Antagonistic Activity of <Emphasis Type="Italic">Nocardia brasiliensis</Emphasis> PTCC 1422 Against Isolated Enterobacteriaceae from Urinary Tract Infections

The main drawback of current antibiotic therapies is the emergence and rapid increase in antibiotic resistance. Nocardiae are aerobic, Gram-positive, catalase-positive, non-motile actinomycetes. Nocardia brasiliensis was reported as antibiotic producer. The purpose of the study was to determine antibacterial activity of N. brasiliensis PTCC 1422 against isolated Enterobacteriaceae from urinary tract infections (UTIs). The common bacteria from UTIs were isolated from hospital samples. Antimicrobial susceptibility test was performed for the isolated pathogens using Kirby–Bauer disk diffusion method according to clinical and Laboratory Standards Institute guideline. Antagonistic activity of N. brasiliensis PTCC 1422 was examined with well diffusion methods. Supernatant of N. brasiliensis PTCC 1422 by submerged culture was analyzed with gas chromatography–mass spectrometry. Isolated strains included Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Proteus mirabilis. The most common pathogen isolated was E. coli (72.5 %). Bacterial isolates revealed the presence of high levels of antimicrobial resistances to ceftriaxone and low levels of resistance to cephalexin. Supernatant of N. brasiliensis PTCC 1422 showed antibacterial activity against all of the isolated microorganisms in well diffusion method. The antibiotic resistance among the uropathogens is an evolving process, so a routine surveillance to monitor the etiologic agents of UTI and the resistance pattern should be carried out timely to choose the most effective empirical treatment by the physicians. Our present investigation indicates that the substances present in the N. brasiliensis PTCC 1422 could be used to inhibit the growth of human pathogen. Antibacterial resistance among bacterial uropathogen is an evolving process. Therefore, in the field on the need of re-evaluation of empirical treatment of UTIs, our present. The study has demonstrated that N. brasiliensis PTCC 1422 has a high potential for the treatment of UTIs.     

Functional Properties of <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis> Strains Isolated from Brazilian Goat Milk

The search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria isolated from traditional fermented stinky bean (<Emphasis Type="Italic">Sataw-Dong</Emphasis>)

The aim of this study was to evaluate the technological and functional potential of lactic acid bacteria (LAB) isolated from fermented stinky bean (Sataw-Dong). Of the 114 LAB colonies isolated from spontaneously fermented stinky bean which showed inhibitory activity against two food-borne pathogens (Staphylococcus aureus DMST 4480 and Escherichia coli DMST 4212), the five isolates (KJ03, KJ15, KJ17, KJ22, KJ23) exhibiting excellent antagonistic activity were subjected to further study. These five strains showed titratable acidity as lactic acid in the range of 1.47–1.55 %, with strains KJ03 and KJ23 additionally exhibiting a high NaCl tolerance of >7 % (w/v). Using 16S rRNA gene sequence analysis, strains KJ03 and KJ23 were identified as Lactobacillus plantarum and L. fermentum, respectively, and further investigated for their functional properties in vitro. Both strains survived well in a simulated gastrointestinal tract environment with <1 log cell decrease over 8 h (>8 log CFU/ml). Lactobacillus plantarum KJ03 showed the best performance with respect to cholesterol removal (53 %), while L. fermentum KJ23 showed the highest cell-surface hydrophobicity (39.5 %). Neither of the two strains showed any hemolysis activity. Both strains hydrolyzed glycodeoxycholic and taurodeoxycholic acids. In terms of antibiotic susceptibility, L. fermentum KJ23 was not sensitive to tetracycline. Taking all of the results into account, L. plantarum KJ03 possessed desirable in vitro functional properties. This strain is therefore a good candidate for further investigation for use in Sataw-Dong fermentation to assess its technological performance as a potential probiotic starter.     

An evaluation of multidrug-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates in urinary tract infections from Aguascalientes,Mexico: cross-sectional study

Background

Uropathogenic Escherichia coli (UPEC) are one of the main bacteria causing urinary tract infections (UTIs). The rates of UPEC with high resistance towards antibiotics and multidrug-resistant bacteria have increased dramatically in recent years and could difficult the treatment.

Methods

The aim of the study was to determine multidrug-resistant bacteria, antibiotic resistance profile, virulence traits, and genetic background of 110 E. coli isolated from community (79 isolates) and hospital-acquired (31 isolates) urinary tract infections. The plasmid-mediated quinolone resistance genes presence was also investigated. A subset of 18 isolates with a quinolone-resistance phenotype was examined for common virulence genes encoded in diarrheagenic and extra-intestinal pathogenic E. coli by a specific E. coli microarray.

Results

Female children were the group most affected by UTIs, which were mainly community-acquired. Resistance to trimethoprim–sulfamethoxazole, ampicillin, and ampicillin–sulbactam was most prevalent. A frequent occurrence of resistance toward ciprofloxacin (47.3%), levofloxacin (43.6%) and cephalosporins (27.6%) was observed. In addition, 63% of the strains were multidrug-resistant (MDR). Almost all the fluoroquinolone (FQ)-resistant strains showed MDR-phenotype. Isolates from male patients were associated to FQ-resistant and MDR-phenotype. Moreover, hospital-acquired infections were correlated to third generation cephalosporin and nitrofurantoin resistance and the presence of kpsMTII gene. Overall, fimH (71.8%) and fyuA (68.2%), had the highest prevalence as virulence genes among isolates. However, the profile of virulence genes displayed a great diversity, which included the presence of genes related to diarrheagenic E. coli. Out of 110 isolates, 25 isolates (22.7%) were positive to qnrA, 23 (20.9%) to qnrB, 7 (6.4%) to qnrS1, 7 (6.4%) to aac(6′)lb-cr, 5 (4.5%) to qnrD, and 1 (0.9%) to qnrC genes. A total of 12.7% of the isolates harbored bla_CTX-M genes, with bla_CTX-M-15 being the most prevalent.

Conclusions

Urinary tract infection due to E. coli may be difficult to treat empirically due to high resistance to commonly used antibiotics. Continuous surveillance of multidrug resistant organisms and patterns of drug resistance are needed in order to prevent treatment failure and reduce selective pressure. These findings may help choosing more suitable treatments of UTI patients in this region of Mexico.

     

Screening and molecular identification of potential probiotic lactic acid bacteria in effluents generated during ogi production

Screening and molecular identification of probiotic lactic acid bacteria (LAB) in effluents generated during the production of ogi, a fermented cereal (maize, millet, and sorghum) were done. LAB were isolated from effluents generated during the first and second fermentation stages in ogi production. Bacterial strains isolated were identified microscopically and phenotypically using standard methods. Probiotic potential properties of the isolated LAB were investigated in terms of their resistance to pH 1.5 and 0.3% bile salt concentration for 4 h. The potential LAB isolates ability to inhibit the growth of pathogenic organisms (Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium) was evaluated in vitro. The pH and LAB count in the effluents ranged from 3.31 to 4.49 and 3.67 to 4.72 log cfu/ml, respectively. A total of 88 LAB isolates were obtained from the effluents and only 10 LAB isolates remained viable at pH 1.5 and 0.3% bile salt. The zones of inhibition of the LAB isolates with probiotic potential ranged from 7.00 to 24.70 mm against test organsisms. Probiotic potential LAB isolates were molecularly identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus brevis. Survival and proliferation of LAB isolates at low pH, 0.3% bile salt condition, and their inhibition against some test pathogens showed that these LAB isolates could be a potential probiotics for research and commercial purposes.     

Long-term adaptation of <Emphasis Type="Italic">Escherichia coli</Emphasis> to methanogenic co-culture enhanced succinate production from crude glycerol

Escherichia coli can hardly grow anaerobically on glycerol without exogenous electron acceptor. The formate-consuming methanogen Methanobacterium formicicum plays a role as a living electron acceptor in glycerol fermentation of E. coli. Wild-type and mutant E. coli strains were screened for succinate production using glycerol in a co-culture with M. formicicum. Subsequently, E. coli was adapted to glycerol fermentation over 39 rounds (273 days) by successive co-culture with M. formicicum. The adapted E. coli (19.9 mM) produced twice as much succinate as non-adapted E. coli (9.7 mM) and 62% more methane. This study demonstrated improved succinate production from waste glycerol using an adapted wild-type strain of E. coli with wild-type M. formicicum, which is more useful than genetically modified strains. Crude glycerol, an economical feedstock, was used for the cultivation. Furthermore, the increase in methane production by M. formicicum during co-culture with adapted E. coli illustrated the possibility of energy-saving effects for the fermentation process.     

Probiotic Potential of Autochthonous Bacteria Isolated from the Gastrointestinal Tract of Four Freshwater Teleosts

In this study, a total of 121 bacterial strains were isolated from the gastrointestinal tract of four teleostean species, namely striped snakehead (Channa striatus), striped dwarf catfish (Mystus vittatus), orangefin labeo (Labeo calbasu) and mrigal carp (Cirrhinus mrigala), among which 8 isolates showed promising antibacterial activity against four potential fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas sobria and Pseudomonas fluorescens and were non-hemolytic. The isolates were further screened in response to fish bile tolerance and extracellular digestive enzyme activity. Two bacterial strains MVF1 and MVH7 showed highest tolerance and extracellular enzymes activities, and selected for further studies. Antagonistic activity of these two isolates was further confirmed by in vitro growth inhibition assay against four selected fish pathogens in liquid medium. Finally, these two bacterial strains MVF1 and MVH7 were selected as potential probiotic candidates and thus identification by partial 16S rRNA gene sequence analysis. The bacterial isolates MVF1 and MVH7 were identified as two strains of Bacillus sp.     

Probiotic potential of novel <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from salted-fermented shrimp as antagonists for <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Lactobacillus strains have been considered good candidates as biological control agents for prevention or treatment of plant and animal infections. One L. plantarum strain FB003 and three strains (FB011, FB081, and FB110) which closed to L. sakei were isolated from fermented and salted shrimp and their abilities in inhibiting growth of Vibrio parahaemolyticus were characterized. These strains were selected as potential probiotics based on their oro-gastro-intestinal resistance, gut colonization, adhesion to Caco-2 cells, antimicrobial activities, antibiotic resistance, and safety aspects. Results of this study revealed that these isolates possessed high aggregation activities against pathogens in host intestines. Strain FB011 strain showed higher coaggregation and immunomodulatory activity in the gastro-intestinal tract than L. plantarum. These difference effects of Lactobacillus strains provide valuable information about using them to prevent Vibrio infections in the aquaculture industry.     

Characterization of bacteriocins produced by strains of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> isolated from Minas cheese

Interest in obtaining bacteriocin-producing strains of lactic acid bacteria (LAB) from different sources has been increasing in recent years due to their multiple applications in health and food industries. This study focused on the isolation and characterization of metabolically active populations of bacteriocinogenic LAB and the evaluation of their antimicrobial substances as well as of some nutritional requirements of them. One hundred and fifty colonies of LAB from artisanal cheeses produced in Minas Gerais state (Brazil) were isolated and screened for their antimicrobial activity. According to their activity against Listeria monocytogenes, ten strains were selected and subsequently identified using biochemical and molecular techniques including 16s rRNA amplification and sequencing as Enterococcus faecalis, Lactobacillus spp., and Pediococcus pentosaceus. Antimicrobial substances produced by four of the selected strains, P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146, and P. pentosaceus 147, were biochemically characterized, and presented sensitivity to proteolytic enzymes (suggesting their proteinaceous nature) and to extreme pH. Antimicrobial activity showed stability after treatment with lipase, catalase, α-amylase, and chemicals. Growth kinetics of the P. pentosaceus selected showed maximal bacteriocin production at 37 °C during the end of the exponential growth phase (25,600 AU/mL) and stable production during 24 h of incubation. Dextrose, maltose, and a mixture of peptone, meat extract, and yeast extract increased bacteriocin production. This study demonstrated that dairy products provide a good alternative for obtaining LAB, with the ability to produce antimicrobial substances such as bacteriocins that have potential use as biopreservatives in food.     

Functional Characterization of Potential Probiotic Lactic Acid Bacteria Isolated from <Emphasis Type="Italic">Kalarei</Emphasis> and Development of Probiotic Fermented Oat Flour

Considerable variations among probiotics with respect to their health benefitting attributes fuel the research on bioprospecting of proficient probiotic strains from various ecological niches especially the poorly unexplored ones. In the current study, kalarei, an indigenous cheese-like fermented milk product, and other dairy-based sources like curd and raw milk were used for isolation of lactic acid bacteria (LAB). Among 34 LAB isolates, 7 that could withstand simulated gastrointestinal (GI) conditions were characterized for functional probiotic attributes, viz. adhesion ability, aggregation and coaggregation, extracellular enzyme producing capability, antibacterial activity against pathogens and antibiotic resistance. The isolate M-13 (from kalarei) which exhibited most of the desirable probiotic functional properties was identified as Lactobacillus plantarum based on 16S ribosomal DNA sequence analysis and designated as L. plantarum M-13. The sequence was submitted to GenBank (accession number KT592509). The study presents the first ever report of isolation of potential probiotic LAB, i.e. L. plantarum M-13 from indigenous food kalarei, and its application for development of potential probiotic fermented oat flour (PFOF). PFOF was analysed for parameters like viability of L. plantarum M-13, acidity and pH. Results show that PFOF serves as a good matrix for potential probiotic L. plantarum M-13 as it supported adequate growth of the organism (14.4 log cfu/ml after 72 h of fermentation). In addition, appreciable acid production by L. plantarum M-13 and consequential pH reduction indicates the vigorous and active metabolic status of the potential probiotic organism in the food matrix. Thus, study shows that fermented oat flour may possibly be developed as a potential probiotic carrier especially in view of the problems associated with dairy products as probiotic vehicles.     

Possible Probiotic Lactic Acid Bacteria Isolated from Oysters (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>)

We attempted to isolate lactic acid bacteria (LAB) from the marine oyster (Crassostrea gigas) and selected several environmental stress-resistant isolates for the development of a future probiotic adjuvant for marine aquaculture. Twenty-six presumptive LAB isolates were extracted from oysters and screened (by an agar diffusion assay) for antimicrobial activity toward various pathogens: Vibrio parahaemolyticus, Streptococcus iniae, and Edwardsiella tarda. Eight isolates had an antibacterial activity toward V. parahaemolyticus; in particular, 6 isolates showed a growth-inhibitory activity, with inhibition zone diameters > 15 mm. Of these, 5 isolates (JL17, JL18, JL28, HL7, and HL32) were also active against S. iniae and E. tarda. Enterococcus faecium HL7 was selected as the isolate most resistant to environmental stressors: the minimum NaCl, ethanol, and hydrogen peroxide concentrations at which HL7 cells lost their viability were 1.9 M, 11%, and 0.013%, respectively. When an antibiotic sensitivity test was performed on E. faecium HL7, this isolate was found to be resistant to trimethoprim/sulfamethoxazole, cephalothin, ampicillin, rifampin, gentamicin, cefotaxime, cefepime, cefotetan, nalidixic acid, and kanamycin. While the oyster model studies provided indication that E. faecium HL7 could be a good candidate as biocontrol agent against V. vulnificus, further optimization is needed in the actual animal rearing situation.     

<Emphasis Type="Italic">In situ</Emphasis> fermentation dynamics during production of <Emphasis Type="Italic">gundruk</Emphasis> and <Emphasis Type="Italic">khalpi</Emphasis>, ethnic fermented vegetable products of the Himalayas

Gundruk is a fermented leafy vegetable and khalpi is a fermented cucumber product, prepared and consumed in the Himalayas. In situ fermentation dynamics during production of gundruk and khalpi was studied. Significant increase in population of lactic acid bacteria (LAB) was found during first few days of gundruk and khlapi fermentation, respectively. Gundruk fermentation was initiated by Lactobacillus brevis, Pediococcus pentosaceus and finally dominated by Lb. plantarum. Similarly in khalpi fermentation, heterofermentative LAB such as Leuconostoc fallax, Lb. brevis and P. pentosaceus initiated the fermentation and finally completed by Lb. plantarum. Attempts were made to produce gundruk and khalpi using mixed starter culture of LAB previously isolated from respective products. Both the products prepared under lab condition had scored higher sensory-rankings comparable to market products.     

Genetic diversity and antimicrobial activity of lactic acid bacteria in the preparation of traditional fermented potato product ‘<Emphasis Type="Italic">tunta’</Emphasis>

Fermentation microorganisms, lactic acid bacteria (LAB) and yeast from 12 samples of tunta production chain were quantified, from the native potatoes used by the process fermentation of potatoes in the river up to the final product. During fermentation, the LAB population steadily increased from 3 to 4 to 8 log CFU/g during the first 8 days in the river and the yeast population increased from 2 to 3 to 3–4 log CFU/g. Overall, 115 LAB strains were isolated using a culture-dependent method. Molecular techniques and 16S rRNA gene sequencing enabled the identification of native species. In LAB isolates, members of the Lactobacillaceae (64%), Leuconostocaceae (9%) and Enterococcaceae (2%) families were identified. The most prevalent LAB species in the tunta production chain was Lactobacillus curvatus, followed by Leuconostoc mesenteroides and Lactobacillus sakei, Lactobacillus brevis and Enterococcus mundtii were also present. Only 13 LAB strains showed anti-listerial activity, and one of them, identified as En. mundtii DSM 4838^T [MG031213], produced antimicrobial compounds that were determined to be proteins after treatment with proteolytic enzymes. Based on these results, we suggest that traditional fermented product-derived LAB strains from specific environments could be selected and used for technological application to control pathogenic bacteria and naturally protect food from post-harvest deleterious microbiota.     

Long-term H<Subscript>2</Subscript> photoproduction from starch by co-culture of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in a repeated batch process

Objectives

To prove the possibility of efficient starch photofermentation in co-culture of heterotrophic and phototrophic bacteria over prolonged period.

Results

Repeated batch photofermentation of starch was demonstrated in co-culture Clostridium butyricum and Rhodobacter sphaeroides under microaerobic conditions. It continued 15 months without addition of new inoculum or pH regulation when using 4–5 g starch l^?1 and 0.04 g yeast extract l^?1. The complete degradation of starch without volatile fatty acids accumulation was shown in this co-culture. The average H₂ yield of 5.2 mol/mol glucose was much higher than that in Clostridium monoculture. The species composition of co-culture was studied by q-PCR assay. The concentration of Clostridium cells in prolonged co-culture was lower than in monoculture and even in a single batch co-culture. This means that Clostridia growth was significantly limited whereas starch hydrolysis still took place.

Conclusion

The prolonged repeated batch photofermentation of starch by co-culture C. butyricum and R. sphaeroides provided efficient H₂ production without accumulation of organic acids under conditions of Clostridia limitation.

     

Antimicrobial properties of endophytic actinomycetes isolated from <Emphasis Type="Italic">Combretum latifolium</Emphasis> Blume,a medicinal shrub from Western Ghats of India

Endophytic actinomycetes were isolated from Combretum latifolium Blume (Combretaceae),Western Ghats of Southern India and identified by its characteristic culture morphology and molecular analysis of 16S rRNA gene sequences. In this survey of endophytic actinomycetes, a total of 117 isolates representing 9 different genera of endophytic actinomycetes were obtained using four different isolation media and several of them seemed to be novel taxa. Streptomyces genera (35%) was the most frequently isolated strains, followed by Nocordiopsis (17%) and Micromonospora (13%). ISP-4 medium recovered more isolates (47%) when compared to rest of the media used. Preliminary antibacterial activity of the isolates was carried out by confrontation test. Ethyl acetate fraction of selected isolates in disc diffusion assay exhibited broad spectrum antimicrobial activity against test human pathogens. All Streptomyces spp. strains displayed significant antimicrobial activity against test pathogens. Strain CLA-66 and CLA-68 which are Nocordipsis spp. inhibited both bacterial and fungal pathogens where as other isolates inhibited atleast three test human pathogens in disc diffusion assay. Antimicrobial screening of endophytic actinomycetes from this host may represent a unique potential niche for antimicrobial compounds of industrial and pharmaceutical applications. This work is the first comprehensive report on incidence of potential endophytic actinomycetes inhabiting C. latifolium Blume.     

Isolation of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains from shellfish for their potential use as probiotics

Microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for their aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate antimicrobial activity against various pathogens, bile salt tolerance, and acid tolerance of 65 presumptive Lactobacillus spp. isolated from shellfish samples. Four strains (HL1, HL12, HL20, and JL28) were selected after qualitatively identifying high levels of antimicrobial activity against bacteria including Staphylococcus aureus, Salmonella typhimurium, Salmonella enteritidis, Escherichia coli O157:H7, Vibrio ichthyoenteri, Edwardsiella tarda, Streptococcus iniae, and V. parahaemolyticus. The sequence analysis of their 16S rRNA genes revealed that the four strains belong to the Lactobacillus plantarum species. In addition, their survivability was tested in bile salt and acidic conditions to show their potential use as probiotics in the gastrointestinal tract.     

Diversity and Probiotic Potential of Lactic Acid Bacteria Isolated from Horreh,a Traditional Iranian Fermented Food

The aim of this study was to evaluate the probiotic potential of lactic acid bacteria (LAB) strains isolated from Horreh. Some probiotic properties, e.g., resistance to acid, bile tolerance, antibacterial activity, and antibiotic susceptibility, were investigated. A total of 140 Gram-positive and catalase-negative isolates from Horreh were subjected to identification and grouping by cultural methods and the 16S rRNA sequencing. The new isolates were identified to be Lactobacillus (fermentum, plantarum, and brevis) Weissella cibaria, Enterococcus (faecium and faecalis), Leuconostoc (citreum and mesenteroides subsp. mesenteroides) and Pediococcus pentosaceus. Probiotic potential study of LAB isolates showed that Lb. plantarum and Leu. mesenteroides subsp. mesenteroides isolates were able to grow at pH 2.5 and 3.5. Lactobacillus plantarum (isolate A44) showed the highest cell hydrophobicity (84.5%). According to antibacterial activity tests, Listeria innocua and Staphylococcus aureus were the most sensitive indicators against the selected LAB strains, while Escherichia coli and Bacillus cereus were the most resistant. In addition, all the isolated LAB species were resistant to vancomycin. The results of the present study suggested that the Lactobacillus fermentum and plantarum isolated from Horreh, characterized in this study, have potential use for industrial purposes as probiotics.     

Isolation and characterization of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains in China

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B₁ (AFB₁) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB₁ biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.     

Phylogenetic analysis of <Emphasis Type="Italic">Plectosphaerella</Emphasis> species based on multi-locus DNA sequences and description of <Emphasis Type="Italic">P. sinensis</Emphasis> sp. nov.

Species in Plectosphaerella are well known as pathogens of several plant species causing fruit, root and collar rot and collapse. In an investigation of endophytic fungi associated with cucurbit plants in China, we isolated 77 strains belonging to the genus Plectosphaerella. To identify the isolated strains, we collected the type or reference strains of all currently accepted species in Plectosphaerella except P. oratosquillae and conducted a phylogenetic analysis. Phylogenetic analysis of the partial 28S rDNA sequences showed that all species in Plectosphaerella were located in one clade of Plectosphaerellaceae. Based on multi-locus phylogenetic analysis of the ITS, CaM, EF1, TUB and morphological characteristics, all species in Plectosphaerella were well separated. Three endophytic strains from stems of Cucurbita moschata, Citrullus lanatus and Cucumis melo from North China were assigned to a new species described as P. sinensis in this paper. The new species differs morphologically from other Plectosphaerella species by irregular chlamydospores, and the dimensions of phialides and conidia. The other endophytic strains from several cucurbit plants were identified as P. cucumerina.