Lactobacilli From Buffalo Milk and Yogurt With Antibacterial Activity Against Gram-Negative Uropathogens

Background: This descriptive cross-sectional study was conducted to determine the antibacterial activity of Lactobacillus strains isolated from buffalo milk and yogurt in Aliabad-e Katul city, Golestan province, north-east of Iran. Methods: Raw milk and yogurt samples were collected and cultured on MRS medium by incubating anaerobically at 37°C for 48 hours. The suspected colonies were identified on the basis of Gram staining, biochemical tests, and carbohydrates fermentation. The antibacterial activity of the cell-free supernatant (CFS) extracted from Lactobacillus strains was determined using the agar well diffusion method against standard strains of Escherichia coli ATCC 11303, Klebsiella pneumoniae ATCC 13883, and Pseudomonas aeruginosa ATCC 15442 as well as gram-negative uropathogens previously isolated from patients with urinary tract infections (UTIs). Three isolates of E. coli (E1, E2, and E3), two isolates of P. aeruginosa (P1 and P2), and two isolates of K. pneumoniae (K1 and K2) were used in this study. Results: A total of 19 Lactobacillus strains were identified as L. plantarum, L. casei, L. acidophilus, and L. helveticus. Based on the results of antibacterial activity test, the isolates had the highest and lowest inhibitory effects on the E. coli and K. pneumoniae isolates, respectively. Among the isolates, only L. casei isolates showed inhibitory activity against K. pneumoniae isolates. Conclusions: In this study, Lactobacilli from buffalo milk and yogurt demonstrated a good inhibitory activity against E. coli as a common cause of urinary tract infection. Therefore, further studies are recommended to elucidate their potential for being used as an alternative to antibiotic therapy.


Background
In recent years, the rise of antibiotic resistance and declining discovery of new antibiotics have created a global health crisis. Overuse and misuse of antibiotics have led to the rapid emergence of antibiotic-resistant bacteria. Of particular concern, no new antibiotics have been approved for treating gram-negative pathogens in decades. Therefore, there is an urgent need to find novel and safe antibacterial substances as alternatives to antibiotics (1,2).
Today, lactic acid bacteria (LAB) due to the potential production of metabolites with antimicrobial activity including diacetyl, hydrogen peroxide, acetaldehyde, organic acids, bacteriocin, and bacteriocin-like substances have received the attention of many researchers (3). Moreover, their antibacterial effects have been attributed to the reduction in pH and competition for nutrients resources (4). Therefore, studies related to the antibacterial activity of these organisms have received much attention to prevent, control, and treat diseases (5). In the past 20 years, the antagonistic effect of LAB on many pathogenic microorganisms has been reported (1,2).
There are lots of LAB in dairy products and many people consume different types of these products as a significant source of protein (6), among which cow, sheep, and goat yogurt and milk are very common in Iran. However, buffalo yogurt and milk are more common in rural areas of the country (7). Compared to other milks, buffalo milk has a low cholesterol content and a high level of calcium, in addition, it is also a source of antimicrobial metabolites such as lactic acid and bacteriocins (6). In Iran, most of the studies have focused on the identification of Lactobacillus strains in bovine dairy products and their antibacterial properties, while buffalo dairy products have received very little attention so far. Therefore, the aim of this study was to isolate and identify different strains of Lactobacillus spp. from buffalo yogurt and milk and determine their antibacterial activity against gramnegative standard strains and uropathogens isolated from patients with urinary tract infections (UTIs).
April to June 2016 to determine the inhibitory effect of Lactobacillus strains isolated from buffalo yogurt and milk collected in Aliabad-e Katul city, Golestan province, north-east of Iran. The raw milk and yogurt samples were collected in sterile screw-capped falcon tubes with ice packs and transported to the laboratory of Islamic Azad University, Gorgan Branch, Iran. Afterwards, 2 g of yogurt and 1 mL of milk were aseptically inoculated into MRS broth and incubated anaerobically at 37°C for 48 hours. Then, the enriched samples in MRS broth were cultured on MRS agar (Conda Pronadisa, Spain). The plates were incubated anaerobically at 37°C for 48 hours. Then, the suspicious colonies were tested using Gram staining and catalase reaction. Gram-positive and catalase-negative bacilli were purified by streaking on MRS agar and identified on the basis of biochemical tests including fermentation of galactose (Merck, Germany), maltose (Conda Pronadisa, Spain), fructose (Merck, Germany), sucrose (Merck, Germany), raffinose (Merck, Germany), sorbitol (Merck, Germany), lactose (Merck, Germany), rhamnose (Merck, Germany(, and mannitol (Merck, Germany), as well as the ability to grow at 10°C and 45°C and in the presence of 6.5% NaCl according to Bergey's Manual of Systematic Bacteriology (6,8). The isolates were stocked in MRS broth containing 20% glycerol (Oxoid, Canada) at -20°C until further used.

Antibacterial Activity Test
The antibacterial activity of Lactobacilli strains against standard strains and gram-negative uropathogens was investigated by well diffusion method (9). In this method, the isolated colonies were inoculated in MRS broth and incubated for 48 hours at 37°C. Subsequently, the MRS broth was centrifuged at 10 000×g for 15 minutes to obtain cell-free supernatants (CFSs). The CFS was sterilized by passage through 0.45 μm Millipore filters. The standard strains used in this study included Escherichia coli ATCC 11303, Klebsiella pneumoniae ATCC 13883, and Pseudomonas aeruginosa ATCC 15442 (provided by the Faculty of Veterinary Medicine, Tehran University). In addition to standard strains, three isolates of E. coli (E1, E2, and E3), two isolates of P. aeruginosa (P1 and P2), and two isolates of K. pneumoniae (K1 and K2) which were previously isolated from patients with UTIs were included in this study (10). Then, 50 μL of the CFS was poured in each 5-mm-deep wells punched into the nutrient agar plates previously seeded with 10 6 CFU/ mL of the test bacteria pre-cultured in LB broth. The plates were then incubated at 37°C for 24 hours. Based on the diameter (mm) of the clear inhibitory zone formed around the wells, antibacterial activity was estimated (3,6). Inhibition zone <15 mm and ≥15 mm was considered moderate and relatively strong activity, respectively (11). Antimicrobial tests were done in triplicate and the mean values were recorded.

Results
A total of 19 Lactobacillus strains were isolated from 10 samples (5 samples of milk and 5 samples of yogurt), forming round creamy white colonies on MRS agar plate. Morphological and biochemical characteristics were employed to identify the isolates (Table 1). Based on the results of sugar fermentation and different growth conditions, Lactobacilli were identified as shown in Table  2. Among the isolates, L. plantarum and L. casei were the most prevalent Lactobacilli.
According to the results of antibacterial activity test, L. plantarum P1-Y (16.8 mm) and L. helveticus H1-Y (16.2 mm) exhibited relatively strong inhibitory effects on E. coli E1 and did not show any inhibitory effect on the growth of K. pneumoniae isolates. Moreover, L. casei C2-Y showed a relatively strong inhibitory effect (15.8 mm) on E. coli E1 but weak activity on K. pneumoniae and P. aeruginosa isolates. L. acidophilus A1-Y had a relatively strong inhibitory effect on the growth of E. coli E2 (15.1 mm) but no inhibitory activity against K. pneumoniae isolates. Among the isolates, only L. casei isolates demonstrated inhibitory activity against K. pneumoniae isolates. However, all of the isolates had the highest and lowest inhibitory effects on the E. coli and K. pneumoniae isolates, respectively. The mean diameters of growth inhibition zones (mm) were given in Tables 3-5.

Discussion
The study was designed for identification of Lactobacillus spp. from buffalo milk and yogurt samples and assessment of their antibacterial activity against gramnegative standard strains and uropathogens. Based on the morphological characteristics, 19 isolates from the samples were identified as Lactobacillus spp. The isolated bacteria were non-spore forming gram-positive rodshaped facultative anaerobes which were indicated to be In this study, among the isolates, L. plantarum (42.1%) had the highest frequency followed by L. casei (26.3%). The CFSs of the isolates showed good and weak inhibitory effects on the E. coli and P. aeruginosa isolates, respectively. They did not have any inhibitory effects on K. pneumoniae isolates except for CFSs from L. casei isolates.
Similar studies have been undertaken in Iran and other countries. In agreement with the present study, in a study conducted by Dorri et al, the most commonly identified strains of Lactobacilli were L. casei, L. acidophilus, and L. plantarum (12). Moreover, the findings of Farahbakhsh et al and Naeemi et al are also consistent with the findings of the present study in which among Lactobacilli isolates, L. plantarum as a predominant isolate had the highest antibacterial activity against the test organisms (13,14). In another study, L. casei had the highest frequency in traditional dairy products followed by L. acidophilus in Gorgan, north-east of Iran (15). Forhad et al also isolated a total of four isolates including L. fermentum, L. casei, L. Acidophilus, and Bifidobacterium longum from buffalo milk in Bangladesh (6). In another study, Eid et al isolated L. fermentum, L. Acidophilus, and L. pentosus from buffalo milk, among which L. pentosus had the highest antibacterial activity against the indicator organisms (3). In a study, Chowdhury et al isolated four L. plantarum strains that inhibited the growth of test pathogens to some extent but the highest and lowest inhibition zones were observed against Bacillus cereus and Staphylococcus aureus, respectively (5).
Hossein Alipour et al isolated L. salivarius from buffalo milk with the most and least inhibitory effect on S. aureus and S. typhimurium, respectively (7). The antagonistic activity of LAB isolated from traditional dairy products including L. plantarum and L. fermentum was studied against E. coli O157:H7 by Rahimpour Hesari et al. Among the isolates, the antagonistic activity of L. plantarum was greater than that of L. fermentum (16). Consistent with the findings of the present study, a study from Nepal showed the antibacterial effect of Lactobacilli isolated from dairy products on E. coli, Salmonella paratyphi, Salmonella typhi, Pseudomonas spp., S. aureus, Proteus spp., Acinetobacter     (18). In a study, L. plantarum and Lactococcus piscium from goat milk were the most common probiotic isolates and L. lactis showed the highest inhibitory effects on drug-resistant Acinetobacter baumannii (19). A study from Pakistan showed the antibacterial effect of LAB on multidrug-resistant uropathogens including Candida albicans, P. aeruginosa, K. pneumoniae, Enterococcus faecalis, and E. coli. The growth inhibition zone was over 10 mm against all the uropathogenic test organisms, while L. fermentum and L. plantarum strains demonstrated great inhibitory activities against E. coli and E. faecalis (20). In a study, L. casei and L. lactis isolates from yogurt showed better inhibitory effects on pathogenic bacteria and the highest and lowest inhibitory effect was observed on Yersinia enterocolitica and B. cereus (21). In a study by Kazemi Darsnaki et al, six LAB strains were isolated from yogurt and probiotic pills and L. acidophilus had the highest antibacterial activity against B. cereus (22). In a study from Egypt, the highest antagonistic activity was observed for Lactobacillus paracasei and L. helveticus against the tested pathogens followed by L. fermentum, while Bifidobacterium longum and L. lactis subsp. lactis showed weak or no antibacterial activity against the tested strains (23).
Considering the results of all studies including the present study, LAB from dairy products presented the inhibitory activity against gram-positive and gramnegative bacteria. Among the LAB, lactobacilli with satisfactory antagonistic activity against the pathogens have been shown to be one of the best alternatives to antibiotic therapy.

Conclusions
In this study, Lactobacilli had the highest antibacterial activity against the E. coli isolates as a common cause of UTIs; therefore, further studies are recommended to elucidate their potential for being used as an alternative to antibiotic therapy.