Prevalence and Antibiotic Susceptibility of Campylobacter species Isolated From Chicken and Beef Meat

Background: To study prevalence of Campylobacter spp. in chicken and beef meat, and determine the drug susceptibility of strains, 450 samples in Tehran, Iran were investigated. Objectives: This study aimed to determine the prevalence and the antimicrobial resistance of entropathogenic Campylobacter strains, especially C. jejuni isolated from raw chicken and beef meat in TehranIran. Materials and Methods: Out of 250 chickens and 200 beef meats, 121(26.8 %) contaminated cases with Campylobacter strains were isolated. Campylobacter was isolated from a significantly larger number of chickens (44%) than beef meats (5.5 %) (P < 0.05). Results: From all isolated Campylobacter organisms, 93 (76.8%) species were identified as C. jejuni and 28 cases (23.1%) as C. coli. Susceptibilities of 121 strains (93 C. jejuni and 28 C. coli) were determined against 12 antimicrobial drugs using the disk agar diffusion method. Resistance to nalidixic acid (75%) and ciprofloxacin (50%) was an alarming finding, moreover, 32.6% of isolates was resistant to tetracycline, 10.8% to ampicillin, 29.3% to colisitin and 26.1% to amoxicillin. The highest sensitivity was seen to erythromycin (95 %) and gentamicin (96%). Conclusions: These results showed that a high proportion of chicken and beef meat in Iran is contaminated with Campylobacter, particularly with Campylobacter jejuni. The high rate of contamination, especially chicken is a significant public health concern. Most of the isolates were resistant; therefore, human infection with Campylobacter spp. via consumption of these products is possible.


Background
Campylobacter is one of the most common causes of acute bacterial gastroenteritis in human worldwide (1).Campylobacteriosis is a zoonotic disease; domestic animals such as poultry, pigs, and cattle may act as reservoirs for Campylobacter spp.(2).The consumption of chicken and chicken products, are implicated in higher number of worldwide outbreaks of acute Campylobacter enterocolitis in both industrialized and developing countries, especially among children, the elderly and immuno-suppressed patients over the recent years (3,4).
Besides, low infection dose of C. jejuni, its rate of infection increases along with the ingested dose (5).C. jejuni and C. coli are isolated mostly from humans.C. jejuni is often implicated as the cause of campylobacteriosis, while C. coli seems to be less frequent in causing human acute diarrhea (6).Recently, food-borne infections with resistant pathogens have emerged as a threat to human health.The concern for this food-borne infection has in-creased because of the frequent isolation of antimicrobial resistant Campylobacter strains in humans and animals (7,8).This is almost a consequence of the massive use of antibiotics in modern intensive animal and poultry production units for therapy and prevention of their diseases (9).Illness caused by Campylobacter is usually self-limiting and therapy is not required, except in severe episodes of disease or in immunocompromised patients, that antibiotic therapy may be necessary.In these specific cases, therapy may be complicated by the fact that antimicrobial resistance in Campylobacter isolates from human infections has become increasingly common (10).
According to different reports from Iran, the prevalence of Campylobacter in clinical samples ranges from 4 to 10 percent (11)(12)(13).However, there is not any information about prevalence and antibiotic susceptibility of different species of Campylobacter isolated from food samples in Iran.

Objectives
This study aimed to determine the prevalence and the antimicrobial resistance pattern of entropathogenic Campylobacter strains, especially C. jejuni isolated from raw chicken and beef meat in Tehran-Iran.

Samples
To investigate the prevalence of Campylobacter, a total of 450 samples, including chicken (n = 250) and beef meat (n = 200) were collected from Tehran, from April 2011 to May 2012.All samples were received by the laboratory of the Department of Microbiology, and microbiological analysis was carried out within 2 hours after the collection.

Isolation and Identification of Campylobacter spp.
To isolate Campylobacter, 25 g of each sample after hemogenizing was pre-enriched in Campylobacter enrichment broth base (Preston enrichment broth base, HIMEDIA, Mumbai, India, M899) supplemented with Campylobacter selective supplement IV (HIMEDIA, Mumbai, India, FD158) and 5% defibrinated sheep blood.After inoculation at 42°C for 24 h in a microaerophilic condition, 0.1 mL of the sample was streaked onto Campylobacter selective agar base (HIMEDIA, Mumbai, India, M994) supplemented with an antibiotic supplement for the selective isolation of Campylobacter species (Campylobacter Supplement-2, Blaser Wang, HIMEDIA, Mumbai, India, FD 006) and 5% (V/V) defibrinated sheep blood and incubated for 48 h at 42°C under the same condition.One presumptive Campylobacter colony from each selective agar plate was subcultured and tested for Gram staining, production of catalase, oxidase and hippurate hydrolysis.One strain from each Campylobacter-positive sample was selected for susceptibility testing.

Statistical Analysis
Statistical analysis of results was performed with SPSS/ PC 11.5 software (SPSS, Chicago, IL).The chi-square test and Fisher's exact two-tailed test were used for statistical analysis.A P value less than 0.05 was considered statistically significant.

Discussion
Awareness of the public health implications of Campylobacter infections has evolved for over a century (15).Campylobacteriosis is a leading cause of gastroenteritis in many countries, and it has been isolated from a considerable number of patient with gastroenteritis in Iran (3)(4)(5)(11)(12)(13).Prevalence of Campylobacter spp. in Iran is lower than developed countries (1,16,17).However, similar finding was reported by Dadi et al. (18).According to our results, Campylobacter was recovered at higher prevalence in chicken samples than beef meat and C. jejuni was the most predominant Campylobacter species recovered from meat and chicken.These data are in accordance with reports from other countries (18)(19)(20)(21).Proportion of C. jejuni and C. coli in chicken and beef meat samples was similar.C. jejuni was significantly more prevalent than C. coli in both chicken and beef meat (P < 0.05).
The increasing rate of human infections caused by antimicrobial-resistant strains of C. jejuni makes clinical management of cases with campylobacteriosis more difficult (12,22,23).Antimicrobial resistance can prolong illness and compromise treatment of patients with bacteremia.The rate of antimicrobial-resistant enteric infections is highest in the developing countries, where the use of antimicrobial drugs in humans and animals is relatively unrestricted (20).
Int J Enteric Pathog.2014;2(2):e17087 Resistance of C. jejuni to quinolones was the most alarming finding in this study.Interestingly, in a recently published study from Iran, clinical isolates of Campylobacter spp.showed high resistance to same antibiotics (12), which may be as a result of consumption of contaminated food that harbors antibiotic resistant Campylobacter.Thus, administration of quinolones as the drug of choice for acute diarrhea in Iran seems to be revised.Current study has employed larger samples and not restricted to a special season.Remarkable difference between antibiotic susceptibility pattern of C. jejuni and C. coli has been reported.The incidence of resistance to most of the tested antimicrobial agent in this study was generally higher for C. jejuni than for C. coli (Table 1).This finding confirmed what has been previously described (8,24), although results from another study demonstrated that C. coli was generally more resistant than C. jejuni (9,18,23,25).Owing to the increased reporting of antimicrobial resistance in Campylobacter worldwide, attempts should be made to control their use in animal husbandry.
In conclusion, the result showed that a high proportion of chicken and beef meat in Iran is contaminated with Campylobacter, particularly with Campylobacter jejuni.The high rate of contamination in chicken meat alarms a significant public health concern.Most of the isolates were resistant; therefore, there is a possible risk of human infection with Campylobacter spp.via consumption of these products.

Table 1 .
Number and Percentages of Antimicrobial Resistant Campylobacter Strains Isolated From Chicken and Beef Meat a a Data are presented as No. (%)