Resistance to the toxicity of conjugative bile salts and Assessment of plasmid borne antibiotic resistance among Indigenous lactobacillus isolates of Pakistan.
S Ajmal, J Akhtar, E Shoeb, N Ahmed
and probiotics., antibiotics, bile salt hydrolase, deconjugation, hypocholesterolemia, lactobacillus, plasmids, precipitation
S Ajmal, J Akhtar, E Shoeb, N Ahmed. Resistance to the toxicity of conjugative bile salts and Assessment of plasmid borne antibiotic resistance among Indigenous lactobacillus isolates of Pakistan.. The Internet Journal of Nutrition and Wellness. 2009 Volume 10 Number 1.
Probiotics are the viable microorganisms, which upon digestion exert health-promoting effects on host. Probiotics bacteria must have the potential to serve as a host of antibiotic resistance genes with the risk of transferring the genes in many lactic acid bacteria and other pathogenic bacteria. Many Lactic acid bacteria may carry potentially transmissible plasmid-encoded antibiotic resistance genes. This antibiotic resistance can cause significant harm and suffering to the host. The study aim was to investigate the antibiotic resistance in indigenous lactobacillus isolates and existence of plasmids in the indigenous lactobacillus isolates and relationship between the antibiotic resistance and plasmid.
The importance of the intestinal microflora has attracted much interest in recent years, particularly with respect to ways in which the microflora can be manipulated to improve human health. One possible way of regulation of the intestinal microflora is the development of new probiotic strains aims at more active beneficial organisms. (Gilliland 1989) but the question of their safety and the risk to benefit ratio have to be assessed.
Generally probiotics can be defined as microbial cells components that have a beneficial effect on the health of host (Fuller 1989; Marteau et al. 1998; Marteau et al. 2002). Lactic acid bacteria (LAB) are resistant to antibiotics and most of them are nontransferable (Salminen et al. 1998). In probiotic applications, selected LAB strains mainly belonging to the genera
The transmission of antibiotic resistance genes to unrelated pathogenic or potentially pathogenic bacteria in the gut is a major health concern, with obvious end product for the selection and safety of probiotic strains. Any strains harbouring antibiotic resistance plasmids are considered unsuitable for use as human or animal probiotics (Salminen et al., 1998a: Saarela et al. 2000). While some researchers suggest that antibiotic-resistant probiotic strains may beneficial for maintaining the normal intestinal microflora of those patients suffering from reduction in microflora due to the administration of various antimicrobial drugs (Salminen et al. 1998b).
Potential characteristics of probiotics include the ability to reduce antibiotic use, the apparently high index of safety, for using it in various therapies as potential probiotic isolates are classified, and generally regarded as safe as opposed to antibiotics, which have a number of recognized adverse effects. The present study was evaluate, to further test the safety and potential effects of indigenous
Materials and Methods
Media and Chemicals
MRS (Oxoid) broth and agar were used in all the experiments were obtained from Sigma, while antibiotics in this study used include Rifampicillin (Rif), Tetracycline (Tetr), Kanamycin (Kan), Neomycin (Neo), Streptomycin (Strep), Chloromphenicol (Chlor), Vancomycin (Van) were also obtained from Sigma. Sodium salt of Taurocholic acid (TCA), Glycocholic acid (GCA) were obtained from Sigma and curing regent Acridine orange (AO) were obtained from Merck. All antibiotics were purchased from Sigma. Stock antibiotic solutions were filter sterilized (0.2 μm) and kept at −20 °C. Working solutions were prepared to specific concentrations at which equal volumes of solutions.
Assay for toxicity of conjugated bile salt.
The isolates were tested for the capacity to resist the bactericidal activity of a conjugated bile salt (TCA) with an assay modified from the assay described by De Smet et al (De Smet et al. 1998). A stationary-phase culture inoculum (1%) was added to MRS broth supplemented with TCA, GCA at a concentration of 0.5%. At zero time and after 24, 48, and 72 h of anaerobic incubation at 37°C samples were removed and dilutions of the bacterial suspensions were prepared. Aliquots of the dilutions were smeared onto MRS agar plates, which were then incubated anaerobically at 37°C for 48 h and 72h. Population estimates were made from viable counts.
Determination of Antibiotic resistance patterns
Commercially available antibiotics used in this study include Rifampicillin (Rif), Tetracycline (Tetr), Kanamycin (Kan), Neomycin (Neo), Streptomycin (Strep), Chloromphenicol (Cm), Vancomycin (Van). Stock solutions of these antibiotics were prepared as described by (Maniatis et al. 1990). MRS agar (1.5%) was prepared and supplemented with 10µg/ml of each antibiotic. The antibiotic supplemented agar was solidified and overnight cultures were than spreaded onto agar plates and incubated for 72h at 37˚C and the growth were observed after 24h, 48h and 72h. Minimal inhibitory concentrations MIC of Rif, Tetra, Kan, Neo, Strepto, Chloro, Van upto 150µl. Antibiotic resistance patterns were studied by replica techniques.
The plasmid analysis was investigated in 10 isolates namely CMGsaM269, CMGsa154, CMGsaM156, CMGsaI248, CMGsaS306, CMGsaM163, CMGsa243, CMGsa168, CMGsa145, CMGsa180, CMGsa127, CMGsa147 was done according to the method of Anderson and McKay (Anderson and McKay 1983; Frere 1994) with minor modifications. Lactobacillus isolates were picked from agar plates, inoculated in 15ml MRS broth. Overnight culture broth than centrifuged at 5000×g for 15 minutes at 4˚C. Cell pellets were washed with TES buffer and resuspended in lysozyme (25mg/ml) and incubated in water bath for 5o minutes. The mixtures were vortexed briefly for 40 seconds and the DNA was extracted using phenol and chloroform: isoamyl alcohol (24:1). Purified DNA were analyzed by electrophoresing (100 Volts for 1h and 30 minutes) on 0.7% agrose gels, stained with ethidium bromide and then photographed. 1 Kb λ DNA Ladder was used as a source of size marker for plasmids DNA in indigenous isolates.
The plasmid curing was done according to (Kullen and. Klaenhammer 2000; Chase et al. 1978) by subjected representative strains to Acridine orange (AO) (Merck) mediated plasmid (bearing resistance markers) elimination using a curing agent Acridine.
The analysis of Plasmid DNA was performed by electrophoresis using 0.7% agrose (Sigma) gel in Tris acetate buffer (40mM Tris, 20mM sodium acetate, 2mM EDTA) adjusted to pH 7.8 with acetic acid. Electrophoresis was carried out at 100 Volts for 1-1.5 hours. The gel stained for 15 minutes with 0.5g/ml ethidium bromide (Sigma) and destained for 5 minutes with water and visualized and photographed on under UV Alpha Imager of Alpha Innotech.
Results and Discussion
Resistance to bile salt toxicity result: About 79 isolates were collected from different food and dairy sources of Karachi were screened for bile resistance pattern. Resistance to Bile Toxicity level of the indigenous isolates against conjugative bile salts such as Glycocholic acid (GCA) and Taurocholic acid (TCA) is shown in table 1. The indigenous
Antibiotic resistance result: Resistance level of indigenous
Plasmid Profile result: The presence of plasmid DNA was investigated in 12 indigenous
Plasmid curing result
The result obtained in curing experiment of five isolates namely CMGsa127, CMGsa147, CMGsa154, CMGsa180 and CMGsa243 with one curing agent Acridine orange. After treatment with Acridine orange for 72hours, the indigenous
Abbreviations: R: Rifampicillin, T: Tetracycline, K: Kanamycin, N: Neomycin, S: Streptomycin, C: Chloromphenicol, V: Vancomycin.
Lactobacilli show a high impact on effective protection to human health, there is obvious evidence that lactobacilli from different origins possess Probiotic. In order to survive and colonize in the gastrointestinal tract, the bacteria should express high tolerance to acidic media and bile and should be able to adhere to the intestinal surfaces (Ouwehand et al. 2002). The antibiotic resistance of pathogenic bacteria is an increasing medical problem (Mazel and Davies 1999) and raises the question of antibiotic resistance among desired probiotic strains. Therefore, the antibiotic susceptibility test therefore should be incorporated for the safety assessment of the desired property of the promising probiotic lactobacilli.
Isolates with certain antibiotic resistances is very important because of the potential transferability of resistance traits to other bacteria, including pathogenic microorganisms. According to the opinion of the FEEDAP panel on the updating of criteria used in the assessment of bacteria for resistance to antibiotics of human, strains carrying an acquired resistance to antimicrobial(s) should not be used as feed additives, unless it can be demonstrated that it is a result of chromosomal mutation(s) (European Food Safety Authority (EFSA) 2005). It is important to realize that acquired antibiotic resistances can be transferred by other mechanisms, such as transformation or transduction, that are more difficult to study under controlled laboratory conditions.
In general, probiotic strain must colonize in GTI of host and must have acid, bile salt – tolerance (Du Toit et al. 1998; Gilliland et al. 1985) and they should not carry transmissible antibiotics resistance genes such genes are undesirable and could lead to the development of antibiotic resistant pathogens (Salminen et al. 1998b; Saarela et al. 2000). In Present study, 79 Indigenous isolates of
And from present result, it was suggested that the finally selected indigenous isolated Lactobacillus strains had an excellent ability to tolerate toxicity of conjugated bile salts. Except five isolates namely CMGsa127, CMGsa147, CMGsa154, CMGsa180 and CMGsa243 antibiotic marker plasmid DNA can not be used as these indigenous isolates harbouring antibiotic resistance plasmids are considered unsuitable to prevent diseases from human health or animal while the mechanisms of other isolates for regulating serum cholesterol and the effect on serum cholesterol level in vivo needs further extensive investigations. Nevertheless, results achieved are positive and promising and the development of new indigenous probiotic products has produced new scientific achievements and a strong demand for improved and scientifically-based selection criteria.