B2 Strains Research Articles

Endophytes in the plant Huperzia serrata: fungal diversity and discovery of a new pentapeptide.

Endophytic fungi are an underexploited resource of natural products and have a capacity to produce diverse classes of plant-derived secondary metabolites. Here, we investigated the diversity of endophytic fungi from Huperzia serrata and the potential for discovering novel fungal natural products. One hundred and fifty-five endophytic fungi isolates obtained from H. serrata, belonging to four classes Dothideomycetes (47.3%), Sordariomycetes (36.8%), Eurotiomycetes (10.6%) and an undefined class (5.3%, Mucoraceae), were grouped into nine genera based on morphological and molecular identification. Colletotrichum, Cladosporium, Sordariomycetes and Guignardia were the dominant genera, whereas the remaining genera were infrequent groups. To our knowledge, the fungal genera Mucor and Neurospora were first reported in Huperzia plant. Among these endophytic fungi isolates, strain B14, belonging to Penicillium oxalicum, gave a gray precipitate with Dragendorff's reagent. A new pentapeptide was isolated from the culture of strain B14, and its chemical structure was elucidated on the basis of spectroscopic data from (1)H NMR, (13)C NMR and ESI-MS/MS. Taken together, H. serrata has a significant diversity of endophytic fungi that could be a rich resource for the discovery of new natural products.

Escherichia coli strains of phylogenetic group B2 and D and bacteriocin production are associated with advanced colorectal neoplasia.

BackgroundColorectal cancer (CRC) is the 3rd most common cancer worldwide and the Czech Republic has the 6th highest incidence of CRC worldwide. Large intestinal microbiota play in its etiopathogenesis important role. Bacteriocins are proteins, produced by bacteria from the Enterobacteriaceae family. The aim of our prospective study was to assess the colonization of large intestinal mucosa by Escherichia coli strains and to investigate their bacteriocin production.MethodsA total of 30 consecutive patients with colorectal adenoma, CRA (17 men, 13 women, aged 39–79, mean age 63 ± 9), 30 patients with CRC (23 men, 7 women, aged 38–86, mean age 67 ± 11) and 20 healthy controls (9 men, 11 women, age 23–84, mean age 55 ± 15) were enrolled into prospective study. Mucosal biopsies were taken in the caecum, transverse colon and rectum during pancolonoscopy. Microbiological culture, isolation and identification of bacteria followed. Bacteriocin production was assessed by growth inhibition of indicator strains E. coli K12-Row, E. coli C6 (phi), and Shigella sonnei 17. Identification of bacteriocin-encoding determinants and E. coli phylogroups was performed using PCR methods.ResultsA total of 622 strains were isolated and further investigated. A significantly higher frequency of simultaneous production of colicins and microcins was revealed in the group of patients with CRC, when compared to patients with CRA, p = 0.031. A significantly higher frequency of E. coli phylogroup D was found in patients with CRC, when compared to controls, p = 0.044. A significantly higher prevalence of bacteriocinogeny was confirmed in patients with advanced adenoma when compared to patients with non-advanced adenoma, p = 0.010. Increasing bacteriocinogeny was associated with an increasing stage of CRC (assessed according to TNM classification). Either E. coli phylogroup B2 or E. coli phylogroup D were isolated in biopsies of patients with right-sided CRC. A statistically higher incidence of E. coli phylogroup B2 was found in patients with right-sided CRC when compared to patients with left-sided CRC, p = 0.028.ConclusionsLarge intestinal mucosa of patients with more advanced colorectal neoplasia is colonized with more virulent strains of E. coli and higher production of bacteriocins is observed in these patients when compared to those with less advanced colorectal neoplasia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0733-7) contains supplementary material, which is available to authorized users.

The conserved nhaAR operon is drastically divergent between B2 and non-B2 Escherichia coli and is involved in extra-intestinal virulence.

The Escherichia coli species is divided in phylogenetic groups that differ in their virulence and commensal distribution. Strains belonging to the B2 group are involved in extra-intestinal pathologies but also appear to be more prevalent as commensals among human occidental populations. To investigate the genetic specificities of B2 sub-group, we used 128 sequenced genomes and identified genes of the core genome that showed marked difference between B2 and non-B2 genomes. We focused on the gene and its surrounding region with the strongest divergence between B2 and non-B2, the antiporter gene nhaA. This gene is part of the nhaAR operon, which is in the core genome but flanked by mobile regions, and is involved in growth at high pH and high sodium concentrations. Consistently, we found that a panel of non-B2 strains grew faster than B2 at high pH and high sodium concentrations. However, we could not identify differences in expression of the nhaAR operon using fluorescence reporter plasmids. Furthermore, the operon deletion had no differential impact between B2 and non-B2 strains, and did not result in a fitness modification in a murine model of gut colonization. Nevertheless, sequence analysis and experiments in a murine model of septicemia revealed that recombination in nhaA among B2 strains was observed in strains with low virulence. Finally, nhaA and nhaAR operon deletions drastically decreased virulence in one B2 strain. This effect of nhaAR deletion appeared to be stronger than deletion of all pathogenicity islands. Thus, a population genetic approach allowed us to identify an operon in the core genome without strong effect in commensalism but with an important role in extra-intestinal virulence, a landmark of the B2 strains.

Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain pathoadaptive mutations in a subset of extraintestinal E. coli strains.

BackgroundAnalysis of the Escherichia coli collection of reference strains (ECOR) for the presence of the gene locus clyA, which encodes the pore-forming protein ClyA (cytolysin A), revealed that a non-functional clyA locus is common among certain extraintestinal pathogenic E. coli (ExPEC). In fact, all 15 ECOR group B2 strains and several additionally examined extraintestinal pathogenic (uropathogenic (UPEC) and neonatal meningitis (NBM)) E. coli strains contained various ΔclyA alleles.ResultsThere are at least four different variants of ΔclyA, suggesting that such deletions in clyA have arisen at more than one occasion. On the basis of this occurrence of the truncated clyA genes, we considered that there may be a patho-adaptive selection for deletions in clyA in extraintestinal pathogenic E. coli. In E. coli K-12 the clyA gene has been viewed as “cryptic” since it is tightly silenced by the nucleoid structuring protein H-NS. We constructed a restored clyA+ locus in derivatives of the UPEC strain 536 for further investigation of this hypothesis and, in particular, how the gene would be expressed. Our results show that the level of clyA+ expression is highly increased in the UPEC derivatives in comparison with the non-pathogenic E. coli K-12. Transcription of the clyA+ gene was induced to even higher levels when the SfaX regulatory protein was overproduced. The derivative with a restored clyA+ locus displayed a somewhat slower growth than the parental UPEC strain 536 when a sub-inhibitory concentration of the antimicrobial peptide Polymyxin B was added to the growth medium.ConclusionsTaken together, our findings show that the clyA+ locus is expressed at an elevated level in the UPEC strain and we conclude that this is at least in part due to the effect of the SfaX/PapX transcriptional regulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0216-4) contains supplementary material, which is available to authorized users.

Correlation between the genomic o454-nlpD region polymorphisms, virulence gene equipment and phylogenetic group of extraintestinal Escherichia coli (ExPEC) enables pathotyping irrespective of host, disease and source of isolation.

BackgroundThe mutS-rpoS intergenic region in E. coli displays a mosaic structure which revealed pathotype specific patterns. To assess the importance of this region as a surrogate marker for the identification of highly virulent extraintestinal pathogenic E. coli (ExPEC) strains we aimed to: (i) characterize the genetic diversity of the mutS gene and the o454-nlpD genomic region among 510 E. coli strains from animals and humans; (ii) delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) identify the most important VAGs for classification of the o454-nlpD region.MethodsSize variation in the o454-nlpD region was investigated by PCR amplification and sequencing. Phylogenetic relationships were assessed by Ecor- and Multilocus sequence- typing (MLST), and a comparative analysis between mutS gene phylogenetic tree obtained with RAxML and the MLST grouping method was performed. Correlation between o454-nlpD patterns and the features described above were analysed. In addition, the importance of 47 PCR-amplified ExPEC-related VAGs for classification of o454-nlpD patterns was investigated by means of Random Forest algorithm.ResultsFour main structures (patterns I-IV) of the o454-nlpD region among ExPEC and commensal E. coli strains were identified. Statistical analysis showed a positive and exclusive association between pattern III and the ExPEC strains. A strong association between pattern III and either the Ecor group B2 or the sequence type complexes known to represent the phylogenetic background of highly virulent ExPEC strains (such as STC95, STC73 and STC131) was found as well. RF analyses determined five genes (csgA, malX, chuA, sit, and vat) to be suitable to predict pattern III strains.ConclusionThe significant association between pattern III and group B2 strains suggested the o454-nlpD region to be of great value in identifying highly virulent strains among the mixed population of E. coli promising to be the basis of a future typing tool for ExPEC and their gut reservoir. Furthermore, top-ranked VAGs for classification and prediction of pattern III were identified. These data are most valuable for defining ExPEC pathotype in future in vivo assays.

Effect of azithromycin on enhancement of methane production from waste activated sludge.

In the methane production from waste activated sludge (WAS), complex bacterial interactions in WAS have been known as a major contribution to methane production. Therefore, the influence of bacterial community changes toward methane production from WAS was investigated by an application of antibiotics as a simple means for it. In this study, azithromycin (Azm) as an antibiotic was mainly used to observe the effect on microbial changes that influence methane production from WAS. The results showed that at the end of fermentation, Azm enhanced methane production about twofold compared to control. Azm fostered the growth of acid-producing bacterial communities, which synthesized more precursors for methane formation. DGGE result showed that the hydrolysis as well as acetogenesis stage was improved by the dominant of B1, B2 and B3 strains, which are Clostridium species. In the presence of Azm, the total population of archaeal group was increased, resulting in higher methane productivity achievement.

Distribución de grupos filogenéticos y factores de virulencia en cepas de Escherichia coli uropatógena productora de β-lactamasa CTX-M-15 aisladas de pacientes de la comunidad en Mérida, Venezuela

In this study, the distribution of phylogenetic groups and the genetic detection of virulence factors in CTX-M-15 β-lactamase-producing uropathogenic Escherichia coli (UPEC) strains were analyzed. Twenty eight strains were isolated between January 2009 and July 2011 from patients with urinary tract infection (UTI) who attended the Public Health Laboratory at Mérida, Venezuela. Determination of phylogenetic groups and detection of six virulence genes, fimH, fyuA, kpsMTII, usp, PAI and papAH, were performed by PCR amplification. Fifteen of the 28 isolates were mainly located in the phylogenetic group A, followed by B2 (12/28) and D (1/28). No direct relationship between the severity or recurrence of UTI and the distribution of phylogroups was observed. All studied virulence factors were found in group B2 strains with the highest frequency. The prevalent virulence profile included the combination of three main genes: fimH, kpsMTII and fyuA and, to a lesser extent, the presence of other determinants such as usp, PAI and/or papAH. These results indicate that virulent UPEC incorporated three important properties: adhesion, iron uptake and evasion of phagocytosis, which favored the production of recurrent UTI. This is the first report describing the association of phylogenetic groups with the potential virulence of CTX-M-15 β-lactamase producing UPEC strains in Venezuela.

Mo1141 Association of Right-Sided Colorectal Cancer and Ulcerative Colitis With Escherichia coli Strains of Phylogenetic Group B2, Which Harbour Genotoxic “PKS” Island

Mo1141 Association of Right-Sided Colorectal Cancer and Ulcerative Colitis With Escherichia coli Strains of Phylogenetic Group B2, Which Harbour Genotoxic “PKS” Island

Tu1760 Functional Evidence for a Causative Role of the Intestinal Microbiota in Crohn's Disease-Like Ileitis Using Antibiotic-Treated and Germ-Free TNFDeltaare/+ Mice

BACKGROUND: An increased prevalence of phylogenetic B2 group Escherichia coli (E. coli) has been associated with active disease in IBD patients (IBDEC). Many of the B2 group strains harbor virulence genes present in extraintestinal pathogenic E.coli including hemolysin (hly) and cytotoxic necrotizing factor type 1 (CNF1) the loci of which is often linked. Here we investigated the potential of B2 strains of E.coli isolated from IBD patients to damage the integrity of the intestinal epithelium and the potential contribution of Hly and CNF1. METHODS: E coli strains isolated from 35 patients with IBD were tested for virulence (Petersen et al 2009) and of those isolate C691-04A showed high cytotoxicity on dendritic cells ( submitted) and a rapidly decrease of the trans-epithelial electric resistance (TEER) of a monolayer of intestinal epithelial cell lines. Genome sequencing of C691-04A confirmed the presence of hly cluster. It is known that some E coli have two hly-clusters. In order to investigate the role of hly and CNF1 in disruption of the epithelial integrity the following mutants were created in strain C691-04A; 1: CAS965, hly cluster II upstream of CNF1; 2: CAS954, hly cluster I mutant; 3: CAS956 is a knock out of both loci of hly; 4: CAS964, CNF1 mutant; 5: CAS966, clbA mutant. Caco-2 cells were grown to confluence in 24-well trans-well devices for 14 days to form a polarized cell monolayer and TER measured constantly during exposure to E. coli strains at MOI 10. The wild type and commensal E. coli were included as controls. RESULTS: E. coli C691-04A WT as well as CNF1, clbA and single loci of hly mutants from cluster I and II were all able to damage Caco-2 cell tight junctions in less than 6 hrs co-culture (see figure). Confocal microscopy of occludin stained trans-well sections revealed loss of occludin from the tight junction within 3 hours. However, this phenotype was lost in a mutant with knock out of both hly loci (p<0.001). Commensal E. coli isolated from healthy controls also did not cause loss of TER in 6 h. CONCLUSION: IBD-associated E. coli such as C691-04A can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect is abolished in a mutant with knock out of both loci of the hly genes and diminished in one of the single knockout mutants. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.

Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23

An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 °C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladAαB23, ladAβB23, and ladB B23, on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladAαB23, ladAβB23, and ladB B23, was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2Δ1. It was found that all three genes were functional in P. fluorescens KOB2Δ1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes.

CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence?

To discern the relevance of ST648 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli as a putative new group of multiresistant and extraintestinal pathogenic strains in animals, its frequency, ESBL types, antimicrobial resistance patterns and virulence gene (VG) profiles should be determined and compared with ST131 strains from the same collection of strains. ESBL-producing E. coli isolates (n = 1152), consecutively sampled from predominantly dogs, cats and horses between 2008 and 2011, were assigned to a phylogenetic group by PCR. Partial multilocus sequence typing was performed for group D and B2 strains and strains presumed to be D-ST648 and B2-ST131 were fully typed. ESBL genes and extraintestinal pathogenic E. coli (ExPEC)-like VGs were characterized by PCR and sequence analysis and antimicrobial resistance was determined by broth dilution. Clonal analysis was done by PFGE. Forty (3.5%) ESBL-producing E. coli were determined as D-ST648, whereas B2-ST131 isolates occurred less frequently (2.8%). Although the predominant ESBL type in both groups was CTX-M-15 (72.5% versus 46.9%), ST648 strains from companion animals and horses displayed a lower variety of ESBL types (CTX-M-1, -3, -14, -15 and -61 versus CTX-M-1,-2,-14,-15,-27 and -55 and SHV-12). In contrast to ST131 strains, a higher proportion of ST648 strains showed resistance to most non-β-lactam antibiotics. Overall, VGs were less abundant in ST648 strains, although some strains had VG profiles comparable to those of ST131 strains. ExPEC-associated serotype O1:H6 was predominant (46.8%) among the ST648 strains. Some PFGE clusters comprised ST648 isolates from pets, horses and wild birds and humans included from previous studies. Our findings demonstrate that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential.

Growth temperature of different local isolates of Bacillus sp. in the solid state affects production of raw starch digesting amylases

Natural amylase producers, wild type strains of Bacillus sp., were isolated from different regions of Serbia. Strains with the highest amylase activity based on the starch-agar plate test were grown on solid-state fermentation (SSF) on triticale. The influence of the substrate and different cultivation temperature (28 and 37?C) on the production of amylase was examined. The tested strains produced ?-amylases when grown on triticale grains both at 28 and at 37?C, but the activity of amylases and the number and intensity of the produced isoforms were different. Significant hydrolysis of raw cornstarch was obtained with the Bacillus sp. strains 2B, 5B, 18 and 24B. The produced ?-amylases hydrolyzed raw cornstarch at a temperature below the temperature of gelatinization, but the ability for hydrolysis was not directly related to the total enzyme activity, suggesting that only certain isoforms are involved in the hydrolysis.

Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India

Human metapneumovirus (HMPV) is an important respiratory virus implicated in respiratory infections. The purpose of this study was to develop a one-step real-time RT-PCR assay that can detect all four lineages of HMPV and to identify the HMPV lineages circulating in Pune, India. Conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. A total of 224 clinical samples that were positive for different respiratory viruses (including 51 samples that were positive for HMPV) were tested using the real time RT-PCR assay, and the specificity of the assay was observed to be 100 %. Using in vitro-synthesized RNA, the sensitivity of the assay was ascertained to be 100 copies of the target gene per reaction. Phylogenetic analysis of the nucleoprotein (N) and attachment glycoprotein (G) genes confirmed that this assay detected all lineages of HMPV. A2, B1 and B2 strains were observed during the study period. Our assay is highly sensitive and specific for all known lineages of HMPV, making it a valuable tool for rapid detection of the virus. A2 and B2 were the predominant subtypes circulating in Pune, Western India.

Identification and characterization of foodborne pathogen Listeria monocytogenes strain Pyde1 and Pyde2 using 16S rRNA gene sequencing

AimListeria monocytogenes acts as a pathogen for humans and animals, mainly causing, neonatal sepsis, abortions in pregnant females and severe infections such as septicemia and meningoencephalitis in susceptible hosts. Current study was aimed to identify novel strains of L. monocytogenes from retail chicken, beef meat and seafood samples. MethodsIn order to identify the strain, extraction and amplification of genomic DNA, 16S rRNA sequence analysis was carried out. Phylogenetic trees were constructed using dnapars and dnaml available in Phylip. The secondary structures of 16S rRNA gene sequence were predicted using UNAFOLD, a Linux based software. ResultsThe results obtained were found to be a novel foodborne pathogens, which was further named L. monocytogenes strain Pyde1 and L. monocytogenes strain Pyde2, after characterization the sequence of isolate was deposited in GenBank with accession numbers ‘KC852899’ and ‘KC852900’ respectively. The Gibb's free energy of the secondary structures of L. monocytogenes strain Pyde1 and Pyde2 were −275.60 and −282.20 kcal/mol seems to be more stable in the present investigation. ConclusionThe described results of phylogenetic distinctiveness and phenotypic disparities indicate that strain 2b represents a novel strain of foodborne pathogens within L. monocytogenes species, for which the name L. monocytogenes strain Pyde1 and L. monocytogenes strain Pyde2 is proposed.

Characterization of an industrially important alkalophilic bacterium, Bacillus agaradhaerens strain nandiniphanse5

AimThe phenotypic and phylogenetic characterization of an alkalophilic bacterium producing two industrially important enzymes – alkaline protease and alkaline amylase, was performed. A Gram-positive, rod-shaped, spore-forming alkalophilic bacterium (2b) isolated from alkaline dairy soil of Indore region of Madhya Pradesh, was selected in this work. MethodsThe alkalophile was subjected to a taxonomic investigation, including standard bacteriological characterization and 16S rRNA gene sequencing. Phylogenetic trees were constructed by different treeing algorithms: neighbour joining, maximum parsimony tree, maximum-likelihood and UPGMA method using MEGA5 software. The secondary structures of 16S rRNA gene sequence were predicted using GeneBee package and UNAFOLD, a Linux based software. ResultsIts overall biochemical, physiological and phylogenetic characteristics indicated that strain 2b is an alkaliphilic Bacillus belonging to the species agaradhaerens. The free energy of the secondary structure of rRNA was −171.7 and −265.13 kcal/mol respectively. The structure obtained with a Gibb's free energy, ΔG −265.13 kcal/mol by UNAFOLD seems to be more stable in the present study. ConclusionThe taxonomic investigation identifies 2b as Bacillus agaradhaerens.

Phylogenetic association of fluoroquinolone and cephalosporin resistance of D-O1-ST648 Escherichia coli carrying bla CMY-2 from faecal samples of dogs in Japan

This study aimed to investigate the genetic association between fluoroquinolone (FQ) and/or cephalosporin (CEP) resistance in Escherichia coli isolates from dogs, and the risk to human health. We characterized E. coli clinical isolates, derived from faecal samples of dogs attending veterinary hospitals, using phylogenetic grouping, determination of virulence factor (VF) prevalence, multilocus sequence typing (MLST) and O serotyping. The D group was the dominant phylogenetic group among strains resistant to FQ and/or CEP. In contrast, the dominant group among susceptible strains was group B2. Group D strains showed a significantly higher prevalence of VFs than strains belonging to groups A and B1, and were resistant to significantly more antimicrobials than group B2 strains. The phylogenetic distribution of FQ-CEP-resistant E. coli groups (FQ-CEPRECs) and FQ-resistant groups was significantly correlated (r = 0.98), but FQ-CEPRECs and CEP-resistant E. coli groups were not correlated (r = 0.58). Data from PFGE, O serotype and MLST analyses indicated that the majority of FQ-resistant strains derived from a particular lineage of phylogenetic group D: serotype O1 and sequence type (ST) 648. Some D-O1-ST648 strains carried blaCMY-2, showed multidrug resistance and possessed a higher prevalence of the VFs kspMT, ompT and PAI compared with other group D strains. Our data indicate that the emergence of FQ-CEP-resistant E. coli is based primarily on FQ-resistant E. coli. Moreover, as strains of the D-O1-ST648 lineage have been found in clinical isolates derived from humans at a relatively high frequency, our findings indicate that the spreading of D-O1-ST648 strains may cause serious difficulties in both veterinary and human clinical fields in the future.

Temporal variations in the concentrations of bacteria and their lytic phages: an example of an indigenous phage host system in Lake Plußsee, Germany

1. We describe the abundance of an autochthonous alphaproteobacteria Sphingomonas sp. and its lytic phages during a period of about 3 months in Lake Plussee, Northern Germany. This is the first report of a specific autochthonous phage host system of a heterotrophic bacterial strain in a natural freshwater environment. 2. The concentration of bacterial hosts (Sphingomonas sp. strain B18) fluctuated between 20 and 1,150 (median 168) colony-forming units (CFU) mL-1 and the concentration of the specific lytic phages ranged from 20 to 680 (median 110) plaque-forming units (PFU) mL-1. Different Sphingomonas sp. strains were isolated, showing different sensitivity against two phage isolates. The specific virus to bacteria ratio (VBR) varied by a factor of about 280 and ranged from 0.03 to about 8.5 (median 0.6). 3. Encounter rates of 0.0007 to 0.198 d-1 (median 0.03 d-1) were calculated from the measured concentrations of Sphingomonas sp. and its lytic phages. From changes of PFU between two consecutive samplings net phage decay rates in a range between 0.003 and 1.3 d-1 (median 0.26 d-1) were calculated. 4. Concentration factors were calculated which enable sufficient encounter rates to explain the concentrations of bacteria and their phages. 5. The results of Kokjohn et al. (1991) support the suggestion that the explanation of the coexistence of lytic phages and their host bacteria by the used encounter rate model needs to be revised.

Isolation and virulence of entomopathogenic fungi against larvae of hazelnut weevil Curculio nucum (Coleoptera, Curculionidae) and the effects of combining Metarhizium anisopliae with entomopathogenic nematodes in the laboratory

An approach to ensure effective pest biocontrol would be to select native isolates of biological control agents (BCAs). A survey to isolate entomopathogenic fungi (EPF) from a hazelnut growing area has been carried out. EPF were recovered from 133 of the 295 soil samples. The main species isolated were Metarhizium anisopliae sensu lato (36%) and Beauveria bassiana sensu lato (42.8%). With regard to controlling abiotic factors, altitude had an effect on the distribution of B. bassiana, but not on M. anisopliae. Cropping system did not have an effect on the occurrence of these EPF, while pH appeared as a predictive variable for both. In addition, we tested the virulence of six of these isolates: three M. anisopliae and three of B. bassiana against larvae of Curculio nucum L. The highest larval mortality (reaching 80%) was due to M. anisopliae (strain 34) when applied in simultaneous combination with four entomopathogenic nematode species: Steinernema carpocapsae (strain B14), Steinernema feltiae (strain D114), Steinernema. sp. (strain D122) and Heterorhabditis bacteriophora (strain DG46). The effect of nematodes was greater and no antagonistic or synergistic effects were observed.

Zoonotic potential of multidrug-resistant extraintestinal pathogenic Escherichia coli obtained from healthy poultry carcasses in Salvador, Brazil

The zoonotic potential to cause human and/or animal infections among multidrug-resistant extraintestinal pathogenic Escherichia coli from avian origin was investigated. Twenty-seven extraintestinal pathogenic E. coli isolates containing the increased survival gene (iss) were obtained from the livers of healthy and diseased poultry carcasses at two slaughterhouses in Salvador, northeastern Brazil. The antimicrobial resistance-susceptibility profiles were conducted with antibiotics of avian and/or human use by the standardized disc-diffusion method. Antimicrobial resistance was higher for levofloxacin (51.8%), amoxicillin/clavulanic acid (70.4%), ampicillin (81.5%), cefalotin (88.8%), tetracycline (100%) and streptomycin (100%). The minimum inhibitory concentrations above the resistance breakpoints of doxycycline, neomycin, oxytetracycline and enrofloxacin reached, respectively, 88.0%, 100%, 75% and 91.7% of the isolates. Strains with high and low antimicrobial resistance were i.p. administered to Swiss mice, and histopathological examination was carried out seven days after infection. Resistance to goat and human serum complement was also evaluated. The results show that Swiss mice challenged with strain 2B (resistant to 11 antimicrobials) provoked a severe degeneration of hepatocytes besides lymphocytic infiltration in the liver, whereas the spleen showed areas of degeneration of the white and red pulp. Conversely, the spleen and liver of mice challenged with strain 4A (resistant to two antimicrobials) were morphologically preserved. In addition, complement resistance to goat and human serum was high for strain 2B and low for strain 4A. Our data show that multidrug resistance and pathogenesis can be correlated in extraintestinal pathogenic E. coli strains obtained from apparently healthy poultry carcasses, increasing the risk for human public healthy.

Molecular Epidemiology of Human Metapneumovirus from 2005 to 2011 in Fukui, Japan

To investigate the molecular epidemiology of human metapneumovirus (HMPV) infections in acute respiratory infections (ARI), we performed genetic analysis of the F gene in HMPV from patients with ARI in Fukui Prefecture from August 2005 to July 2011. HMPV was detected in 53 of 741 nasopharyngeal swabs (7.2%). Phylogenetic analysis helped us assign 31 strains to subgroup A2, 1 strain to subgroup B1, and 21 strains to subgroup B2. The prevalence of HMPV was peaked between January and June. A high degree of nucleotide identity was seen among subgroup A2 strains (95.6-100%) and subgroup B2 strains (97.5-100%). In addition, no positively selected sites (substitutions) were found in the F gene in these HMPV strains. The results suggest that the prevalent HMPV strains in Fukui were associated with various ARI in Japan during the investigation period.