alexa Multidrug Resistant-Proteus Mirabilis Isolated from Chicken Droppings in Commercial Poultry Farms: Bio-security Concern and Emerging Public Health Threat in Bangladesh
ISSN: 2380-5439
Journal of Health Education Research & Development
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Multidrug Resistant-Proteus Mirabilis Isolated from Chicken Droppings in Commercial Poultry Farms: Bio-security Concern and Emerging Public Health Threat in Bangladesh

Arifatun Nahar1, Mashuk Siddiquee1, Shamsun Nahar1, Kazi Selim Anwar2 and Salequl Islam1,3*
1Department of Microbiology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh
2Microbiology Unit, Faculty of Medicine, AIMST University, Kedah D/A, Malaysia
3Department of Epidemiology, Johns Hopkins School of Public Health, MD 21205, USA
Corresponding Author : Salequl Islam
Department of Epidemiology
Johns Hopkins School of Public Health, USA
Tel: +1-410-614-0274
E-mail: [email protected]
Received June 28, 2014; Accepted August 20, 2014; Published August 22, 2014
Citation: Nahar A, Siddiquee M, Nahar S, Anwar KS, Islam S (2014) Multidrug Resistant-Proteus Mirabilis Isolated from Chicken Droppings in Commercial Poultry Farms: Bio-security Concern and Emerging Public Health Threat in Bangladesh. J Biosafety Health Educ 2:120. doi:10.4172/2332-0893.1000120
Copyright: © 2014 Nahar A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Poultry is a rampantly expanding agro-industry in Bangladesh like other developing countries. Several studies have detected multidrug resistance (MDR) Proteus mirabilis from poultry meat globally; however, no similar data was available for poultry samples in Bangladesh. P. mirabilis is a zoonotic human pathogen of urinary tract infection (UTI), nosocomial infection and wound infection, therefore, a potential threat to public health. We isolated P. mirabilis from chicken droppings collected from local commercial poultry farms and examined their antimicrobials susceptibilities. Chicken droppings were streak-cultured onto xylose lysine deoxycholate agar plates after enriching in buffered peptone water. Selective colonies were identified by biochemical test and API20E kits. Antimicrobial susceptibilities were tested by Kirby-Bauer disk diffusion method. Total 36 P. mirabilis were isolated from 39% (27 of 70) chicken droppings. Tetracycline evidenced as the highest individually-resistant (94%, 34/36) antibiotic (AB) while ciprofloxacin was the lowest (17%, 6/36). Hazard lies when 83% P. mirabilis were proved to be MDR (30/36), being resistant to three or more
AB. Findings provide a baseline data on MDR P. mirabilis circulating around these PFs, it would assist the veterinarian in rational treatment and biosafety planning. More detail studies will be required to clarify their antimicrobial resistance and clinical relevance.

Keywords
Proteus mirabilis; Antimicrobial resistance; Poultry; Chicken droppings; Bangladesh
Introduction
Poultry remains the largest domestic animal stock in the world in terms of the number of animals [1]. The industry has expanded extensively in commercial levels as well as in household traditional levels in Bangladesh. More than three million peoples are employed directly in poultry sector, which provides the largest supply of meat and eggs [2], so as to meet up the major protein sources for entire population of the country. Since raising small-scale commercial poultry farms (PFs) demands low investment, it has been expanding at a high rate, mostly at the rural and semi-urban areas which contribute in national economic growth, considerably. These PFs are often run by unskilled, non-professional managers having poor knowledge on biosecurity alike other developing countries [3]. Since most of these PFs neither do have a good surveillance systems nor well-documented monitoring mechanisms to record potential pathogenic microorganisms or other poultry-hazards claiming serious public health implications. Recent data from various poultry based studies in Bangladesh evidences high prevalence of human pathogens like, Escherichia coli, non-typhoidal Salmonella enterica and Enterobacter spp [2,4,5], being similar to other countries reporting various enterobacteriaceae in eggs and meats [6,7]. Several authors reported presence of P. mirabilis in poultry meat [8,9]; none of similar type of data had been reported from in Bangladesh. Thus the aforementioned facts prompted us to investigate the presence of P. mirabilis in chicken droppings of Bangladeshi poultry that might be transmitting this zoonotic pathogen [10] to vulnerable workers while handing infected chicken directly or through fecal-contaminated poultry products as similarly enterobacteriaceae have been reported to transmit [11,12].
In Bangladesh, wide-spectrum antibiotics are often used irrationally (misused/overused) due to lack of adequate education and shortfall in mass awareness among most of the work-forces. Poor surveillance, less quality assurance parameters, lacks in monitoring and gaps in regulatory mechanisms has thrown the overall public health situation in greater threat [2]. Such malpractices in antimicrobial uses have been reported to exert selection pressure of antimicrobial resistance to gutmicrobial flora in poultry [13,14]; including recent evidences of multi drug resistance (MDR) P. mirabilis [9]. This P. mirabilis is known human pathogen as a common cause of human urinary tract infection (UTI), nosocomial infection, wound infection [15] and showed clear history of zoonosis in wide host ranges with emergence of MDR in recent years [10,16]. MDR P. mirabilis may therefore be transmitted among PFworkers who in turn may transmit that in surrounding environment thus infecting the catchment population at large. Therefore, we wanted to measure susceptibilities/resistance of the detected P. mirabilis to selected antimicrobials (AB) in our limited settings.surveillance and regular monitoring in these PFs thus augmenting the disease prevention and control strategies against poultry based Proteusinfection, effectively.
Methods
Study area description and sample collection
More than half of the PFs in Bangladesh are situated at the periphery of Dhaka among the 64 districts of the country [17]. Savar, a subdistrict, located about 30 kilometer north-west of Dhaka city (Figure 1), is an industrial area. The vast majority of industries including small-, medium- and large- scale poultry and related agro-farms have been established in this region. Seven small-scale poultry farms were randomly selected from 4 distinct semi/peri-urban areas of Savar (Khas Mahal, South Rajason, Pan Dhoya and Islamnagar) for sampling of chick-dropping during a four months period (Apr-Jul, 2012). Three of those farms were cultivating broiler type chickens, three with layerchickens and one with pre-starter broiler chickens. We collected 70 different samples, 10 from each farm, aseptically, when a unique code number for each farm and an identity-number for respective sample was assigned carefully (Table 1).
Sample preparation
Following utmost precautions chicken droppings were collected so as to prevent probable cross contaminations. Samples were immediately stored in pre-labeled plastic containers in insulated ice-boxes and were transported to Jahangirnagar University laboratory within an hour where all microbiological examination were carried out. One loopful from each chicken dropping was diluted aseptically within 200μl sterile PBS in Eppendorf tubes and mixed properly using vortex mixture. Diluted samples were inoculated on specific culture media and the left-out samples were safely stored overnight at 4°C safely to repeat microbiological analyses, if required.
Culture procedures and identification techniques
We modified the traditional Salmonella isolation protocol to isolate Proteus spp. Diluted poultry droppings were enriched in buffered peptone water, (HIMEDIA, India) and one loopful of broth was streaked onto xylose lysine deoxycholate (XLD) agar plates (MERCK, India). Following incubation for 24 h at 37°C, three to four Salmonella-like colonies (red with or without black centre on XLD) were picked with needle and stabbed-streaked in nutrient agar (NA) slant to examine the swarming phenotype of the isolates. The purified Salmonella-typical colonies were examined further for their detail biochemical properties to identify presumptive P. mirabilis and finally confirmed by API20E (bioMe´rieux, Durham, NC).
Antimicrobial susceptibility testing (AST)
AST for P. mirabilis isolates was done following Kirby-Bauer disk diffusion method [18] and the zone of inhibition were interpreted according to instructions from the Clinical and Laboratory Standards Institute (CLSI, 2010). About 20 different antibiotics were reported to be used frequently by Bangladeshi poultry farmers [4]. Of them, six antimicrobials (ABs) from five generic groups, namely, β-lactam, quinolone, tetracycline, aminoglycosides and synthetic antibiotic (trimethoprim-sulfamethaxole), were tested to characterize P. mirabilis in this study. Commercially available antimicrobial discs (Oxoid, UK) and Mueller-Hinton agar (MHA, Oxoid, UK) media were used for the assay.
In this method, P. mirabilis isolates grown on XLD were then inoculated into nutrient broth for 18–24 hours at 37°C. Then, one loopful of inocula was added onto 9 mL of MH-broth (Oxoid, UK.) and again incubated aerobically at 37°C but for 5–6 hours only to reach standard turbidity of growth near 108 colony forming unit per milliliter (CFU/mL). The inocula were then lawned evenly using sterile cotton swabs on MHA plates. After air drying (under a safety hood), all the 6 AB-discs such as ampicillin (AML) 10 μg, ciprofloxacin (CIP) 5 μg, gentamycin (GN) 10 μg, nalidixic acid (NA) 30 μg, tetracycline (TE) 30 μg and trimethomprime- sulfamethoxazole (SXT) 25 μg were placed on MHA aseptically and kept at 4°C for 30–60 minutes for adequate diffusion. The plates containing AB-disks were incubated overnight at 37°C keeping in upright position and the diameter of zone of inhibition were read to interpret as resistant, intermediate/moderately sensitive, sensitive (susceptible) according to the reference inhibition zone by respective antibiotic. Reference non-pathogenic Escherichia coli (E. coli) were used as control strain.
Results
Isolation and confirmation of Proteus mirabilis
To isolate and identify Proteus mirabilis, a sum of 70 chicken droppings were collected from Savar area of Bangladesh (Figure 1). Colony characteristics of P. mirabilis on XLD medium (red with black centre colonies), were similar to those of Salmonella spp, whereas the negative control, E. coli showed different colony characteristics; yellowish to while without black-centre on XLD. When we examined those 81 Salmonella like colonies (on XLD), 36 (44%) were identified as P. mirabilis based on their detailed biochemical properties (slant: alkaline/red, and butt: acidic/yellow, producing H2S in Kligler Iron Agar [KIA] test). All those were oxidase negative, urease positive; and catalase positive. Moreover, these isolates showed typical biochemical markers of ‘IMViC’ as ‘- + - +/-’ (indicates: indole negative; methyl red positive; VP positive/negative and citrate variable [+/-]). Under light microscopy, these showed gram-negative short rod (under 100x oil emersion magnification). Isolates of P. mirabilis were further confirmed by the API 20E system.
The isolation rates of Proteus mirabilis in tested poultry samples
This study revealed the presence of P. mirabilis in samples of chickendroppings in five of seven farms; therefore, the overall farm prevalence for P. mirabilis became 71% (Table 1). All the 20 samples (ID CK001 to CK020) collected from PF1 (broiler type) and PF2 (layer type) chicken, both being located at ‘South Rajason’ area, showed growth of plentiful droppings-typical bacteria, but not P. mirabilis. In contrast, samples from other PFs yielded P. mirabilis, with intra-sample presence of 30% in PF4 to 90% in PF7 (Table 1). Thus, the overall presence among 70 samples of chicken droppings yielded 38.6% P. mirabilis (27/70). We tested 36 isolates from 27 culture-positive samples for AST.
Antimicrobial resistance patterns (AST) of Proteus mirabilis
The pattern of AST for 36 P. mirabilis isolates varied in its susceptibility/resistance pattern using six different AB-disks belonging to five different generic groups. The antimicrobial resistance profile showed tetracycline as the highest resistant AB (94.4%; 34 of 36) followed by nalidixic acid (89%, 32 of 36) (Table 2). Isolates showing higher level of resistance to β-lactam antibiotic were ampicillin (66.7%, 24 /36) and trimethoprim-sulfamethaxole (66.7%, 24/36). Similarly, aminoglycoside (Gentamycin) were resistant to 53% (19 of 36 isolates). In contrary, ciprofloxacin revealed relatively lower resistance (16.7%, 6 of 36) (Table 2). Combining the results together, we observed 83.3% P. mirabilis (30 of 36) were found resistant to 3 or more ABs, followed by 11% isolates (4/36) showing resistance to 2 ABs evidencing multidrugresistant (MDR) (Figure 2). However, only 5.6% (2 of 36) were found to be resistant to single AB. Alarmingly, no isolate was found to be susceptible to all AB tested.
Discussion
Ever growing global migratory trend, rapid industrialization and extensive growth in poultry production are thought to contribute the possible rapid dissemination of zoonotic pathogens posing public health a potential concern [19-21] and a big threat. Few studies have been reported from Bangladesh on the zoonotic infections among poultry, despite rampantly expansion of the PF industries [2,4,5].
In spite of indispensable mandate to follow stringent biosecurity guidelines to limit and prevent transmission of potential pathogens via poultry products [22,23], the benchmark guidelines of biosecurity are often not practiced in majority of PFs in Bangladesh as latest reports revealed [12]. This factor plausibly contribute in transmitting potential zoonotic pathogens to PF workers directly (while handing fecal contaminated meat/eggs) or indirectly (while processing/dressing infected poultry) [19-21] which ultimately spread out among the catchment population and surrounding environment. Evidences from latest reports show that poultry-borne Proteus spp were associated with zoonotic UTI [24] and that lead to rheumatoid arthritis (RA) very commonly in developing countries [25], with particularly on P. mirabilis causing nosocomial/ wound infections [15].
Findings of our study (38.6% P. mirabilis in chicken droppings) remain a potential public health concern in poultry industry of Bangladesh. This study has covered small number of poultry farms from focal area and detected 71% farm-prevalence for P. mirabilis and thereby has generated the baseline prevalence-data for the bacteria in the poultry industry of this country. The outcomes of this study would be helpful in designing further extensive investigations covering more poultry farms from expanded areas of the country to find nationwide prevalence of this zoonotic human pathogen. Emerging of MDR poultry-origin P. mirabilis has increased over the recent years [8,10,16,26], remains comparable to that of ours. In this study, isolates from chicken droppings showed high resistance properties towards tetracycline, nalidixic acid, ampicillin trimethoprim-sulfamethaxole and gentamycin. Individually, the high resistant of P. mirabilis to individual AB like tetracycline does attest the findings of earlier study [27]. More importantly, emergence of resistance against β-lactam antibiotic (we studied here ampicillin only) becoming alarming, since β -lactams are often remain the typical choice by the clinicians in treating a wide range of infections caused by Proteus spp. as a recent report says [9]. We believe that a more detailed study on extended-spectrum β-lactam (ESBL) antimicrobials would assist further in clarifying issue on emergence of resistance by P. mirabilis. However, we observed, unlike other antimicrobials, ciprofloxacin, a quinolone antimicrobial revealed much better potency (>80% were susceptible) against P. mirabilis.
We postulate several plausible factors, like environmental degradation (grossly polluted), disease profile (much higher prevalence of communicable/infectious disease), natural disasters (round the year flood, or cyclone, or tidal bores, etc.) may have augmented in the emergence of MDR issues directly or indirectly. In Bangladesh, indiscriminant use of AB (either irrational prescription by unqualified village-doctors/quacks or improperly taking AB or non-compliance of correct AB-dosage taken by the patients) and overuse/misuse of easily available AB by the PFs/allied industries to prevent their poultryflocks from unexpected diseases or deaths. Therefore, the acquisition of antimicrobial resistance may occur due to the selective pressure of AB abuses.
The drug-administration approved antimicrobial compounds are easily available in open markets to treat broiler/layer chickens in Bangladesh. These include ciprofloxacin, streptomycin, gentamicin, erythromycin, tetracycline, furazolidone and many others [4]. These are used singly or in combination with 2 or more ABs, which has been reported to contribute significantly in the emergence of MDRresistance in chicken isolates [28]. We therefore postulate further, that high percentages of MDR P. mirabilis in chicken droppings that we observed here, may threaten the total public health in Bangladesh at large through spreading that out among adjacent communities (oralfecal route of contamination). This may also be augmented by common unhygienic defecation practice (with inadequate post-toileting hand washing) by the vast majority rural inhabitants hindered by grossly inadequate water and sanitary system in Bangladesh.
High population density of Bangladesh may also contribute in spreading P. mirabilis following person to person transmission (PF environment/community to adjacent healthy populations) much faster to contaminate the surrounding population. This remains more true to hospital environment where this might subsequently result in nosocomial mediated P. mirabilis infections, as reported by Ebringer et al. [25]. Staying apart from the aforementioned postulates, a more logical stronger pre-hypothetical nod exists in chick-guts: MDR P. mirabilis in the gut of chicken may favor well in transmitting MDR genetic traits dangerously to AB-susceptible- P. mirabilis strain even to other Gram-negative gut microbiota through interspecies horizontal transfer as evidenced earlier [29,30]. More studies will be needed to evaluate and transmission of the antimicrobial resistance via poultry borne P. mirabilis. The resistant pattern of poultry originated P. mirabilis has not been compared and standardized with that of human clinical isolates. Establishment of clonal relationship between these poultry and clinical isolates would be noteworthy to translate our preliminary observation in PF into human-clinical applications, more effectively. We strongly recommend further large scale multi-center research encompassing larger sample size, involving heterogenic poultry farms from diversified areas to examine details of phenotypic and genotypic characterization of this zoonotic pathogen P. mirabilis.
Conclusion
To our knowledge, it is the first study of its type describing the presence of Proteus mirabilis in Bangladeshi poultry samples. We believe the data that this study generated will contribute to serve as the baseline information on the emerging communicable diseases in PFs in Bangladesh. Our finding demands stringent surveillance system to be developed in Bangladesh for antimicrobial resistance monitoring and biosafety on P. mirabilis and other pathogens found in poultry products.
Author Contributions
AN, collected samples, performed major experiments, prepared the results and helped in manuscript preparation; MS, helped samplecollection, assisted laboratory experiments and data acquisition; SN, contributed reagents and assisted data analysis; KSA, conceived the research idea, editing of first two drafts of this report and finalized the manuscript; SI, conceived, designed and coordinated the experiments, wrote the manuscript.
Competing Interests
None of the authors declared competing or conflict of interest
Ethics Statement
Verbal consents were obtained from farm owners for collection of respective chicken droppings and anonymity was strictly maintained.
Acknowledgements
This work was supported in part by University Grant Commission (UGC), Bangladesh. We are thankful to Dr. Munirul Alam, Senior Scientist, Center for food and waterborne diseases, International Center for Diarrheal Disease Research, Bangladesh (ICDDR,B) for providing us reagents and laboratory supports to characterize Proteus mirabilis.
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