OppA,the ecto-ATPase of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>induces ATP release and cell death in HeLa cells

Background  

In the facultative human pathogen Mycoplasma hominis, which belongs to the cell wall-less Mollicutes, the surface-localised substrate-binding domain OppA of the oligopeptide permease was characterised as the main ecto-ATPase.     

Multi-locus analysis of human infective <Emphasis Type="Italic">Cryptosporidium</Emphasis> species and subtypes using ten novel genetic loci

Background  

Cryptosporidium is a protozoan parasite that causes diarrheal illness in a wide range of hosts including humans. Two species, C. parvum and C. hominis are of primary public health relevance. Genome sequences of these two species are available and show only 3-5% sequence divergence. We investigated this sequence variability, which could correspond either to sequence gaps in the published genome sequences or to the presence of species-specific genes. Comparative genomic tools were used to identify putative species-specific genes and a subset of these genes was tested by PCR in a collection of Cryptosporidium clinical isolates and reference strains.     

P80, the HinT interacting membrane protein,is a secreted antigen of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>

Background  

Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT.     

Probiotic potential of Staphylococcus hominis MBBL 2–9 as anti‐Staphylococcus aureus agent isolated from the vaginal microbiota of a healthy woman

Aims: To isolate and characterize an antagonist for use as probiotic agent in the biocontrol of Staphylococcus aureus. Methods and Results: Bacteria that exhibited antimicrobial activity against Gram‐positive bacteria including Staph. aureus were isolated from 12 healthy women, with Staphylococcus hominis MBBL 2–9 showing the strongest activity. The bacteriocin produced by Staph. hominis MBBL 2–9 was purified by 60% ammonium sulfate saturation, ultrafiltration, HLB cartridge and reverse‐phase HPLC. The molecular weight was estimated as 2038·2 Da by MALDI–TOF mass spectrometry. The antagonist survived up to 2 h in artificial gastric juice (pH 2·5) and grew in the presence of 1% porcine bile extract. In addition, Staph. hominis MBBL 2–9 adhered effectively to HT‐29 epithelial cell line. Conclusion: Staphylococcus hominis MBBL 2–9 exhibited desirable probiotic traits such as acid tolerance, bile resistance and adherence to epithelial cell line. The bacterium also produced a bacteriocin with unique molecular weight and high antimicrobial activity similar to traditional antibiotics. Significance and Impact of the Study: This study is the first report of a bacteriocin‐producing Staph. hominis MBBL 2–9 that has potential for use as a probiotic agent against Staph. aureus.     

<Emphasis Type="Italic">Ureaplasma urealyticum</Emphasis> and <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> Sensitivity to Bacteriocins Produced by Two Lactobacilli Strains

The purpose of the present study was to determine the inhibitory activities of two bacteriocins, produced by lactobacilli, against genital mycoplasmas. In this study, infections produced by genital mycoplasmas were studied; of these, 1.3% were caused by Mycoplasma hominis, 10.7% by Ureaplasma urealyticum and 5.6% by U. urealyticum + M. hominis. U. urealyticum was isolated from 75 out of 123 patients with genital mycoplasmas, while M. hominis was isolated from 9 patients (7.3%) and both U. urealyticum and M. hominis from 39 patients (31.7%). Bacteriocins, L23 and L60, produced by Lactobacillus fermentum and L. rhamnosus, respectively, appear to be two novel inhibitors of bacterial infection with potential antibacterial activity. Both bacteriocins proved to be active against 100% of strains tested; MICs of bacteriocin L23 ranged between 320 and 160 UA ml⁻¹ for 78% of the M. hominis strains and between 320 and 80 UA ml⁻¹ for 95% of the U. urealyticum strains. In addition, bacteriocin L60 was still active at 160 UA ml⁻¹ for a high percentage (56%) of M. hominis strains, and at 80 UA ml⁻¹ for 53% of the U. urealyticum strains. Interestingly, these antimicrobial substances produced by lactobacilli showed an inhibitory activity against genital mycoplasmas even when diluted. Altogether, our study indicates that the bacteriocins, L23 and L60, are good candidates for the treatment or prevention of genital infections in women.     

Test method development to evaluate hot,humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores

Aims

To develop test methods and evaluate the survival of Bacillus anthracis ?Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air.

Methods and Results

Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH‐adjusted bleach decontamination.

Conclusions

Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ?Sterne and B. thuringiensis Al Hakam spores with similar kinetics.

Significance and Impact of the Study

Hot, humid air is a potential alternative to conventional chemical decontamination.     

Use of DNA melting simulation software for <Emphasis Type="Italic">in silico</Emphasis> diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes

Background  

DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria.     

Effect of a disinfection strategy on the methicillin‐resistant Staphylococcus aureus CC398 prevalence of sows,their piglets and the barn environment

Aims

To assess, in a cleaned and disinfected barn environment, the efficacy of an animal disinfection strategy to reduce the livestock‐associated methicillin‐resistant Staphylococcus aureus (LA‐MRSA) prevalence in sows, their offspring and the barn environment.

Methods and Results

On each farm, six sow rounds were sampled; sows were divided into either a test or control group. Per round, 20 sows and 40 of their piglets were sampled at different time points together with the barn environment. The disinfection strategy of the test groups consisted of washing the sows with a shampoo followed by disinfection of the skin with a solution containing chlorhexidine digluconate and isopropanol. On the first day of disinfection and 6 days after stopping the disinfection, a significant decrease (P < 0·01) of on average 68 and 66% in sow MRSA prevalence was observed on both farms, whereas no decrease was seen in the control groups. Just before weaning, 21–28 days after the end of the disinfection strategy, the difference in MRSA prevalence between both groups was reduced to 4% and no longer significant (P = 0·20). The MRSA prevalence of the piglets in the test groups was significantly lower (26%; P < 0·01) 6 days after the end of disinfection. Just before weaning, this difference was reduced to 5% but still significant (P < 0·01). In the swine nursery unit, no significant difference (P = 0·99) was seen between both groups. Based on semi‐quantitative counts, a relationship (r² > 0·6; P < 0·01) was seen between MRSA contamination in the barn environment and the MRSA prevalence in pigs.

Conclusion

Results show that the tested disinfection strategy reduces temporarily the sow and piglet MRSA status, but does not result in a final reduction in MRSA at weaning or in the nursery unit.

Significance and Impact of the Study

First report on the efficacy of an animal disinfection strategy to reduce LA‐MRSA prevalence in sows, their offspring and the barn environment.     

Mixed infections with <Emphasis Type="Italic">Chlamydia</Emphasis> and porcine epidemic diarrhea virus - a new <Emphasis Type="Italic">in vitro</Emphasis> model of chlamydial persistence

Background  

Chlamydiae induce persistent infections, which have been associated with a wide range of chronic diseases in humans and animals. Mixed infections with Chlamydia and porcine epidemic diarrhea virus (PEDV) may result in generation of persistent chlamydial infections. To test this hypothesis, an in vitro model of dual infection with cell culture-adapted PEDV and Chlamydia abortus or Chlamydia pecorum in Vero cells was established.     

Brief report: Lactobacillus bulgaricus GLB44 (Proviotic™) plus esomeprazole for Helicobacter pylori eradication: A pilot study

Background

Recent studies of Lactobacillus delbrueckii subsp. bulgaricus GLB44 plus a proton‐pump inhibitor (PPI) reported cures of more than 90% of patients with active Helicobacter pylori infections.

Aim

To confirm the high H. pylori cure rates reported previously.

Method

A pilot study was done in healthy H. pylori‐infected volunteers using 3‐gram sachet (3 billion cells) of L. delbrueckii GLB44 plus 22.3 mg of esomeprazole b.i.d., for 14 days. The result was determined by urea breath testing 4 weeks after therapy. Stopping rules required for ending enrollment if less than 3 of the first 10 subjects were cured.

Results

Nine subjects were entered and because all failed to achieve negative urea breath test, the stopping rule required the study to end.

Conclusion

We were unable to confirm reports of achieving a high H. pylori cure rate with L. delbrueckii GLB44 plus a PPI.     

Environmental amoebae do not support the long‐term survival of virulent mycobacteria

Aims

To test the hypothesis that Mycobacterium bovis can persist in the environment within protozoa.

Methods and Results

In this study, we used a novel approach to detect internalized mycobacteria in environmental protozoa from badger latrines. Acid‐fast micro‐organisms were visualized in isolated amoebae, although we were unable to identify them to species level as no mycobacteria were grown from these samples nor was M. bovis detected by IS6110 PCR. Co‐incubation of Acanthamoeba castellanii with virulent M. bovis substantially reduced levels of bacilli, indicating that the amoebae have a negative effect on the persistence of M. bovis.

Conclusions

The internalization of mycobacteria in protozoa might be a rare event under environmental conditions. The results suggest that amoebae might contribute to the inactivation of M. bovis rather than representing a potential environmental reservoir.

Significance and Impact of the Study

Protozoa have been suggested to act as an environmental reservoir for M. bovis. The current study suggests that environmental amoebae play at most a minor role as potential reservoirs of M. bovis and that protozoa might inhibit persistence of M. bovis in the environment.     

A method for the identification of guinea pig blood meal in the Chagas disease vector, <Emphasis Type="Italic">Triatoma infestans</Emphasis>

Background  

In a SINE-based PCR assay, a primer set specific for guinea pig genome short interspersed elements DNA was used to test the utility of genomic markers for identifying the source of vertebrate blood meals of Triatoma infestans.     

Comparative phylogeography of two co‐distributed but ecologically distinct rainbowfishes of far‐northern Australia

Aim

To test the influence of historical and contemporary environment in shaping the genetic diversity of freshwater fauna we contrast genetic structure in two co‐distributed, but ecologically distinct, rainbowfish; a habitat generalist (Melanotaenia splendida) and a habitat specialist (M. trifasciata).

Location

Fishes were sampled from far northern Australia (Queensland and Northern Territory).

Methods

We used sequence data from one mitochondrial gene and one nuclear gene to investigate patterns of genetic diversity in M. splendida and M. trifasciata to determine how differences in habitat preference and historical changes in drainage boundaries affected patterns of connectivity.

Results

Melanotaenia splendida showed high levels of genetic diversity and little population structure across its range. In contrast, M. trifasciata showed high levels of population structure. Whereas phylogeographic patterns differed, both species showed a strong relationship between geographical distance and genetic differentiation between populations. Melanotaenia splendida showed a shallower relationship with geographical distance, and genetic differentiation was best explained by stream length and a lower scaled ocean distance (11.98 times coast length). For M. trifasciata, genetic differentiation was best explained by overwater distance between catchments and ocean distance scaled at 1.16 × 10⁶ times coast length.

Main conclusions

Connectivity of freshwater populations inhabiting regions periodically interconnected during glacial periods appears to have been affected by ecological differences between species. Species‐specific differences are epitomized here by the contrast between co‐distributed congeners with different habitat requirements: for the habitat generalist, M. splendida, there was evidence for greater historical genetic connectivity with oceans as a weaker barrier to gene exchange in contrast with the habitat specialist, M. trifasciata.     

The influence of acetyl phosphate on DspA signalling in the Cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803

Background  

The dspA (hik33) gene, coding for a putative sensory histidine kinase, is conserved in plastids (ycf26) and cyanobacteria. It has been linked with a number of different stress responses in cyanobacteria.     

Independent Origins of New Sex-Linked Chromosomes in the <Emphasis Type="Italic">melanica</Emphasis> and <Emphasis Type="Italic">robusta</Emphasis> Species Groups of <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

Recent translocations of autosomal regions to the sex chromosomes represent important systems for identifying the evolutionary forces affecting convergent patterns of sex-chromosome heteromorphism. Additions to the sex chromosomes have been reported in the melanica and robusta species groups, two sister clades of Drosophila. The close relationship between these two species groups and the similarity of their rearranged karyotypes motivates this test of alternative hypotheses; the rearranged sex chromosomes in both groups are derived through a common origin, or the rearrangements are derived through at least two independent origins. Here we examine chromosomal arrangement in representatives of the melanica and the robusta species groups and test these alternative hypotheses using a phylogenetic approach.     

Exocytosis and protein secretion in <Emphasis Type="Italic">Trypanosoma</Emphasis>

Background  

Human African trypanosomiasis is a lethal disease caused by the extracellular parasite Trypanosoma brucei. The proteins secreted by T. brucei inhibit the maturation of dendritic cells and their ability to induce lymphocytic allogenic responses. To better understand the pathogenic process, we combined different approaches to characterize these secreted proteins.     

<Emphasis Type="Italic">Post hoc</Emphasis> pattern matching: assigning significance to statistically defined expression patterns in single channel microarray data

Background  

Researchers using RNA expression microarrays in experimental designs with more than two treatment groups often identify statistically significant genes with ANOVA approaches. However, the ANOVA test does not discriminate which of the multiple treatment groups differ from one another. Thus, post hoc tests, such as linear contrasts, template correlations, and pairwise comparisons are used. Linear contrasts and template correlations work extremely well, especially when the researcher has a priori information pointing to a particular pattern/template among the different treatment groups. Further, all pairwise comparisons can be used to identify particular, treatment group-dependent patterns of gene expression. However, these approaches are biased by the researcher's assumptions, and some treatment-based patterns may fail to be detected using these approaches. Finally, different patterns may have different probabilities of occurring by chance, importantly influencing researchers' conclusions about a pattern and its constituent genes.     

Microbiological and Molecular Characterization of Staphylococcus hominis Isolates from Blood

Background

Among Coagulase-Negative Staphylococci (CoNS), Staphylococcus hominis represents the third most common organism recoverable from the blood of immunocompromised patients. The aim of this study was to characterize biofilm formation, antibiotic resistance, define the SCCmec (Staphylococcal Chromosomal Cassette mec) type, and genetic relatedness of clinical S. hominis isolates.

Methodology

S. hominis blood isolates (n = 21) were screened for biofilm formation using crystal violet staining. Methicillin resistance was evaluated using the cefoxitin disk test and the mecA gene was detected by PCR. Antibiotic resistance was determined by the broth microdilution method. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) and SCCmec typed by multiplex PCR using two different methodologies described for Staphylococcus aureus.

Results

Of the S. hominis isolates screened, 47.6% (10/21) were categorized as strong biofilm producers and 23.8% (5/21) as weak producers. Furthermore, 81% (17/21) of the isolates were methicillin resistant and mecA gene carriers. Resistance to ampicillin, erythromycin, and trimethoprim was observed in >70% of isolates screened. Each isolate showed a different PFGE macrorestriction pattern with similarity ranging between 0–95%. Among mecA-positive isolates, 14 (82%) harbored a non-typeable SCCmec type: eight isolates were not positive for any ccr complex; four contained the mec complex A ccrAB1 and ccrC, one isolate contained mec complex A, ccrAB4 and ccrC, and one isolate contained the mec complex A, ccrAB1, ccrAB4, and ccrC. Two isolates harbored the association: mec complex A and ccrAB1. Only one strain was typeable as SCCmec III.

Conclusions

The S. hominis isolates analyzed were variable biofilm producers had a high prevalence of methicillin resistance and resistance to other antibiotics, and high genetic diversity. The results of this study strongly suggested that S. hominis isolates harbor new SCCmec structural elements and might be reservoirs of ccrC1 in addition to ccrAB1 and mec complex A.     

Characterization of proteome alterations in <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in response to lead exposure

Background  

Total soluble proteome alterations of white rot fungus Phanerochaete chrysosporium in response to different doses (25, 50 and 100 μM) of Pb (II) were characterized by 2DE in combination with MALDI-TOF-MS.