Antibiotic Resistance Of Lactic Acid Bacteria Research Articles

ANTIBIOTIC RESISTANCE OF LACTIC ACID BACTERY LEAVEN «VIVO PROBIOYOGURT»

Lactic acid bacteria play a key role in human microecology and biotechnology – form organoleptic characteristics of products, increase the nutritional, including biological value of functional foods. Natural resistance to antibiotics is one of the important factors that determine the probiotic properties of lacto- and bifidobacteria. Aim. Study of the antibiotic resistance of functionally active probiotic cultures of "VIVO probioyogurt" leaven to determine the possibility of using a fermented milk product, which is prepared on its basis, during antibiotic therapy to maintain and restore normal intestinal microflora. Methods. Pure cultures of lactic acid bacteria (LAB) were selected for the study: (Lactobacillus delbrueckii ssp., L. acidophilus, L.casei, L. rhamnosus, L.paracasei, Streptococcus thermophilus, Bifidobacterium lactis (2 strains), B. infantis), which are part of leaven "VIVO probioyogurt" the quality of which is confirmed by certificates of the International Organization for Standardization ISO 9001: 2008, as well as ISO 22000: 2005. The method of the experiment consisted of the following stages: preparation of nutrient media ("Lactobacagar", "Bifidoagar", glucose-peptone medium), working solutions of antibiotics; working suspension of LAB; suspensions of cultures (lacto- and bifidobacteria), cultivation LAB on elective nutrient media with the addition of antibiotics and evaluation of research results. Determination of antibiotic resistance of LAB was performed by the method of double dilutions. Results. The use of this technique enabled to establish the minimum inhibitory concentration (MIC) of antibiotics of different groups relative to the LAB. The results of the research were processed using a licensed computer program Microsoft Excel. Conclusions. Evaluation of the results of studies to determine the MIC of antibiotics – benzylpenicillin, azithromycin, lincomycin, gentamicin sulfate, ceftriaxone, norfloxacin, amoxil, streptomycin, tetracycline, erythromycin in relation to IBD; fermented milk product, which was prepared on the basis of this starter culture, it was advisable to use during antibiotic therapy to restore and maintain normal intestinal microflora.

Antibiotic resistance in vancomycin‐resistant lactic acid bacteria (VRLAB) isolated from foods of animal origin

In this study, antibiotic resistance in 47 vancomycin-resistant lactic acid bacteria (VRLAB), isolated from 340 foods of animal origin obtained in Turkey, were investigated. VRLAB isolates were molecularly identified as Enterococcus gallinarum (44.68%), Pediococcus pentosaceus (25.53%), P. acidilactici (12.77%), E. casseliflavus (6.38%), E. casseliflavus/E. gallinarum (4.26%), Weissella cibaria (2.13%), W. confusa (2.13%), and W. minor (2.13%). The disk diffusion test results showed that 57.45%, 53.19%, 44.68%, and 44.68% of VRLAB were resistant to ciprofloxacin, norfloxacin, teicoplanin, and vancomycin, respectively. It was found that all the Pediococcus and Weissella isolates and 53.85% of the Enterococcus isolates had multiple antibiotic resistance. Various transferable antibiotic resistance genes were found in the VRLAB using polymerase chain reaction (PCR). The most common antibiotic resistance genes in the VRLAB isolates were vanC1 (46.81%), tetM (23.4%), and ermB (19.15%). Practical applications Lactic acid bacteria (LAB) play a key role in food fermentations. However, antibiotic resistance in LAB is a concern because these bacteria can, potentially, be a reservoir that facilitates the spread of antibiotic resistance genes to pathogenic bacteria. Vancomycin is a glycopeptide antibiotic that is used as a last resort in the cure of multidrug-resistant enterococci. In this study, vancomycin-resistant Enterococcus, Pediococcus, and Weissella strains were isolated from foods of animal origin. Several transferable antibiotic resistance genes were detected in these isolates. The results of this study indicated that VRLAB strains isolated from foods of animal origin may be the reservoirs for antibiotic resistance genes. Our findings suggest that antibiotic resistance profiles and resistance genes, in addition to industrial characteristics, should be investigated when selecting LAB from foods of animal origin to be used as starter cultures.

Effect of probiotics during vonoprazan-containing triple therapy on gut microbiota in Helicobacter pylori infection: A randomized controlled trial.

Probiotics are beneficial to patients with Helicobacter pylori infections by modulating the gut microbiota. Biofermin-R (BFR) is a multiple antibiotic-resistant lactic acid bacteria preparation of Enterococcus faecium 129 BIO 3B-R and is effective in normalizing the gut microbiota when used in combination with antibiotics. This study aimed to determine the effect of BFR in combination with vonoprazan (VPZ)-based therapy on gut microbiota. Patients with positive urinary anti-H pylori antibody test (primary test) and fecal H pylori antigen test (secondary test) were examined. Patients in group 1 (BFR- ) received VPZ (20mg twice daily), amoxicillin (750mg twice daily), and clarithromycin (400mg twice daily) for 7days. Patients in group 2 (BFR+ ) received BFR (3 tablets/day) for 7days, in addition to the aforementioned treatments. Following treatment, the relative abundance, α-diversity, and β-diversity of gut microbiota were assessed. Supplementation with BFR prevented the decrease in a-diversity after eradication therapy (Day 7). β-diversity was similar between groups. The incidence rate of diarrhea was non-significantly higher in the BFR- than in the BFR+ group (73.1% vs 56.5%; P=.361). Stool consistency was comparable in the BFR+ group on Days 7 and 1 (3.86±0.95 vs 3.86±1.46; P=.415). Biofermin-R combined with VPZ-based therapy resulted in higher microbial α-strain diversity and suppressed stool softening during H pylori eradication therapy.

Identification and antibioticresistance of lactic acid bacteria isolated from the gastrointestinal tract of a rabbit

The probiotic preparations, used in rabbit breeding, contain bacteria, isolated from various econiches. Therefore, these drugs are universal and recommended for different types of animals. One of the ways to increase the effectiveness of probiotics, used in breeding rabbits is the selection of strains of biologically active representatives of the obligate microflora of the gastrointestinal tract of these animals.Purpose. Identify lactic acid bacteria, isolated from the gastrointestinal tract of a rabbit, promising for the creation of probiotic preparations. Investigate antibiotic resistance of the most biologically active isolates.Methods. The identification of lactic acid bacteria to the genus Lactobacillus was carried out according to generally accepted methods. Molecular genetic identification was performed using a polymerase chain reaction. For the most promising strains, the sensitivity to antibiotics was studied using the disco-diffusion method and the minimum inhibitory concentration of antibiotics, which completely retarded the growth of bacteria.Results. 250 bacterial isolates, isolated from the gastrointestinal tract of rabbits were identified to the Lactobacillus genus, 10 most active of which were selected for the further work. It has been established, that 40% of selected isolates of lactic acid bacteria are capable of fermenting arabinose, dulcite; 50% beckoning; 70% sorbitol; 80% - mannose, fructose; 90% - lactose; 100% - galactose, maltose, sucrase, glucose; 20% - raffinose. Molecular genetic studies have established that the isolate Lactobacillus sp. 13/2 does not have genes typical for L. acidophilus and L. helveticus species. It has been found, that the lowest value of the minimum inhibitory concentration of antibiotics of the studied isolates was revealed to prolonged-action penicillins, which act bactericidally, disrupting the synthesis of the bacterial cell wall. The highest is for ampicillin, which is active against gram-positive bacteria that are affected by benzylpenicillin. The results indicate the absence of acquired antibiotic resistance. However, isolate L-13/2 showed resistance to oxacillin, kanamycin, streptomycin, nalidixic acid.Findings. According to a complex of morphological, cultural, and physiological and biochemical properties, the ten most active isolates of lactic acid bacteria, isolated from the gastrointestinal tract of rabbits, of 250 ones are previously assigned to phylogenetic groups: L. acidophilus, L. lactis, L. casei, L. plantarum. For use the selected isolate Lactobacillus sp. 13/2 as a basis for the probiotic preparation, it is necessary to use additional identification methods to determine the species.It has been established, that the studied isolates as a whole did not exhibit antibiotic resistance. Perspective isolate Lactobacillus sp. 13/2 was resistant to some antibiotics. Therefore, there is a need to study in greater detail the antibiotic resistance of the isolate under study in order to exclude the possibility of horizontal transfer of resistance genes

Sublichenin, a new subtilin-like lantibiotics of probiotic bacterium Bacillus licheniformis MCC 2512T with antibacterial activity

Probiotic bacteria with antibacterial activity is of desirable trait since they can check the growth of pathogenic bacteria besides exhibiting health benefits to host. Aim of this study was to characterize Bacillus licheniformis MCC 2512T (MCC 2512), a potential probiotic culture for its ability to produce subtilin-like antibiotics. The anti-microbial compound produced by MCC 2512 was identified and characterized using subtilin-specific cell reporter, Bacillus subtilis 168:BS2 (BS2). Induction of β-gal by the test culture suggested the ability of B. licheniformis to produce subtilin-like lantibiotic. Subsequently, DNA sequencing of major lanS-operon was carried out, wherein sequencing results showed that lan cluster of MCC 2512 resembles entianin (etn) type. Upon lan-S disruption, the bacterial culture lost antimicrobial activity as well as ability to induce β-gal with BS2 reporter. High amount of succinylated form of antibiotics produced by wild type and un-succinylated form by engineered strain of B. subtilis 15029p clearly indicates that MCC 2512 is indeed an inter-specific subtilin-like (named as sublichenin) lantibiotic producer. Partially concentrated sublichenin preparation exhibited strong antibacterial activity against food-borne pathogens and antibiotic resistant (AR) lactic acid bacteria (LAB) with a minimum inhibitory concentration in the range of 6–10 and 0.5–1.5 μg/ml, respectively. Production of lantibiotic, sublichenin by a probiotic bacterium of B. licheniformis MCC 2512T and its antibacterial activity against food associated AR LAB is a new information reported in this study.

Antibiotic resistance of lactis acid bacteria and the risk of its transmission with fermented dairy products

Bacterial resistance to antimicrobials is a global health problem that affects not only on humane and veterinary medicine, but also on food products. The food chain can be by transmission of antibiotic resistance from bacterial populations to animals and humans. Literary data on the current state of the problem of antibiotic resistance of lactic acid bacteria (LAB) in Ukraine and in the world are given in the review. Possible ways of transferring resistance to antibiotics through fermented dairy products are shown. The main aspects of the danger of transmission of antibiotic resistance genes through the LAB and fermented dairy products are revealed. The main modern approaches to the definition of antibiotic resistance of microorganisms with the use of classical and modern research methods are described. The article provides the main sources of information on the safety of use of LAB as starter cultures and probiotics for the production of fermented dairy products. The hypothesis of the resistance gene's reservoir suggests that LAB can be a reservoir of sustainability genes, and the subsequent transfer of such genes to pathogenic and opportunistic microorganisms. The presence of antibiotic resistance genes transposed horizontally is inadmissible for lactobacilli, which are used as commercial bacterial agents for the production of fermented dairy products. According to the literature data, the absence of acquired antimicrobial resistance has become an important criterion for assessing the safety of lactobacilli, which are used as starting cultures for the production of fermented dairy products or probiotics. It has been established that it is obligatory to study the antibiotic resistance gene in addition to clinical and laboratory methods of studying the antibiotic resistance of LAB. To minimize the formation of antibiotic-resistant bacteria in food products of plant and animal origin it is possible by careful monitoring of residues of antibiotics in raw materials and finished products. This will prevent the entry of antibiotic resistant strains into the natural cycle.

The Association of Biofilm Formation with Antibiotic Resistance in Lactic Acid Bacteria from Fermented Foods

AbstractFifty‐three lactic acid bacteria (LAB) strains from five species of bacteria were isolated and identified from yogurt and Chinese pickled vegetables. Susceptibility of all the isolates to five antibiotics (ampicillin, erythromycin, tetracycline, vancomycin, streptomycin) was tested with disc diffusion methods. All LAB were resistant to streptomycin. Thirty‐seven strains were resistant to vancomycin, seven strains were resistant to tetracycline and erythromycin, and six strains were resistant to ampicillin. The corresponding genetic determinants were characterized by polymerase chain reaction. No antibiotic resistant gene was detected among all resistant strains. Biofilm‐forming ability was monitored by a modified crystal violet staining method. Among the 53 strains, 32 strains showed different levels of biofilm‐forming ability, while 21 strains did not form biofilms. The minimal inhibitory concentration of antibiotics (MIC) and the biofilm inhibitory concentration (BIC) were determined in the 32 strains which can form biofilms. There are significance correlations between biofilm formation and BIC/MIC for all antibiotics (P < 0.05) except vancomycin. Biofilms exhibited increased antimicrobial resistance compared with planktonic isolates, but the extent of enhanced antibiotic resistance in biofilms is different with different antibiotics.Practical ApplicationRecently, there are increasing researches focusing on the safety of LAB, due to their antibiotic resistance. However, effects of antibiotic resistance in LAB were different in different application fields. So it is necessary to understand the mechanism of resistance all sidedly. In this study, we found those LAB isolates, which exhibiting antibiotics resistance in the susceptive tests, carried no transferable antibiotic resistance determinant, while there was a significant positive correlation between four antibiotics resistance and biofilm‐forming ability. Data derived from this study can be used as a basis for assessing the safety of LAB applied in fermented food.

Oral administration of Bifidobacterium longum in a gastro-resistant seamless capsule decreases serum phosphate levels in patients receiving haemodialysis.

Oral administration of Bifidobacterium longum in a gastro-resistant seamless capsule decreases serum phosphate levels in patients receiving haemodialysis.

Susceptibility of Spore-forming Butyric Acid Bacteria to Antimicrobial Agents

Antimicrobial agents occasionally cause certain adverse effects, such as diarrhea and loose stool, by altering the composition of the intestinal flora. Antibiotic-resistant lactic acid bacteria are used to prevent these adverse effects. Although these bacteria are not resistant to several recently introduced antimicrobial agents, bacterial preparations are still sometimes prescribed concomitantly with these antimicrobial agents. In this study, we investigated whether the administration of the spore-forming butyric acid bacteria Clostridium butyricum improves the adverse clinical effects by preventing diarrhea. Inhibition of C. butyricum growth was observed with 17 of the 20 antimicrobial agents used. However, dilution of 11 of these 17 agents resulted in the regrowth of C. butyricum. These results suggest that C. butyricum may survive exposure to several antibiotic agents by forming spores. Further, a decrease in the antimicrobial agent concentration in the gastrointestinal tract permits the vegetative growth of C. butyricum, which functions as a probiotic.

Assessment of antibiotic resistance of lactic acid bacteria in Chinese fermented foods

Lactic acid bacteria isolated from 11 Chinese fermented foods were investigated for their resistance incidences of 7 clinically important antibiotics, including chloramphenicol, kanamycin, tetracycline, ciprofloxacin, ampicillin, clindamicin and erythromycin. It was found that antibiotic resistant lactic acid bacteria are widespread among traditional Chinese fermented foods and their resistance incidences depended on the raw material and manufacturing area of the foods. Resistance incidences of fermented sausages were much higher than that of fermented vegetables. Among 202 antibiotic resistant lactic acid bacterial isolates, 14 were further identified and their minimum inhibitory concentrations against 7 antibiotics were examined. Multi-resistance was observed in these 14 strains, and the presence of genes leading resistance was examined by PCR. Genes tetM and ermB were detected on both plasmid and chromosome in certain strains; gene aphA3 was only detected on plasmid while gene mefA was only on chromosome; genes gyrA, blaZ and catpIP501 were not detected. These results indicate the possible role of lactic acid bacteria as reservoirs for dissemination of antibiotic resistance in foods and the environment.

Characterization of the Tn 916 Conjugative Transposon in a Food-Borne Strain of Lactobacillus paracasei

Food-borne antibiotic-resistant lactic acid bacteria have received growing attention in the past few years. We have recently identified tetracycline-resistant Lactobacillus paracasei in samples of milk and natural whey starter cultures employed in the manufacturing process of a typical Italian fermented dairy product, Mozzarella di Bufala Campana. In the present study, we have characterized at the molecular level the genetic context of tetracycline resistance determinants in these natural strains, which we have identified as tet(M). This gene was present in 21 independent isolates, whose fingerprinting profiles were distributed into eight different repetitive extragenic palindromic groups by cluster analysis. We provide evidence that the gene is associated with the broad-host, conjugative transposon Tn916, which had never before been described to occur in L. paracasei. PCR analysis of four independent isolates by use of specifically designed primer pairs detected the presence of a circular intermediate form of the transposon, carrying a coupling sequence (GGCAAA) located between the two termini of Tn916. This novel coupling sequence conferred low conjugation frequency in mating experiments with the recipient strain JH2-2 of Enterococcus faecalis.

Isolation and Molecular Characterization of Antibiotic-Resistant Lactic Acid Bacteria from Poultry and Swine Meat Products

The transfer via the food chain from animals to humans of microbes that are resistant to antimicrobial agents is of increasing concern. To determine the contributions of nonpathogenic microflora to the occurrence and spread of antibiotic resistance (AR) genes in the food chain, 123 lactic acid bacteria were isolated from 29 samples of raw and processed pork and chicken meat products that had previously tested positive for one or more AR genes that encode clinically relevant ARs: tet(M), tet(O), tet(K), erm(A), erm(B), erm(C), aac (6′)-Ie aph (2′′)-Ia, mecA, and blaZ. All of the isolates were initially tested for their AR gene profiles by PCR. The 59 isolates carrying a tet, erm, or blaZ gene were taken through molecular identification, analyzed by determination of the MIC, and subjected to genetic fingerprinting. Lactococcus garvieae was the predominant species (28 isolates), followed by Lactobacillus plantarum (11 isolates) and L. salivarius (6 isolates), whereas Lactococcus lactis subsp. lactis, Lactobacillus johnsonii, L. reuteri, L. crispatus, and L. brevis were identified at lower frequencies. The tet(M) and erm(B) genes were the most frequently detected. Assessment of multiple resistances in 18 tet positive (tet+) isolates revealed that tet(M) plus erm(B) and tet(K) plus erm(B) were the most frequent AR gene patterns. Partial sequencing of the tet(M) open reading frame of three selected strains showed high sequence similarities (>99%) with tet(M) genes previously found in human pathogens (Listeria monocytogenes and Neisseria meningitidis). Southern hybridization with plasmid profiles revealed these strains contained tet(M)-carrying plasmids.

Investigation for Proper Use of Probiotics in Nagasaki University Hospital of Medicine and Dentistry

Antimicrobial agents sometimes cause the adverse effects of diarrhea and loose stool. Antibiotic-resistant lactic acid bacteria are used to prevent these adverse effects. The bacteria are not resistant to several antimicrobial agents, although the bacterium preparations are sometimes prescribed the antimicrobial agents concomitantly. Therefore this paper reports that the minimal inhibitory concentration of three new antimicrobial agents against antibiotic-resistant lactic acid bacteria were determined using a microdilution method with cation-adjusted Mueller-Hinton broth. Furthermore, we investigated antimicrobial agents that are prescribed concomitantly with antibiotic-resistant lactic acid bacterium preparations or a clostridium butyricum preparation. The bacteria were susceptible to the three new antimicrobial agents. Approximately 50% of the bacterium preparations were prescribed alone, and 30% were prescribed concomitantly with antimicrobial agents that show antimicrobial activity against the bacteria. Consequently, we suggest that pharmacists need to confirm prescriptions and to provide more drug information on antibiotic-resistant lactic acid bacterium preparations.

Acquired antibiotic resistance in lactic acid bacteria from food.

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. theta-type replicating plasmids of the pAMbeta1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29,871 bp resistance plasmid detected in Lactococcus lactis subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabolic traits to generate food-grade vectors.