Risk factors assessment and antimicrobial resistance of Salmonella isolates from apparently healthy and diarrheal dogs in Baghdad, Iraq

1. Salmonella has been isolated from both healthy and diarrheal dogs in Baghdad.
2. Three species of Salmonella were isolated. Further, S. Muenchen was isolated for the first time from dogs in Iraq.
3. The importance of associated risk factors of Salmonella isolated from dogs in Salmonella prevalence is well known, and this study was the first report in Iraq focusing on these parameters.
4. Detection of drug and multidrug resistance in Salmonella isolates from dogs examined.
5. Salmonella identified in dogs in Baghdad reflects a potential risk to public healing.

Introduction

Salmonellosis is a critical disease-causing high morbidity and mortality, leading to major global economic problems (1). The dog is one of the pet animals that humans are increasingly breeding and purchasing globally, and due to the contact between these animals and humans, they have been considered a possible source of many zoonotic diseases such as Salmonellosis (2-5). Dogs may be asymptomatic carriers of Salmonella spp. and harbour it in their gastrointestinal tract and mesenteric lymph nodes, where they may shed the Salmonella intermittently for more than six weeks (3,6). The clinical signs appear after 3-5 days of infection and include anorexia, fever, diarrhea or bloody diarrhea, abdominal pain, septicemia, and abortion (7-9). Salmonella infections in dogs can be influenced by variable factors, such as feeding, geographic area, the socioeconomic status of the owners, age, gender, breed, and public awareness of canine zoonosis (6,10). These pathogens in animals that live near populations of humans require strategies for monitoring and controlling them to protect both animal and human populations (11). Antimicrobial drugs are commonly used in both humans and animals. Antimicrobial resistance and Multidrug Resistance (MDR) among Salmonella spp. have increased globally, especially for clinically significant antibiotics such as cephalosporins and fluoroquinolones, now an increasingly significant issue. Antimicrobial resistance in Salmonella isolates from people and animals (including dogs) has been the subject of several publications (6-13). Salmonella has been isolated in Iraq from food samples (12,14), milk (15), animals (15,16), and humans (13,17).

There is an absence of adequate studies in Iraq evaluating Salmonella prevalence in dogs, the risk factors influencing the prevalence rate, or Salmonella antimicrobial resistance to antibiotics. Determining Salmonella prevalence in dogs with diarrhea and otherwise healthy dogs was the goal of the present experiment, as was identifying the risk factors associated with the Salmonella prevalence and determining the antimicrobial resistance profiles of Salmonella species isolates.

Materials and methods

Ethical approve

This Study was approved by Animal Care and Use Committee of College of Veterinary Medicine, Baghdad university No. 2190/PG in 10-10-2022

Sample and data collection

Between October 2022 and February 2023, 165 rectal swabs were collected from apparently healthy (90) and diarrheal (75) dogs when they visited the Baghdad veterinary teaching hospital, clinical veterinary clinic, and market sale. The diarrheal dogs were selected based on history and clinical signs suspected of Salmonella infections. The information from each dog was collected by questionnaire to assess the possible risk factors connected to Salmonella infection occurrences, such as gender, breed, age, bodily condition, feeding, and educational status of owners. The breeds of dogs were classified according to the classification used in the Fédération Sinologue International (FCI) as small, including Terrier (30), French Bulldog (13), Poodle (12), Pomeranian (15), Poo (25), Lolo Fox (10), and large, including pointers (6), Belgian Malinois (10), Hasky (15), Fox (1), German Shepherd (24), and Giant Schnauzer (4). According to age, dogs are divided into young (1 year) and adults (>1 year). Transport media transported samples to the Baghdad Veterinary College (Zoonotic Diseases Unit) in a box containing ice packs.

Salmonella isolation and characterization

Salmonella species isolation and identification from fecal swabs were done according to the International Standard Organization (ISO) (18). The samples were transferred into buffered peptone water (Hi Media/India) at 42°C for 24 hours of incubation. Then, the pre-enriched sample was transferred (1 ml) from its container to tetrathionate broth and incubated for 24 hours at 42 °C. Following this, ten μl of tetrathionate broth was streaked onto Salmonella-Shigella (SS) agar (Hi Media/India) and xylose lysine deoxycholate (XLD) agar and then incubated for 24 hours at 37°C for further analysis. Colonies that had pink coloration with or without black cores on the XLD agar were picked up and sub-cultured on Hi Crome^TM Salmonella agar (HI Media/India), followed by incubation for 24 hours at 37°C. The mauve colonies observed on Hi Crome^TM agar were transferred onto nutrient agar (Oxoid/UK) and incubated at 37°C for 24 hours to facilitate further identification. Colonies from nutrient agar were picked and examined for oxidase, catalase, and Gram stain. In addition, the following biochemical test agars: Klingler's Iron agar, urea agar, and Simmon's citrate agar (Hi Media in India), were inoculated and then incubated for 24 hours at a temperature of 37°C. The Salmonella colonies exhibited an alkaline slant and acid bottom formation on Klingler's Iron agar, along with hydrogen sulfide production. Additionally, these colonies demonstrated positive results for citrate utilization and negative results for the Indole test and the urease test were more identified using the Analytical Profile Index 20E (API 20E) identification Kits (bio Merieux, France), VITEK 2 system compact (bio Merieux, France), and serotyped were performed by slide and tube agglutination tests in the Central Health Laboratory in Baghdad, Iraq, using identification kits (bio Merieux, l'Etoile, France)

Antimicrobial resistance testing, multidrug resistance (MDR), and multidrug antibiotic resistance index (MARI)

Salmonella isolates were evaluated for resistance to 10 antimicrobial drugs, including cefotaxime (30mg), amoxicillin (20 mg)/ clavulanic acid (10mg) (AUG30C), ciprofloxacin (5 mg), norfloxacin (10mg), trimethoprim/sulfamethoxazole (1.25/23.75mg), tetracycline (30mg), azithromycin (15mg), gentamycin (10mg), ampicillin (10), and chloramphenicol (30mg) using the antibiotic disc diffusion method according to the Clinical and Laboratory Standards Institute guideline (CLSI) (19). Bacterial suspensions (1.5*10⁵ CFU/ml) were prepared by selecting 4-5 colonies of each bacterium from nutrient agar and suspending them in a sterile test tube containing 4 ml of normal saline using a McFarland 0.5 tube. A sterile cotton swab was carefully dipped into the bacterial suspension and then evenly spread over Mueller-Hinton agar. It was left for 10 minutes to absorb the bacterial suspension. The antimicrobial discs were then put on the agar using sterile forceps and firmly pressed against the medium to confirm contact with the medium's surface. Subsequently, the plates were inverted and incubated at 37 °C for 24 hours. The measurement of the inhibitory zones surrounding the antimicrobial discs was conducted using a metric ruler, with the values recorded in millimeters and the characteristics of Salmonella spp. as resistant (R) and susceptible (S), according to CLSI (19). The phenomenon of multidrug resistance (MDR) was identified by observing that the isolates exhibited resistance to two or more classes of antimicrobial agents. The Multidrug Antibiotic Resistance Index (MARI) was computed for all Salmonella isolates using the formula A / B, where "A" represents the count of antimicrobials to which an isolate exhibited resistance, and "B" denotes the total number of antimicrobials to which the isolate was exposed (20,21).

Statical analysis

The Static Analysis System (SAS, 2018) was used to compute the data to assess the impact of various factors on the research parameters. A chi-squared test (X²) was employed to significantly compare percentages (0.01 and 0.05 probability).

Results

Prevalence rate of Salmonella in dogs

Of the 165 dogs examined, ten (6.06%) were positive for Salmonella; in diarrheal dogs, it was 8/75 (10.6%); and in apparently healthy dogs, it was 2/90 (2.22%). Three Salmonella serovars were identified: S. Typhimurium was the most isolated at 6/10 (60%), followed by S. Enteritidis at 3/10 (30%) and S. Muenchen at 1/10 (10%).

Risk factors linked to Salmonella in dogs

The analysis of factors associated with dog fecal shedding of Salmonella included gender, breed, age, body condition, diarrhea problem occurrence, feeding, and educational status of owners recorded in Table (1). Salmonella isolates were higher in diarrheal dogs 10.66% than in non-diarrheic dogs 2.22%, with significant differences. The odds ratio (OR) of Salmonella shedding in sick dogs was 5.25 times higher than in apparently healthy dogs (OR = 5.25, 95% CI: 1.08–25.55, P-Value = 0.04). According to the breed, the Salmonella prevalence rate in large breeds was 13.3% higher than in small breeds 1.9%, with significant differences, and the OR was 7.92 times higher in large dogs (OR = 7.92, 95% CI: 1.62–38.65, P-value = 0.01) as compared with small animals. Concerning body condition, thin-body dogs were high isolates at 16.98% compared to fat-body animals at 1.25%. In contrast, medium-body dogs were not isolated. Moreover, there was a significant difference between thin and fat body conditions in dogs. The OR in thin dogs was 13.87 times and 16.15 times greater than in both medium and fat body condition dogs (OR = 16.15, 95% CI: 1.98–131.78, P-value = 0.07; OR = 13.87, 95% CI: 0.78–247.01, P-value = 0.07), respectively. The result of feed processing showed that the difference between uncooked and mixed food was significant. Salmonella in dogs fed uncooked food was at 12.9% compared to dogs fed mixed food at 1.49%, and the OR in dogs with uncooked food was 7.48 times greater than that in dogs with mixed food (OR = 7.48, 95% CI: 1.53–36.48, P-value = 0.01). The Salmonella prevalence rate in females was 9.33% higher than in males 3.33%, with no significant differences, and the OR in females was 2.98 times higher than in male dogs (OR = 2.98.95% CI: 1.62–38.65, P = 0.01). In regards to age, the Salmonella prevalence rate in the young dogs did not differ significantly compared with the old dogs; young dogs reported higher isolates at 8% than old dogs at 3%, and the OR of the young dogs was 2.74 times that of the old dogs (OR = 2.74, 95% CI: 0.56–13.33, P = 0.21). According to the educational status of dog owners, no significant differences were found; Salmonella was identified in dogs’ owners with high school and above education at (6/115) 8% than those of owners with below high school education at (4/50) 5.21% (OR=0.63, 95CL:0.17-2.35, P-value=0.49) (Table 1).

Table 1: Analysis of risk factors for dogs' Salmonella isolated from dogs

NS: non-significant *: significant **: highly significant

Antimicrobial resistance patterns of Salmonella isolates from dogs

Results of antibiotic resistance showed the isolates had complete resistance of 100% to both amoxicillin/clavulanic acid and cefotaxime, 70% resistance against azithromycin, and 60% resistance against each of ampicillin, gentamycin, and tetracycline. In comparison, resistance was 50%, 40%, and 30% against trimethoprim-sulfamethoxazole, chloramphenicol, and ciprofloxacin, respectively. S. muenchen was 100% resistant to amoxicillin, clavulanic acid, and cefotaxime, whereas it was 100% susceptible to other antimicrobial agents. S. Typhimurium had 100% resistance to cefotaxime and amoxicillin/clavulanic acid, 66.6% resistance to ampicillin, azithromycin, and tetracycline, 50% resistance to gentamycin, 33.3% resistance to chloramphenicol and trimethoprim-sulfamethoxazole, and 16.6% resistance to ciprofloxacin. S. enteritidis was resistant 100% to amoxicillin/clavulanic acid, azithromycin, cefotaxime, gentamycin, and trimethoprim-sulfamethoxazole, and 66.6% to ampicillin, chloramphenicol, ciprofloxacin, and tetracycline. All of the Salmonella serovars were MDR 100%, except for S. Muenchen, and the isolates were high-risk (MAR Index of S. Typhimurium ranged from 0.4 - 0.7; S. Enteritidis from 0.7 - 0.8 and S. Muenchen was 0.2 recorded; also, nine of the ten nontyphoidal Salmonella isolates were resistant to three or more antimicrobials, and two (20%) were resistant to eight antimicrobials; furthermore, four of the six isolates of S. Typhimurium exhibited resistance to at least five antimicrobials (Tables 2 and 3).

Table 2: Antimicrobial resistance pattern of all dogs Salmonella isolates in Baghdad, Iraq

AMP: Ampicillin, AMC: Amoxicillin/Clavulanic Acid, AZM: Azithromycin, CTX: Cefotaxime, CHL: Chloramphenicol, CIP: Ciprofloxacin, GEN: Gentamycin, NOR: Norfloxacin, TET: Tetracycline, SXT: Trimethoprim-Sulfamethoxazole.

Table 3: MDR profiles and MAR index for isolates of dogs Salmonella serovars in Baghdad, Iraq

Discussion

Salmonella Typhimurium and Salmonella Enteritidis are extensively distributed and often linked with diseases in humans and animals (22, 23, 24). In the current study, Salmonella Typhimurium was the highest isolate from dogs, followed by Salmonella Enteritidis, which was similar to other findings: 63.63% of Salmonella Typhimurium and 36.36% of Salmonella Enteritidis were identified in dogs (25). Also, Salmonella Typhimurium was isolated from dogs at 50% (26). While Salmonella Enteritidis was reported as having the most isolates with a rate of 47.61% (10/21) compared to Salmonella Typhimurium at a rate of 19.04% (4/21) (27), Salmonella Muenchen was previously isolated from children and frozen beef meat in Iraq (28,29); in the current study, this serovar has been isolated from diarrheal dogs for the first time in Iraq, whereas globally, it was reported in dogs (30, 31). This serovar has a broad range of hosts, causing severe infections and complications (32).

In the current study, 6.06% of dogs were positive for Salmonella in Baghdad city, which in diarrheal dogs was significantly higher than in apparently healthy dogs. It has been observed that dogs suffering from diarrhea have a higher likelihood of testing positive for Salmonella and shedding Salmonella in their feces compared to non-diarrheic dogs. Similarly, Salmonella was recorded in diarrheal and apparently healthy dogs at 6.4% (26). Also, Usmael et al. (10) mentioned that the Salmonella prevalence in dogs was 6.3%; in diarrheal dogs, it was elevated compared to dogs without diarrhea. The prevalence observed in this study was more significant than that documented by Reimschuessel et al. (33) stated that the dog's Salmonella occurrence rate was 2.47% and higher in diarrheal dogs than in non-diarrheic dogs, with significant differences. In addition, a study found the Salmonella prevalence rate in diarrheal dogs at 3.5% (22), and in other studies, the Salmonella prevalence rate in apparently healthy dogs was reported at 1.85, 8.2, 11, and 11.7% (6,27,31,34), respectively. The variation in the findings of the current investigation, when compared with other information, may be attributed to the variation in sample size, research time, technique and methods used for diagnosis, geographical area, and study season (46,47).

The higher rate of Salmonella prevalence in large breeds and higher risk in comparison with small breeds, with significant differences in the present study, corresponds with Chapple et al. (30), who recorded a higher rate of Salmonella infection in large breeds than in small breeds. No significant difference in dog breeds was observed by Gebremedhin et al. (34) and Núñez Castro et al. (35). The higher rate of Salmonella in large breeds than in small breeds may be due to physiological and anatomical differences; the weight of the intestines in large dogs is 3% of the body weight compared to 7% in smaller breeds; this means there is a small intestinal area for digestion and absorption of the food, and the food spends a long time in the colon for large-breed dogs. Moreover, the low capacity of the stomach in small dogs creates sensitivity to disease (36, 37). This research found that thin-body-conditioned dogs shed significantly more Salmonella and were at a higher risk than fat and medium-body-conditioned dogs. These findings are consistent with the results of Usaeml et al. (10), as they reported that the Salmonella levels in thin-body conditioned dogs were higher than those in fat-body conditioned dogs. Also, the results were incompatible with those of Núñez Castro et al. (35), who recorded no significant differences according to body condition in dogs. It is difficult to untangle the relationship between a body condition and a bacterial infection; dogs with weakened body conditions are more susceptible to infections, which are more likely to occur when they have reduced access to food and compromised immunity (38).

Dogs fed uncooked were infected higher than those fed a mixed diet, with a significant difference; these findings match another published paper, revealing a higher Salmonella rate in dogs fed on offal than in dogs fed a mixed diet (6, 10). According to the Public Health Agency of Canada (PHAC), Salmonella contamination was commonly observed in uncooked meat and meat-based products. Raw diets were made at home and were recognized as a substantial reservoir of this bacterial pathogen (39). Focuses on the effect of gender; our data showed that female dogs had a higher incidence rate of Salmonella, with no significant difference in the current study. The findings of our study were consistent with those of previous research (6, 34). Correspondingly, Jajere et al. (30) noted that the gender of the dogs affected the probability of a dog harboring Salmonella. The variation in the isolation rates found in this study compared to others may primarily be due to differences in the sampling period and the techniques used for isolation (40).

Regarding Dogs' ages, which is yet another risk factor for shedding Salmonella, this study indicated a higher Salmonella rate in young dogs than in old dogs, with no significant difference, and young dogs were more at risk than old dogs. Similarly, Salmonella in young dogs was higher than in adults, with no significant differences reported (10,31,41). Moreover, Salmonella was found infected in dogs under one year compared to dogs older than one year, with a significant difference reported by Núñez Castro et al. (35). It can be hard to compare data due to different age patterns being seen in different studies and because the selected society's lifestyles and dog care practices differ. Young animals usually have undeveloped immune systems, making them more vulnerable to bacterial infections (42,43). According to the educational status of dog Owners, our finding shows no significant difference in dog Salmonella prevalence depending on the owners' educational levels below high school or high school and above. These findings concord with an earlier study that reported no significant difference regarding the educational status of dog owners (34).

In the present work, the isolates of Salmonella dogs were very resistant to amoxicillin/clavulanic acid, cefotaxime, azithromycin, ampicillin, gentamycin, tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, and ciprofloxacin. In contrast, all isolates were susceptible to norfloxacin. These findings were more significant than those reported by Usmael et al. (10) as they documented that the resistance rates of Salmonella isolated from dogs were 41.7% for ampicillin, 21.2% for tetracycline, 12.5% for amoxicillin/clavulanate, and 4.2% for trimethoprim-sulfamethoxazole. In contrast, Kiflu et al. (31) report that 14 different Salmonella serotypes in dogs have a low level of resistance compared to our results, as they showed that the resistance rate was 26.2% for amoxicillin/clavulanic acid, 30.9% for ampicillin, 7.1% for chloramphenicol, 2.4% for gentamicin, 9.5% for sulfamethoxazole and trimethoprim, and 0% for ciprofloxacin; furthermore, they observed that Salmonella Muenchen and Salmonella Typhimurium isolated from apparently healthy dogs were sensitive to all these antibiotics mentioned above. Al–Rubaye and Al-Doori (26) observe that 62.5% of dog Salmonella isolates resist ampicillin, 50% to tetracycline, and 37.5% to chloramphenicol and gentamicin. The Salmonella isolates found in dogs have shown the greatest resistance to ampicillin 100%, tetracycline 93.3%, and chloramphenicol 20%. Nonetheless, they were entirely susceptible to norfloxacin, with a 100% percentage (34). Developing germs resistant to conventional antibiotics is a serious health hazard; it has rapidly and considerably increased in recent decades (49). The elevated resistance to cefotaxime and amoxicillin/clavulanic acid in this work may result from their widespread use in pet medicine in Baghdad.

The MAR index is a proactive, functional, and less expensive method for identifying the origins of bacteria that are resistant to antibiotics, and MAR index values of 0.2 or higher may reveal that the use of antibiotics is the contamination source with the most significant risk (21). There is a concerning incidence of multidrug resistance within Salmonella serotypes isolated from dogs, which poses a high risk. MDR patterns were observed among Salmonella isolates from dogs at 45.2%; other researchers found that the MDR of dog Salmonella isolates was between 6% and 46% (31,34). In the same circumstance, a study of a high risk of Salmonella isolated from other models, such as humans, was reported (13). The increase in Salmonella resistance is assigned to the indiscriminate utilization of antibiotics in the veterinary and human fields (44,45).

Conclusion

This study revealed that diarrheal dogs have a higher prevalence of Salmonella than apparently healthy dogs, and dogs fed uncooked feed are at high risk of becoming infected with Salmonella. Apparently healthy dogs act as potential sources of antimicrobials, and the pathogenic strains of Salmonella can be transmissible to humans and other animals.

Conflict of interest

There are no conflicts among the authors; there is interest.

Acknowledgments

The research was conducted in the College of Veterinary Medicine's United Zoonotic Diseases laboratories at the University of Baghdad, Iraq. My sincere gratitude to the workers at these laboratories for supplying the necessary tools, specifications, and space