as saprophytes and are mostly found in manure, soil, and human and animal faeces (Senior et al., 1997), while enterobacter are prototrophic in nature and are commonly found on a number of different plants and seeds (Francine and Patrick, 2006).Hence, these pathogens could enter frequently in commercial broilers as well as backyard chickens.The common infections of backyard poultry that were treated by antibiotics could be associated with these pathogens.
It is obvious that backyard poultry hardly receive antibiotics but they may be interconnected through a numerous paths with other organisms like humans and exotic poultry that had been formerly wide-open to a number of antibacterial agents, e.g., in many rural communities the people commonly defecate and urinate around surroundings where the backyard chicken move freely for picking feed.Such type of unhygienic human excretes disposal leads to

Table 1 :
Number and percentage of enterobacteriaceae isolates recovered from broilers and back-yard chickens *Results were considered significant when P < 0.05

Table 2 :
Prevalence of Extended-Spectrum-β-Lactamase-Producing enterobacteriaceae in broilers and back-yard chickens *Results were considered significant when P < 0.05