Prevalence and multidrug resistance pattern of Salmonella isolated from resident wild birds of Bangladesh

Aim: Salmonellosis is one of the most common zoonotic diseases, and the presence of antimicrobial resistant Salmonella in wild birds is global public health threat. Throughout the last decades, multidrug resistance of Salmonella spp. has increased, particularly in developing countries. Therefore, a cross-sectional study was conducted to investigate the prevalence of Salmonella spp. and antimicrobial resistance pattern against Salmonella spp. from two species of resident wild birds namely house crow (Corvus splendens) and Asian pied starling (Gracupica contra). Materials and Methods: Samples were collected from cloacal swabs of house crows and Asian pied starling for isolating Salmonella spp. (bacteriological culture methods) followed by antimicrobial susceptibility testing (disk diffusion method) against Salmonella spp. isolates during March to December 2014. Results: The prevalence of Salmonella in Asian pied starling and house crows were 67% and 65%, respectively. Within the category of samples from different species, the variation in prevalence was not varied significantly (p>0.05). Isolated Salmonella spp. was tested for resistance to six different antimicrobial agents. Among six antimicrobial tested, 100% resistance were found to penicillin, oxacillin, and clindamycin followed by erythromycin (50-93%), kanamycin (7-20%), and cephalothin (30-67%) from both species of birds. Kanamycin remained sensitive in (70-73%), cephalothin (26-70%), and erythromycin appeared to be (0-30%) sensitive against Salmonella spp. isolates. Isolated Salmonella spp. was multidrug resistant up to three of the six antimicrobials tested. Conclusion: It can be said that the rational use of antimicrobials needs to be adopted in the treatment of disease for livestock, poultry, and human of Bangladesh to limit the emergence of drug resistance to Salmonella spp.


Introduction
Salmonella has been renowned as a significant pathogen that can contaminate human and animals consequential in major morbidity and mortality [1].Salmonella spp. was recognized as a major cause of food and waterborne illnesses in many countries that are closely associated with the presence of resident wild birds in the vicinity of domestic one where wild birds were a major carrier for this Salmonella spp.Most of the Salmonellosis is zoonotic in origin [2].Salmonellosis has been found to be major infectious diseases of all ages of birds.This disease is endemic in many developing countries, particularly the Asian subcontinent and, South and Central America.
Salmonellosis is a major problem in poultry industries of Bangladesh and its prevalence ranged from 7% to 53.3% [3][4][5].The probable cause for spreading of this disease can be Salmonella carrying wild birds, from them Salmonella spread into livestock and human.In recent years, problems related to Salmonella have increased significantly, both in terms of the incidence and severity of cases of human Salmonellosis.Heavy use of antibiotics for medical and veterinary purposes [6,7], as well as the domestic and agricultural use of pesticides and related compounds [7], caused significant antibiotic contamination of the natural environment and consequent development of resistance in communities.
In veterinary practice, antibiotics are used in livestock production, disease prevention, and as Available at www.onehealthjournal.org/Vol.2/7.pdfgrowth-promoting feed additives, and due to indiscriminate use of antimicrobials, the drug residues were deposited in the environment and also in the food animals tissues [8,9] which ultimately causes antimicrobial resistance.The use of antibiotics in animals disrupts normal flora of intestine, resulting into emergence of antibiotic-resistant Salmonella and their prolonged fecal shedding into the environment.Such practice has led to misuse of antibiotics with the associated high prevalence of antibiotic resistance among isolates from animal and food sources [10].Effluents from environments (hospitals and slaughterhouses) are alarming risk for clinician to treat the patient with antibiotics due to harboring antibiotic resistance bacteria [11][12][13].A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes, antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health.There are reports of a high prevalence of resistance in Salmonella isolates from countries such as Taiwan, India, and The Netherlands and resistant isolates from France, Canada, and Ethiopia.Similarly, there are various reports of multidrug resistant Salmonella organisms isolated in Bangladesh.Recently, antimicrobial resistance Salmonella spp.isolated from commercial layer eggs, eggshell surface, and egg-storing trays in Bangladesh [12].However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife [14,15].Most research on the epidemiology of antibiotic resistance spreading has paying attention on human and veterinary medicine, but there is a rising interest to recognize how bacterial resistance is transferred within reservoirs in natural environments [16].Due to their diversity in migratory patterns and ecological niches, and their ease in picking up human/environmental bacteria, they act as mirrors of human activities.In addition, bird migration provides a possible mechanism for the establishment of new endemic foci of disease at great distances from where an infection was first acquired [17].Besides, due to large range of migration and flying of wild birds close to the human settlement may cause drug resistance.
Some opportunistic and nonstructural studies had been conducted for Salmonella in commercial poultry in Bangladesh.Very few studies were conducted on isolation and drug resistance in Salmonella spp.throughout the world from wild birds but none in Bangladesh.This study, therefore, aimed to investigate the prevalence of Salmonella spp. in two species of wild birds (house crow and Asian pied starling) and antimicrobial resistance pattern against Salmonella isolates from those resident wild birds.

Ethical approval
The current study was approved by the Animal Ethical Experimentation Committee (CVASU-AEEC) of Chittagong Veterinary and Animal Sciences University, Bangladesh.

Study design
A cross-sectional study was conducted in two areas of Chittagong.The current study was conducted at Pahartoli and Bakolia of Chittagong City Corporation (CCC) during March to December 2014.

Sample collection and preservation
A total of 100 cloacal samples were collected from the resident wild bird that were trapped using misnet and from which 40 samples were house crows and 60 were Asian pied starling.A sterile cotton swab stick was used to collect the cloacal sample and then put the stick immediately into a sterile vial containing 6 ml amines transport media (Oxoid) and kept in a cool box (4°C).The samples were then transferred to the Poultry Research and Training Center (PRTC) laboratory under sterile conditions and processed for the isolation of Salmonella spp.

Salmonella isolation and identification procedures
The study was conducted utilizing the conventional methods for the detection of Salmonella spp.following the standard guidelines.There were four definite sequential steps: (1) Nonselective preenrichment, (2) selective enrichment, (3) plating out and identification, and (4) confirmation using triple sugar iron (TSI) agar.Buffered peptone water was used as nonselective preenrichment broth.Xylose lysine deoxycholate (XLD) was used as selective enrichment broth.After incubation, a loop full of broth was streaked on XLD medium and incubated at 37°C for 24 h.Colonies with black centers were considered presumptive Salmonella spp., for plating out and identification Salmonella-Shigella (SS) agar was used.The depth of the agar in the Petri dishes were maintained approximately at 4 mm.The plates were incubated at 37°C±1°C for 24±3 h.The plates were incubated in an inverted position.After incubation, the plates were checked for the development of typical Salmonella colonies.Typical colonies of Salmonella on SS agar were blackish.TSI agar was used to see the typical colonies and biochemical reactions for the identification of Salmonella spp.typical colonies grown on the SS agar plates were transferred and inoculated in TSI agar slant and incubated at 37°C±1°C for 24±3 h.The TSI agar slant surface was streaked, and the butt was stabbed and incubated at 37°C±1°C for 24±3 h.

Selection of antimicrobials for antimicrobial susceptibility testing
In the present research, the Salmonella isolates were tested with antimicrobial sensitivity testing to see whether they are resistant to antimicrobials (penicillin, erythromycin, clindamycin, oxacillin, kanamycin, and cephalothin) used in Bangladesh.

Antimicrobial sensitivity test
The antimicrobial sensitivity testing was done by the disk diffusion method as described by NCCLS Available at www.onehealthjournal.org/Vol.2/7.pdf2000, now known as the Clinical and Laboratory Standards Institute [18].Mueller-Hinton agar was used for this testing.The agar was prepared as per the instructions provided by the manufacturer.McFarland 0.5 turbidity standards were prepared as per the standard guidelines described by the CLSI.The predetermined battery of antimicrobial disks such as penicillin (10 units/disc), cephalothin (30 µg/ disc), oxacillin (1 µg/disc), kanamycin (30 µg/disc), erythromycin (15 µg/disc), and clindamycin (2 µg/ disc) were chosen and placed centrally onto the surface of the inoculated agar plate.The disk placed in the agar surface was not closer than 24 mm from center to center.A total of six disks were placed on one 150 mm plate.The plates were inverted and placed in an incubator set to 35°C within 15 min after the disks were applied.After 16-18 h of incubation, each plate was examined.
The resulting zone of inhibition was uniformly circular with a confluent lawn of growth.The diameters of the zones of complete inhibition were measured, including the diameter of the disk.Zones were measured to the nearest whole millimeter, using a suitable scale, which held on the back of the inverted Petri plate.The sizes of zones of inhibition were interpreted by referring to zone diameter interpretive standards from NCCLS 2000 [19], and the isolates were considered as sensitive, intermediately sensitive, or resistant to these agents tested according to the standard [18].

Data analysis
Field and laboratory data were stored and then cleaned in the MS Excel-2007 program before exporting to STATA/IC-13.0 for analysis.Descriptive analysis was performed to know the frequency and distribution of Salmonella and antibiotic resistance pattern.

Results
Salmonella spp. was isolated from cloacal sample on two species of wild birds such as crow and Asian pied starling at Pahartoli and Bakolia under CCC areas of Bangladesh to evaluate antimicrobial susceptibility to estimate the prevalence and pattern of antimicrobial resistance among Salmonella spp.isolates.
Table-1 shows the prevalence of Salmonella spp.within two species of resident wild birds.The prevalence of Asian pied starling and house crow was 67% and 65%, respectively.Within the category of samples from different species, the variation in prevalence was not varied significantly (p>0.05).The strength of association between the prevalence of Salmonella spp.within house crow and Asian pied starling were 1.4%, where the strength of association was 1.4% higher in Asian pied starling than house crows.
Among the six tested antimicrobials resistance pattern against Salmonella isolates penicillin, oxacillin, and clindamycin turned out as the highest level of resistance (100%) followed by erythromycin (50-93%), cephalothin (30-67%), and kanamycin (7-20%) from house crows and Asian pied starling.The rate of sensitivity to individual antibiotics against Salmonella isolates from two different species of birds was highest in kanamycin ranged (70-73%) followed by cephalothin (26-70%) and erythromycin (0-30%).Penicillin, oxacillin, and clindamycin showed no variation of resistance level; it was (100%) resistance in two species of birds and no isolates were found to sensitive against these three antibiotics.In case of erythromycin, 93% of Salmonella isolates from Asian pied starling and 50% from house crows were resistance to erythromycin.Antimicrobial resistance of kanamycin was found in crows (20%) and Asian pied starling (7%).The resistance level of cephalothin was higher in Asian pied starling (67%) and lower in house crows (30%).The sensitivity of kanamycin and cephalothin to isolated Salmonella spp. was better within two species of birds among all used antibiotics.Highest resistance pattern for intermediate type was in kanamycin ranged (10-20%) followed by erythromycin (7-20%), cephalothin (0-7%), and (0%) for other three antibiotics to isolated Salmonella for both species of birds.Highest intermediate type resistance was found (20%) to isolated Salmonella spp.from Asian pied starling in both kanamycin and erythromycin .

Discussion
House crow (Corvus splendens) and Asian pied starling (Gracupica contra) are very much common in our country, especially in the major city areas.They are usually carrion eaters and take their foods from the garbage of city areas.Nowadays, for the easy and frequent access of these two species in rural and urban area where different antibiotics are used regularly for human and livestock treatment, house crow and Asian pied starling got some antibiotic residue or antibiotic resistant bacteria from those human and livestock source anyway and they developed antibiotic resistance against some bacterial pathogens, such as Salmonellosis, which have important public health significance.
The prevalence of Salmonella in crow was 65% which was very much higher than the result of previous research isolated from crows cloaca [20].The prevalence of Salmonella in Asian pied starling was 67% which was also very much higher than the previous studies [21,22] on European starlings.Research conducted by isolation of S. enterica from gastrointestinal tract samples of European starling showed 2.5% prevalence [23] which is much lower than the current study.The higher prevalence rate of the present study may be due to overpopulated environment of Bangladesh and more frequent presence of house crow and Asian pied starling as a store house for microorganisms including Salmonella spp.[11,13].The favorable climatic condition and substandard hygienic condition of Bangladesh may also contribute to a higher prevalence rate of Salmonella.
Approaches to prevent and control Salmonellosis in the food animal industry by extensive use of antibiotics [24] has led to emergence of resistant bacteria [25] through mutation and acquisition of resistance encoding genes [26].The situation in developing countries such as Bangladesh may be exaggerated by easy accessibility of antimicrobials at a lower price and their extensive use in poultry production system [27].The current study recorded multiple antimicrobial resistances against Salmonella spp.(up to six) and in most cases, estimated 100% resistance to 2-4 antimicrobials across the study sites.These threatening correspond to the many nonepidemiological and opportunistic earlier studies in Bangladesh [28,29], India [30], Nepal, and Bhutan.The results of present study revealed that all the Salmonella isolates from cloacal swab of crows and Asian pied starling were resistance to penicillin which has the similarity with earlier studies [31][32][33].Several previous researches [32,34,35] found 100 resistance of oxacillin against Salmonella both in house crow and Asian pied starling.Erythromycin was resistance more against Salmonella spp.isolated from Asian pied starling (93%) than the resistance against Salmonella spp.isolated from house crows (50%).This results varied with many other previous studies where they said erythromycin were totally resistant to Salmonella spp.[32,36,37].The lower resistance of kanamycin found in the current study has more or less similarity with the result of different scientist where kanamycin resistance to Salmonella spp. was 18-60% [38][39][40][41], and it is good news for Bangladesh because it may be used effectively against Salmonella.Clindamycin showed 100% resistance against Salmonella, which found the similarity with the result of others similar researches [42][43][44][45].Cephalothin was resistance more against Salmonella spp.isolated from Asian pied starling (67%) than the resistance against Salmonella spp.isolated from house crows (30%).This result was more or less similar with many other previous studies where they said cephalothin was slightly resistant to Salmonella spp.[46].Penicillin, oxacillin, erythromycin, kanamycin, clindamycin, and cephalothin antibiotics are commonly used in livestock, poultry, and human treatment in Bangladesh.Therefore, residues of these antibiotics could have been passed through different environmental sources to the birds or they found antibiotic resistant Salmonella spp.from those sources during their migration.The high resistance of antibiotic to Salmonella spp.from cloacal swabs of house crow and Asian pied starling of the present study might be due to indiscriminate use of these antibiotics to human as well as in livestock.That's why wild birds got the antibiotic resistant Salmonella spp. or residues of those antibiotics by taking the dung or stool or other wastage of those livestock, poultry, or human.In addition, sometimes farmers select drugs by their own or rely on neighbor experienced farmers or nonveterinarians and these malpractices may not always ensure proper drugs doses, frequency of drug administration and complete course of drug treatment those can be a major cause for the establishment of antibiotic resistant Salmonella spp.

Conclusion
Salmonellosis is a leading, zoonotic, and widely distributed disease throughout the world.The level of resistance of Salmonella to antibiotics is an alarming issue to the human and livestock.Therefore, it is necessary to inform people about the future alarming condition of the antibiotic resistance.There is need for more rational use of antibiotics in animal production and more careful use in humans.It is important to take concerted action to improve antibiotic resistance surveillance capacity worldwide with a view to monitoring the emerging resistance genes and their transfer in both animal and human.In addition, alternatives to antibiotics should be explored such as the application of probiotics in poultry for production of safe edible products.To control Salmonella infection of wild birds in Bangladesh detailed epidemiological investigation is needed.Proper disposal of dustbin content, sewage water, dead carcass, waste food, and human stool should be needed to prevent transmission of Salmonella infection.
Copyright: Faruq, et al.This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Table - 1
: Prevalence of Salmonella spp.within two species of resident wild birds.

Table - 2
: Antimicrobial resistance pattern of Salmonella isolates from house crows.

Table - 3
: Antimicrobial resistance pattern of Salmonella isolates from Asian pied starling.

Table - 4
: Patterns of multidrug resistance in isolates of Salmonella from two different species.