maryam royan

Identifying the strains carrying acquired tetracycline resistance genes is one of the important points in evaluating the safety of probiotic strains. The aim of the present study is to comprehensively evaluate tetracycline resistance in lactobacillus spp. isolated from the digestive system of Iranian backyard chickens.

In the present study, the phenotypic sensitivity patterns of 36 lactobacillus isolates belonging to four species L. reuteri, L. salivarius, L. crispatus and L. johnsonii were evaluated by measuring the Minimum Inhibitory Concentration (MIC) to tetracycline. After that, four tetracycline resistance genes tet(L), tet(M), tet(W) and tet(O) were detected in isolates with phenotypic resistance to this antibiotic based on the polymerase chain reaction method.

As a result, four isolates of L. reuteri, three isolates of L.salivarius, two isolates of L. crispatus and four isolates of L. johnsonii among the studied lactobacillus spp. showed phenotypic resistance to tetracycline. After the detection of tetracycline resistance genes in isolates with phenotypic resistance, the presence of tet(W) gene was confirmed in all investigated isolates. The simultaneous presence of tet(M), tet(L) and tet(W) genes was observed in three studied L. salivarius isolates. It was also found that the phenotypic resistance observed in three isolates of L. johnsoniiABRIG7, L. johnsoniiABRIG14 and L. johnsoniiABRIG24 is caused by the simultaneous presence of tet(O) and tet(W) genes.

This study showed that tetracycline resistance among Lactobacilli isolated from digestive system of Iranian backyard Chickens is due to the presence of single or multiple resistance genes.. We also found that tet(W) is the most widespread tetracycline resistance gene among investigated Lactobacillus isolates. Therefore, local poultry breeders should refrain from arbitrarily using tetracycline, and when the digestive system of domestic chickens is the source for probiotic microorganism selection, the evaluation of resistance to tetracycline from the phenotypic and molecular point of view should be taken into consideration in order to select safe strains.

Introduction: 

Feed additives are commonly used in poultry feed to enhance performance, promote health, and increase nutrient efficiency. The use of antibiotics as growth promoters in poultry feed has been prevalent for years. However, due to concerns regarding the accumulation of antibiotic residues in poultry products and the development of antibiotic-resistant bacterial strains, antibiotics are no longer considered desirable additives in poultry feed. As a non-therapeutic alternative to antibiotics, probiotics have been introduced as suitable candidates to promote growth. Probiotics have beneficial effects on poultry digestive enzymes, improve intestinal absorption, and neutralize toxins produced by harmful microorganisms, ultimately improving the immune system and economic performance. Among the most popular probiotic bacteria are Lactobacilli, as they are generally classified as safe bacteria.

Materials and methods:

 Lactobacillus reuteri (L. reuteri ABRIG25 (MF686485)) and salivarius (L. salivarius NABRII59 (MH595987) isolated from the digestive tract of Guilan’s native chicks and Mazandaran’s duck  respectively, were prepared up to 1.36×109 CFU using MRS medium at 37 ° C, under anaerobic conditions. 300 one-day-old male Arbor-Acres chicks were distributed in a completely randomized design, with 5 treatments, 4 replications (15 chicks per replicate). Experimental treatments were: 1- Basic diet as control group (Cont), 2- Basic diet + 100 g / ton Avilamycin as antibiotic group (Anti), 3- Basic diet + 200 g / ton commercial probiotic (Lacto-feed®) (Plac), 4- Basic diet + 1 g / Kg of L. salivarius NABRII59 (MH595987) bacterial powder (Pls1), and 5- basic diet + 1 g / kg of L. reuteri ABRIG25 (MF686485) bacterial powder (Plr1). Daily feed intake, weight gain, and feed conversion ratio of broilers were determined and recorded in starter, grower, and finisher  periods. On day 42, two chicks were slaughtered from each replicate and the weight of internal organs and carcass cuts were recorded as a percentage of carcass weight. Chicken antibody reaction were determined using SRBS suspension. On days 22 and 35 of the rearing period, two chicks were randomly selected from each cage and 0.1 cc of  SRBC solution was injected into to the wing’s vein, intravenously. Humoral immunity test was applied on days 29 and 42, using 1 cc of blood taken from the wing vein of chickens. The hemagglutination reaction was recorded based on the last two dilutions as SRBC’s antibody using the logarithm and the antibody titer against Newcastle was determined by hem agglutination inhibition (HI) test. On day 42, blood samples were taken randomly from 2 birds per replication to evaluate blood-serum parameters including glucose, cholesterol, triglyceride, HDL, LDL and VLDL. All data were analyzed using SAS software v.9.1 (2012) in GLM procedure using a completely randomized design, and comparison of statistical means was performed, using Duncan's method at the level of 0.05.

The present study investigated the effect of native probiotic isolates (Plr1, Pls1) compared to commercial probiotics (Plac) and antibiotics (Anti) on the growth performance of chickens. The results showed no significant differences in daily feed intake, weight gain, and feed conversion ratio between experimental treatments. However, two native probiotic isolates (Plr1, Pls1), the commercial probiotic (Plac), and the antibiotic (Anti) resulted in a significant reduction (P <0.05) in proventriculus weight, while there were no significant differences in the relative weight of other organs. Both the commercial probiotic (Plac) and the native Lactobacillus isolates (Plr1 and Pls1) significantly improved total immunoglobulin and immunoglobulin G (IgG) levels after both the first and second injections (P <0.05). Lactobacillus salivarius (Pls1) isolate also significantly improved immunoglobulin M (IgM) levels after the second injection. However, there were no significant differences between treatments and the control group in terms of antibody titer against Newcastle disease vaccine, blood glucose, cholesterol, triglyceride, VLDL, and LDL levels. Probiotics can affect gut microbiota by competing for nutrients and attachment sites on the intestinal epithelial cells. Additionally, they may improve blood parameters and stimulate immune system cells to produce cytokines, which play an important role in inducing and regulating immune responses in poultry. Probiotics support lactic acid-producing bacteria and stabilize the gut microflora, which has beneficial effects on feed conversion ratio by stimulating the production of digestive enzymes. Furthermore, probiotics may reduce cholesterol synthesis by fermenting indigestible carbohydrates, leading to the production of short-chain fatty acids and ultimately lowering cholesterol levels in the host bird. Discrepancies in results reported by different researchers may partly be due to differences in chick’s age and breed, level of stress, diet composition, consumption period and duration, type of commercial probiotics, dose or amount of probiotic intake, management skills, and environmental conditions in different experiments.

The general conclusion is that the probiotic isolates used in this study are competitive with the commercial type of probiotics as well as the antibiotic used and are promising as probiotic candidate with beneficial effects on broiler’s performance, blood biochemical parameters and immune system

In the recent years, many countries have banned or restricted the usage of antibiotics as growth-promoting, due to their undesirable effects on human and animal health. Using probiotics as an alternative to growth-promoting antibiotics play an important role in improving performance, health and increasing the quality of poultry products. Lactobacilli are the most popular probiotic bacteria that have beneficial effects on host’s performance and health. In this study, the effect of two native strains of Lactobacillus ruteri isolated from the gastrointestinal tract of native chickens in north-western and south-western of Iran were investigated on performance, carcass characteristics, blood parameters and immune response of broilers.

In this experiment, 300 Arbor-Acres® Plus+ male broiler chickens were divided in to 5 treatments, 4 replications (15 chickens per replication), in a completely randomized design. The experimental treatments include: 1- Basic diet as a control treatment, 2- Basic diet + Avilamycin antibiotic, 3- Basic diet + commercial probiotic (Bio-Poul®), 4- Basic diet + Lactobacillus reuteri isolate MG547731 (Plr2), and 5- Basic diet + Lactobacillus reuteri isolate MG547727 (Plr3).

Under the condition of this experiment, commercial probiotics, antibiotic and either Lactobacillus reuteri (Plr2) isolates, significantly improved daily weight gain in final period (p<0.05). Native isolates probiotics and commercial probiotics significantly (p<0.05) reduced the relative weight of gizzard and proventriculus, though no significant (p<0.05) differences were recorded for other carcass components in broiler chickens. After the first injection of SRBC, treatments include antibiotic and Lactobacillus reuteri (Plr2) isolate caused a significant increase of IgM, and after the second injection, Lactobacillus reuteri (Plr2) and commercial probiotic treatments, showed a significant (p<0.05) increase in immunoglobulin G and Total immunoglobulin. The antibody titer against Newcastle vaccine did not show a significant difference between treatments and the control group. There was no significant difference between serum glucose, cholesterol, triglyceride, VLDL and LDL between treatments and the control group. No significant difference was obtained in serum HDL level in probiotic treatments, while antibiotic treatment appeared with a significant decrease in HDL.

Addition of native Lactobacillus reuteri isolates to poultry diets can have beneficial effects similar to commercial probiotics, on performance, immune response and blood parameters of broilers.

This study aimed to investigate the phenotypic and genotypic patterns of antibiotic resistance in two potential probiotic lactic acid bacteria, Lactococcus lactis subsp. cremoris (NABRII64 and NABRII66) isolated from the rainbow trout intestine. The phenotypic susceptibility pattern of the strains was studied based on the Minimum Inhibitory Concentration (MIC) of eight most commonly used antibiotics in medicine and veterinary including ampicillin, kanamycin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. After comparing the MICs with standard values recommended by European Food Safety Authority (EFSA), the nature of phenotypic resistance observed in bacterial strains was investigated by polymerase chain reaction (PCR) through plasmid DNA extraction. The results of the phenotypic evaluation indicated the tetracycline resistance in both bacterial strains (MIC<4mg/L). Genotyping of antibiotic resistance genes including tet (S), tet (L), tet (M), tet (O), tet (W) and tet (K) indicated the presence of tet (S) and tet (M) resistance genes in plasmid DNA of both bacterial strains. These results exhibited the acquired resistance and the presence of two tetracycline resistance genes in the plasmid DNA of two bacterial strains, NABRII64 and NABRII66. However, further studies are required to understand the nature of the acquired resistance mechanism in the future.

This study was conducted to survey the antimicrobial activity of Lactococcus lactis subsp. cremoris NABRII66 lactic acid bacterial strain against common bacterial pathogens in aquaculture and human populations by in vitro experiments. L. lactis subsp. cremoris NABRII66 has been isolated from rainbow trout intestine. Two methods including double layer agar and microtiter plate assay were used to evaluate the inhibition activity of selected bacterial strain against two bacterial pathogens L. garvieae and Aeromonas salmonicida species affecting salmonids and other fish speciesand two other bacterial pathogens including Escherichia coli and Staphylococcus aureus, as two most common source of infection in human population. The results of double layer agar method showed the antimicrobial activity of L. lactis subsp. cremoris NABRII66 bacterial strain against all the tested pathogens and the highest significant growth inhibition zone was observed against A. salmonicida and S. aureus bacterial pathogens (p<0/05). The results of the microtiter plate assay method showed that cell-free supernatant of L. lactis subsp. cremoris NABRII66 bacterial strain could significantly inhibit the growth of A. salmonicida (p<0/05). This study showed that Lactococcus lactis subsp. cremoris NABRII66 bacterial strain isolated from rainbow trout intestine had antimicrobial activity against four aquaculture and human's bacterial pathogens by producing some extracellular inhibitory compounds, which should be evaluated in future studies.

The purpose of this study was to investigate the effects of the use of three lactobacillus strains (L. reuteri ABRIG17 (MF686477), L. reuteri ABRIG23 (MF686483), L. reuteri ABRIG3 (MF686463)), Isolated from duodenum and Jejunum sections of the native Guilan chickens and one isolate of L. salivarius NABRII58 (MH595986) isolated from the digestive system of the native ducks of Mazandaran, on serum lipids and immune parameters of broiler chickens. The strains were isolated from 383 gram-positive and catalase negative lactic acid bacteria in a screening procedure for the detection of bacteria with probiotic potential. In the experiment, 500 male Ross 308 male broiler chicks were used in a completely randomized design with 5 treatments, 5 replicates and 20 birds per replicate. The treatments consisted of: 1-The basal diet as control group (treatment C), 2- The basal diet + 1 g / kg of mixed powder containing MF686463 (L. Reuteri ABRIG3 (LR1), 3- The base diet + 1 g /kg of L. Reuteri ABRIG23 (MF686483) (LR2 treatment), 4- The basal diet + 1 g / kg of L. Reuteri (MF686477) ABRIG17 (LR3 treatment) and 5- The basal diet + 1 g / kg of L. Salivarius (MH595986) NABRII58 (Treatment LS). The use of bacterial strains in LR1 and LS treatments resulted in improved weight gain at the end of the experiment (P <0.05). The bacterial strains in LR1 treatment improved daily weight gain in whole experimental period (P <0.05). All three strains of Lactobacillus used, namely, LR1, LR2 and LR3 treatments, caused a significant decrease in abdominal fat pad (P <0.05). In LR3 treatment, the increase in carcass weight was observed (P <0.05). Measuring serum immunoglobulins in broiler chicks after two stages of sheep red blood cell injection showed that LR3 had the highest total immunoglobulin level after the second injection (P <0.05), and IgG levels in the LR1 treatment increased after the first injection compared to the control group (P <0.05). After the second injection, the IgG of all the four experimental groups were higher than control group (P <0.05), but IgM showed no significant difference between experimental treatments and control group. Total serum cholesterol concentration of LR2 treatment was significantly (P <0.05) lower than other treatments. There was no significant difference in serum triglyceride level between control and the LR1, LR2 and LS treatments, and only LR3 triglyceride level was higher than others (P <0.05). The level of HDL or good cholesterol in LS treatment was higher than the control group (P <0.05). Serum LDL level did not show any significant difference between the experimental and control groups. The present study revealed that probiotic bacteria can be isolated from native poultry microbial populations and isolated bacteria can improve the production and immunity of broiler chicks. It is worthy of note that the positive effect of Lactobacillus salivarius strain isolated from the native duck gastrointestinal tract on the weight gain and serum immunoglobulins in broiler chickens suggested that the gastrointestinal bacteria of a species of poultry could be used as a probiotic in other types of poultry.

In this study, the probiotic potential of lactic acid bacteria isolated from rainbow trout intestine with inhibitory activity against Streptococcus iniae was evaluated by in vitro experiments. For this purpose, gram-positive and catalase negative lactic acid bacteria were screened based on probiotic characteristics such as acid and bile salt tolerance, antimicrobial activities against S. iniae and bio-safety assay including haemolytic activity and antibiotic susceptibility to eight major commercial antibiotics. At last, genotypic identification of candidate lactic acid bacteria isolates carried out by 16S rRNA gene sequence analysis. Based on results, among 148 bacterial isolates, five lactic acid bacteria strains belonged to the Lactococcus lactis subsp. were tolerant to pH=3-7.5, fish bile extract (10%) and had antimicrobial activity against S. iniae. Also, the bio-safety assay results revealed that none of the bacterial strains showed beta-haemolytic activity and all candidate bacterial strains exhibited phenotypic resistance to tetracycline. The multiple antibiotic resistant (tetracycline and chloramphenicol) was observed only in L. lactis NABRII61. This study showed that the five lactic acid bacteria isolated from rainbow trout intestinal microflora could be considered as probiotic strains in aquaculture. However, further experiments are required to confirm the nature of antibiotic resistance.

Liver fatty acid-binding protein (L-FABP) is the main cytosolic binding site for long chain fatty acids in hepatocytes. FABPs enhance the uptake of fatty acids into the cell by increasing their concentration due to decreasing concentration of unbound fatty acids inside the cell. The aim of this study was to evaluate the effects of dietary unsaturated to saturated fatty acid ratio and feed restriction on L-FABP mRNA expression. A total of 720, 10-day old male Ross 308 broiler chicks were fed diets with unsaturated to saturated fatty acid ratio (U/S) of 2, 3.5, 5, or 6.5 as ad libitum or skip-a-day feeding schedule (during 18–28 days of age). Relative expression of L-FABP mRNA in hepatocytes of broilers was determined using quantitative reverse transcription real-time polymerase chain reaction. Our results show that feed restriction induced the expression of L-FABP gene in the liver of broilers. Moreover, L-FABP gene expression was increased by dietary U/S ratio of 6.5. There was no interaction between dietary U/S and feed restriction on the L-FABP gene expression. Results indicate that birds have a mechanism for regulation of fatty acid transfer under different nutritional conditions.