Multidrug Resistant Providencia stuartii in Chicken Droppings: Public Health Implications for Poultry Workers and Associated Communities in nearby Dhaka Metropolis, Bangladesh

Introduction: Poultry farms (PFs) have appeared successful and wide spread business-industry in Bangladesh, which often remains contaminated with various hazardous microorganisms when standard hygiene practices are compromised. We sought to investigate a zoonotic human pathogen, Providencia stuartii and their antibiotic resistance pattern in chicken droppings collected from local poultry farms in Savar area, Dhaka, Bangladesh. Materials and Methods: We conducted a cross-sectional study to find the prevalence of antibiotic resistance in bacteria from chicken droppings in linked to antibiotic-uses and abuses in PFs. Random chicken droppings were collected from broiler type chickens, layer-chickens, and pre-starter broiler chickens to make samples representative. Following standard bacteriological culture, Original Research Article semisolid chicken-droppings were diluted aseptically, enriched in buffered peptone water, and then streaked onto a xylose-lysine-deoxycholate agar plate. Selected P. stuartii colonies were identified biochemically using API 20E ( BioMe´rieux ) and antimicrobial susceptibility testing was performed following Kirby-Bauer (disk-diffusion) method. Results: We reported the isolation of P. stuartii for the first time in Bangladesh in chicken droppings collected from randomly selected local PFs. Red colored colonies without black centre on XLD medium were considered as presumptive Providencia stuartii that were subsequently confirmed by API 20E system. Six chicken-droppings revealed the presence of P. stuartii from a total 70 samples tested, showed a prevalence of 8.6% with overall farm prevalence is 71.4%. We took 11 isolates from the six positive samples to examine their antibiotic resistance and found 82% of them were resistant to nalidixic, 73% to ampicillin, and 54.5% to trimethoprim- sulfamethoxazole. Relatively ciprofloxacin and gentamicin appeared more functional, where only 27.3% and 18.2% strains showed resistant, respectively. Over 54% of the isolates appeared resistant to >3 antibiotics and 36.4% with two different antibiotics. None of the isolates remained susceptible to all the 6 antibiotics tested. Conclusion: Detected MDR P. stuartii in chicken-droppings from local poultry farms may contribute their transmission to surrounding communities and may implicate serious biosecurity concern in environmental and food-safety issues in resource constraint countries, like, Bangladesh.


INTRODUCTION
Poultry farms (PFs) have commenced as one of the most wide-spread industries in Bangladesh because of their low-investment requirements [1]. These PFs generate high employment opportunities in poultry value chain [2], thereby, are contributing to national economy notably. Consequently, broiler meats and layer farm eggs fulfill the maximum demand of animal protein for the entire population of Bangladesh [2,3]. Apart from these, most of the PFs in Bangladeshi are run by unskilled staff and non-professional management having lack of proper knowledge on concerned biosecurity, environmental hygiene practices and food safety regulations [4]. These farms neither possess a robust surveillance system nor a stringent regulatory body. Even these PFs don't have a well-documented data archiving mechanism to record pathogenic microorganisms, systematically [5]. Bangladeshi poultry sectors usually do misuse and/or overuse of several wide-spectrum antibiotics and thereby, have generated selection pressure towards antimicrobial resistance [6]. However, this important aspect is often ignored or overlooked in Bangladesh [7] and some other countries [8][9][10]. The World Health Organization (WHO) is much concerned on the spreading of antibiotic resistance in the South-East Asian countries, where medicine uses are not very strict [11].
Bangladesh is a small and resource-constraint country that suffers from serious environmental pollution. The country lacks decent sewerage management and water sanitation systems, particularly in sub-urban and village areas. Practices exist of spreading chicken droppings into nearby environments from PFs that may disseminate potential zoonotic pathogens into the surrounding communities quite easily. High dense population may also augment transmitting the infections person-to-person and aggravate in threatening public health. Hazardous microorganisms that are reported from Bangladeshi poultry contamination include Enterobacter spp., Escherichia coli and non-Salmonella enterica, Proteus mirabilis and other species of Enterobacteriaceae [12][13][14]. We sought to investigate the zoonotic pathogen, Providencia stuartii in chicken droppings collected from local poultry farms under this study. The genus Providencia are opportunistic pathogens that include six species, namely, P. stuartii, P. rettgeri, P. alcalifaciens, P. rustigianii, P. heimbachae, and P. vermicola [15]. They are Gram negative, facultative anaerobe and motile bacteria, reportedly have multiple animal reservoirs. Providencia spp. were reported to cause a large outbreak of foodborne illnesses [16], pericarditis [17], meningitis [18], endocarditis [19] and diarrhea [20]. P. stuartii remains the second most common cause of catheter-associated urinary tract infection (Ca-UTI) and, was reported to co-colonize with P. mirabilis [21,22]. To our knowledge, there is no baseline data available yet on the contamination rate and antimicrobial resistance pattern of P. stuartii in Bangladeshi PFs/chicken droppings. Therefore, this study was a footstep initiative to fill up the knowledge gap. The isolation of P. stuartii from poultry source will authenticate formulating awareness program for preventing zoonotic infections to vulnerable workers and associated communities in Bangladesh.

Description of Study Area and Collection of Samples
The fast growing PFs have flourished in both urban and rural areas of Bangladesh and abundantly around capital city, Dhaka. More than half of the PFs in the country are situated at the periphery of Dhaka district. We conducted this study in one of its sub-district 'Savar'-located nearly 30 km north-west of Dhaka city (Fig. 1). Seventy chicken droppings were sampled (10 each of 7 PFs surveyed) from structurallydifferent semi-urban areas and a unique identification code was assigned for each sample based on area-specific individual PFs (Table 1). We conducted a cross-sectional study to find the prevalence of antibiotic resistance in bacteria from chicken droppings in linked to antibioticuses and abuses in PFs. Representative chicken droppings were collected from broiler type chickens, layer-chickens, and pre-starter broiler chickens from different racks randomly. The sampling PFs were selected from two different communities of Savar area to cover the interpretive population.

Preparation of Samples
Following all safety precautions and aseptic techniques, chicken droppings were collected to avoid probable cross contaminations. Samples were stored immediately in pre-labeled clean plastic containers in insulated ice-boxes and transported to the bacteriology lab of the Department of Microbiology, Jahangirnagar University, Savar, where all microbiological tests were carried out. One loopful of semisolid chicken dropping was diluted aseptically within 200 µl sterile phosphate buffered saline (PBS) in micro-centrifuge tubes and vortexed to mix homogenously, and then, inoculated on preselected culture media. The remaining sampleportions were stored overnight safely at 4°C to repeat the microbiological analyses.

Culture and Identification Techniques
For efficient bacteriological culture, we adopted modified techniques of traditional Salmonella isolation protocol to for isolating Providencia stuartii. Briefly, the diluted chick-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). After an overnight incubation (24 h at 37°C), three to four Salmonella-like typical colonies (reddish butt with/without a black dot in centre) were picked-up with flame-sterilized needle and stab-streaked into nutrient agar slants to examine specific phenotype characteristic of swarming growth by Proteus species. The purified colonies were identified further based on detailed biochemical properties for the colonies of Providencia stuartii using a rapid biochemical-test kit (API 20E, BioMe´rieux, Durham, NC) consisting of a set of chromogenic panel, carbohydrate batteries and enzymatic substrates [23].

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing (AST) for Providencia stuartii isolates was performed following the standard Kirby-Bauer disk diffusion method [24] and zones of inhibition were interpreted following CLSI (Clinical & Laboratory Standards Institute-2010) recommendations. Six different antimicrobials, which are commonly used by Bangladeshi poultry farmers [7], were tested against P. stuartii in order to characterize their resistance potential. Antimicrobials belonged to five different genera were tested (beta-lactam, quinolone, tetracycline, aminoglycosides, trimethoprim-sulfamethoxazole) in this study.
P. stuartii isolates grown on XLD were first transferred into nutrient agar and incubated overnight at 37°C to get pure culture. Then, one loopful of inocula was added onto 9 mL Mueller-Hinton (MH) broth and incubated aerobically at 37°C for 5-6 hours so as to get a turbidity equivalent to 0.5 McFarland standard. Then, these inocula were lawned homogenously with a sterile cotton swabs on the MHA plates (Oxoid, UK). After air drying under a safety hood,

Fig. 1. Location of sampling sites in the map of Bangladesh
A total of 70 chicken dropping samples were collected from seven poultry farms located at Savar area, which is located about 30 kilometer north-west of the capital city of Bangladesh, Dhaka. Savar is semi-urban area that have been established with different scale poultry farms and other industries. Out of the seven sampling farms, three were cultivating broiler type chickens, three with layer-chickens and one with pre-starter broiler chickens Bangladesh antibiotic discs (amoxicillin, 10 µg, Ciprofloxacin, 5 µg, Gentamicin, 10 µg, Nalidixic acid, 30 µg, Tetracycline, 30 µg and trimethoprimsulfamethoxazole, 25 µg) were placed on those evenly-lawned MHA-plates, aseptically. Then, the plates were kept at 4°C for 30-60 minutes for adequate diffusion and then incubated overnight keeping upright position at 37°C aerobically. The diameter of the zone of inhibition of each disk was read to interpret as resistant, moderately sensitive or sensitive (susceptible) employing standard reference data provided by CLSI.

Isolation, Identification and Confirmation of Providencia stuartii
Red colored colonies without black centre on XLD medium were considered as presumptive Providencia stuartii resembling Salmonella spp.
Of the 81 Salmonella-typical colonies (on XLD) examined, 11 were identified as Providencia stuartii based more on their detail biochemical properties: slant alkaline (red), butt acidic (yellow), H 2 S negative, gas negative; indole positive; methyl red positive; VP negative; citrate test positive; urease positive/negative; oxidase negative; catalase positive. Subsequently, they were further confirmed as Providencia stuartii employing API 20E system ( Supplementary  Fig. 1c). All the isolates were Gram negative and short rod shaped under light microscope.

Prevalence of P. stuartii
Culture findings revealed the presence of P. stuartii among the chicken-droppings in five of seven PFs, thus yielding an overall farm prevalence for P. stuartii as 71.43% (Table 1). All the samples designated CK011-CK020 and CK051-CK060 collected from PF2 and PF6 respectively showed abundant growth for typical faecal microorganisms in both aerobic and anaerobic conditions. However, no growth of P. stuartii were found in PF2 cultivating layer-type chicken (located in 'S. Rajason' area), and PF6 cultivated pre-starter broiler-type chicken (located in 'Pandhoya'). Samples from remaining farms revealing the growth of P. stuartii demonstrated an intra-sample prevalence of 10% in PF1, PF4, PF5 and PF7 to 20% in PF3 (Table 1). Thus, the overall prevalence of P. stuartii among 70 chicken-droppings examined was about 9%, (6/70), when 11 of the isolates from 6 positive-samples were examined for subsequent AST.

DISCUSSION
The growth of poultry production by setting up chicken farms has increased extensively over the last 15 years in developing countries including Bangladesh [5]. PFs presumly contribute in the dissemination of various zoonotic pathogens, thus, spreading in working areas and surrounding communities leading to potential public health hazards [8,9]. Parallel to the rapid expansion of the business, sufficint biosecurity measures are not being practiced in these Bangladeshi PFs despite existing guideline on the prevention and control of transmission of associated pathogens [25,26]. A few studies have been carried out to identify zoonotic pathogens in Bangladeshi PFs [13,14,27]. There was a clear knowledge gap about many potential zoonotic pathogens that could be transmitted via poultry industries [28,29]. The promising poultry industry in Bangladesh may emerge as potential epicenters of zoonotic epidemics in absence of proper attentions and care. The reporting of Providencia stuartii from the poultry samples will be a flagship findings in this arena for the country.
Over 55% of the P. stuartii isolates became MDR against six most commonly used antibiotics we tested in this study. Most of these antibiotics are often used by the PF-workers/farmers to treat chickens in Bangladesh, including erythromycin and streptomycin [7] and often in a combination of two or more antibiotics. These aforementioned facts remain one of the most prudent contributing factors in acquiescing emergence of 9% 36% 55% 1 antimicrobial 2 antimicrobials ≥3 antimicrobials antimicrobial resistance in chicken-population in adjunct to certain selective pressure owing to indiscriminant use of antibiotics in these PFs [30]. Notably, antibiotics are easily available in Bangladesh beyond prescriptions that dispense without proper dosage, regimen directions of usage to treat human patients or to protect diseased poultry-flocks [11,31]. These misuses were reported to promote emergence of MDR strains in many countries [32]. Earlier studies reported P. stuartii to be resistant to penicillins, aminoglycosides, tetracyclines, sulphamethoxazole, polymyxin B, colistin, nitrofurantoin, older-cephalosporins, fosfomycin and chloramphenicol [33,34]. Our antibiotic resistance outcome is congruent to the earlier research that concedes P stuartii as the most resistant species in Providencia genus [35]. Their multidrug-resistant trait is plasmid mediated [35], however, further study will be required to affirm it for our isolates.
Sub-standard sanitation practice in a improvised and densely populated country like Bangladesh may aggravate the antibiotic resistance lineage easily to population level. So, MDR P. stuartii in chicken droppings may aggravate spreading both by person-to-person transmission [17][18][19] and via human to animal interface [36]. They may impose a threat of cross-resistance to other pathogens [37], by changing in ways that lead to the reduced effectiveness of antibiotics to cure or prevent infections. Consequently, attributes of MDR-resistance may also emerge in other dominant common flora under the selective pressure of antibiotics and lead a serious clinical implications. Transmitting the PF-origin MDR-Providencia spp. into surrounding environment and adjacent communities could make a huge public health challenge for Bangladesh in near future as happened in other communities [35,38]. Therefore, Government health authority should administer stringent biosecurity measures and formulate national guidelines for controlling MDR and abuses of antibiotics in animal husbandry.
This study has several inherent limitations. All of the chicken dropping samples in the study were collected from Savar area (Dhaka). Samples from other areas of the country were not included in this study. The results obtained from this analysis might appear some variation to the samples in other regions of the country. The small sample size was a major constraint to convey meaningful outcomes by statistical analyses. Therefore, a prospective design of longitudinal study involving larger sample from diverse geographic areas may provide more detail insight of P. stuartii as zoonotic pathogens. The identification of P. stuartii was done based on detail biochemical followed by API 20E confirmation. However, parallel molecular detection could provide extra strength in our findings. Moreover, the establishment of a clonal relationship between PF-originated and a clinical sample remains important to translate our preliminary findings into clinical applications more effectively.

CONCLUSION
This study has provided baseline prevalence data on the presence of P. stuartii and their antimicrobial resistance pattern in the chicken droppings in poultry farms in a region of Bangladesh. The report will contribute initial findings to make awareness on the emerging zoonotic infections by P. stuartii in PFs in Bangladesh. More in-depth study covering other regions of the country can authenticate our preliminary findings and can be helpful in developing biosecurity concern in environmental and food-safety issues nationwide and in abroad.

ETHICS STATEMENT
Consents were obtained from respective farm owners before the collection of respective chicken droppings.