Human-specific E. coli single nucleotide polymorphism (SNP) genotypes detected in a South-East Queensland waterway, Australia

The World Health Organization recommends that the majority of water monitoring laboratories in the world should test for E. coli daily since thermotolerant coliforms and E. coli are key indicators for risk assessment of recreational waters. Recently, we developed a new SNP method for typing E. coli strains, by which human-specific genotypes were identified. Here, we report the presence of these previously described specific SNP profiles in environmental water, sourced from the Coomera River, located on South East Queensland, Australia, over a period of two years. This study tested for the presence of human-specific E. coli to ascertain whether hydrologic and anthropogenic activity plays a key role in the pollution of the investigated watershed or whether the pollution is from other sources. We found six human-specific SNP profiles and one animal-specific SNP profile consistently across sampling sites and times. We have demonstrated that our SNP genotyping method is able to rapidly identify and characterise human- and animal-specific E. coli isolates in water sources.

SNP diversity of Enterococcus faecalis and Enterococcus faecium in a South East Queensland waterway, Australia, and associated antibiotic resistance gene profiles

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are associated with faecal pollution of water, linked to swimmer-associated gastroenteritis and demonstrate a wide range of antibiotic resistance. The Coomera River is a main water source for the Pimpama-Coomera watershed and is located in South East Queensland, Australia, which is used intensively for agriculture and recreational purposes. This study investigated the diversity of E. faecalis and E. faecium using Single Nucleotide Polymorphisms (SNPs) and associated antibiotic resistance profiles. RESULTS: Total enterococcal counts (cfu/ml) for three/six sampling sites were above the United States Environmental Protection Agency (USEPA) recommended level during rainfall periods and fall into categories B and C of the Australian National Health and Medical Research Council (NHMRC) guidelines (with a 1-10% gastrointestinal illness risk). E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles (validated by MLST analysis) respectively. This study showed the high diversity of E. faecalis and E. faecium over a period of two years and both human-related and human-specific SNP profiles were identified. 81.8% of E. faecalis and 70.21% of E. faecium SNP profiles were associated with genotypic and phenotypic antibiotic resistance. Gentamicin resistance was higher in E. faecalis (47% resistant) and harboured the aac(6')-aph(2') gene. Ciprofloxacin resistance was more common in E. faecium (12.7% resistant) and gyrA gene mutations were detected in these isolates. Tetracycline resistance was less common in both species while tet(L) and tet(M) genes were more prevalent. Ampicillin resistance was only found in E. faecium isolates with mutations in the pbp5 gene. Vancomycin resistance was not detected in any of the isolates. We found that antibiotic resistance profiles further sub-divided the SNP profiles of both E. faecalis and E. faecium. CONCLUSIONS: The distribution of E. faecalis and E. faecium genotypes is highly diverse in the Coomera River. The SNP genotyping method is rapid and robust and can be applied to study the diversity of E. faecalis and E. faecium in waterways. It can also be used to test for human-related and human-specific enterococci in water. The resolving power can be increased by including antibiotic-resistant profiles which can be used as a possible source tracking tool. This warrants further investigation.

Hormone-dependent bacterial growth persistence and biofilm formation - A pilot study investigating human follicular fluid collected during IVF cycles

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.

The phylogeny of Ureaplasma urealyticum based on the mba gene fragment

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from an alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types which we have designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3, and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a, and 6b) thus facilitating high resolution typing of U. urealyticum. Inclusion of the reference strains for serovars 1, 3, 6, and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however this fragment was invariant within the parvo biovar. This study has shown that the sequence of the mba can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Intra-amniotic ureaplasma infection : adverse outcomes vary depending on gestation

Background: Ureaplasmas are the most prevalent bacteria isolated from preterm deliveries and the prognosis for neonates varies depending on the gestation at delivery. Ureaplasmas vary their surface-exposed antigen (MBA, a virulence mechanism) during chronic intra-amniotic infections, but it is not known when changes first occur during gestation. Method: U. parvum serovar 3 (2x10e7CFU) was injected intra-amniotically (IA) into six experimental cohorts of pregnant ewes (of n=7), 3 days (d) or 7d before delivery at either: 100d, 124d or 140d gestation (term=145d). Control ewes received IA 10B broth. Fetuses were delivered surgically and ureaplasmas cultured from amniotic fluid (AF), chorioamnion, fetal lung (FL) and umbilical cord. Ureaplasmas were tested by western blot to demonstrate MBA variation. Results: The highest number of ureaplasmas were recovered from FL at 100d gestation after 3 days of infection (p<0.03). Six of 7(86%) 100d–3d FL demonstrated an ureaplasma MBA variant, but only 17% and 15% of FL showed an MBA variant after 3d infection at 124d and 140d gestation respectively. Greatest variation of the MBA occurred in AF and FL at 124d gestation after 7d infection. The least MBA variation was observed at 140d; however, at this time the most severe histological chorioamnionitis was observed. Conclusions: After intra-amniotic ureaplasma injections, higher numbers of ureaplasmas gained access to the FL at 100d gestation than observed at later gestations. This may exacerbate the adverse outcomes for neonates delivered early in gestation. In late gestation, ureaplasma MBA variation was minimal, but chorioamnionitis was the most severe. Adverse pregnancy outcomes associated with IA ureaplasma infection may vary depending on the duration of gestation, the number of ureaplasmas isolated from the fetal tissues and the degree of MBA variation.

Ureaplasma speioces are associated with chorioamnionitiis in late preterm placentas

Background: Preterm birth is a major cause of neonatal morbidity and mortality, with 75% of preterm births occurring late preterm. Previous studies have investigated the microbial diversity within placentas delivered early preterm but there has been no investigation of the prevalence of bacteria, particularly Ureaplasma spp. in late preterm placentas. Method: Women giving birth late preterm (320 – 366 weeks of gestation) in Cincinnati were recruited for this study. Samples of chorioamnion were collected aseptically at the time of delivery, shipped to QUT and tested for Ureaplasma spp. and other bacteria by culture and/or PCR assays. The presence of bacteria was correlated with adverse pregnancy outcomes, including histological chorioamnionitis (tissue sections read by US pathologists). Results: To date, Ureaplasma spp. have been detected in 15/270 (5.5%) of placentas by culture and 19/270 (7%) by PCR. Ureaplasma presence correlated with histological chorioamnionitis (12/19%; 63%). However, the presence of other bacteria was not associated with chorioamnionitis (5%). Chorioamnionitis was unevenly distributed in ethnic groups, with a higher incidence in African-Americans’ (6/7; 86%), compared to Caucasians’ (6/12; 50%) who were colonised with ureaplasmas. Conclusion: This study is the first to report the prevalence of ureaplasmas in women (7%) who deliver late preterm. Ureaplasma spp. were associated with a higher incidence of chorioamnionitis (63% compared to 15% for non-infected women). This data strongly suggests that ureaplasmas are a cause of late preterm deliveries and African-American women are at greater risk of chorioamnionitis.

The role of HtrA as a chaperone and protease in bacterial pathogenisis

HtrA (High Temperature Requirement A) is a critical stress response protease and chaperone for many bacteria. HtrA is a multitasking protein which can degrade unfolded proteins, conduct specific proteolysis of some substrates for correct assembly, interact with substrates to ensure correct folding, assembly or localisation, and chaperone unfolded proteins. These functions are critical for the virulence of a number of bacterial pathogens, in some cases not simply due to the broad activities of HtrA in protection against the protein stress conditions which occur during virulence. But also due to the role of HtrA in either specific proteolysis or assembly of key protein substrates which function directly in virulence. Remarkably, these activities are all conducted without any requirement for ATP. The biochemical mechanism of HtrA relies both on the chymotryptic serine protease active site as well as the presence of two PDZ (protein binding) domains. The mechanism is a unique combination of activation by substrate motifs to alter the confirmation of the active site, and assembly into a multimeric complex which has enhanced degradation and may also act as a protective cage for proteins which are not degraded. The role of this protease in the pathogenesis of a number of bacteria and the details of its distinctive biochemical activation and assembly mechanisms are discussed in this chapter.

Water microbiology

Microbiology is the science devoted lo the study of organisms that are too small to be seen by the naked eye. These microorganisms are a large and diverse group of free-living forms that exist as single cells or cell clusters. Being free-living, microbial cells are distinct from the cells of animals and plants as the latter are not able to live alone in nature but only in characteristic groups. A single microbial cell, generally, is able to carry out its life processes of growth, respiration and reproduction independently of other cells, either of the same kind or of different kinds. There are five subdisciplines of microbiology: (a) the study of bacteria (bacteriology); (b) the study of viruses (virology); (c) the study of algae (phycology); (d) the study of fungi (mycology); and (e) the study of protozoa (protozoology). In the examination of the environment, all five areas of microbiology are studied. This becomes obvious when discussing the significance of each of these groups of organisms in relation to human health.

Repeated intrauterine exposures to inflammatory stimuli attenuated transforming growth factor-β signaling in the ovine fetal lung

Background: Bronchopulmonary dysplasia (BPD) is one of the most common complications after preterm birth and is associated with intrauterine exposure to bacteria. Transforming growth factor-β (TGFβ) is implicated in the development of BPD. Objectives: We hypothesized that different and/or multiple bacterial signals could elicit divergent TGFβ signaling responses in the developing lung. Methods: Time-mated pregnant Merino ewes received an intra-amniotic injection of lipopolysaccharide (LPS) and/or Ureaplasma parvum serovar 3 (UP) at 117 days' and/or 121/122 days' gestational age (GA). Controls received an equivalent injection of saline and or media. Lambs were euthanized at 124 days' GA (term = 150 days' GA). TGFβ1, TGFβ2, TGFβ3, TGFβ receptor (R)1 and TGFβR2 protein levels, Smad2 phosphorylation and elastin deposition were evaluated in lung tissue. Results: Total TGFβ1 and TGFβ2 decreased by 24 and 51% after combined UP+LPS exposure, whereas total TGFβ1 increased by 31% after 7 days' LPS exposure but not after double exposures. Alveolar expression of TGFβR2 decreased 75% after UP, but remained unaltered after double exposures. Decreased focal elastin deposition after single LPS exposure was prevented by double exposures. Conclusions: TGFβ signaling components and elastin responded differently to intrauterine LPS and UP exposure. Multiple bacterial exposures attenuated TGFβ signaling and normalized elastin deposition.

TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes : the effect of microorganisms within human follicular fluid collected during IVF cycles

Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours’ incubation within human follicular fluids (n = 5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (<29%) were associated with extensive fragmentation in mouse oocytes (80–100%). Bacteria colonizing human follicular fluid in vivo may cause DNA fragmentation in mouse oocytes following 12 h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes.

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental E. faecalis and E. faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E and esp were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linozolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d/r) or inverted-repeat (i/r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i/r T-DNA insertions is a primary inducer of transgene silencing in plants. © CSIRO 2005.

Heterogeneity of clinical and environmental isolates of Mycobacterium fortuitum using repetitive element sequence-based PCR: municipal water an unlikely source of community-acquired infections

M. fortuitum is a rapidly growing mycobacterium associated with community-acquired and nosocomial wound, soft tissue, and pulmonary infections. It has been postulated that water has been the source of infection especially in the hospital setting. The aim of this study was to determine if municipal water may be the source of community-acquired or nosocomial infections in the Brisbane area. Between 2007 and 2009, 20 strains of M. fortuitum were recovered from municipal water and 53 patients’ isolates were submitted to the reference laboratory. A wide variation in strain types was identified using repetitive element sequence-based PCR, with 13 clusters of ≥2 indistinguishable isolates, and 28 patterns consisting of individual isolates. The clusters could be grouped into seven similar groups (>95% similarity). Municipal water and clinical isolates collected during the same time period and from the same geographical area consisted of different strain types, making municipal water an unlikely source of sporadic human infection.