Introduction

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 Lactobacillus and Bifidobacterium are used as food supplements that may favourably influence the intestinal flora of human and animal hosts, e.g. by competitive exclusion of gastrointestinal pathogens, stimulation of the immune response or antimutagenic and anticancerogenic activities (Mercenier et al. 2003; Servin 2004). On the other hand, lactobacilli, pediococci and lactococci have also been implicated in rare cases of human infections (Davies et al. 1986; Cannon et al. 2005). Lactic acid bacteria widely used as probiotics potential to serve as a host of antibiotic resistance genes with the great risk of transferring the genes in many lactic acid bacteria and other pathogenic microorganisms. However, some LAB may carry potentially transmissible plasmid-encoded antibiotic resistance genes (Tannock et al.1989; Tannock et al. 1994). The spontaneous loss of a phenotypic characteristic from a bacterial strain is frequently linked with the loss of an extrachromsomal DNA (Kullen and. Klaenhammer 2000) while Ishiwa and Iwata have shown the curing of plasmid on basis of drug resistance (Ishiwa and Iwata 1980; Du Toit et al. 1998 Saxelin 1997; Orrhage and Nord 2000; Cummings and others 2001).}

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 lactobacillus isolates, by investigating their antibiotic susceptibilities and plasmid profiles so that the selection and safety of probiotics isolates would be determined.

Materials and Methods

Media and Chemicals

Bacterial Strains

Determination of Antibiotic resistance patterns

Plasmid analysis

Curing experiments

Gel electrophoresis

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 Lactobacillus isolates were also determined for their resistance to toxicity of conjugated bile salts. Fifty two isolates were able to resist the toxicity of GCA and twenty seven were sensitive to GCA toxicity, while in case of conjugated bile salt TCA, fifty eight have shown resistance to TCA toxicity and twenty one were sensitive to TCA toxicity (Table 1).

Antibiotic resistance result: Resistance level of indigenous Lactobacillus isolates against different antibiotics at different concentrations are shown in Table 2 indicates the cumulative antibiotic resistance patterns offered by the isolates at 100µl while antibiotic resistance pattern of various concentrations are shown in Figure1.

Plasmid Profile result: The presence of plasmid DNA was investigated in 12 indigenous Lactobacillus isolates from food and faeces. Figure 2 show the plasmid profiles of these indigenous isolates. The Indigenous lactobacillus isolates contained varying number of plasmids DNA bands. The number of plasmids present in a single isolate varied from 1 to 2 ranging between 1kb in size. On the other hand, the isolate CMGsaM269, CMGsaM156, CMGsaI248, CMGsaS306, CMGsaM163, CMGsa168, CMGsa145, were devoid of plasmid DNA.

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 Lactobacillus isolates CMGsa180, CMGsa154, CMGsa127 showed resistance upto 1800µl AO but sensitive to 2000µl AO Table 3a, 3b, 3c, 3d and 3e respectively. The minimal inhibitory concentration of AO was checked for the loss of antibiotic markers and plasmid DNA. The curing agent found effective with respect to the loss of antibiotic markers in indigenous Lactobacillus isolates CMGsa127, CMGsa147, CMGsa154, CMGsa180 and CMGsa243.

Figure 1

Table 1: Resistance to the toxicity of Conjugative bile salts by Indigenous Lactobacillus Isolates of Pakistan

Figure 2

Table 2: Antibiotic resistance patterns (100µg/ml) by Indigenous Lactobacillus isolates of Pakistan

Abbreviations: R: Rifampicillin, T: Tetracycline, K: Kanamycin, N: Neomycin, S: Streptomycin, C: Chloromphenicol, V: Vancomycin.

Figure 3

Figure 1: Determination of Antibiotic Resistance offered by Indigenous Lactobacillus Isolates of Pakistan at Various concentrations of Antibiotics.

Figure 4

Figure 2: Plasmid Profile of Indigenous Lactobacillus isolates containing plasmid DNA.

Figure 5

Table 3a: Growth Pattern of Indigenous Isolates of Pakistan CMGsa127 at various concentration of Acridine Orange

Figure 6

Table 3b: Growth Pattern of Indigenous Isolates of Pakistan CMGsa147 at various concentration of Acridine Orange

Figure 7

Table 3c: Growth Pattern of Indigenous Isolates of Pakistan CMGsa154 at various concentration of Acridine Orange

Figure 8

Table 3d: Growth Pattern of Indigenous Isolates of Pakistan CMGsa180 at various concentration of Acridine Orange

Figure 9

Table 3e: Growth Pattern of Indigenous Isolates of Pakistan CMGsa243 at various concentration of Acridine Orange

Discussion

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 lactobacilli were collected from different food and dairy sources of Karachi were screened for bile and drug 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 I. The indigenous isolates were also determined for their resistance to toxicity of conjugated bile salts. Fifty two isolates were able to resist the toxicity of GCA and twenty seven were sensitive to GCA toxicity, while in case of conjugated bile salt TCA, fifty eight have shown resistance to TCA toxicity and twenty one were sensitive to TCA toxicity (Table I).

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.