PREVALENCE OF SOME ANTIMICROBIAL RESISTANCE GENES IN MULTIDRUG RESISTANT SALMONELLA ISOLATED FROM BROILER CHICKENS

Assiut University web-site: www.aun.edu.eg

PREVALENCE OF SOME ANTIMICROBIAL RESISTANCE GENES IN MULTIDRUG RESISTANTSALMONELLAISOLATED FROM BROILER CHICKENS

WALID H. HASSAN¹, AHMED H. ABED¹, ABD EL RADY THABET²

and EMAN A.M. EL NADY².

¹ Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Beni-Suef University.

² Department of Bacteriology, Animal Health Research Institute, Assiut Branch.

Received: 28 June 2017;       Accepted: 31 July 2017

INTRODUCTION

Salmonellosis is one of the major bacterial problems in the poultry industry worldwide. salmonellaspecies are responsible for a variety of acute and chronic diseases in both poultry and humans (Okwori et al., 2013).

Although more than 2500 serotypes of salmonellahave been identified, in recent years, S. enterica serovar Enteritidis (S. Enteritidis) and S. enterica serovar Typhimurium (S. Typhimurium) have been recognized as the two major causative agents of salmonellosis in birds, mammals and humans (Darwin and Miller, 1999).

Although antimicrobials are valuable tools to treat clinical disease and to maintain healthy and productive birds, antimicrobial drug use has been implicated as a risk factor in the development and dissemination of drug resistance (Gosh and LaPara, 2007). Food of animal origin and their production environments are reservoirs of both resistant bacteria and   resistance   genes  that  could   be   transferred to

Corresponding author: Dr. EMAN A.M. EL NADY

E-mail address: elnadyeman@yahoo.com

Present address: Department of Bacteriology, Animal Health Research Institute, Assiut Branch.

humans either by direct contact or indirectly via the food production chain (WHO, 2011). Therefore, the appropriate antibiotic should better be selected on the basis of its sensitivity which could be detected by laboratory examination. The recovery of antimicrobial-resistant salmonellain foods of animal origin has raised concerns that the treatment of human salmonellosis may be compromised because antimicrobial-resistant strains appear to be more often associated with severe disease than are susceptible isolates. Of significant concern is the isolation of salmonellaexhibiting decreased susceptibility to fluoroquinolones (e.g., ciprofloxacin) and extended-spectrum cephalosporins (e.g., ceftiofur and ceftriaxone), because these two antimicrobial classes are important in treating salmonellainfections in adults and children, respectively (Gupta et al., 2003).

Resistance to antimicrobial agents within nontyphoidal salmonella serotypes is considered a serious problem worldwide. Surveillance data demonstrates an obvious increase in overall antimicrobial resistance among salmonellae from 20-30% in the early 1990s to as high as 70% in some countries in the 2000s (Su et al., 2004). More than 340 beta-lactamases have been described in salmonellaand the prevalence of genes encoding for them varies region by region (Hasman et al., 2005).

The purpose of this study was genetic analysis of some β-lactamase resistance genes in salmonellae isolates from broiler chicken.

MATERIALS AND METHODS

1 - Samples

Five hundred samples were collected from diseased broiler chickens (suspected to harbor salmonella) of different ages (3-6 weeks). Sampling were carried out from different farms in Assiut Governorate during the period from January 2015 to December 2015. These chickens were subjected to clinical and postmortem examinations. Samples were collected aseptically from the lesions in the internal organs including liver, heart, lung, air sacs, and kidney.

2 - Isolation and identification of Salmonella species

Samples were cultured into selenite-F broth and incubated at 37ºC for 18-24 hrs. Then, a loopful of this culture was streaked out onto MacConkey's agar then the non-lactose fermenter (pale) colonies were streaked onto xylose lysine deoxycholate (XLD) and Salmonella-Shigella (SS) agar media and incubated at 37ºC for 18-24 hours. All isolates were identified as salmonellaspecies based on their colony morphology and biochemical tests according to schemes described by Collee et al. (1996) and Quinn et al. (2002). The salmonellaisolates were also confirmed biochemically by using the API 20E system (BioMérieux, Marcy-l’Étoile, France).

3 - Serotyping of Salmonella species

Salmonella isolates were serotyped by slide agglutination test carried out according to Kauffman-White scheme (Kauffman, 1974) for the determination of Somatic (O) and flagellar (H) antigens using salmonella antiserum (DENKA SEIKEN Co., Japan).

4 - Antimicrobial susceptibility testing

All Salmonella isolates were tested for their antimicrobial susceptibility to 14 different antimicrobial discs including erythromycin (15μg), nalidixic acid (30μg), penicillin (10 IU), amoxicillin (30μg), oxytetracycline (30μg), sulphamethoxazole-trimethoprime (25μg), ampicillin (10μg), streptomycin (10μg), neomycin (30μg), chloramphenicol (30μg), norfloxacin (10μg), ciprofloxacin (5μg), kanamycin (30μg) and gentamicin (10μg) (Oxoid Limited, Basing Stoke, UK). Antimicrobial susceptibility testing was performed using disc diffusion method on Muller Hinton agar according to CLSI (2015). The antimicrobial susceptibility was based on the induced inhibition zones according to the guidelines of the CLSI (2015). Resistance to two/or more antimicrobials of different classes was taken as multidrug resistant (MDR) (Chandran et al., 2008).

5 - Multiplex-PCR for detection of β-lactamases resistance genes

5.1- DNA extraction:

DNA was extracted by using bacterial DNA e

xtraction kits (Qiagen, Germany, GmbH) according to the manufacturer instructions.

5.2- Screening of β-lactamases resistance genes by multiplex-PCR:

The multiplex-PCR assay was applied on 26 salmonella isolates for detection of 2 β-lactamase resistance genes bla_CTX (responsible for cefotaxime resistance) and bla_CMY (responsible for cephalosporin resistance) according to (Ahmed et al., 2009). Targeted genes and their primer sequences are listed in (Table 1).

Table 1: Primer sequences and amplified products for the β-lactamase resistance genes blaCTX and blaCMY.

RESULTS

1 - Bacteriological isolation of salmonella species.

Out of 500 broiler chicken samples, 26 salmonella isolates were recovered with a prevalence rate of 5.2%.

2 - Serotyping of salmonella isolates.

The 26 salmonella isolates were serotyped as 13 S. Enteritidis (50%), 8 S. Typhimurium (30.8%) and 5 S. Kentuky (19.2%).

3 - Antimicrobial susceptibility testing.

Results of in-vitro sensitivity tests showed that salmonella isolates were completely resistant to erythromycin and penicillin while they were highly resistant to amoxicillin and streptomycin (92.3% for both), nalidixic acid (80.8%), sulphamethoxazole-trimethoprime (76.9%), ampicillin (69.2%) and oxytetracycline (65.4%). On contrary, salmonella strains were highly sensitive to kanamycin (96.2%) then gentamycin (73.1%) (Table 2). MDRsalmonellaisolates were 24 isolates (92.3%).

Table 2: Distribution of Antimicrobial susceptibility for 26 salmonella isolates.

4 - Multiplex-PCR for detection of β-lactamases resistance genes (bla_CTX and bla_CMY).

Multiplex-PCR assay was done for 26 salmonella strains for detection of 2 β-lactamase resistance genes bla_CTX and bla_CMY. The results revealed that bla_CTX gene was found in 14/26 isolates (53.9%) arranged as follow; 8 S. Enteritidis (30.8%), 4 S. Typhimurium (15.4%) and 2 S. Kentucky (7.7%). Meanwhile, bla_CMY gene was found in 9/26 isolates (34.6%) arranged as follow; 4 S. Enteritidis (15.4%), 3 S. Typhimurium (11.5%) and 2 S. Kentucky (7.7%) (Tables 3& 4 and Fig. 1& 2).

Table 3: Multiplex-PCR results for β-lactamase resistance genes in salmonella isolates.

Table 4: Resistance phenotype and prevalence of β-lactamase resistance genes in salmonella isolated from diseased broilers.

DISCUSSION

Salmonellosis causes great mortalities and various morbidity changes as well as economic losses in poultry industry and a serious public health problem throughout the world(Pedersen et al., 2002). Identification and genotyping of salmonellaisolates are essential for epidemiological surveillance and investigations of outbreak.

In the current work, 26 salmonella species were recovered from the 500 samples of internal organs (liver, heart, lung, airsacs, and kidney) of broiler chickens with prevalence rate of 5.2%. This result was similar to that obtained by reported by Abd El-Galil et al. (1995); 6%, and nearly similar to those of Abd El Fattah (2014); 8.72%, and Radwan et al. (2016); 12%, while it was much lower than that reported by Sharada et al. (1999); 30.5%.

Serotyping of 26salmonella isolates showed that S.Enteritidis, S.Typhimurium and S.Kentuky were identified at rates of 50%, 30.8% and 19.2%, respectively. The obtained results run parallel to that obtained by Hegazy (2002) who detected S.Enteritidis and S. Kentucky at rates of 62.16%, and 5.41% respectively.

Antimicrobial therapy is one of the primary control for reducing both the incidence and mortality associated with avian diseases therefore reducing their enormous losses in the poultry industry(Radwan et al., 2016). Increasing antimicrobial resistance is an important public health concern, and the emergence and spread of antimicrobial resistance is a complex problem driven by numerous interconnected factors. In-vitro antimicrobial susceptibility testing of veterinary pathogens can provide valuable guidance to the veterinarian in the choice of appropriate chemotherapy (Radwan et al., 2016). Moreover, it is very useful to detect the multidrug resistant isolates.

In the present study, the recovered salmonella isolates (n=26) were subjected to in-vitro antimicrobial susceptibility tests against 14 different antimicrobial drugs to detect MDR isolates for further analyses of the isolates. Results illustrated in table (2) revealed that Salmonella isolates showed complete resistance against erythromycin and penicillin while they were highly resistant to amoxicillin and streptomycin (92.3% for both), nalidixic acid (80.8%), sulphamethoxazole-trimethoprime (76.9%), ampicillin (69.2%) and oxytetracycline (65.4%). On the other hand, they were highly sensitive to kanamycin (96.2%) then gentamycin (73.1%). MDRsalmonellaisolates were 24 isolates with prevalence rate of 92.3%. Comparable results have been reported worldwide; in Egypt (Ahmed et al., 2009₎, in USA (Frye and Fedorka-Cray, 2007), and in Italy (Mammina et al., 2002), and in Portugal (Antunes et al., 2006).

Beta-lactams belong to a family of antibiotics, the members of which have a β-lactam ring. Penicillins, cephalosporins, clavams (or oxapenams), cephamycins and carbapenems are members of this family. In Gram-negative bacteria, resistance to β-lactam antibiotics is primarily mediated by β-lactamases, which hydrolyze the β-lactam ring and thus inactivate the antibiotic. Many different β-lactamases have been described, but TEM-, SHV-, OXA-, CMY- and CTX-M-β-lactamases are the most predominant in Gram-negative bacteria. bla_CTX-Marise resistance to penicillins, extended-spectrum cephalosporins, and monobactams, and the enzymes are inhibited by clavulanate, sulbactam, and tazobactam. Typically, the CTX-M-ases hydrolyze cefotaxime more efficiently than ceftazidime, which is reflected in substantially higher MICs to cefotaxime than to ceftazidime (Livermore and Woodford, 2006).

In the current study 2 β-lactamase resistance genes (bla_CTX and bla_CMY) were evaluated in all salmonella isolates (n=26) using multiplex-PCR assay. The results illustrated in tables (3& 4) and Fig. (1& 2) revealed that bla_CTX gene was found in 14/26 isolates (53.9%) arranged as follow; 8 S. Enteritidis (30.8%), 4 S. Typhimurium (15.4%) and 2 S. Kentucky (7.7%). The resistance gene bla_CTX has previously been identified and reported increasingly in Gram-negative rods (Bradford, 2001; Bonnet, 2004; Eckert et al., 2004; Lartigue et al., 2005; Naas et al., 2005 and Pitout et al., 2005) and was characterized and isolated in Germany and Italy (Barthelemy et al., 1992 and Bauernfeind et al., 1996). On the other hand, bla_CMY gene was found in 9/26 isolates (34.6%) arranged as follow; 4 S. Enteritidis (15.4%), 3 S. Typhimurium (11.5%) and 2 S. Kentucky (7.7%). The gene bla_CMY has previously been identified in S. Typhimurium and other serovars isolated from animals in Canada (Allen and Poppe, 2002), Egypt (Ahmed et al., 2009) and USA (Doublet et al., 2004; Frye and Fedorka-Cray, 2007 and Zhao et al., 2007).

CONCLUSION

The presence of multidrug resistance pathogens occurred due to the misuse of the antibiotics and it is considered a great problem. In this study, multidrug-resistant strains of S. Enteritidis, S. Typhimurium and S. Kentucky from diseased broilers were recovered and identified. Furthermore, 2 important antimicrobial resistance genes (bla_CTX and bla_CMY) were characterized using multiplex-PCR. These resistance genes confer resistance to many antimicrobial agents regularly used in poultry farming and hospitals.

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