Bacterial and fungal community composition over time in chicken litter with high or low moisture content

1. Changes in bacterial and fungal communities in chicken litter with high and low moisture content over a five week period during a single chicken grow out cycle in a poultry shed in subtropical Australia were investigated to study the association between specific microbes and odour production. 2. Microbial biomass, as indicated by DNA yields, was higher and community composition was more dynamic over time in moist compared with dry chicken litter. 3. Bacillus, Atopostipes and Aspergillus species increased in relative abundance in moist chicken litter samples over time reflecting the relatively high fitness and hence activity of these specific bacteria and this specific fungus in this environment.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Impact of the Human Bacterial Environment on Mycobacteriosis and Allergy

My work describes two sectors of the human bacterial environment: 1. The sources of exposure to infectious non-tuberculous mycobacteria. 2. Bacteria in dust, reflecting the airborne bacterial exposure in environments protecting from or predisposing to allergic disorders. Non-tuberculous mycobacteria (NTM) transmit to humans and animals from the environment. Infection by NTM in Finland has increased during the past decade beyond that by Mycobacterium tuberculosis. Among the farm animals, porcine mycobacteriosis is the predominant NTM disease in Finland. Symptoms of mycobacteriosis are found in 0.34 % of slaughtered pigs. Soil and drinking water are suspected as sources for humans and bedding materials for pigs. To achieve quantitative data on the sources of human and porcine NTM exposure, methods for quantitation of environmental NTM are needed. We developed a quantitative real-time PCR method, utilizing primers targeted at the 16S rRNA gene of the genus of Mycobacterium. With this method, I found in Finnish sphagnum peat, sandy soils and mud high contents of mycobacterial DNA, 106 to 107 genome equivalents per gram. A similar result was obtained by a method based on the Mycobacterium-specific hybridization of 16S rRNA. Since rRNA is found mainly in live cells, this result shows that the DNA detected by qPCR mainly represented live mycobacteria. Next, I investigated the occurrence of environmental mycobacteria in the bedding materials obtained from 5 pig farms with high prevalence (>4 %) of mycobacteriosis. When I used for quantification the same qPCR methods as for the soils, I found that piggery samples contained non-mycobacterial DNA that was amplified in spite of several mismatches with the primers. I therefore improved the qPCR assay by designing Mycobacterium-specific detection probes. Using the probe qPCR assay, I found 105 to 107 genome equivalents of mycobacterial DNA in unused bedding materials and up to 1000 fold more in the bedding collected after use in the piggery. This result shows that there was a source of mycobacteria in the bedding materials purchased by the piggery and that mycobacteria increased in the bedding materials during use in the piggery. Allergic diseases have reached epidemic proportions in urbanized countries. At the same time, childhood in rural environment or simple living conditions appears to protect against allergic disorders. Exposure to immunoreactive microbial components in rural environments seems to prevent allergies. I searched for differences in the bacterial communities of two indoor dusts, an urban house dust shown to possess immunoreactivity of the TH2-type and a farm barn dust with TH1-activity. The immunoreactivities of the dusts were revealed by my collaborators, in vitro in human dendritic cells and in vivo in mouse. The dusts accumulated >10 years in the respiratory zone (>1.5 m above floor), thus reflecting the long-term content of airborne bacteria at the two sites. I investigated these dusts by cloning and sequencing of bacterial 16S rRNA genes from dust contained DNA. From the TH2-active urban house dust, I isolated 139 16S rRNA gene clones. The most prevalent genera among the clones were Corynebacterium (5 species, 34 clones), Streptococcus (8 species, 33 clones), Staphylococcus (5 species, 9 clones) and Finegoldia (1 species, 9 clones). Almost all of these species are known as colonizers of the human skin and oral cavity. Species of Corynebacterium and Streptococcus have been reported to contain anti-inflammatory lipoarabinomannans and immunmoreactive beta-glucans respectively. Streptococcus mitis, found in the urban house dust is known as an inducer of TH2 polarized immunity, characteristic of allergic disorders. I isolated 152 DNA clones from the TH1-active farm barn dust and found species quite different from those found from the urban house dust. Among others, I found DNA clones representing Bacillus licheniformis, Acinetobacter lwoffii and Lactobacillus each of which was recently reported to possess anti-allergy immunoreactivity. Moreover, the farm barn dust contained dramatically higher bacterial diversity than the urban house dust. Exposure to this dust thus stimulated the human dendritic cells by multiple microbial components. Such stimulation was reported to promote TH1 immunity. The biodiversity in dust may thus be connected to its immunoreactivity. Furthermore, the bacterial biomass in the farm barn dust consisted of live intact bacteria mainly. In the urban house dust only ~1 % of the biomass appeared as intact bacteria, as judged by microscoping. Fragmented microbes may possess bioactivity different from that of intact cells. This was recently shown for moulds. If this is also valid for bacteria, the different immunoreactivities of the two dusts may be explained by the intactness of dustborne bacteria. Based on these results, we offer three factors potentially contributing to the polarized immunoreactivities of the two dusts: (i) the species-composition, (ii) the biodiversity and (iii) the intactness of the dustborne bacterial biomass. The risk of childhood atopic diseases is 4-fold lower in the Russian compared with the Finnish Karelia. This difference across the country border is not explainable by different geo-climatic factors or genetic susceptibilities of the two populations. Instead, the explanation must be lifestyle-related. It has already been reported that the microbiological quality of drinking water differs on the two sides of the borders. In collaboration with allergists, I investigated dusts collected from homes in the Russian Karelia and in the Finnish Karelia. I found that bacterial 16S rRNA genes cloned from the Russian Karelian dusts (10 homes, 234 clones) predominantly represented Gram-positive taxa (the phyla Actinobacteria and Firmicutes, 67%). The Russian Karelian dusts contained nine-fold more of muramic acid (60 to 70 ng mg-1) than the Finnish Karelian dusts (3 to 11 ng mg-1). Among the DNA clones isolated from the Finnish side (n=231), Gram-negative taxa (40%) outnumbered the Gram-positives (34%). Out of the 465 DNA clones isolated from the Karelian dusts, 242 were assigned to cultured validly described bacterial species. In Russian Karelia, animal-associated species e.g. Staphylococcus and Macrococcus were numerous (27 clones, 14 unique species). This finding may connect to the difference in the prevalence of allergy, as childhood contacts with pets and farm animals have been connected with low allergy risk. Plant-associated bacteria and plant-borne 16S rRNA genes (chloroplast) were frequent among the DNA clones isolated from the Finnish Karelia, indicating components originating from plants. In conclusion, my work revealed three major differences between the bacterial communtites in the Russian and in the Finnish Karelian homes: (i) the high prevalence of Gram-positive bacteria on the Russian side and of Gram-negative bacteria on the Finnish side and (ii) the rich presence of animal-associated bacteria on the Russian side whereas (iii) plant-associated bacteria prevailed on the Finnish side. One or several of these factors may connect to the differences in the prevalence of allergy.

Bacterial wilt of ginger in Queensland: Reappraisal of a disease outbreak

In 1955 a severe wilt disease occurring on ginger in the Near North Coast district of Queensland was incorrectly attributed to infection by a Fusarium sp., and later shown to be caused by a strain of Ralstonia solanacearum, now reclassified as R. sequeirae. The disease was brought from China into Australia on latently infected rhizomes, and possibly also with associated soil. Several DNA-based diagnostic methods have shown that the pathogen causing bacterial wilt of ginger in parts of China is indistinguishable from the pathogen uniquely associated with the disease in Queensland. © 2012 Australasian Plant Pathology Society Inc.

A convenient sample preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage

Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM) provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving pre-fixation with 75 mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained from stems stood into water. The lysine-based protocol was compared with four other processing protocols of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with CPD. The non-fixed control. FD and the glutaraldehyde fixation with HMDS drying gave poor preservation of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was superior to these three methods in terms of better preserving the EPS. However, this fourth method gave condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for examination of bacterial stem-end blockage in cut ornamentals. (C) 2012 Elsevier B.V. All rights reserved.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Boronia heterophylla vase life is influenced more by ethylene than by bacterial numbers or vase solution pH

Bacterial proliferation in both vase solutions and in cut flower stems has been implicated in reducing the vase life of numerous genera. Boronia heterophylla F. Muell. (Red Boronia) vase life was assessed at two stages of floral maturity for nine vase solution treatments covering a pH range of 2.5-5.7. Vase life for advanced harvest maturity stems ranged from 4.2 d in 10 mM citric acid + 50 mg L-1 chlorine (pH 2.5) to 12.9 d after STS pulsing (pH 5.7). For normal harvest maturity stems, the corresponding range was 5.8-19.0 d, respectively. Vase solutions containing 50 mg L-1 chlorine biocide resulted in decreased longevity. In contrast, pulsing with the ethylene-binding inhibitor, STS, significantly increased vase life. The number of bacteria in the vase solutions after 11 d was determined in stems of advanced maturity. The solution with the greatest number of bacteria, 4.0 x 10(10) cfu mL(-1), was water used after STS pulsing and in which the flowers lasted longest. Vase solution bacteria were enumerated on days 0,3, 6, 9 and 12 of the vase period with stems of normal harvest maturity. There was no relationship between vase life and vase solution bacterial numbers ((R) over bar (2) = 0.000). Moreover, there was a negative relationship between numbers of bacteria in basal 0-5 cm stem segments and vase life. As no correlations were evident between longevity and either the pH or vase solution bacterial numbers, B. heterophylla vase life was evidently limited principally by ethylene action. (C) 2013 Elsevier B.V. All rights reserved.

Programming rumen bacterial communities in newborn Merino lambs

Establishment of the rumen microbiome can be affected by both early-life dietary measures and rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate the effects of inclusion of dietary fat type and the effects of rumen inoculum from different sources on ruminal bacterial communities present in early stages of the lambs’ life. Two different diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month pre-lambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses, with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4% Megalac® provided as a source of rumen-protected fat (PF). One of three inoculums was introduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet; (2) rumen fluid from donor ewes fed CO; or (3) a control treatment of MilliQ-water. After weaning at 3 months of age, each of the six lamb treatment groups were grazed in spatially separated paddocks. Rumen bacterial populations of ewes and lambs were characterised using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Species richness and biodiversity of the bacterial communities were found to be affected by the diet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinate analysis and analysis of similarity (ANOSIM) showed between diet differences in bacterial community groups existed in ewes and differential bacterial clusters occurred in lambs due to both diet and neonatal inoculation. Diet and rumen inoculation acted together to clearly differentiate the bacterial communities through to weaning, however the microbiome effects of these initial early life interventions diminished with time so that rumen bacterial communities showed greater similarity 2 months after weaning. These results demonstrate that ruminal bacterial communities of newborn lambs can be altered by modifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changed by diet while they are suckling or by inoculating their rumen, and resulting changes in the rumen bacterial microbiome can persist beyond weaning.

Investigation of an emerging bacterial disease in wild Queensland groper, marine fish and stingrays with production of diagnostic tools to reduce the spread of disease to other states of Australia : final report

This project describes how Streptococcus agalactiae can be transmitted experimentally in Queensland grouper. The implications of this research furthers the relatedness between Australian S. agalactiae strains from animals and humans. Additionally, this research has developed diagnostic tools for Australian State Veterinary Laboratories and Universities, which will assist in State and National aquatic animal disease detection, surveillance, disease monitoring and reporting

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.