Muddy puddles - the microbiology of puddles located outside tertiary university teaching hospitals.

In the British Isles, the frequency of rain results in the formation of puddles on footpaths and roads in/around hospitals. No data are available demonstrating the microbiological composition of such puddles and therefore a study was undertaken to examine the microbiology of puddles in the grounds of two tertiary university-teaching hospitals (18 sites) and compared with control puddles from non-hospital rural environments (eight sites), estimating (i) total viable count; (ii) identification of organisms in puddles; (iii) enumeration of Escherichia coli: (iv) detection of Extended Spectrum ß-Lactamase producing organisms and (v) direct antimicrobial susceptibility testing. A mean count of 2·3 × 103  CFU per ml and 1·0 × 109  CFU per ml was obtained for hospital and non-hospital puddles respectively. Isolates (n = 77; 54 hospital and 23 non-hospital) were isolated comprising of 23 species among 17 genera (hospital sites), where the majority (10/16; 62·5%) of genera identified were Gram-negative approximately, a fifth (20·6%) were shared by hospital and non-hospital rural samples. Escherichia coli was detected in half of the hospital puddles and under-half (37·5%) of the rural puddles extended spectrum ß-lactamase organisms were not detected in any samples examined. Rainwater puddles from the hospital and non-hospital environments contain a diverse range of bacteria, which are capable of causing infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the presence of a wide diversity of bacterial taxa associated with rainwater puddles around hospitals, many of which are capable of causing human disease. Of clinical significance is the presence of Pseudomonas aeruginosa isolated from a hospital puddle, particularly for patients with cystic fibrosis. The presence of potentially disease-causing bacteria in puddles in and around hospitals identifies a new potential environmental reservoir of bacteria. Furthermore work is now needed to define their potential of entering or exiting hospital wards by contaminated footwear.

Campylobacter and Salmonella are prevalent in broiler farms in Kyushu, Japan: results of a 2-year distribution and circulation dynamics audit.

AIM: To elucidate the distribution and circulation dynamics of Campylobacter and Salmonella in Japanese chicken broiler flocks. METHODS AND RESULTS: A 2-year investigation of the distribution of Campylobacter and Salmonella was conducted in 25 broiler flocks at nine farms in Japan from 2013 to 2014. Campylobacter and Salmonella tested positive in 11 (44·0%) and 24 (96·0%) broiler flocks respectively. One hundred and ninety-five Campylobacter and 184 Salmonella isolates were characterized into 12 Campylobacter (including two novel genotypes) and three Salmonella MLST genotypes. Only Salmonella isolation between caecal and environmental samples were significantly correlated. Further, one litter sample tested positive for Salmonella before new chicks were introduced. The Campylobacter strains rapidly lost culturability within 2-18 days; in contrast, the Salmonella strains survived from 64-211 days in artificially inoculated water samples. CONCLUSION: No persistent circulation-mediated Campylobacter contamination was observed. In contrast, circulation of Salmonella in broiler houses was seen, apparently due to the litter excreted from broiler flocks, as well as Salmonella-contaminated water and feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the distribution, genotypic data and circulation dynamics of Campylobacter and Salmonella as recently observed in Japanese chicken broiler farms.

Molecular characterisation of a type III restriction-modification system in Campylobacter upsaliensis.

Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.

Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model.

Iron overload of a chronic nature has been associated with a wide variety of human diseases, including infection, carcinogenesis, and atherosclerosis. Recently, a highly specific turn-on fluorescent probe (RhoNox-1) specific to labile ferrous iron [Fe(II)], but not to labile ferric iron [Fe(III)], was developed. The evaluation of Fe(II) is more important than Fe(III) in vivo in that Fe(II) is an initiating component of the Fenton reaction. In this study, we applied this probe to frozen sections of an established Fenton reaction-based rat renal carcinogenesis model with an iron chelate, ferric nitrilotriacetate (Fe-NTA), in which catalytic iron induces the Fenton reaction specifically in the renal proximal tubules, presumably after iron reduction. Notably, this probe reacted with Fe(II) but with neither Fe(II)-NTA, Fe(III) nor Fe(III)-NTA in vitro. Prominent red fluorescent color was explicitly observed in and around the lumina of renal proximal tubules 1 h after an intraperitoneal injection of 10-35 mg iron/kg Fe-NTA, which was dose-dependent, according to semiquantitative analysis. The RhoNox-1 signal colocalized with the generation of hydroxyl radicals, as detected by hydroxyphenyl fluorescein (HPF). The results demonstrate the transformation of Fe(III)-NTA to Fe(II) in vivo in the Fe-NTA-induced renal carcinogenesis model. Therefore, this probe would be useful for localizing catalytic Fe(II) in studies using tissues.

Uneven distribution of the luxS gene within the genus Campylobacter.

Polymerase chain reaction (PCR) amplification was performed on 20 isolates of five Campylobacter species using a degenerate primer pair designed in silico to generate a product of the luxS gene or its homologue from Campylobacter organisms. Although the primer pair successfully amplified products of approximately 500 base pairs (bp) with the eight isolates of C. jejuni and C. coli and some of C. upsaliensis and C. fetus, it failed to amplify fragments with all four isolates of C. lari (two urease-negative C. lari; two urease-positive thermophilic campylobacters). When Southern blot hybridisation analysis was carried using the mixed luxS gene fragments prepared from the C. jejuni, C. coli, C. upsaliensis and C. fetus strains as a probe, all C. jejuni, C. coli, C. upsaliensis and C. fetus isolates gave positive signals, but no positive signal was detected with any C. lari isolate. These results clearly indicate that C. jejuni, C. coli, C. upsaliensis and C. fetus carry the luxS gene or its homologue. However, no luxS gene or its homologue was identified to occur in the C. lari genome. Although autoinducer-2 assays were positive in C. jejuni, C. coli, C. upsaliensis and C. fetus isolates, it was negative with all the C. lari isolates examined. In addition, a biofilm formation assay demonstrated that biofilm formation in the C. lari species does not appear to correlate with the occurrence of the luxS gene because biofilm formation occurred among some isolates of C. lari.

Influence of pH of TSI medium on the detection of hydrogen sulfide production by Campylobacter hyointestinalis.

AIMS: To examine the influence of pH of triple sugar iron (TSI) agar medium on the detection of hydrogen sulfide (H(2)S) production in Campylobacter hyointestinalis ssp. hyointestinalis (CHH). METHODS AND RESULTS: TSI medium was adjusted by the addition in HCl or NaOH to cover a pH 6.0-9.0. One loopful of bacterial growth of CHH strain ATCC 35217 was inoculated into each different pH medium, and incubated at 37 degrees C under micro-aerobic conditions. The H(2)S production was not detectable even after incubation for 72 h in acidic medium pH; however, TSI with alkaline pH (8.0-9.0) allowed detection as early as 3 h of incubation. A total of 20 CHH strains from various animal sources were examined for the detection of H(2)S production in TSI medium with pH 9.0. The H(2)S was detected in all the strains examined within 12 h, and the judgment was unambiguous. CONCLUSION: The results showed that the detection of H(2)S production by CHH was influenced by medium pH, and TSI with alkaline condition is highly sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that TSI medium with alkaline pH contributes to fast detection and led to unambiguous judgment of H(2)S production by CHH.

Characterization of Lancefield group C Streptococcus dysgalactiae isolated from farmed fish.

A Lancefield group C streptococcal (GCS) infection caused by Streptococcus dysgalactiae that is characterized by severe necrotic lesions of the caudal peduncle has been an increasing cause of mortality in farmed fish such as amberjack, Seriola dumerili, and yellowtail, Seriola quinqueradiata, in the southern part of Kyushu, Japan. In this study, enzymatic profiles of GCS strains from fish and mammals were investigated using the API ZYM system, and genotypic characterization of GCS strains was performed using biased sinusoidal field gel electrophoresis (BSFGE). The partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish and mammals was also compared. The API ZYM test indicated that it is difficult to differentiate isolates of S. dysgalactiae from fish and animals based on enzymological variations. In the BSFGE analysis, the macrorestriction profiles, which were obtained using SmaI or ApaI as a restriction enzyme, revealed variations between the fish and animal isolates. The partial sequence of the 16S-23S rDNA intergenic spacer region of all the tested fish isolates differed from all mammalian isolates in one or two nucleotides. The possibility of a clonal expansion of S. dysgalactiae strains in farmed fish was also suggested by the BSFGE profiles of fish isolates.

Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan.

A Lancefield serological group C Streptococcus sp. was isolated from cultured amberjack, Seriola dumerili Risso, and yellowtail, Seriola quinqueradiata Temminck and Schlegel, immunized with Lactococcus garvieae commercial vaccines in Japan. The isolated bacteria were Gram-positive cocci, auto-aggregating in saline, morphologically long chains in growth medium, catalase negative and alpha-haemolytic on blood agar. An almost complete gene sequence of the 16S rDNA of two isolates was determined and compared with that of bacterial strains in the database. The isolates were identified as Streptococcus dysgalactiae based on the results of the 16S rDNA sequence, the bacteriological properties and the Lancefield serological grouping. Oligonucleotide primers specifically designed for the 16S-23S rDNA intergenic spacer region of S. dysgalactiae amplified a gene from all the fish isolates, as well as the type strains alpha-haemolytic S. dysgalactiae subsp. dysgalactiae ATCC430738 and beta-haemolytic S. dysgalactiae subsp. equisimilis ATCC35666, but not those of S. equi ATCC33398, Lactococcus garvieae ATCC43921 and L. garvieae KG9408. The severe necrotic lesions of the caudal peduncle seen in experimentally infected fish were similar to those seen in naturally infected fish.

Development of a combined filtration-enrichment culture followed by a one-step duplex PCR technique for the rapid detection of Campylobacter jejuni and C. coli in human faecal samples.

A new combined filtration-enrichment culture followed by a PCR technique for the rapid detection of Campylobacterjejuni and C. coli in human faeces has been developed. Only bacteria that passed through the membrane could multiply in the enrichment culture; target bacteria were detected by a one-step duplex PCR technique with combinations of primers that are specific for different Campylobacter spp., which should allow for the detection of a mixed infection in a single patient. A Falcon cell-culture insert and 24-well tissue-culture plates were used. After 2 days, both C. jejuni and C. coli were reliably detected in diluted faeces that were seeded with as few as 10 cells which corresponds to a concentration of 10(3) cfu/g. Even allowing for the dilution of faecal samples, this represents an increase in sensitivity of two-to-three orders of magnitude over the conventional filtration method.

DNA diversity of the wla gene cluster among serotype HS:19 and non-HS:19 Campylobacter jejuni strains.

Campylobacter jejuni infection is an important trigger of Guillain-Barré syndrome (GBS), and serotype HS:19 strains are over-represented among GBS-associated isolates. Structures in C. jejuni lipooligosaccharide (LOS) resemble human gangliosides, suggesting that molecular mimicry could be important in triggering the neural injury. We assessed the genetic diversity among 36 C. jejuni serotype HS:19 and non-HS:19 strains by analysis of PCR-based restriction fragment length polymorphism (RFLP) patterns of 12 LOS biosynthesis-related genes (wla cluster). PCR amplification revealed that the size, order, and direction of each wla gene was identical among all strains tested. However, an additional ORF, located between wlaI and wlaK, was detected in 28 of the 36 isolates examined, and nucleotide sequence analysis revealed that the gene was identical to orfE in C. jejuni strain NCTC 11168. An inverted repeat motif was found downstream of the wlaI stop codon and upstream of the orfE stop codon, an organization allowing pairing of repeated sequences that could lead to deletion of the internal segment. Digestion of the PCR products with restriction endonuclease DdeI or AluI and cluster analysis of RFLP banding patterns showed that all HS:19 strains were closely related and distinct from non-HS:19 strains, consistent with earlier analyses, suggesting that HS:19 strains represent a highly clonal population. RFLP analysis of wla genes also may be useful for epidemiological studies.

Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus, produces both neurosporene and lycopene.

Biosynthetic pathways for carotenoids in the purple photosynthetic bacterium, Rubrivivax gelatinosus, which synthesizes spirilloxanthin in addition to spheroidene and OH-spheroidene, were investigated by means of genetic manipulation. A phytoene desaturase gene (crtI) found in the photosynthesis gene cluster of this bacterium was expressed in an Escherichia coli strain that can produce phytoene. Both neurosporene and lycopene were synthesized in the recombinant, probably by three- and four-step desaturation reactions of CrtI. A mutant of RVI: gelatinosus lacking the crtI gene produced only phytoene, indicating that this organism had no other phytoene desaturases. When the crtI deletion mutant was complemented by the three-step phytoene desaturase of Rhodobacter capsulatus, spirilloxanthin and its precursors were not synthesized, although spheroidene and OH-spheroidene were accumulated. It was concluded that neurosporene and lycopene are produced by a single phytoene desaturase in RVI: gelatinosus resulting in the synthesis of spheroidene and spirilloxanthin, and that there are no pathways for spirilloxanthin synthesis via spheroidene.

Biotransformation of phenanthrene and 1-methoxynaphthalene with Streptomyces lividans cells expressing a marine bacterial phenanthrene dioxygenase gene cluster.

The phdABCD gene cluster in a marine bacterium Nocardioides sp. strain KP7 codes for the multicomponent enzyme phenanthrene dioxygenase. phdA encoding an iron-sulfur protein large subunit alpha, phdB encoding its small subunit beta, phdC encoding ferredoxin, and phdD encoding ferredoxin reductase, were replaced in such a way that the termination codons of the preceding open reading frames were overlapped with the initiation codons of the following genes. This manipulated phdABCD gene cluster was positioned downstream of the thiostrepton-inducible promoter PtipA in a high-copy-number vector pIJ6021, and introduced into the gram-positive, soil-inhabiting, filamentous bacterium Streptomyces lividans. The recombinant S. lividans cells converted phenanthrene into a cis-diol form, which was determined to be cis-3,4-dihydroxy-3,4-dihydrophenanthrene by its UV spectral data as well as HPLC property, using the authentic sample for comparison. This biotransformation proceeded very efficiently; 200 microM and 2 mm of phenanthrene were almost completely converted to its cis-diol form in 6 h and 32 h, respectively. In addition, the S. lividans cells carrying the phdABCD gene cluster were found to transform 1-methoxynaphthalene to two products, which were identified to be 8-methoxy-2-naphthol in addition to 8-methoxy-1,2-dihydro-1,2-naphthalenediol by their EI-MS, 1H- and 13C-NMR spectral data.

Differentiation of Fusobacterium necrophorum subspecies from bovine pathological lesions by RAPD-PCR.

Nineteen strains from bovine abscesses identified as Fusobacterium necrophorum by the VPI method were examined by other methods. The API 20A test kit characterized all 19 strains as F. necrophorum. Seven of the strains had haemagglutinating activity and were classified as F. necrophorum subspecies necrophorum, and the remaining, 12 nonhaemagglutinating strains, were classified as F. necrophorum subspecies funduliforme. We used RAPD-PCR with a 10-mer oligonucleotide primer, W1L-2, to confirm this differentiation of the two subspecies. These results suggest that random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with a suitable primer can be used as a new tool for the differentiation of F. necrophorum subspecies isolated from bovine pathological lesions.

Immunization against intestinal bacterial endotoxin prevents alcoholic fatty liver in rats.

Accumulating evidences indicate that an endotoxin originating from intestinal gram-negative bacteria may be involved in alcohol-induced liver injury including fatty liver. Therefore, whether immunization against intestinal bacterial endotoxin blocked fatty liver induced by chronic alcohol and diet including much-unsaturated fatty acid was investigated in rats. The titer of antibody against the endotoxin increased significantly after 13 weeks of continuous immunization. Daily alcohol treatment was initiated at 12 weeks and continued for 4 weeks. Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and triglyceride (TG) levels increased significantly in non-immunized rats receiving alcohol, but not in immunized rats. Continuous alcohol treatment gradually decreased the survival rate to 60% from 13 days after beginning administration in non-immunized, but not immunized, rats. A histochemical study revealed that continuous treatment with alcohol and unsaturated fatty acids caused fatty liver in non-immunized, but not immunized, rats. This study strongly supports the hypothesis that alcohol-induced fatty liver is due to a circulating endotoxin, and suggests that immunization for endotoxin prevent the alcoholic fatty liver.

Critical contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to apoptosis of human CD4+ T cells in HIV-1-infected hu-PBL-NOD-SCID mice.

Apoptosis is a key for CD4+ T cell destruction in HIV-1-infected patients. In this study, human peripheral blood lymphocyte (PBL)-transplanted nonobese diabetic (NOD)-severe combined immunodeficient (SCID) (hu-PBL-NOD-SCID) mice were used to examine in vivo apoptosis after HIV-1 infection. As the hu-PBL-NOD-SCID mouse model allowed us to see extensive infection with HIV-1 and to analyze apoptosis in human cells in combination with immunohistological methods, we were able to quantify the number of apoptotic cells with HIV-1 infection. As demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), massive apoptosis was predominantly observed in virus-uninfected CD4+ T cells in the spleens of HIV-1-infected mice. A combination of TUNEL and immunostaining for death-inducing tumor necrosis factor (TNF) family molecules indicated that the apoptotic cells were frequently found in conjugation with TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD3+CD4+ human T cells. Administration of a neutralizing anti-TRAIL mAb in HIV-1-infected mice markedly inhibited the development of CD4+ T cell apoptosis. These results suggest that a large number of HIV-1-uninfected CD4+ T cells undergo TRAIL-mediated apoptosis in HIV-infected lymphoid organs.

Oxygenation reactions of various tricyclic fused aromatic compounds using Escherichia coli and Streptomyces lividans transformants carrying several arene dioxygenase genes.

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-dihydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.

Quenching of singlet oxygen by carotenoids produced in escherichia coli - attenuation of singlet oxygen-mediated bacterial killing by carotenoids.

We examined the viability of Escherichia coli transformants harboring various carotenoids synthesizing genes in a medium containing an enzymatic singlet oxygen generating system, which contained myeloperoxidase, hydrogen peroxide and Br(-) at pH 4.5. Singlet oxygen quenching activities of various carotenoids in phosphatidyl choline micelles in aqueous media were also studied using the same enzymatic singlet oxygen generating system. Viability of the transformants producing carotenoids was higher than that of the wild type E. coli in the singlet oxygen generation mixture. Of the transformants tested, the viability of zeaxanthin-diglucoside producing transformant was the highest. Carotenoids in increasing order of k(q) values were beta-carotene, a cyclic carotene