Drug resistance patterns of Salmonella isolates of equine origin from India

Introduction: Salmonellosis is a zoonosis, and one of the most serious public health and animal health problems. Methodology: We studied 111 isolates of Salmonella belonging to 14 S. enterica subspecies enterica serovars namely S. Abortusequi (45), S. Weltevreden (1), S. Dumfries (2), S. Tshiongwe (1), S.I. 4,5,12:r,i:1,5 (12), S. Bovismorbificans (3), S. Drogana (8), S. Lagos (4), S. Kottbus (3), S. Richmond (1), S. Typhimurium (6), S. Newport (7), S. Paratyphi B var Java (17) and S. Saintpaul (5) isolated from equids in India. Results: All strains studied were resistant to one or more antimicrobials. Strains were resistant to ampicillin (18, 16%), ampicillin+cloxacillin (6, 5%), cefotaxime (6, 5%), chloramphenicol (2, 2%), ciprofloxacin (9, 8%), gentamicin (27, 24%), kanamycin (37, 33%), nalidixic acid (10, 9%), furazolidone (97, 87%), streptomycin (33, 30%), sulphamethoxazole (91, 82%), tetracycline (48, 43%) and trimethoprim (5, 4.5%). Multiple-drug-resistance was detected in 84 (75.7%) isolates and was seen in isolates of all serovars except of S. Kottbus, a rare serovar in India. Salmonella isolates could be classified into 51 resistotypes but 47 (42.3%) isolates belonged to six major resistotypes. Resistotype 13 (resistant to furazolidone, sulphamethoxazole and tetracycline) was most common, followed by resistotype 19 (resistant to nalidixic acid, sulphamethoxazole and tetracycline), resistotype 28 (resistant to furazolidone, streptomycine, sulphamethoxazole and tetracycline) and resistotype 40 (resistant to furazolidone, gentamycin, kanamycin, streptomycine, sulphamethoxazole and tetracycline) including 11, 8, 8 and 7 strains of different serovars, respectively. Conclusions: This study revealed that antimicrobial drug resistance was common in Salmonella isolates from equids even towards those drugs not used in equids.


Introduction
Salmonellosis is a zoonosis, and one of the most serious public health and animal health problems.Almost all warm-blooded and many cold-blooded animals are its natural hosts.It may vary in severity from undetectable infections to acute disease, which may be fatal to the very young, the old, or the debilitated individual.Salmonellosis in equids is a global problem with varying severity and prevalence varying from less than 2% to about 27% depending upon the topography and climatic conditions [1,2,3,4].
The earliest report of salmonellosis in equids was from cases of abortion in mares [5,6]; causative organism was identified as Salmonella abortusequi, now known as S. enterica subsp.enterica serovar Abortusequi (S.Abortusequi).Earlier S. Abortusequi was found globally; however, today the infections are mostly limited to Asian and African countries.
The majority of Salmonella infections in equids are iatrogenic [13].Important risk factors, which lead to precipitation of Salmonella infection in equids, include administration of antimicrobials, colic, surgery, and transportation stress.Animals with severe neutropenia (due to either any immunodeficiency problem or to prolonged anticancer chemotherapy) are also at increased risk for bacterial and fungal infections.[14][15][16].
Antimicrobial resistance in disease-causing bacteria results in increased illness, deaths, and increased health-care costs [17][18].Emergence of multi-drug-resistance (MDR) in empirical antimicrobial agents is a global problem, particularly in pathogens causing nosocomial infections.The increasing use of indwelling devices, as well as widespread and indiscriminate use of antibacterial agents in hospital settings, contributes to antimicrobial resistance among pathogens causing nosocomial infections [17,19].Although, much work has been conducted on the emergence of antimicrobial resistance to Salmonella in human health, [20][21][22], little is known about antimicrobial resistance in Salmonella strains prevalent in equids in India.We conducted antimicrobial drug sensitivity tests on 111 Salmonella isolates from equids isolated from different parts of India [12] and detected MDR in more than75% of the isolates.

Discussion
In absence of regulations regarding the use of antimicrobials in domestic animals in India, there is no restriction on use of antimicrobials intended for human use and choice of drug often depends on economic availability of the medicine [25,26].In equids, penicillins, tetracyclines, gentamicin, cotrimoxazole, cefotaxime, ampicillin and kanamycin are often the drugs of choice, but many veterinarians also use quinolones, third-generation cephalosporins, and new generation aminoglycosides [25,26].MDR in Salmonella isolates and other bacteria is increasingly common in clinical as well as nonclinical samples of equine origin in India [26,27] and it might be associated with indiscriminate drug use of antimicrobials in animals and potentially in human beings in India [25,26].
Although use of sulfonamides, furazolidone and tetracycline is minimal in equids [25,26,28], the majority of Salmonella isolates of equine origin were resistant to sulfamethoxazole (82%), furazolidone (87%) and tetracycline (43%).This pattern of MDR might be attributed to widespread use of these antimicrobials as empirical therapy of febrile syndromes in humans and other animals, as well as to the use of these antimicrobials as growth-enhancers in animal production [25,26,29,30].Resistance to sulfamethoxazole in MDR Salmonella has been prevalent for three decades among human isolates in India [30,31] and communicability of Salmonella between human and animals [32,33] might be responsible for the similar pattern in Salmonella isolates from equids and from other animals.Resistance to similar drugs is reported in Salmonella isolated from food, water [21,22] and in different animals [34] in India.
Horse meat is rarely consumed in India and neighbouring countries, but spread of salmonellosis from horses carrying MDR Salmonella to other livestock reared for meat or milk or other products and even to persons caring for horses is a possibility facilitating circulation of MDR strains [35].Animals scavenging on dead horses, or dogs and other pet animals fed on horse meat, may also acquire Salmonella infection from contaminated horse meat/ Table 2. Multiple drug resistance (MDR) among Salmonella of equine origin in India.flesh and may have a significant role in the spread of salmonellosis in the environment [32,33].Prevalence of Salmonella isolates from equids resistant to drugs used in human patients and also in the strains belonging to serovars often causing salmonellosis in human but not in equids (S.Paratyphi B) indicates the similar possibility of the circulation of MDR strains in the environment and among horses, one of the natural hosts for this dreaded pathogen.Moreover, the possibility of the spread of MDR Salmonella infection through horse meat exported to the consumer countries cannot be excluded, and one such outbreak of MDR S. Newport (the serovar also present in Indian horses) recently occurred in France [36].Salmonella serovars similar to those in equids of India have also been reported frequently from meat and excretions of horses in other countries [32,33,[35][36][37][38].
Apart from resistance to sulfamethoxazole and furazolidone, S. Abortusequi isolates were more often resistant to ciprofloxacin, gentamicin, kanamycin and streptomycin but rarely to tetracycline (Table 1).Ciprofloxacin, gentamicin, kanamycin, and streptomycin are commonly used and tetracyclines are rarely used drugs in equids in India [25,26].The presence of Salmonella Abortusequi, being a hostadapted Salmonella rarely isolated from other animals and sources [12], indicated that empirical drug use affects the emergence of drug resistance in Salmonella [30,39,40] even in cases when the causal agent is not directly hit by the treatment.Resistance to chloramphenicol in only S. Paratyphi B and S. Saintpaul, common serovars in human patients in India, further indicates that Salmonella probably circulates in different animals and humans; and empirical drug use resulting in emergence of resistant strains in one host might be responsible for similar resistance in other hosts where the concerned drug is not used.Chloramphenicol is rarely used in animals, but it is the most commonly used antibiotic in humans for treatment of typhoid and paratyphoid fever [30,31].
Nalidixic acid and ciprofloxacin resistance in isolates of S. Abortusequi, S. Typhimurium, S. Paratyphi B and S. Weltevreden is also significant because it is associated with higher MICs for other fluoroquinolones rendering them clinically ineffective in spite of sensitivity shown by disc diffusion method in vitro [30,41].Ciprofloxacin and enerofloxacin are the commonly used drugs in equids in India [25,26] which might have selected for resistance to fluoroquinolones.
Predominance of MDR among Salmonella isolates from equids is of particular importance because of the apparent risk to humans acquiring zoonotic Salmonella through contact with horses.Although there is no microbiologically proven link between antimicrobial resistance and virulence for humans in zoonotic Salmonella, increased rates of hospitalisation have been reported for patients with infections with MDR S.Typhimurium [29].Multiple drug resistance to empirically used drugs is anticipated; however, resistance to chloramphenicol and tetracyclines, rarely used in horses, is surprising because emergence of drug-resistant Salmonella is thought to be due to selective pressure from the use traits, even in absence of antimicrobial selection pressure, might be due to either the ability of Salmonella to build up multidrug resistance as an integral part of its genome [29] or to recent acquisition of MDR strains by equids from other animals and human beings.Cefotaxime resistance in only 5% of isolates of Salmonella of equine origin supports recommending this drug as the safest choice for treatment of salmonellosis in equids [28].Low levels of resistance to cefotaxime in isolates of Salmonella from equids might be attributed to either less use of this thirdgeneration cephalosporin in equids [26,28] or in other animals and human beings probably due to its comparatively high cost compared to broad-spectrum and cheaper fluoroquinolones [42].
The study concludes that MDR is common among Salmonella isolates of equine origin in India.Detection of MDR in more than 75% of Salmonella strains, even in the serovars rarely causing any disease in equids, indicated that the horse is a natural host of Salmonella.Resistance to antimicrobial drugs not used in equids also suggests that either Salmonella maintains the MDR gene pool on chromosomes even in absence of selection pressure, or that Salmonella is frequently communicable between different host species or that both of the factors required in MDR development and maintenance might be important.

Table 1 .
Antimicrobial drug resistance in Salmonella isolates of equine origin in India.

Table 3 .
Resistotypes (R-types) among Salmonella isolates of equine origin in India.