QUINOLONE RESISTANT ESCHERICHIA COLI ISOLATED FROM CHICKEN MEAT IN BOSNIA AND HERZEGOVINA.

Vildana Hadzic 1 and Anesa Jerkovic Mujkic 2 . 1. Hospital for Respiratory diseases and TBC, 72 270 Travnik, Bosnia and Herzegovina. 2. Faculty of Science, University of Sarajevo, 71000 Sarajevo, Bosnia and Herzegovina. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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antimicrobial agents (Amundsen et al., 1988). In Europe, concern for the spread of antimicrobial-resistant bacteria from the large reservoirs in food animals led the countries in the European Union to abandon the use of antimicrobial agents for growth promotion in food animals by January 1 st 2006 (Anderson et al., 2003). Although withdrawal of antimicrobial agents for growth promotion may significantly reduce the total amount of antimicrobial agents used in animals, available data indicate that the consumption of antimicrobial agents for clinical therapy in animals is considerable and may even exceed the consumption in humans (Arathy et al., 2011). Most of the antimicrobial agents used for growth promotion in animals are mainly active against gram-positive bacteria, whereas many of the antimicrobial agents used in veterinary clinical therapy are broad spectrum and are mainly active against gram-negative bacteria, such as Salmonella and E. coli.
Fluoroquinolones are broad-spectrum antimicrobial agents that are highly effective for the treatment of a variety of infections in humans and animals. The use of fluoroquinolone (FQ) agents in food animal production is suspected of selecting for FQ-resistant gram-negative bacteria, such as Salmonella enterica, Campylobacter jejuni, and Escherichia coli, that can be transmitted to humans via the food supply (Garau et al., 1999;Gorbach, 2001;Smith et al., 1999, Chiu et al., 2002. The past two decades have witnessed major increases in emergence and spread of multidrug-resistant bacteria and increasing resistance to newer compounds, such as fluoroquinolones and certain cephalosporins (Levy & Marshall, 2004). Resistance to fluoroquinolones develops more rapidly in E. coli than in other members of the Enterobacteriaceae (Gales et al., 2000). Retail poultry products are routinely heavily contaminated with avian fecal E. coli (Gorbach, 2001;Smith et al., 1999). Such E. coli, which can be antibiotic resistant (including to FQs) (Chiu et al., 2002), widely contaminates kitchen surfaces during meal preparation, is not readily removed from these surfaces by standard cleaning procedures, and can subsequently be isolated from the feces of persons preparing the meals (Cogan et al., 1999;Linton et al., 1977). Thus, the possibility of foodborne transmission of FQ-resistant E. coli from poultry to humans is highly plausible. Studies of farms have shown an association of multidrug-resistant E. coli with chronic antimicrobial drug exposure (McEwen & Fedorka-Cray, 2002; Jensen et al., 2006), there are few data on temporal trends of antimicrobial drug resistance in food animal E. coli isolates, particularly those recovered before 1980. The aim of the study is to evaluate the fluoroquinolone resistance of E. coli isolated from the chicken meat in Bosnia and Herzegovina.

Materials and Methods:-
The study included 55 chicken meat samples collected randomly from retail supermarkets, butcheries and slaughterhouses. Meat samples were aseptically collected and then packaged in sterile polythene zip bags and carried to the laboratory in aseptic conditions in a cold box within two hours from the time of purchase. Duplicate samples were obtained whenever possible. All samples were analyzed within 2-4 hours after their arrival to the laboratory. A sharp sterile knife was used to cut sample from surface in sterile tray. To isolate bacteria, a 25-g portion of sample was placed into sterile 225 ml buffered peptone water. For the preparation of samples, we used the guidelines given in the Microbiology of Food and Feeding Standards -Preparation of test samples, initial suspensions and decimal dilutions for microbiological tests (EN ISO 6887-1: 1999, IDT, ISO 6887-1: 1999, IDT). The time between the end of the preparation of the initial suspension and the point when the inoculum is in contact with the nutrient medium should not be more than 45 minutes, while the time between preparation of the initial suspension and the beginning of the preparation of decimal dilutions is limited to 30 minutes.
After the preparation of the samples, the microbiological analysis was carried out according to the ISO standard Microbiology of food and animal feedingstuffs -Horizontal method for counting <beta> glucuronidase positive Escherichia coli -Part 2: Colony counting technique at 44 ° C using 5-bromo-4-chloro -3-indolyl-beta] -Dglucuronide (ISO 16649-2: 2001, IDT). Volume of 1 ml of the initial solution is aseptically transferred by micropipette to a sterile Petri dish. Two plates were inoculated after dilution. The procedure is repeated with other decimal dilutions, using new sterile pipettes for each dilution. In each Petri dish, 15 ml of TBX medium was poured, which was previously cooled to 44 -47 °C in a water bath. The inoculum was carefully mixed with the medium and left to harden on the horizontal surface. The time that flows between the distribution of the inoculum in the vessel and the spillage of the media should not exceed 15 minutes.
After incubation for 24 h, all Petri plates were examined for the rise of Escherichia coli colonies. The ATCC strain of E. coli 25922 was used as a control strain. All the bacteriological media and antimicrobial disks were purchased from HiMedia Laboratories, Mumbai, India. Susceptibility tests were performed using the Kirby-Bauer method on Mueller-Hinton agar in accordance with Clinical and Laboratory Standards Institute, 2017 (CLSI, 2017) and using ampicillin (10 µg), amoxicililn (25µg), ciprofloxacin (5µg) and nalidixic acid discs (30µg) (Mast group, UK). The 971 E. coli isolates were inoculated in nutrient broth and incubated at 35+2 °C for 24 h. The broth was diluted in normal saline solution to a density of 0.5 McFarland turbidity standard. Cotton swabs were used for streaking the diluted broth onto Mueller-Hinton agar plates. After 15 minutes, antibiotic discs were placed 30 mm apart and 10 mm away from the edge of the plate. Plates were inverted and incubated aerobically at 35+2 °C for 16 to 18 hours. The zones of inhibition was measured, recorded, and interpreted according to the recommendation of the CLSI [CLSI, 2017].

Results:-
Out of 55 tested chicken meat samples, E. coli was isolated from 40 (72.73%) samples (Table 1). E. coli was recovered from 21 frozen and 19 fresh chicken meat samples. The resistance rates for each tested antibiotic for 40 isolates are given in Graph 1. Occurrence of resistance among E. coli isolates from chicken meat for all antimicrobiotics was extremely high. Of the antimicrobiotics included, the highest rates of resistance were found for quinolones. Resistance to nalidixic acid was determined by 100% of the tested chicken samples Total resistance is the absence of the inhibition zone around the nalidixic acid disc was recorded for 38 tested samples. An inhibition zones were detected only for two isolates: one isolate from mechanically seperated chicken meat (12 mm) and one isolate from frozen chicken drumstick (4 mm).

Graph 1:-Graphical presentation of resistant isolates of Escherichia coli from chicken meat samples
Resistance to fluoroquinolone agents ciprofloxacin was demonstrated in 95% of the E. coli isolates. Isolate from a sample of chicken drumstick showed sensitivity to the tested antibiotic with an inhibition zone of 25 mm. Also, isolate from a sample of mechanically eliminated chicken meat showed sensitivity to the antibiotic tested, and the measured inhibition zone was 26 mm. Resistance to antibiotics from the group of aminopenicillins, beta-lactam antibiotics that act on the synthesis of the bacterial cell wall was also established in most of the tested samples. E. coli that are resistant to quinolones and fluoroquinolones contaminate many retail meat products, particularly poultry, corresponding with the use of fluoroquinolones in food animals, particularly chickens and turkeys (Johnson et al., 2003).
In the present study all of the E. coli isolated from chicken meat (100%) were resistant to nalidixic acid (quinolone) and 92.5% were resistant to ciprofloxacin (fluoroquinolone). Rate of resistance to aminopenicillines (amoxicillin and ampicillin) was also extremely high. For amoxicillin and ampicillin the resistance levels were identical (90%). Similar findings were reported for nalidixic-resistance for E. coli isolates recovered from chickens in Iran (Moniri & Dastehgoli, 2005), where 100% isolates were resistant to nalidixic acid, but a lower percentage of resistance (41.9% ) was found for ciprofloxacin. Extremely high resistance to ciprofloxacin was found in Bogota (Colombia). All of 182 E. coli strains obtained from chicken feces and retail poultry meat were resistant to ciprofloxacin (Arias et al., 2005). The rates of resistance found for tested antimicrobials in E. coli isolates obtained from the chicken meat in Bosnia and Herzegovina tend to be higher than the levels reported in European Union countries. In EU during 2016, quinolone resistance was the most common trait and occurrence was overall very high in indicator E. coli from broilers and their meat at 59.8% and 64.0% for nalidixic acid and ciprofloxacin, respectively and rate of resistance to ampicillin was also very high (58.0%) in the EU member states ( The extremely high recovery rate of (fluoro)quinolone-resistant E. coli from broiler meat in Bosnia and Herzegovina are of concern, because fluoroquinolones are recognised by WHO as critically important antibiotics in human medicine which are used for treating severe and invasive infections (World Health Organization, 2017). Therefore, we must focusing on measures to maximaly reduce their use in poultry and other farm animals.