mecA locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone

An emerging public health phenomenon is the increasing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of mecA in combination with a single nucleotide polymorphism (SNP) defined by the S. aureus multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms-interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of mecA were determined. The CA-MRSA isolates were found to contain a truncated mecA downstream region consisting of mecA-HVR-IS431 mec-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different mecA downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the S. aureus MLST database, and the arcC 2726 polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated mecA downstream region in combination with the interrogation of arcC position 272 provided an unambiguous identification of WSPP isolates.

Cytokine gene polymorphisms in idiopathic pulmonary fibrosis

Pro- and anti-fibrotic cytokine gene polymorphisms may affect expression of idiopathic pulmonary fibrosis (IPF). The aims of the present case-control study were to examine polymorphisms in the IL-6, transforming growth factor (TGF)-beta1, tumour necrosis factor (TNF)-alpha and interleukin-1 (IL-1)Ra genes in patients with IPF (n=22)-compared to healthy controls (n=140). Genotyping was performed on DNA extracted from peripheral blood lymphocytes, using polymerase chain reaction-restriction fragment length polymorphism with gene polymorphisms determined according to-published techniques. The following sites were examined: (i) IL-1Ra*1-5 (86 bp variable tandem repeat intron 2), (ii) IL-6 (-174G>C), (iii) TNF-alpha (-308G>A) and (iv) TGF-beta1 (Arg25Pro). The TNF-alpha (-308 A) allele was over-represented in the IPF (p(corr)=0.004) group compared to controls. Risk of IPF was significant for heterozygotes for: (i) the TNF-alpha (-308 A) allele (A/G) (odds ratio (OR) 2.9; 95% confidence interval (CI) 1.2-7.2; P=0.02), (ii) homozygotes (A/A) (OR 13.9; 95%CI 1.2-160; P=0.04) and (iii) carriage of the allele (A/A+A/G) (OR 4; 95%CI 1.6-10.2; P=0.003). The distribution of alleles and genotypes for IL-6, TGF-beta1 and IL-1Ra between the two groups was not significantly different. This is the third study to independently confirm that there is a significant association of the TNF-alpha (-308 A) allele with IPF. Further research is needed to assess the utility of cytokine gene polymorphisms as markers of disease-susceptibility.

Human metapneumovirus, Australia, 2001-2004

We examined 10,025 respiratory samples collected for 4 years (2001-2004) and found a 7.1% average annual incidence of human meta pneumovirus. The epidemic peak of infection was late winter to spring, and genotyping showed a change in predominant viral genotype in 3 of the 4 years.

Application of DNA parentage analyses for determining relative growth rates of Penaeus japonicus families reared in commercial ponds

The ability to track large numbers of individuals and families is a key determinant of the power and precision of breeding programs, including the capacity to quantify interactions between genotypes and their environment. Until recently, most family based selective breeding programs for shrimp, and other highly fecund aquaculture species, have been restricted by the number of animals that can be physically tagged and individually selected. Advances in the development of molecular markers, such as microsatellite loci, are now providing the means to track large numbers of individuals and families in commercial production systems. In this study microsatellites, coupled with DNA parentage analyses, were used to determine the relative performance of 22 families of R japonicus reared in commercial production ponds. In the experimental design 6000 post-larvae from each of 22 families, whose maternal parents had been genotyped at 8 microsatellite loci, were stocked into each of four I ha ponds. After 6 months the ponds were harvested and a total of 6000 individuals were randomly weighed from each pond. Mean wet weight of the shrimp from one pond was significantly lower than that of the other three ponds demonstrating a possible pond effect on growth rate. The representation of families in the top 10% of each pond's weight distribution was then determined by randomly genotyping up to 300 individuals from this upper weight class. Parentage analyses based on individual genotypic data demonstrated that some families were over-represented in the top 10% in all ponds, while others were under-represented due to slower growth rates. The results also revealed some weak, but significant, male genotype x environment (G x E) interactions in the expression of shrimp growth for some families. This indicates that G x E effects may need to be factored into future R japonicus selective breeding programs. This study demonstrated the utility of DNA parentage analyses for tracking individual family performance in communally stocked shrimp pond populations and, its application to examining G x E effects on trait expression under commercial culture conditions. Crown Copyright (c) 2005 Published by Elsevier B.V. All rights reserved.

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n=140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n=18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.

Isolation and characterization of microsatellite loci from Macadamia

Thirty-three microsatellite loci were isolated for the Australian rainforest tree Macadamia integrifolia. Genotyping across a test panel of 43 commercial cultivars generated an average polymorphic information content of 0.480. Five loci showed no polymorphism across cultivars. Significant linkage disequilibrium was detected in 10 pairwise comparisons, including two pairs of loci identified from the same clone sequence. The 33 microsatellite loci represent a significant tool for genome mapping and population genetic studies.

HLA and Genomewide Allele Sharing in Dizygotic Twins

Gametic selection during fertilization or the effects of specific genotypes on the viability of embryos may cause a skewed transmission of chromosomes to surviving offspring. A recent analysis of transmission distortion in humans reported significant excess sharing among full siblings. Dizygotic (DZ) twin pairs are a special case of the simultaneous survival of two genotypes, and there have been reports of DZ pairs with excess allele sharing around the HLA locus, a candidate locus for embryo survival. We performed an allele-sharing study of 1,592 DZ twin pairs from two independent Australian cohorts, of which 1,561 pairs were informative for linkage on chromosome 6. We also analyzed allele sharing in 336 DZ twin pairs from The Netherlands. We found no evidence of excess allele sharing, either at the HLA locus or in the rest of the genome. In contrast, we found evidence of a small but significant (P = .003 for the Australian sample) genomewide deficit in the proportion of two alleles shared identical by descent among DZ twin pairs. We reconciled conflicting evidence in the literature for excess genomewide allele sharing by performing a simulation study that shows how undetected genotyping errors can lead to an apparent deficit or excess of allele sharing among sibling pairs, dependent on whether parental genotypes are known. Our results imply that gene-mapping studies based on affected sibling pairs that include DZ pairs will not suffer from false-positive results due to loci involved in embryo survival.

Neurobiological alterations in the human alcoholic brain: effect of genotype

Long-term alcohol abuse by human subjects leads to selective brain damage that is restricted in extent and variable in severity. Within the cerebral cortex, neuronal loss is most marked in the superior frontal cortex and relatively mild in motor cortex. Cirrhotic alcoholics and subjects with alcohol-related Wernicke-Korsakoff syndrome show more severe and more extensive damage than do uncomplicated cases. Accumulating evidence suggests that the likelihood of developing alcohol dependency is associated with one or more genetic markers. In previous work we showed that GABAA receptor functionality, and the subunit isoform expression that underlies this, differed in region- and disease-specific ways between alcoholics and controls. By contrast, glutamate receptor (NMDA, KA, AMPA) differences were muted or absent. Here we asked if genotype differentiated the form, pharmacology, or expression of glutamate and GABA receptors in pathologically vulnerable and spared cortical regions, with a view to determining whether such subject factors might influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy under informed, written consent from uncomplicated and alcoholic-cirrhotic Caucasian (predominantly Anglo-Celtic) cases, together with matched controls and cases with cirrhosis of non-alcoholic origin. All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were processed for synaptic membrane receptor binding, mRNA analysis by quantitative RT-PCR, and protein analysis by Western blot. Genotyping was performed by PCR methods, in the main using published primers. Several genetic markers differentiated between our alcoholic and control subjects, including the GABAA receptor 2 subunit (GABB2) gene ( 2 (3) 10.329, P 0.01), the dopamine D2 receptor B1 (DRD2B) allele ( 2 (3) 10.109, P 0.01) and a subset of the alcohol dehydrogenase-3 (ADH3) alleles ( 2 (2) 4.730, P 0.05). Although neither the type-2 glutamate transporter (EAAT2) nor the serotonin transporter (5HTT) genes were significantly associated with alcoholism, only EAAT2 heterozygotes showed a significant association between ADH3 genotype and alcoholism ( 2 (3) 7.475, P 0.05). Other interactions between genotypes were also observed. DRD2A, DRD2B, GABB2, EAAT2 and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters, although in combined subjects there was a significant DRD2B X Area Interaction with glutamateNMDA receptor efficacy (F(1,57) 4.67; P 0.05), measured as the extent of glutamate-enhanced MK801 binding. In contrast, there was a significant Case-group X ADH3 X Area Interaction with glutamateNMDA receptor efficacy (F(3,57) 2.97; P 0.05). When GABAA receptor subunit isoform expression was examined, significant Case-group X Genotype X Area X Isoform interactions were found for EAAT2 with subunit mRNA (F(1,37) 4.22; P0.05), for GABB2 with isoform protein (F(1,37) 5.69; P 0.05), and for DRD2B with isoform protein (F(2,34)5.69; P0.05). The results suggest that subjects’ genetic makeup may modulate the effectiveness of amino acid-mediated transmission in different cortical regions, and thereby influence neuronal vulnerability to excitotoxicity.