Antimicrobial and antiadhesive effects of Lactobacillus isolates of healthy human gut origin on Enterotoxigenic Escherichia coli (ETEC) and Enteroaggregative Escherichia coli (EAEC).

PURPOSE: Diarrhea is one of the five leading causes of mortality in children under the age of five, especially in developing countries. Nowadays, by increasing the resistance of pathogens to antibiotics, employment of probiotics as novel therapeutic method, could be considered as a necessity.The aim of this study was to examine the features and antagonistic action of Lactobacillus strains, against the growth and adhesion of Enterotoxigenic Escherichia coli (ETEC) and Enteroaggregative Escherichia coli (EAEC) strains creating diarrhea in children. Then, we introduced new strains of Lactobacillus as probiotic candidates, to prevent diarrheal infections in children. METHODS: Stool samples were collected from healthy individuals, and Lactobacillus strains were isolated. The antimicrobial effect of the isolates against ETEC and EAEC strains investigated by agar well diffusion method and their resistance to acidic and bile conditions. The potency of selected isolates in adhesion to HT-29 epithelial cells and their ability to inhibit the adhesion of ETEC and EAEC strains to this cell were measured. At the end, identification of the optimally efficient Lactobacillus isolates was performed by 16S rDNA sequencing and making Phylogenetic tree using MEGA (version 4.0) software. RESULTS: In total, 157 isolates suspected to Lactobacillus were isolated from 115 stool samples. In antimicrobial activity test, ETEC and EAEC growth was inhibited by 132 and 84 isolates respectively, while 17 isolates showed resistance to Bile. Of 17 Bile resistant Lactobacillus isolates, 15 isolates were resistant to pH: 3.2. Further, among 15 isolates, only two isolates, were resistant to pH: 2.5. In the adhesion assay, five isolates had more adhesion tendency to HT-29 epithelial cells than L. rhamnosus GG, which was considered as a positive control. Investigation of isolates that inhibit adhesion of ETEC and EAEC strains to HT-29 cells showed that four isolates were able to inhibit ETEC adhesion. However, only one out of four isolates was relatively able to have an impact on EAEC adhesion. CONCLUSION: In conclusion, three species of Lactobacillus including L. paracasei (two strain), L. fermentum (two strain) and L. plantarum showed good probiotic properties compared to other isolates that were identified by sequencing. In this study, strain L. fermentum 61.1 had the highest adhesion ability to HT-29 cells and strain L. paracasei 47.2 had the highest potency to inhibit ETEC adhesion to HT-29 cells. These isolates have good probiotic properties and are likely to be effective in preventing or treating diarrheal infections, especially in children.

Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria.

Lactobacilli are normal microflora of the gastrointestinal (GI) tract and are a heterogeneous group of lactic acid bacteria (LAB). Lactobacillus strains with Probiotic activity may have health Benefits for human. This study investigates the probiotic potential of Lactobacillus strains obtained from the feces of healthy infants and also explores antibacterial activity of Lactobacillus strains with probiotic potential against enteropathogenic bacteria. Fecal samples were collected from 95 healthy infants younger than 18 months. Two hundred and ninety Lactobacillus strains were isolated and assessed for probiotic potential properties including ability to survive in gastrointestinal conditions (pH 2.0, 0.3% oxgall), adherence to HT-29 cells and antibiotic resistance. Six strains including Lactobacillus fermentum (4 strains), Lactobacillus paracasei and Lactobacillus plantarum showed good probiotic potential and inhibited the growth of enteropathogenic bacteria including ETEC H10407, Shigella flexneri ATCC 12022, Shigella sonnei ATCC 9290, Salmonella enteritidis H7 and Yersinia enterocolitica ATCC 23715. These Lactobacillus strains with probiotic potential may be useful for prevention or treatment of diarrhea, but further in vitro and in vivo studies on these strains are still required.

Antimicrobial effect of different physical and chemical compounds of zinc oxide and graphene oxide added to composite resins.

Background: Graphene oxide (GO), a product of graphite, is a candidate for nano-reinforcing cement-based materials due to its good water dispersibility and excellent mechanical properties. On the other hand, zinc oxide (ZnO) is well-known for its antibacterial characteristics as well. Therefore, we aimed to evaluate the impacts of adding ZnO and GO nanoparticles on the antibacterial properties of flowable composites. Materials and Methods: In this, in vitro experimental study was designated into five groups containing: (1) no nanoparticles as control group, (2) 1 wt.% ZnO nanoparticle, (3) 1 wt.% GO, (4) 1 wt.% physical compound of ZnO and GO, and (5) 1 wt.% chemical compound of ZnO and GO. The antibacterial properties of composite resin discs were evaluated by direct contact test. Data were analyzed using a one-way analysis of variance, followed by Tukey' post hoc tests (P = 0.05). Results: Streptococcus mutans colony counting in the first 24 h showed the least growth rate in the chemical compound group (2.2 × 10[5]). However, in 7 days, the least colony number was observed in the GO group (2 × 10[3]). Moreover, the physical compound showed the least bacterial adhesion. Conclusion: Adding GO alone to composites, compared to adding ZnO or physical and chemical compounds of GO-ZnO, was more helpful to increase the antimicrobial characteristics.

Some probiotic properties of Lactobacillus species isolated from honey and their antimicrobial activity against foodborne pathogens.

Lactobacilli commonly used as a probiotic and they can be isolated from various sources such as fermented foods and gastrointestinal tracts of humans and animals. The aims of this study were isolation and identification of lactobacilli from honey and investigation of some probiotic properties and antimicrobial effects against foodborne bacterial pathogens. A total of 88 honey samples were collected from different areas in Iran. About 1.00 g of each honey was cultured in de Man, Rogosa, and Sharpe (MRS) broth and then sub-cultured on MRS agar. The isolates were assessed for probiotic potentials such as tolerance to acid and bile. Then, antimicrobial activity of isolates against seven foodborne pathogens including Listeria monocytogenes, Shigella flexneri, Staphylococcus aureus, Salmonella enteritidis, Enteropathogenic Escherichia coli, Escherichia coli O157 H7 and Bacillus cereus was investigated. From 88 honey samples, 39 isolates were identified by 16S rDNA gene sequencing method. Fructophilic lactic acid bacteria (FLAB) with 29 (74.00%) isolates were dominant identified bacteria (27 L. kunkeei and two Fructobacillus fructosus). Also, four L. plantarum, two L. paracasei, one L. brevis, one L. rhamnosus, one L. casei and one L. fermentum were identified. Two L. kunkeei isolates and one F. fructosus isolate were resistant to acid and bile salt. Two L. rhamnosus isolates and one L. paracasei isolate inhibited all pathogens (100%). This is the first study in Iran that isolated lactobacilli from honey. The FLAB especially L. kunkeei were isolated as dominated species from honey. Some lactobacilli isolates have probiotic potential and may be useful for the prevention and treatment of infections, but more investigations are needed.

Antimicrobial properties of lactic acid bacteria isolated from traditional yogurt and milk against Shigella strains.

Background: Lactic acid bacteria (LAB) are normal flora of the mouth, intestines and the female genital tract. They are also frequently found in meat, vegetables, and dairy products. Most of probiotic bacteria belong to the LAB group. Some probiotic LAB are useful in prevention and treatment of diarrheal diseases. The aim of this study was to investigate the antimicrobial properties of LAB isolated from traditional yogurt and milk against Shigella strains. Materials and methods: Forty LAB strains were isolated from traditional yogurt and milk. The antimicrobial activity of LAB against Shigella strains (eight S. flexneri, four S. sonnei) was examined using the agar-well diffusion assay. LAB strains with antimicrobial effect against all Shigella strains were identified by 16S rRNA gene sequencing. Results: Six LAB strains inhibited the growth of all 12 Shigella strains. Lb. paracasei Y1-3, Lb. paracasei Y8-1 and Lb. fermentum Y2-2 were isolated from yogurt. Lb. paracasei M18-1, Lb. parelimentarius M4-3 and Lb. plantarum M19-1 were isolated from milk. Conclusion: This study showed that Lactobacillus strains with good inhibitory activity against S. flexneri and S. sonnei could be isolated from traditional yogurt and milk.

Diagnosis of Clostridium difficile infection by toxigenic culture and PCR assay.

BACKGROUND AND OBJECTIVES: Clostridium difficile is responsible for 15-25% of nosocomial antibiotic associated diarrhea (AAD) cases and all cases of pseudomembranous colitis. C. difficile has two major virulence factors, toxin A (enterotoxin) and toxin B (cytotoxin). The aim of this study was to determine the frequency of C. difficile strains in patients with diarrhea in Babol' hospitals with toxigenic culture and PCR assay. MATERIALS AND METHODS: One hundred stool specimens were taken from diarrheal patients in hospitals of the city of Babol. All patients had a history of antibiotic use. The samples were cultured on CCFA medium. In the next stage, toxigenic culture was performed for isolated C. difficile strains. Then, PCR assay was used to identify gdh, tcdA and tcdB genes among isolated C. difficile strains. RESULTS: From the 100 stool samples, eight (8%) samples were positive in C. difficile culture. In toxigenic culture, two (2%) of these strains had cytopathic effects on Vero cells. All eight strains had the gdh gene. This gene is specific for C. difficile. Two strains that had cytopathic effects on toxigenic culture were positive for toxin genes. CONCLUSION: The frequency of toxigenic strains in different parts of the world is variable, and needs to be continually investigated. In the present study, the PCR method had a good correlation with toxigenic culture. Thus, it can replace the laborious and costly cell culture method.

Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus Isolates in Fishery Products in Iran.

Staphylococcus aureus is one of the most common causes of seafood-borne diseases worldwide, which are attributable to the contamination of food by preformed enterotoxins. In this study, a total of 206 (34.3%) Staphylococcus aureus strains were obtained from 600 fish and shrimp samples and were tested for their antimicrobial susceptibility. We assessed the prevalence of the genes responsible for the staphylococcal enterotoxins (SEA, SEB) and toxic shock syndrome toxin 1 (TSST-1) genes. The results indicated that 34% of aqua food samples were contaminated with S. aureus, and 23.8% of these isolates were mec-A-positive. Sixty-four percent of the strains isolated from contaminated seafood was enterotoxigenic S. aureus, and 28.2% of SEs were MRSA-positive. The most prevalent genotype was characterized by the presence of the sea gene (45.2%), followed by the seb gene (18.5%), and the tst gene encoding TSST-1 was found in eight strains (3.9%). Of the 206 S. aureus isolates, 189 strains (84.9%) were resistant to at least one antibiotic. Given the frequent outbreaks of enterotoxigenic MRSA, it is necessary to make revisions to mandatory programmes to facilitate improved hygiene practices during fishing, aquaculture, processing, and sales to prevent the contamination of fishery products in Iran.

Phenotypic and Genotypic Characterization of Enteroaggregative Escherichia coli Strains Isolated From Diarrheic Children in Iran.

BACKGROUND: Several studies performed in developed and developing countries have identified enteroaggregative Escherichia coli (EAEC) as the emerging cause of pediatric diarrhea. OBJECTIVES: This study investigated the phenotypic and genetic characteristics of EAEC strains isolated from children with diarrhea between 2007 - 2008 in Tehran, Iran. MATERIALS AND METHODS: EAEC strains were examined for virulence plasmid genes (aap, aggR, and aatA), biofilm formation, and drug resistance. In addition, pulsed-field gel electrophoresis (PFGE) profiles of these strains were determined. RESULTS: Significant percentage of local EAEC strains carried the virulence plasmid genes and formed biofilms. In addition, these strains showed high resistance to ampicillin (100%), tetracycline (65.7%), streptomycin (58.7%), chloramphenicol (52.6%), and trimethoprim/sulfamethoxazole (51.7%) and had different PFGE patterns. CONCLUSIONS: These results indicated that EAEC strains isolated from Iranian children with diarrhea were heterogeneous and showed high resistance rates against commonly used antibiotics, which was similar to that reported in studies performed in other countries.

The Detection of Mupirocin Resistance and Nasal Carriage of Methicillin Resistant Staphylococcus aureus among Healthcare Workers at University Hospitals of Tehran, Iran.

BACKGROUND: Nasal mupirocin is found to eradicate effectively methicillin-resistant Staphylococcus aureus (MRSA) from colonized patients, but there are concerns about resistant strains. The aim of this study was to detect the mupirocin resistance and nasal carriage of methicillin resistant S. aureus among healthcare workers (HCWS) at the university hospitals of Tehran. METHODS: Totally 270 nasal swabs were collected and S. aureus were identified by confirmatory tests in 2013. Determination of oxacillin and mupirocin resistance was performed by disk diffusion method and the mupirocin MIC assessed using E-test. The ileS-2 (mupA) and mecA genes were detected in DNA extracts by multiplex PCR. RESULTS: The prevalence of S. aureus nasal carriage among HCWs was 14.44%. E-test and disk diffusion methods showed 5 and 4 mupirocin resistant isolates, respectively. Statistically significant difference was observed between sex (P=0.035), hospitals (P=0.0001) and occupation (P=0.009) with nasal carriage of S. aureus. A significant difference was found between sex (P=0.041) and occupation (0.034) with regard to MRSA carriage. All MRSA isolates were susceptible to linezolid, fusidic acid and vancomycin. CONCLUSION: Since the HCWs play an important role as a reservoir for resistant isolates in the hospital setting, regularly screening should be performed for identification of nasal carriers.

Antimicrobial Activity of Lactobacillus spp. Isolated From Fecal Flora of Healthy Breast-Fed Infants Against Diarrheagenic Escherichia coli.

BACKGROUND: Among the enteric pathogens, diarrheagenic Escherichia coli are important causes of diarrhea in children in both developing and industrialized countries. Some Lactobacillus species are commonly used as probiotics, with effects especially against acute diarrhea in childhood. OBJECTIVES: The aim of this study was to explore antimicrobial activity of Lactobacillus strains isolated from fecal flora of healthy breast-fed infants against five diarrheagenic E. coli pathotypes such as enteroaggregative E. coli (EAEC), enterohaemorrhagic E. coli (EHEC) enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). MATERIALS AND METHODS: Fecal samples were collected from seven healthy breast-fed infants between 1 to 18 months of age in Tehran city, Iran. Identification of Lactobacillus isolates was performed by biochemical and 16S rRNA gene sequencing methods. An agar well diffusion assay was used for detection of antimicrobial activity of Lactobacillus isolates against five diarrheagenic E. coli pathotypes. RESULTS: A total of 20 Lactobacillus isolates were identified from stool samples. Lactobacillus fermentum was the most frequently isolated strain, followed by L. plantarum and L. rhamnosus. Seven Lactobacillus strains including L. fermentum (four isolates), L. paracasei (one isolate), L. plantarum (one isolate) and L. rhamnosus (one isolate) had a mild inhibitory activity against diarrheagenic E. coli. The mechanism of inhibitory activity of Lactobacillus strains appeared to be due to the production of organic acids or hydrogen peroxide. CONCLUSIONS: Our findings show that Lactobacillus strains with human origin had a mild inhibitory activity against the diarrheagenic E. coli, and these strains may be useful as probiotic candidates in prevention of intestinal infections caused by diarrheagenic E. coli.