Prevalence of antibiotic resistance in lactic acid bacteria isolated from the faeces of broiler chicken in Malaysia

Background Probiotics are commonly used as feed additive to substitute antibiotic as growth promoter in animal farming. Probiotic consists of lactic acid bacteria (LAB), which enhance the growth and health of the animal. Probiotic also have higher possibility to become pathogenic bacteria that may carry antibiotic resistant gene that can be transmitted to other LAB species. The aim of this study was to identify the LAB species in the faeces of broiler chicken and to determine the prevalence of antibiotic resistant in LAB of broiler chicken. Methods Sixty faeces samples were collected from wet markets located in Klang Valley of Malaysia for the isolation of LAB using de-Mann Rogosa Sharpe medium. Thirteen species of LAB were obtained in this study and the identification of LAB was performed by using API test kit on the basis of carbohydrate fermentation profile. Antibiotic susceptibility assay was then carried out to determine the prevalence of LAB antibiotic resistance. Results Lactococcus lactis subsp lactis was found in nine out of sixty faecal samples. Lactobacillus paracasei was the second common LAB species isolated from chicken faecal. No significant difference (P > 0.05) was found between the occurrence of Lactobacillus brevis, Lactobacillus curvatus, Lactobacillus plantarum, Leuconostoc lactis mesenteroides subsp mesenteroides/dectranium and Pediococcus pentosaceus isolated from 5 different locations. Most of the isolated LAB was resistant to antibiotic and high variability of the antibiotic resistance was observed among the LAB against 15 types of antibiotics. Penicillin, amoxicillin, chloramphenicol, and ampicillin had significant higher (P< 0.05) inhibitory zone than nalidixic acid, gentamycin, sulphamethoxazole, kanamycin, and streptomycin. Conclusions Many species of LAB were isolated from the faecal samples of broiler chicken that resistance to the common antibiotics used in the farm. The development of resistant against antibiotics in LAB can be attributed to the long term exposure of antibiotic as growth promoter and therapeutic agents. Thus, it is essential to advise farmer the safety measure of antibiotic application in animal farming. Additionally, the supplementation of probiotic in animal feeding also needs more attention and close monitoring.


Background
Antibiotic are normally used to treat microbial diseases since 50 years ago. However, excessive use of antibiotic may lead to the development of antibiotic resistance in pathogenic bacteria. The report of antibiotic resistance is significantly increased due to the overuse and misuse of antibiotics, which has created an enormous selective pressure on the recessive bacteria [1]. Antibiotic resistant bacteria have the ability to resist toward the actions of naturally occurring or synthetically produced compounds inimical to their survival [2].
Scott [3] reported the identical resistance gene present in bacterial species isolated from different hosts. Antibiotic resistant may acquires through the in-vivo gene transfer between normal flora of gastrointestinal and antibiotic resistant pathogenic bacteria [4]. In fact, the antibiotic resistant pathogenic bacteria pose a great potential threat to human health, especially when the immunity system is not functioning well. Most of the developed countries have prohibited the use of antibiotic as animal growth promoter. Thus, various alternatives have been explored to replace antibiotic as growth promoter. One of the most common and popular alternatives is the application of probiotic as growth promoter in livestock animals. In addition, the probiotic effects of postbiotic metabolites produced by probiotic strains have been shown in various animal species, such as rats [5,6], broilers [7,8], laying hens [9] and post weaning piglets [10], suggesting they have great potential to be used as growth promoter for livestock animals.
Probiotic comprises of beneficial bacteria such as lactic acid bacteria (LAB). LAB is a group of gram-positive anaerobic bacteria which produce predominantly lactic acid from carbohydrate fermentation. Many farmers use probiotic extensively and those bacteria have potential to serve as a host of antibiotic resistance genes with a risk of transferring those genes into many LAB and other pathogens [11].
The antibiotic resistant LAB has been detected by using DNA sequences which is responsible for antibiotic resistance traits. Egervärn et al. [12] reported the emergence of antibiotic resistant in Lactobacillus reuteri and Lactobacillus plantarum. Moreover, Lactobacilli, Pediococci and Leuconostoc spp. have been reported to be highly resistant to vancomycin [11,13] and some Lactobacilli have high resistance to bacitracin, cefoxitin, ciprofloxacin, fusidic acid, streptomycin, sulphadiazine, teicoplanin and vancomycin [14]. Most of the reported LAB that resistant to antibiotics was isolated from food sources. These include the most commonly used probiotic species such as Lactobacillus casei, Lactobacillus acidophilus, L. reuteri, or Lactobacillus rhamnosus, among others, or the yogurt starter bacteria Lactobacillus delbrueckii [15][16][17]. However, the information of antibiotic resistant LAB isolated from the gut of broiler chickens in Malaysia is very limited. Thus, the objective of this study was to investigate the antibiotic resistance profile of LAB isolated from the gut of broiler chickens in Klang Valley, Malaysia.

Sample origin and collection
A total of 60 faeces samples were collected from chicken purchased from wet markets located nearby Universiti Putra Malaysia: Serdang Jaya, Seri Kembangan, Dengkil, Putrajaya, Kajang, Pantai Dalam, and Jenderam Hilir. The age of the chicken was 42 days. The faeces were collected directly from caeca junction to the end of the cloaca of large intestine. The faeces samples (1 g each) were kept at −20°C until further analysis. The experimental design has obtained approval from the Ethics Committee of Universiti Putra Malaysia.

Enumeration and isolation of lactic acid bacteria
The faeces sample was mixed homogeneously at a ratio of 1 g sample with 9 ml of peptone water in the universal bottle and incubated for one hour at room temperature. The samples were then subjected to 10-fold serial dilution using 0.1% (v/v) peptone water [18] and 0.1 ml of each appropriate diluted sample was then plated onto de Man Rogosa Sharpe (MRS) agar and incubated under anaerobic condition at 30°C for 48 hours [19]. The colony forming unit (CFU) per gram of sample was expressed as logarithm at the base of 10 (Log 10 CFU/g). The enumeration of LAB was conducted in triplicates. After 48 hours of incubation, a colony was picked and streaked onto MRS agar and incubated for additional 48 h at 30°C. This process was repeated twice and a pure colony was then transferred to MRS broth and incubated at 30°C for 24 h. The pure LAB culture was then kept in MRS broth supplemented with 20% (v/v) glycerol and incubated at −20°C until further analysis.

Identification of lactic acid bacteria
LAB culture was identified phenotypically on the basis of carbohydrate fermentation profile by using API 50CH kit (BioMerieux, France) according to the instruction of manufacturer. The carbohydrate fermentation profile was then analysed by using APILAB Plus software version 3.3.3 (BioMerieux, France) to identify the species of each isolated LAB culture.

Statistical analysis
The data was analysed using one way analysis of variance (ANOVA) with probability level of 0.05 (P < 0.05) using SAS statistical software [21].  Note: A total of 6 samples were collected from each location.

Identification of lactic acid bacteria
All LAB species isolated from faeces of broiler chicken were identified by API Kit. It has been reported that L. acidophilus and Lactobacillus salivarius have higher chance to be isolated among the Lactobacillus strains colonizing in the crop intestine [22]. However, L. acidophilus and L. salivarius were only found in faeces samples 1 and 4, respectively; indicating L. acidophilus and L. salivarius were not a common LAB found in the faeces of chicken sold in Klang valley of Malaysia.
Antibiotic resistance of lactic acid bacteria Table 2 shows the inhibitory zone of antibiotic susceptibility for nine LAB regardless of species obtained from faecal samples. Penicillin, amoxicillin, chloramphenicol, and ampicillin had significant higher (P < 0.05) inhibitory zone than nalidixic acid, gentamycin, sulphamethoxazole, kanamycin, and streptomycin. These results were similar with the previous report, where lactobacilli were most sensitive to penicillin and ampicillin except for amoxicillin and chloramphenicol [23]. However, there was no significantly different (P > 0.05) between vancomycin and bacitracin. Similar inhibitory zone was also found in bacitracin, nalidixic acid, gentamycin, sulphamethoxazole, kanamycin, and streptomycin. LAB was sensitive to clindamycin, chloramphenicol, amoxicillin, penicillin, erythromycin, and ampicillin in current study. However, it was resistant against nalidixic acid, bacitracin, gentamycin, ciprofloxacin, sulphamethoxazole, kanamycin, tetracycline, streptomycin and vancomycin. These results indicate that LAB is not sensitive to beta-lactams group of antibiotics due to the absence of peptidoglycan in LAB cell. However, Lavanya et al. [24] reported recently that most of the LAB isolated from fermented milk were resistant to penicillin G and only 10% were susceptible to ampicillin. Betalactams antibiotics were the most effective drugs for the treatment of Staphylococci infections. However, Tyler et al., [25] reported that beta-lactams antibiotics have no longer effective to treat Staphylococcus aureus infections such as methicillin-resistant S. aureus and vancomycinresistant S. aureus.
The development of antibiotic resistance in LAB has revealed the fact that the common use of antibiotic as growth promoter in the farm has allowed LAB to develop resistance against antibiotics [26]. The antibiotic resistance in LAB may also acquire through horizontal transferring of resistance gene to the normal LAB by conjugative transposons [1]. As reported by Gilchrist et al. [26], antibiotic resistance in LAB was partly due to poor control of antibiotic as therapeutic and growth promoter in animal feeding. This may contribute to the build-up reservoir of antibiotic resistant bacteria in the gut. Hence, the current result suggests that antibiotic resistance screening is essential before any LAB strains are selected as feed additive for animal feeding. Table 3 shows the diameter of inhibitory zone of antibiotic susceptibility among the LAB species. The inhibitory zone for Leu. lactis mesenteroides subsp mesenteroides/dextranicum was significantly higher (P < 0.05) than other LAB species for the tested antibiotics. Bassam [27] reported that Leu. lactis mesenteroides was resistant to 100% vancomycin, 75% teicoplanin, 87.5% clindamycin, 75% chloramphenicol, erythromycin, gentamicin and streptomycin, 62.5% ampicillin, penicillin G and tetracycline, 50% kanamycin and trimethoprim. L. acidophilus was  resistant (P < 0.05) against nalidixic acid, gentamycin, ciprofloxacin, sulphamethoxazole, kanamycin, streptomycin and vancomycin. However, Sieo et al. [28] claimed that L. acidophilus isolated from chicken gastrointestinal tract was resistant to 100% chloramphenicol (≥200 μg/ ml), 58% to erythromycin (≥200 μg/ml) and 17% to tetracycline (≥200 μg/ml). L. brevis and L. curvatus had similar trend of resistance against the antibiotics. However, L. brevis was more resistant to ciprofloxacin (a fluoroquinolone), tetracycline, and vancomycin as found in the study of Fukao et al. [29]. L. delbrueckii subsp delbrueckii, L. fermentum, L. rhaminosus, L. salivarious, Leu. mesenteroides, P. damnosus, P. pentosaceus were sensitive to all the tested antibiotics. According to Ouoba et al. [30], L. acidophilus L. fermentum, and L. rhaminosus contained positive amplicons of resistance genes encoding aminoglycoside (aph (3′)-III, aadA, aadE) and tetracycline tet (S), and hence they had higher prevalence of phenotypic resistance for aminoglycoside. This can be attributed to intrinsic resistance of different species since none of these bacteria were resistant to penicillin (first generation βlactam). These organisms have the potential to act as reservoir of antimicrobial resistant genes and which have potential to be transferred to other bacteria. Moreover, intrinsic resistance to vancomycin was confirmed for L. paracasei, L. salivarius and L. plantarum, and L. salivarius was also typically resistant to erythromycin [31]. In other study, L. salivarius had the highest resistance to kanamycin and neomycin, but demonstrated the lowest resistance to penicillin [32]. L. paracasei subsp paracasei was usually used as probiotic in lactic acid fermented food for human consumption [33].

Profile of lactic acid bacteria antibiotic resistance
Vankerckhoven et al. [34] reported that L. paracasei 8700:2 strain was not resistant to antibiotic. However, L. paracasei subsp paracasei was found to be more resistant against nalidixic acid and sulphamethozole as compared to other antibiotics as found in this study. Furthermore, L. plantarum was sensitive to all the tested antibiotics except nalidixic acid, gentamycin, sulphamethoxazole, kanamycin and streptomycin. In contrast, Toomey et al. [35] claimed that L. plantarum harbored erm (B) and msrA/B genes, and tet (M) gene that resistant to erythromycin and tetracycline, respectively. Additionally, it is also intrinsically resistant to vancomycin. Similar trend was also found in Lc. lactis except for sulphamethoxazole in current study. Tetracycline and erythromycin-resistance genes were found in Lc. lactis and this microbe is normally representing the fermenting microflora of typical Italian traditional cheese Mozzarella di Bufala Campana [36]. It has been reported that L. fermentum 1, Lc. lactic subsp. lactic 1, L. paracasei subsp. paracasei 1, L. rhamnosus and Lc. lactic subsp. lactic 2 isolated from raw poultry meat were resistant to polymycin B (PB 100), trimethoprim (TM 5), tetracycline (TE 30), oxacillin (OX 1), kanamycin (K 30), erythromycin (E 15), gentamycin (CN 10), ciprofloxacin (CIP 5) and cephalothin (CL 30) [37].

Conclusions
In conclusions, many species of LAB were found in the faecal samples of broiler chicken. Each species of LAB has different inhibitory zone related to antibiotic susceptibility. These results indicate that LAB may develop resistance against antibiotic that may result from the horizontal transferring of resistant gene to other microflora in the gut. The development of resistance to antibiotic can be attributed to long term usage of antibiotic as therapeutics and growth promoter. Thus, it is important and essential to advise the farmer a proper way of antibiotic use as therapeutic and growth promoter agents. Additionally, supplementation of unknown source of probiotic as feed additive needs to be monitored closely in animal feeding.