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Frequency of Salmonella Infection among Backyard and Commercial Duck Flocks in Iran, Serotyping and Drug Resistance Profile of Salmonella Isolated from Ducks

Authors
Javad Javidnezhad 1 iD
Jamshid Razmyar 1 iD
Abbas Barin 2 iD
Affiliations
1Department of Avian Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Overview

Abstract

Salmonella is one of the most important foodborne infections with worldwide distribution that infects humans and a wide range of animals. Poultry and poultry products are considered major sources of Salmonella infections for humans. Like chickens, ducks can play an important role in the transmission of Salmonella bacteria to humans. This study aimed to gain a deeper understanding of the frequency of Salmonella infection in ducks in Iran, as well as to determine the serovar and antimicrobial resistance patterns of Salmonella isolates. In this study, fecal samples were collected from four provinces of Iran. Each of the six fecal samples was pooled, resulting in a total of 352 samples. To isolate Salmonella, all samples were cultured using the standard techniques previously described. From a total of 352 stool samples, 20 Salmonella isolates (17.6%) were isolated and serotyped by using the slide agglutination test. Then, they were subjected to a polymerase chain reaction using Salmonella genus-specific primers and species-specific primers for serovars Enteritidis, Typhimurium, and Infantis. The disc diffusion method was used to determine the sensitivity of isolates to 21 antimicrobial agents. Serotyping identified 15 serovars of Enteritidis and three serovars of Typhimurium. Two isolates remained unknown. No isolate was resistant to ceftazidime, ceftriaxone, lincospectin, fosfomycin, and colistin. The resistance to other agents was variable. There were 17 resistance patterns to 21 antimicrobial agents. Among the resistant isolates, the occurrence of multiple resistance was very significant, as they showed resistance to at least one and up to 11 drugs. By comparing the findings of this study with those of other investigations in this field, it was shown that, like chicken flocks, Salmonella are circulating in duck flocks in Iran.

Salmonellosis, Antimicrobial susceptibility, Salmonella Enteritis, Salmonella Typhimurium, Salmonella Infantis, Duck, Iran

Introduction

In 2022, the first and second most reported zoonosis in humans were campylobacteriosis and salmonellosis, respectively (1). The genus Salmonella belongs to the Enterobacteriaceae family. The members of these gram-negative bacilli are facultatively aerobic and anaerobic, and with the exception of two serovars, Salmonella Pullorum and Salmonella Gallinarum, all other serovars have peri-trich flagella and are motile (2). Members of this genus can grow in the temperature range of 5 to 45 °C, but the optimum growth occurs at 37 °C (2). Bacteria of this genus are classified into two species, enterica and bongori. Enterica includes six sub-species and it is considered an important species because pathogenic Salmonella for humans and warm-blooded animals are under species I (enterica). To date, more than 2600 Salmonella serovars have been identified, and most of those serovars are classified under type I (2). The main way of Salmonella transmission is through the digestive tract, but its transmission through the mucous membranes of the eye conjunctiva or the upper respiratory tract has also been reported (2). Salmonella and in particular S. Enteritidis remained the most frequently reported causative agent for foodborne outbreaks (2). This genus is considered as an important factor in foodborne diseases, which is mostly transmitted through the consumption of meat, milk and eggs and is considered an important indicator for the water and food safety of a country (2). Many researches on the prevalence of Salmonella have shown that the most important and common food sources of Salmonella infection in humans are poultry and poultry products (3, 4). Gastroenteritis, which is accompanied by fever, abdominal cramps and diarrhea, is the most common clinical form of salmonellosis in humans (1). Causing a high economic loss on one hand and the occurrence of food poisoning on the other hand are considered the reasons for the importance of Salmonella for individuals and societies (1).

In addition to horizontal transmission, Salmonella serovars are also transmitted by vertical transmission, which can lead to the contamination of day-old chickens as well as the contamination of other birds. In processing plants, the slaughtering and packaging steps may also spread the Salmonella infection and pose a major risk for consumers. Penetration into the shell and transmission through the shell is also possible for Salmonella, and hence, it can also contaminate the produced eggs (5).

Breeding ducks for the purpose of producing meat and eggs dates back to several hundred years ago (6). The connection between duck and Salmonella has been known for many years and it is referred to as a cause-and-effect situation because it has been indicated that the consumption of duck eggs is very likely to be associated with stomach upset (6). This possibility is mainly due to the presence of Salmonella Typhimurium, which is transmitted to the egg by a clinically healthy duck (6). Meanwhile, Salmonella food poisoning due to consumption of duck meat is a rare event, which is probably due to its cooking methods and the dietary habits of the not so large population that consumes duck meat (6).

Salmonella control will require the adoption of a surveillance program for regular sampling and appropriate control measures. In this regard, the first step is to establish surveillance, determine the status of infection and the underlying factors of infection (7).

The aims of this study were to determine the frequency of Salmonella infection among backyard and commercial duck flocks in Iran, serotyping and drug resistance profile of Salmonella isolated form ducks.

Materials and Methods

Sampling and bacteriological procedures

This study was conducted from August 2022 to August 2023 in four Iranian provinces of Tehran, Golestan, Mazandaran and Guilan, where the duck population was higher than that of in other provinces. During this study, a total of 2112 fresh stool samples were taken, each six samples pooled, and a total of 352 pooled samples were obtained. The share of each province of total and pooled samples were as follows: Tehran, 978 (163 pooled) samples and the other three northern provinces, each 378 (63 pooled) samples. In Tehran province, samples were taken from 53 commercial duck breeding farms, in which, in each flock, a house was divided into six areas and samples were taken from all six areas and the whole house was considered as one sample and the rest of 110 samples of this province were provided from domestic ducks in different areas of the province. In Tehran province, due to the importance and commercial production of ducks in Varamin city, all samples were provided from flocks located in Varamin area. It has been estimated that Varamin area has a population of two million ducks. Breeding flocks of Varamin area are the major sources of ducklings for northern provinces. In three norther provinces, samples from domestic ducks were taken from different cities of Golestan (Kordkoi, Gorgan, Kalaleh, Agh-Ghala, Bandar-Gaz, Bandar-Turkman), Mazandaran (Sari, Behshahr, Jouybar, Babol, Amol, Chalus) and Guilan (Rudsar, Langrood, Amlash, Lahijan, Bandar Anzali, Rasht). After sampling and pooling, samples were transferred to the university laboratory in a closed container and cold condition in less than 24 hours.

All fecal samples were cultured for the isolation and identification of Salmonella according to standard procedures that have been previously described (8). Briefly, the selective enrichment of samples in selenite F at 41°C for 24 h was followed by sub-cultivation on Salmonella-Shigella (SS) and MacConkey agar plates at 37°C for 24 h. Then, the suspect colonies were selected, isolated and further characterized by biochemical identification. Positive samples were kept at -70°C freezer for future use.

Serogroup determination

The serogroup of each Salmonella isolate was determined by slide agglutination test using antisera against O antigen (Poly A-I & Vi) according to the instructions of manufacturer (Difco, USA).

Molecular identification of Salmonella isolates by PCR

In this study, invA gene specific primers were used to confirm the Salmonella genus (Table 1). Also, in order to identify serovars Enteritidis, Typhimurium and Infantis, three pairs of specific primers sdf-1, fli-C and fli-B were used respectively (Table 1). The characteristics of the primers used to detect the genus Salmonella (9) and serovars Typhimurium (10), Enteritidis (11) and Infantis (12) are shown in Table 1. All primers used in PCR reaction were provided from Pishgam Biotech Co. (Tehran, Iran). Other materials were purchased from Yekta Tajhiz Azma Co (Tehran, Iran).

To extract bacterial DNA, one ml pure overnight culture of each Salmonella isolate grown at 37 °C for 16 h was transferred to a clean 1.5 ml microtube and centrifuged for five min at 10000 x g. The supernatants were carefully removed and discarded. The pellet was re-suspended in 300 μl sterile double distilled water by vertexing, incubated for 15 min at 100 °C, chilled on ice immediately, and centrifuged again for five min at 14000 x g in 4 °C. The supernatant was removed and used as template DNA. The concentration of DNA was determined by Biophotometer (Eppendorff, Germany) and adjusted to approximately 200 ng for each PCR reaction. The supernatant was stored at -20 °C for further use.

Amplification reactions for Salmonella genus and three serovars confirmation were carried out in a 25 μl reaction volume containing 12.5 μl of 2x Mastermix (Taq 2x Red Master Mix, Ampliqon, Denmark), 0.5 μl each of forward and reverse primers (10 pmol/μl), 2 μl of DNA template, and 9.5 μL nuclease-free water. Negative controls (dH2O instead of template DNA) were included in all PCR reaction sets. Amplifications were programmed in a thermocycler (SensoQuest, Germany) as described below. For Salmonella genus, 95 °C for one min followed by 38 cycles of 95 °C for 30 sec, 64 °C for 30 sec, 72 °C for 30 sec, and a final extension at 72 °C for 4 min was used (9). For serovar Enteritidis, program was as follows: 95 °C for 2 min followed by 30 cycles of 95 °C for 60 sec, 57 °C for 60 sec, 72 °C for 2 min, and a final extension at 72 °C for 5 min (11). For serovar Typhimurium, 94 °C for 5 min followed by 34 cycles of 94 °C for 60 sec, 58 °C for 60 sec, 72 °C for 90 sec, and a final extension at 72 °C for 10 min was programmed in thermocycler (10) and for serovar Infantis, 95 °C for one min followed by 35 cycles of 95 °C for 60 sec, 56 °C for 15 sec, 72 °C for 60 sec, and a final extension at 72 °C for 270 sec was applied (12). The amplified products were detected by gel electrophoresis in 1% agarose gel at 70 V for 80 min in 1 x TAE buffer.

Drug susceptibility test

The susceptibility of the Salmonella isolates to a panel of antimicrobial agents was determined by the agar disk diffusion method and the interpretation of results was carried out according to the National Committee for Clinical Laboratory Standards guidelines (13). The antimicrobial agents that were tested and their concentrations (µg) were: ciprofloxacin (5), difloxacin (10), ofloxacin (5), norfloxacin (10), enrofloxacin (5), levofloxacin (5), nalidixic acid (30), flumequine (30), ceftazidime (30), ceftriaxone (30), cefixime (5), ampicillin (10), Co-amoxiclav (30), neomycin (30), streptomycin (10), gentamicin (10), lincospectin (15/200), florfenicol (30), colistin (10), trimethoprim-sulfamethoxazole (1.25/23.75), and fosfomycin (200 µg). All antibacterial disks were provided from Padtan Teb Co (Tehran, Iran). The ATCC reference strains Escherichia coli ATCC 25922, Pseudomonas aeruginosa, ATCC 27853, and E. coli ATCC 35218 were used for quality control purposes. In this study, the Salmonella isolates with intermediate susceptibility classification were considered not to be resistant to that drug and the multi-resistance was defined as resistance to more than one drug.

Results

Bacteriological, biochemical tests and genus-specific PCR confirmed the isolation and identification of 20 (17.6%) Salmonella isolates (Figure 1-A). The frequency of Salmonella isolates in provinces of Tehran, Golestan, Guilan and Mazandaran were 10, 5, 3, and 2, respectively. Serovar-specific PCR revealed that out of 20 isolated Salmonella, 15 and 3 isolates were identified as Serovars Enteritidis and Typhimurium, respectively (Figures 1-B and 1-C). No isolates were identified as Salmonella Infantis. Two remaining Salmonella isolates were not positive in any of three serovar-specific PCR and agglutination tests performed with a number of available antisera against Salmonella O antigen and, therefore, remained unknown.

In antimicrobial susceptibility test, no isolate was resistant to ceftazidime, ceftriaxone, lincospectin, fosfomycin, and colistin (Table 2). The rate of resistance to Co-amoxiclav, nalidixic acid, flumequine, and streptomycin was above 50%, in which, resistance to Co-amoxiclav ranked first with 95%. The resistance to the rest of the agents was below 25% (Table 2). There was a high variation in the resistance pattern of among 20 Salmonella isolates of this study, so that 17 resistance patterns to 21 antimicrobial agents were found among isolates (Table 3). Among the resistant isolates, the occurrence of multiple resistances were very significant, so that they showed resistance to at least 1 and at most 11 drugs (Table 4).

Discussion

In this study, the frequency of Salmonella infection among backyard and commercial duck flocks in Iran, the serotypes and drug resistance patterns of isolated Salmonella were evaluated. There is a paucity of information on duck infections in Iran especially on Salmonella. The duck flock contamination rate in this study was found to be 17.6%. The contamination of duck carcasses and eggs to Salmonella may lead to infection of human populations and, therefore, investigating the extent of contamination and isolation of Salmonella from ducks are of important public health concerns. It is noteworthy to mention that a duck can be infected with Salmonella without any apparent symptoms (14).

Few studies have been done in Iran on the prevalence of Salmonella in ducks. In a study of duck intestinal content, 291 samples were collected from meat shops located in Varamin city. The amount of Salmonella infection was reported in 84 cases (28.9%) (15). During a study on wild mallard ducks (Anas platyrhynchos) in the suburbs of Semnan city, out of 247 freshly collected feces samples, 18 cases (7.29%) were positive for Salmonella (16).

Several studies on Salmonella infections in ducks have been reported from other countries. In Taiwan, out of 2000 cloacal swab samples from ducks, in 100 different flocks, the overall infection rate was reported as 4.6% (17). In Vietnam, by examining the internal organs of ducks, the contamination rate varied from 6.3% to 14.3% in different regions of country, while, the incidence rate in dead embryos varied from 3.2% to 31.7% (18). In a survey conducted in Egypt, 40 samples of clinically sick duck feces and 120 wasted ducks were obtained to determine the frequency of different pathogens. The most common bacterial agent was Salmonella with a frequency rate of 3.3% (19). In a Malaysian study in Penang area in 2009, the prevalence and resistance of Salmonella isolated from ducks were estimated and among 531 collected samples, 125 (23.5%) Salmonella isolates were recovered (20). The prevalence of Salmonella in 159 South Korean duck flocks was studied and it was found that Salmonella was common among flocks, so that 69 flocks (43.4%) were Salmonella-infected (21). In another South Korean study, 400 samples of cecal content were taken from duck farms in order to estimate the prevalence and level of antimicrobial resistance of Salmonella isolates. The results showed that 83 isolates (20.75%) were Salmonella (22). In a study in Bangladesh, 120 fecal samples were taken from 12 small to medium breeding farms and 28 native duck flocks using cloacal swabs. In this study, 32 samples (26.67%) were infected with Salmonella. The prevalence rate was 36% in native flocks and 40.2% in breeding farms (23). In another Egyptian research, 100 samples of ducklings suffering from diarrhea and 50 samples from the litter were collected. The prevalence rate of Salmonella in the pooled samples of liver, spleen, cecum and gall bladder was 7% and the rate of litter contamination was 6% (24). In a recent study in China, 180 Salmonella isolates (25.7%) were isolated from a total of 701 samples taken from all stages of the duck production chain (25).

The prevalence of various Salmonella serovars in ducks of Tehran, Golestan, Mazandaran and Guilan provinces was variable. In this research, 20 Salmonella isolates were isolated from duck flocks, and the most isolated serovar was Salmonella Enteritidis, which accounted for 15 isolates (75%) of the total isolates. While in Jamali et al.'s study in 2014, 84 isolates (28.86%) were isolated from a total of 291 samples of duck feces, of which 56 isolates were Salmonella Thompson (59.6%). and only 8 isolates (8.5%) were Salmonella Enteritidis (15). This change in the predominant Salmonella serovar in ducks can be a warning for its transmission to humans through duck eggs and meat because Salmonella Enteritidis is the most transmitted Salmonella from infected eggs to humans among different Salmonella serovars all over the world (with the exception of Australia). It is noteworthy to mention that this in turn can be considered a warning for more cases of human infection (26).

During a study in the United States backs to sixty’s, in a period of 10 years, 491 Salmonella isolates were isolated from 7029 duck carcasses, of which 457 (93%) were Salmonella Typhimurium. The diversity of Salmonella serovars in this study was less than 10 different serovars (27). In 2011, in India, two serovars of Salmonella Typhimurium and Salmonella Enteritidis were isolated by examining duck eggs (28). In Belgium, during a 32-month study period, 95 Salmonella isolates were recovered from 100 duck flocks, which included 11 different Salmonella serovars such as Indiana (42.1%) and Regent (36.8%), Typhimurium (1.1%) and Enteritidis (1.1%) (29). In South Korea, 51 isolates (61.45%) of Salmonella Typhimurium were found among 400 samples collected from the cecal contents of ducks which was the most dominant serovars identified (22). In a study conducted in England, the contamination rate of 1.4% was reported from 145 combined samples of eggs and in which serovar Typhimurium was the predominant serovar (30). In Shandong Province of China, 49 Salmonella isolates were detected among 2,342 samples collected from four duck farms and the serovar Enteritidis was found to be the most dominant serovar (20 out of 49) (31). However, in a recent from China, the most dominant serovar was reported to be serovar Typhimurium (25). Studies completed in Vietnam, have also reported Salmonella Typhimurium as the most common serovar in Vietnamese ducks (32, 33). Salmonella serovar Potsdam also has been found in studies conducted in Taiwan (17) China (34) and has been reported as the most prevalent Salmonella serovar among many samples provided from duck flocks. In Thailand, during a survey, on Salmonella contamination of egg shells and contents, 23 different serovars were identified among 133 Salmonella isolates and four dominant serovars of Typhimurium (5.5%), Cerro (4.1%), Tennessee (2.8%), and Amsterdam (2.1%) were confirmed (35). Salmonella serovar Kentucky also has been reported by Abou Zeid et al. as the most dominant Salmonella serovar in Salmonella isolates recovered from litter and pooled samples of liver, spleen, cecum, and gall bladder (24). In general, studies throughout the world shows the dominance of S. Enteritidis and S. Typhimurium among duck populations.

Antimicrobial susceptibility evaluation of Salmonella isolates form poultry sources has been the subject of many investigations in recent years. In Arak city, in research by Ezzatpanah et al. (2013), 75 Salmonella isolates were obtained from chicken abattoirs. All of isolates were sensitive to enrofloxacin, gentamicin and ceftriaxone and all were resistant to nalidixic acid (36). In another study, 8 Salmonella isolates were obtained from 20 broiler flocks in Guilan province and it was found that all isolates were resistant to sulfamethoxazole + trimethoprim, streptomycin and nalidixic acid and all were sensitive to gentamicin, ceftriaxone and chloramphenicol (37). During a study in Golestan province, after isolating 25 Salmonella isolates from broiler flocks, 100% of these isolates were sensitive to ceftriaxone and cefixime and also were resistant to flumequine and nalidixic acid (38). In a large study that was conducted in Mazandaran and Guilan provinces, 32 resistance patterns were found in Salmonella isolates from poultry (39). They reported no resistance to levofloxacin and ofloxacin, while in our study, 15% of the isolates were resistant to these two antibiotics. In the present study, 17 resistance patterns to 21 antimicrobial drugs were observed among 20 Salmonella isolates. Similar multi-drug resistance (MDR) patterns were found among ducks of different flocks. Resistance to ampicillin, flumequine and nalidixic acid among 10 isolates; or resistance to nalidixic acid, flumequine and Co-amoxiclav among 10 isolates, or resistance to amoxiclav and streptomycin among 7 isolates or resistance to streptomycin, flumequine, nalidixic acid and Co-amoxiclav among 6 isolates were noticeable.

In the current study, the highest resistance rate among isolates was observed against Co-amoxiclav (95%), nalidixic acid (65%), flumequine (55%) and streptomycin (50%). Considering that amoxiclav is not widely used in veterinary medicine, this level of high resistance to Co-amoxiclav may have negative impact on its usefulness for the treatment of human salmonellosis. On the other hand, flumequine and streptomycin are widely used in veterinary medicine and these results demonstrated significantly increased resistance compared to the past as described in the scientific literature. These facts prompt the veterinarians to replace these antimicrobial agents with other compounds such as probiotics and prebiotics. All investigated isolates were sensitive to fosfomycin, lincospectin, ceftazidime, ceftriaxone and colistin. Although no resistance was observed against agents such as colistin and ceftriaxone, the number of isolates with intermediate sensitivity can be a warning for the not so far future because it is clear that Salmonella is able to gain resistance against these antibiotics. In this study, it was shown that the lowest level of drug resistance was against ceftazidime, sulfamethoxazole + trimethoprim, ciprofloxacin, ofloxacin, norfloxacin, levofloxacin and gentamicin. Of course, it was noted that the number of isolates with intermediate sensitivity against ampicillin and third-generation cephalosporins such as ceftriaxone and ceftazidime was on the rise, so that it may be suggested that in the coming years, these antibiotics may not be so effective in the treatment of salmonellosis. Among the third-generation fluoroquinolones, the level of resistance to enrofloxacin was higher than resistance to other members of fluoroquinolones that were tested. The widespread use of enrofloxacin in veterinary medicine compared to other members of this family may be the reason for the occurrence of this high resistance. Also, the number of isolates with intermediate sensitivity to enrofloxacin was relatively high that may affect the clinical efficacy of this drug in the coming years. The transfer of resistance genes to human isolates, especially in the case of antibiotics of the fluoroquinolone and cephalosporin families, which are important antimicrobial agents for the treatment of human salmonellosis, is the most important issue in antimicrobial susceptibility evaluation of bacterial isolates. All of 20 Salmonella isolates of this study were resistant to more than one antimicrobial agent, so that they showed resistance to at least one and at most 11 agents at the same time. One of the important challenges of treating infections caused by microorganisms in humans and animals is the emergence of multi-drug resistances among bacterial isolates in Iran and around the world (40-42).

Comparison of the current study and other studies conducted in different parts of the country, as well as considering or ignoring different species of birds, it can be concluded that the resistance patterns among Salmonella isolates, despite having similarities, may demonstrate great diversity so that it can be different not only between different countries but also from one province to another in one country. For this reason, it is suggested is that targeted studies be conducted by the control and prevention centers of food-borne diseases in order to estimate the association between the prevalence of resistance in birds and cases of human disease throughout the country. A regular program for the periodic monitoring of Salmonella infection in the flocks should be established and also restrictions on the use of antimicrobial agents in animals that are raised for human consumption should be regulated and implemented.

Conflict of Interest

The authors declare no conflict of interest.

Authors Contributions

JJ drafted the manuscript. SMP, JR and AB critically reviewed and revised it. All authors have read and approved the final manuscript and agreed to the published version of the manuscript.

Data Availability Statement

Data are available from the first author upon reasonable request.

Funding

This research was funded by a grant (7508007-6-47) from the University of Tehran Research Council.

Table 1. Characteristics of the primers used to detect Salmonella genus and serovars Enteritidis, Typhimurium and Infantis

Bacteria

Target gene

Nucleotide sequence (5’-3’)

Amplicon size (bp)

Reference

Salmonella genus

invA

F: GTG AAA TTA TCG CCA CGT TCG GGC AA

R: TCA TCG CAC CGT CAA AGG AAC C

284

9

Salmonella Enteritidis

sdf-1

F: TGT GTT TTA TCT GAT GCA AGA GG

R: CGT TCT TCT GGT ACT TAC GAT GAC

293

11

Salmonella Typhimurium

fliC

F: CCC CGC TTA CAG GTG GAC TAC

R: AGC GGG TTT TCG GTG GTT GT

433

10

Salmonella Infantis

fliB

F: TTG CTT CAG CAG ATG CTA AG

R: CCA CCT GCG CCA ACG CT

413

12

Table 2. Antimicrobial susceptibility profile of 20 Salmonella isolates to 21 antimicrobial drugs

No. (%) of susceptible isolates

No. (%) of intermediate susceptible isolates

No. (%) of resistant isolates

Drugs

16 (80)

2 (10)

2 (10)

Ciprofloxacin (CIP)

6 (30)

10 (50)

4 (20)

Difloxacin (DFX)

7 (35)

9 (45)

4 (20)

Enrofloxacin (NFX)

17 (85)

0 (0)

3 (15)

Levofloxacin (LEV)

17 (85)

0 (0)

3 (15)

Norfloxacin (NOR)

17 (85)

0 (0)

3 (15)

Ofloxacin (OFX)

6 (30)

1 (5)

13 (65)

Nalidixic acid (NA)

7 (35)

2 (10)

11 (55)

Flumequine (FM)

9 (45)

9 (45)

2 (10)

Cefixime (CFM)

16 (80)

4 (20)

0 (0)

Ceftazidime (CAZ)

11 (55)

9 (45)

0 (0)

Ceftriaxone (CRO)

0 (0)

1 (5)

19 (95)

Co-Amoxiclav (AMC)

6 (40)

11 (55)

3 (15)

Ampicillin (AM)

9 (45)

1 (5)

10 (50)

Streptomycin (S)

15 (75)

0 (0)

5 (25)

Gentamicin (GM)

8 (40)

10 (50)

2 (10)

Neomycin (N)

18 (90)

2 (10)

0 (0)

Linco-spectin (LP)

7 (35)

11 (55)

2 (10)

Florfenicol (FF)

20 (100)

0 (0)

0 (0)

Fosfomycin (FOS)

3 (15)

17 (85)

0 (0)

Colistin (CL)

19 (95)

0 (0)

1 (5)

Trimethoprim- Sulfamethoxazole (SXT)

Table 3. Drug resistance patterns among 20 Salmonella isolates.

Pattern #

Resistant toa

No. (%) of isolate

1

AM, FM, NA

10 (50)

2

AMC, FM, NA

10 (50)

3

AMC, S

7 (35)

4

AMC, FM, NA, S

6 (30)

5

AMC

3 (15)

6

AM, AMC, GM, S

3 (15)

7

AMC, FM, NA, NFX

3 (15)

8

AMC, FM, GM, NA, S

3 (15)

9

AMC, FF

2 (10)

10

AMC, CIP, DFX, FM, LEV, NA, NFX, NOR, S

2 (10)

11

N, NA

Each pattern

included only one isolate (5%)

12

AMC, N, NA, S

13

AMC, CFM, CIP, DF, FM, NA, LEV, NFX, NOR, S

14

AM, AMC, FF, FM, GM, NA, S, SXT

15

AMC, DFX, FM, GM, LEV, NA, NFX, OFX, S

16

AMC, FM, DFX, NA

17

AM, AMC, GM, NFX, NOR, S

a For abbreviations of drugs refer to Table 2

Table 4. Multi-drug resistancea among 20 Salmonella isolates of this study.

No. of antimicrobial drugs used

No (%) of resistant isolates

≥ 1

20 (100)

> 2

17 (85)

> 3

14 (70)

> 4

11 (55)

> 5

6 (30)

> 6

5 (25)

> 7

5 (25)

> 8

4 (20)

> 9

3 (15)

> 10

2 (10)

> 11

1 (5)

a Multi-drug resistance was defined as resistance to more than one drug.

A

C:\Users\javid\Pictures\spp.jpg

B

C:\Users\javid\Pictures\S,E.jpg

C

Figure 1Gel electrophoresis of PCR products for amplification of invA (for Salmonella genus), sdf-1 (for Salmonella Enteritidis), and fliC (for Salmonella Typhimurium). A: M, 100 bp ladder; columns 1-7, positive Salmonella samples; columns 8 and 9, positive and negative controls, respectively. B: M, 100 bp ladder; columns 1-7, positive Salmonella Enteritidis samples; columns 8 and 9, positive and negative controls, respectively. C: M, 100 bp ladder; columns 2 and 3, positive Salmonella Typhimurium samples; columns 5 and 6, negative and positive controls, respectively.
C:\Users\javid\Pictures\TYPH.jpg

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