Characterization of culturable Paenibacillus spp. from the snow surface on the high Antarctic Plateau (DOME C) and their dissemination in the Concordia research station

Culturable psychrotolerant bacteria were isolated from the top snow on the high Antarctic Plateau surrounding the research station Concordia. A total of 80 isolates were recovered, by enrichment cultures, from two different isolation sites (a distant pristine site [75° S 123° E] and a site near the secondary runway of Concordia). All isolates were classified to the genus Paenibacillus by 16S rRNA gene phylogenetic analysis and belonged to two different species (based on threshold of 97 % similarity in 16S rRNA gene sequence). ERIC-PCR fingerprinting indicated that the isolates from the two different sites were not all clonal. All isolates grew well from 4 to 37 °C and were resistant to ampicillin and streptomycin. In addition, the isolates from the secondary runway were resistant to chromate and sensitive to chloramphenicol, contrary to those from the pristine site. The isolates were compared to 29 Paenibacillus isolates, which were previously recovered from inside the Concordia research station. One of these inside isolates showed ERIC- and REP-PCR fingerprinting profiles identical to those of the runway isolates and was the only inside isolate that was resistant to chromate and sensitive to chloramphenicol. The latter suggested that dissemination of culturable Paenibacillus strains between the harsh Antarctic environment and the inside of the Concordia research station occurred. In addition, inducible prophages, which are potentially involved in horizontal dissemination of genes, were detected in Paenibacillus isolates recovered from outside and inside the station. The highest lysogeny was observed in strains harvested from the hostile environment outside the station.     

Bacteriocin Production, Antibiotic Susceptibility and Prevalence of Haemolytic and Gelatinase Activity in Faecal Lactic Acid Bacteria Isolated from Healthy Ethiopian Infants

The objective of this study was to characterise lactic acid bacteria (LAB) isolated from faecal samples of healthy Ethiopian infants, with emphasis on bacteriocin production and antibiotic susceptibility. One hundred fifty LAB were obtained from 28 healthy Ethiopian infants. The isolates belonged to Lactobacillus (81/150), Enterococcus (54/150) and Streptococcus (15/150) genera. Lactobacillus species were more abundant in the breast-fed infants while Enterococcus dominated the mixed-fed population. Bacteriocin-producing LAB species were isolated from eight of the infants. Many different bacteriocins were identified, including one new bacteriocin from Streptococcus salivarius, avicin A (class IIa) from Enterococcus avium, one class IIa bacteriocin from Enterococcus faecalis strains, one unknown bacteriocin from E. faecalis and two unknown bacteriocins from Lactobacillus fermentum strains and the two-peptide gassericin T from Lactobacillus gasseri isolate. Susceptibility tests performed for nine antibiotics suggest that some lactobacilli might have acquired resistance to erythromycin (3 %) and tetracycline (4 %) only. The streptococci were generally antibiotic sensitive except for penicillin, to which they showed intermediate resistance. All enterococci were susceptible to ampicillin while 13 % showed penicillin resistance. Only one E. faecalis isolate was vancomycin-resistant. Tetracycline (51 %) and erythromycin (26 %) resistance was prevalent among the enterococci, but multidrug resistance was confined to E. faecalis (47 %) and Enterococcus faecium (33 %). Screening of enterococcal virulence traits revealed that 2 % were β-haemolytic. The structural genes of cytolysin were detected in 28 % of the isolates in five enterococcal species, the majority being E. faecalis and Enterococcus raffinosus. This study shows that bacteriocin production and antibiotic resistance is a common trait of faecal LAB of Ethiopian infants while virulence factors occur at low levels.     

Identification and nematicidal activity of bacteria isolated from cow dung

Two hundred and nineteen bacterial strains were isolated from cow dung. Among these, 59 isolates displayed nematicidal activity against the model nematode Caenorhabditis elegans. Of the 59 bacterial strains, 17 killed >90 % of the tested nematodes within 1 h. Based on their 16S rRNA sequences, these 17 strains were identified as Alcaligenes faecalis, Bacillus cereus, Proteus penneri, Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas otitidis, Staphylococcus sciuri, Staphylococcus xylosus, Microbacterium aerolatum, Pseudomonas beteli. Among these 17 strains, 14 produced volatile organic compound(s) that inhibited the mobility of the C. elegans nematodes. These 14 strains also showed nematicidal activity against a plant pathogenic nematode Meloidogyne incognita. This is the first report demonstrating nematicidal activity for strains in genera Proteus, Providencia and Staphylococcus.     

Species diversity and antibiotic resistance properties of Staphylococcus of farm animal origin in Nkonkobe Municipality,South Africa

The occurrence and antibiotic susceptibility profile of Staphylococcus isolates of healthy farm animal origin in Nkonkobe Municipality as well as the prevalence of putative antibiotic resistance genes were investigated using phenotypic and molecular methods. A total of 120 Staphylococcus isolates were isolated from 150 animal samples and consisted of Staphylococcus haemolyticus (30 %) and Staphylococcus aureus (23.3 %) from pig, Staphylococcus capitis (15 %) from goat, S. haemolyticus (5 %) and Staphylococcus xylosus (15 %) from cattle, and other staphylococci (11.7 %) from dead chicken and pigs. Besides this, the presence of these isolates was observed from the animal dung, showing that the organisms are shed to the environment. About 23.3 % of these isolates were coagulase-positive and 76.7 % were coagulase-negative Staphylococcus. Between 75 and 100 % of the isolates were resistant to penicillin G, tetracycline, sulfamethoxazole, and nalidixic acid; about 38 % were methicillin-resistant staphylococci, including 12.6 % methicillin-resistant S. aureus from pigs. In total, 12 % of all isolates were vancomycin resistant. Also, 12 % of the isolates were erythromycin resistant, while 40.2 % were resistant to ceftazidime. Only the genes mecA and mphC could be confirmed, whereas the genes vanA, vanB, ermA, ermB, and ermC could not be detected. The high phenotypic antibiotic resistance and the presence of some associated resistance genes is a potential threat to public health and suggest the animals to be important reservoirs of antibiotic resistance determinants in the environment.     

Skin Commensal Staphylococci May Act as Reservoir for Fusidic Acid Resistance Genes

We analyzed the occurrence and mechanisms of fusidic acid resistance present in staphylococci isolated from 59 healthy volunteers. The fingers of the volunteers were screened for the presence of staphylococci, and the collected isolates were tested for resistance to fusidic acid. A total of 34 fusidic acid resistant staphylococcal strains (all were coagulase-negative) were isolated from 22 individuals (22/59, 37.3%). Examination of the resistance genes revealed that acquired fusB or fusC was present in Staphylococcus epidermidis, Staphylococcus capitis subsp. urealyticus, Staphylococcus hominis subsp. hominis, Staphylococcus warneri and Staphylococcus haemolyticus. Resistance islands (RIs) carrying fusB were found in S. epidermidis and S. capitis subsp. urealyticus, while staphylococcal chromosome cassette (SCC)-related structures harboring fusC were found in S. hominis subsp. hominis. Genotypic analysis of S. epidermidis and S. hominis subsp. hominis indicated that the fus elements were disseminated in diverse genetic strain backgrounds. The fusC elements in S. hominis subsp. hominis strains were highly homologous to SCCfusC in the epidemic sequence type (ST) 239/SCCmecIII methicillin-resistant S. aureus (MRSA) or the pseudo SCCmec in ST779 MRSA. The presence of acquired fusidic acid resistance genes and their genetic environment in commensal staphylococci suggested that the skin commensal staphylococci may act as reservoir for fusidic acid resistance genes.     

Multiresistance of Staphylococcus xylosus and Staphylococcus equorum from Slovak Bryndza cheese

Staphylococcus xylosus, Staphylococcus equorum, and Staphylococcus epidermidis strains were isolated from Bryndza cheese and identified using PCR method. The antimicrobial susceptibility of these strains was assessed using disc diffusion method and broth microdilution method. The highest percentage of resistance was detected for ampicillin and oxacillin, and in contrary, isolates were susceptible or intermediate resistant to ciprofloxacin and chloramphenicol. Fourteen of the S. xylosus isolates (45 %) and eleven of the S. equorum isolates (41 %) exhibited multidrug resistance. None of the S. epidermidis isolate was multiresistant. The phenotypic resistance to oxacillin was verified by PCR amplification of the gene mecA.     

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Aims: The objective of this study was to investigate the detection of SEE, SEG, SEH and SEI in strains of Staphylococcus aureus and coagulase‐negative staphylococci (CNS) using RT‐PCR. Methods and Results: In this study, 90 Staph. aureus strains and 90 CNS strains were analysed by PCR for the detection of genes encoding staphylococcal enterotoxins (SE) E, G, H and I. One or more genes were detected in 54 (60%) Staph. aureus isolates and in 29 (32·2%) CNS isolates. Staphylococcus epidermidis was the most frequently isolated CNS species (n = 64, 71·1%), followed by Staphylococcus warneri (n = 8, 8·9%) and other species (Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus saprophyticus and Staphylococcus xylosus: n = 18, 20%). The genes studied were detected in Staph. epidermidis, Staph. warneri, Staph. haemolyticus, Staph. hominis, Staph. simulans and Staph. lugdunensis. The highest frequency of genes was observed in Staph. epidermidis and Staph. warneri, a finding indicating differences in the pathogenic potential between CNS species and highlighting the importance of the correct identification of these micro‐organisms. RT‐PCR used for the detection of mRNA revealed the expression of SEG, SEH and/or SEI in 32 (59·3%) of the 90 Staph. aureus isolates, whereas expression of some of these genes was observed in 10 (34·5%) of the 90 CNS isolates. Conclusions: Staphylococcus epidermidis was the most toxigenic CNS species. Among the other species, only Staph. warneri and Staph. lugdunensis presented a positive RT‐PCR result. PCR was efficient in confirming the toxigenic capacity of Staph. aureus and CNS. Significance and Impact of the Study: This study permitted to confirm the toxigenic capacity of CNS to better characterize the pathogenic potential of this group of micro‐organisms. In addition, it permitted the detection of SEG, SEH and SEI, enterotoxins that cannot be detected by commercially available immunological methods.     

Prevalence, distribution and antibiotic resistance pattern among enterococci species in two traditional fermented dairy foods

The prevalence, distributions and antibiotic resistance pattern among enterococci species were determined. A total of 30 samples of Nigerian traditional fermented dairy food were positive to presence of enterococci, with viable counts of 4.17 log CFU/g in nunu, lower than 4.55 log CFU/g observed in wara samples. Twenty-five representative strains were characterized by a combination of phenotypic and genomic typing based on 16S rRNA gene and multi-locus sequencing analysis (MLSA) of RNA polymerase A (rpoA) and phenylanaline synthase (pheS) genes sequencing; these strains were identified as Enterococcus faecium (84 %) and Enterococcus faecalis (16 %). All the 95 enterococci isolated from wara and nunu samples were alpha haemolytic with multi-drug resistance to 10 antimicrobials regardless of class. Four strains were sensitive to chloramphenicol (30 μg) while 33.7 % of the total isolates were resistant to vancomycin from 5 μg. This information will enhance understanding of Enterococcus drug resistance and distribution in traditional fermented foods to support safety and guarantee quality of traditional foods in West Africa.     

CRISPRs of Enterococcus faecalis and E. hirae Isolates from Pig Feces Have Species-Specific Repeats But Share Some Common Spacer Sequences

Clustered regularly interspaced short palindromic repeats (CRISPR) are currently a topic of interest in microbiology due to their role as a prokaryotic immune system. Investigations of CRISPR distribution and characterization to date have focused on pathogenic bacteria, while less is known about CRISPR in commensal bacteria, where they may have a significant role in the ecology of the microbiota of humans and other animals, and act as a recorder of interactions between bacteria and viruses. A combination of PCR and sequencing was used to determine prevalence and distribution of CRISPR arrays in Enterococcus faecalis and Enterococcus hirae isolates from the feces of healthy pigs. Both type II CRISPR–Cas and Orphan CRISPR (without Cas genes) were detected in the 195 isolates examined. CRISPR–Cas was detected in 52 (46/88) and 42 % (45/107) E. faecalis and E. hirae isolates, respectively. The prevalence of Orphan CRISPR arrays was higher in E. faecalis isolates (95 %, 84/88) compared with E. hirae isolates (49 %, 53/107). Species-specific repeat sequences were identified in Orphan CRISPR arrays, and 42 unique spacer sequences were identified. Only two spacers matched previously characterized pig virome sequences, and many were apparently derived from chromosomal sequences of enterococci. Surprisingly, 17 (40 %) of the spacers were detected in both species. Shared spacer sequences are evidence of a lack of species specificity in the agents and mechanisms responsible for integration of spacers, and the abundance of spacer sequences corresponding to bacterial chromosomal sequences reflects interspecific interactions within the intestinal microbiota.     

Characterization of the Survival Ability of Cupriavidus metallidurans and Ralstonia pickettii from Space-Related Environments

Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C_254nm radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5–4 μM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 μM AgNO₃ in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.     

Expression of cagA,virB/D Complex and/or vacA Genes in Helicobacter pylori Strains Originating from Patients with Gastric Diseases

In order to better understand pathogenicity of Helicobacter pylori, particularly in the context of its carcinogenic activity, we analysed expression of virulence genes: cagA, virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, virD4) and vacA in strains of the pathogen originating from persons with gastric diseases. The studies were conducted on 42 strains of H. pylori isolated from patients with histological diagnosis of non-atrophic gastritis—NAG (group 1, including subgroup 1 containing cagA⁺ isolates and subgroup 2 containing cagA^- strains), multifocal atrophic gastritis—MAG (group 2) and gastric adenocarcinoma—GC (group 3). Expression of H. pylori genes was studied using microarray technology. In group 1, in all strains of H. pylori cagA⁺ (subgroup 1) high expression of the gene as well as of virB/D was disclosed, accompanied by moderate expression of vacA. In strains of subgroup 2 a moderate expression of vacA was detected. All strains in groups 2 and 3 carried cagA gene but they differed in its expression: a high expression was detected in isolates of group 2 and its hyperexpression in strains of group 3 (hypervirulent strains). In both groups high expression of virB/D and vacA was disclosed. Our results indicate that chronic active gastritis may be induced by both cagA⁺ strains of H. pylori, manifesting high expression of virB/D complex but moderate activity of vacA, and cagA^- strains with moderate expression of vacA gene. On the other hand, in progression of gastric pathology and carcinogenesis linked to H. pylori a significant role was played by hypervirulent strains, manifesting a very high expression of cagA and high activity of virB/D and vacA genes.     

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.     

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.     

A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens

Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674^T and S. equorum subsp. linens DSM 15097^T, respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA–DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.     

Multidrug resistance and high virulence genotype in uropathogenic Escherichia coli due to diffusion of ST131 clonal group producing CTX-M-15: an emerging problem in a Tunisian hospital

A collection of 201 Escherichia coli strains isolated from urine of patients in a Tunisian hospital between January 2006 and July 2008 was studied. Microbial identification was done by conventional methods, and antibiotic susceptibility with disk diffusion method was performed according to the Clinical Laboratory and Standards Institute guidelines. Detection of extended-spectrum beta-lactamase (ESBL) was performed by double-disk synergy test (DDST) and identification was done by PCR and sequencing. ESBL-producing isolates were subjected to molecular typing by random amplified polymorphic DNA (RAPD) and ST131 detection by PCR. Four phylogenetic groups (A, B1, B2 and D), 18 virulence genes and CTX-M group were individualized using PCR. Statistical analysis was done by Pearson χ2 test and Mann–Whitney U test. The strains were recovered primarily from urology (28 %), maternity (19 %) and medicine (16 %) wards. Antibiotic resistance rates were ampicilin (72.1 %), nalidixic acid (41.8 %), ciprofloxacin (38.8 %), gentamicin (23.9 %) and cefotaxime (17.4 %). Thirty-one of cefotaxime-resistant isolates (n?=?35) had a positive DDST and harboured bla _CTX-M-15 gene. Twenty of them (64.5 %) belonged to the ST131 clone and showed the same RAPD DNA profile. Ciprofloxacin- and cotrimoxazole-susceptible isolates were significantly associated with phylogenetic group B2, whereas isolates that were resistant to these molecules were associated with B1 and D phylogenetic groups, respectively. Virulence genes were significantly more frequent among ciprofloxacin- and cotrimoxazole-susceptible strains than those resistant to these antibiotics. However, CXT-M-15-producing isolates were associated with many virulence genes. Isolates concomitantly susceptible to the three antimicrobials agents (ciprofloxacin, cefotaxime and cotrimoxazole) were significantly associated with group B2 and high virulence score, whereas isolates with resistance patterns especially those including resistance to ciprofloxacin belonged predominantly to B1 phylogroup and haboured few virulence genes. The emergence of virulent and multidrug-resistant E. coli is a concerning development that deserves close attention in our institution.     

Comparative Analysis of the Orphan CRISPR2 Locus in 242 Enterococcus faecalis Strains

Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.     

The Diversity and Antimicrobial Activity of Preussia sp. Endophytes Isolated from Australian Dry Rainforests

Limited knowledge currently exists regarding species diversity and antimicrobial activity of endophytic isolates of Preussia within Australia. This report describes endophytic Preussia species that were identified through molecular analysis of the internal transcribed spacer region. Screening for antimicrobial secondary metabolites was determined by testing crude ethyl acetate (EtOAc) extracts derived from fungal mycelia against a panel of ATCC type strains which included Bacillus cereus, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Serratia marcescens, methicillin-resistant Staphylococcus aureus (MRSA) and the opportunist yeast pathogen Candida albicans. Subsequently, high-performance liquid chromatography generated fractions of bioactive EtOAc extracts which were subject to confirmatory testing using the Clinical and Laboratory Standards Institute reference microdilution antimicrobial activity assay. A total of 18 Preussia were isolated from nine host plants with 6/18 having a <97 % sequence similarity to other known species in Genbank, suggesting that they are new species. In preliminary screening, 13/18 Preussia isolates revealed antimicrobial activity against at least one of the microbes tested, whilst 6/18 isolates, including 4/6 putative new species showed specific antimicrobial activity against MRSA and C. albicans. These results highlight the antimicrobial potential of Australian Preussia spp. and also the importance of Australian dry rainforests as an untapped repository of potentially significant bioactive compounds.     

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.     

Safety assessment of commensal enterococci from dogs

Enterococci form a complex, diverse, and very important group of bacteria from the technological and food safety aspect, or from the health-improving aspect as probiotics. Generally, enterococci are considered to be of low pathogenic potential, which is associated mostly with clinical strains. In these strains, production of virulence factors as well as resistance to many antimicrobial drugs could complicate treatment of nosocomial infections. Because there is a lack of information on incidence of these attributes in animal commensal enterococci, we screened 160 strains originating from feces of clinically healthy dogs in Eastern Slovakia (n = 105). The predominant species were Enterococcus faecium (57.5%) followed by Enterococcus faecalis (21.9%), and Enterococcus hirae (17.5%), while Enterococcus casseliflavus (1.9%) and Enterococcus mundtii (1.2%) rarely occurred. Among the tested antibiotics, gentamicin (high level) was the most effective drug against canine enterococci (95% of isolates were sensitive). In contrast, the highest resistance recorded (71.9%) was to teicoplanin. PCR screening showed the highest incidence of virulence genes in E. faecalis species. The most frequently detected were genes encoding adhesins efa _Afm and efa _Afs and sex pheromone cpd. IS16 gene, a marker specific for hospital strains, appeared in nine E. faecium strains. No strain was positive for DNase activity, 8.8% of the isolated strains showed gelatinase activity, and almost 100% strains produced tyramine. It seems commensal-derived enterococci from dogs could also to some extent be potential reservoir of risk factors for other microbiota or organisms.     

Aureobasidium pullulans as a source of liamocins (heavy oils) with anticancer activity

Liamocins are structurally unique, heavier-than-water “oils” produced by certain strains of Aureobasidium pullulans. The aim of the current study is to identify new sources of liamocins and evaluate their potential as anticancer agents. Nine strains of A. pullulans from phylogenetic clades 8, 9, and 11 were examined for the first time for production of liamocins. Strains in these clades have only been isolated from tropical environments, and all strains tested here were from various locations in Thailand. Strains RSU 9, RSU 21, and RSU 29, all from clade 11, produced from 7.0 to 8.6 g liamocins/l from medium containing 5 % sucrose. These are the highest yields of liamocins that we have found thus far. These strains also produced from 9.4 to 17 g pullulan/l. The structural identity of liamocins was confirmed by matrix-assisted laser desorption/ionization mass spectrometry; differential spectra were obtained in which the dominant ion was either at about m/z 805.5 or m/z 949.6, consistent with the structure of liamocins. Liamocins from A. pullulans strains RSU 9 and RSU 21 inhibited two human breast cancer cell lines and a human cervical cancer cell line (IC₅₀ values of 32.2 ± 1.4 to 63.1 ± 2.4 μg liamocins/ml) but were not toxic to a normal cell line. Liamocins weakly inhibited a strain of Enterococcus faecalis, but did not inhibit strains of Lactobacillus fermentum, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Thus, A. pullulans phylogenetic clade 11 is a promising source of liamocins, and these compounds merit further examination as potential anticancer agents.