Whole genome sequencing reveals extensive community-level transmission of group A Streptococcus in remote communities.

Impetigo is common in remote Indigenous children of northern Australia, with the primary driver in this context being Streptococcus pyogenes [or group A Streptococcus (GAS)]. To reduce the high burden of impetigo, the transmission dynamics of GAS must be more clearly elucidated. We performed whole genome sequencing on 31 GAS isolates collected in a single community from children in 11 households with ⩾2 GAS-infected children. We aimed to determine whether transmission was occurring principally within households or across the community. The 31 isolates were represented by nine multilocus sequence types and isolates within each sequence type differed from one another by only 0-3 single nucleotide polymorphisms. There was evidence of extensive transmission both within households and across the community. Our findings suggest that strategies to reduce the burden of impetigo in this setting will need to extend beyond individual households, and incorporate multi-faceted, community-wide approaches.

Progressive increase in community-associated methicillin-resistant Staphylococcus aureus in Indigenous populations in northern Australia from 1993 to 2012.

Hospital-based studies have determined high rates of community-associated methicillin-resistant Staphylococcus aureus (MRSA) in Indigenous populations. However, there is a paucity of community-based data. We obtained 20 years (1993-2012) of data on S. aureus isolates (N = 20 210) collected from community clinics that provide services for Indigenous communities in the Northern Territory, Australia. Methicillin resistance increased from 7% to 24%, resistance to macrolides remained stable at ~25%, and there was a slight increase in resistance to trimethoprim-sulfamethoxazole. The increase in methicillin resistance is concerning for the Indigenous communities represented by this data, but it is also of significance if virulent MRSA clones emerge and spread more widely from such settings.

Differing epidemiology of two major healthcare-associated meticillin-resistant Staphylococcus aureus clones.

BACKGROUND: Two meticillin-resistant Staphylococcus aureus (MRSA) clones, sequence type (ST) 22 and ST239, have successfully spread globally. Across Australia, ST22 has supplanted ST239 as the main healthcare-associated MRSA. To understand the reasons underlying this shift, the epidemiology and clinical features of infections due to ST22 and ST239 MRSA isolates from a tertiary hospital in Melbourne, Australia were compared. METHODS: Over six months, consecutive MRSA isolates with clinical data were collected from specimens referred to Alfred Health Pathology (AHP). Isolates were genotyped by a multi-locus-sequence-typing-based high-resolution melting method. FINDINGS: Three hundred and twenty-eight of 1079 (30%) S. aureus isolated by AHP were MRSA. Of these, 313 were genotyped; 78 (25%) were clonal complex (CC) 22 (representing ST22) and 142 (45%) were CC239 (representing ST239). Common clinical syndromes included skin or soft tissue, respiratory tract and osteo-articular infections. On multi-variate logistic regression, compared with CC239, CC22 was associated with older patients [adjusted odds ratio (aOR) 1.04 for each year increase, 95% confidence interval (CI) 1.02-1.07)], and patients from subacute hospitals (aOR 2.7, 95% CI 1.2-5.8) or long-term care facilities (LTCFs; aOR 5.5, 95% CI 2.0-14.5). Median time from patient admission to MRSA isolation was nine days for CC239 and one day for CC22 (P < 0.01). MRSA strain epidemiology varied according to hospital unit. CONCLUSIONS: CC22 and CC239 MRSA have differing ecological niches. CC22 is associated with elderly patients in LTCFs, and CC239 is associated with nosocomial acquisition. Infection control strategies involving LTCFs and their residents will likely be required to achieve continued MRSA control.

Characterization of levJ, a sucrase/fructanase-encoding gene from Actinomyces naeslundii T14V, and comparison of its product with other sucrose-cleaving enzymes.

A library of Actinomyces naeslundii T14V DNA was constructed in plasmid pUC18 and from this several sucrose-positive clones were isolated. Evidence was obtained that all these clones contained the same gene. One clone, which carried a plasmid that was named pPNG102, was chosen for further study. It was found that the enzyme specified by this plasmid hydrolyzed sucrose, raffinose, inulin and levan, but not dextran, and did not synthesize fructan or glucan from sucrose. The sequence of the insert in pPNG102 was determined and was found to contain a large ORF that specifies a polypeptide of 99,319 Da with similarity to other sucrases. This gene was named levJ. The deduced amino acid (aa) sequence contained both a potential signal sequence and potential C-terminal cell envelope attachment domain. Alignments revealed an internal 331-aa domain not present in other levanases and sucrases. A neighbour-joining tree showed that sucrases of eukaryotic origin form a cluster with eubacterial sucrase/fructanases, and this cluster does not include other eubacterial sucrases. It is postulated that certain eukaryotic sucrase-encoding genes are of eubacterial origin.

Genetic studies of the phs locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism and pH homeostasis.

In Escherichia coli a pleiotropic mutation, phs, has been reported to affect Na+-linked metabolic functions and pH homeostasis. The phs mutation was previously mapped by its proximity to a met marker, presumed to be metB at 89 min. We have shown that a second mutation to auxotrophy, cymX, which is satisfied by either methionine or cysteine, is closely linked to phs. The cymX and phs lesions map close to trkB and rpsL at 73.5 min and we postulate that they are alleles of cysG and crp, respectively. The basis of the pH sensitivity of DZ3 is discussed in the light of this new information.

Roles of the trkB and trkC gene products of Escherichia coli in K+ transport.

Mutations at the trkB and trkC loci of Escherichia coli produce an abnormal efflux of K+. The mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. The mutations can be reverted by insertion of Tn10 into the mutated gene, and spontaneous revertants are fully recessive to the mutant allele in diploids. K+ efflux produced by NEM* and by DNP* persists in strains with presumed null mutations at either locus, indicating neither gene product is the primary target for the effect of these inhibitors on K+ efflux. The results are consistent with the view that trkB and trkC encode independent systems for K+ efflux. Mutations at these loci alter regulation of the process so that K+ efflux occurs inappropriately. A second mutation to the null state abolishes this abnormal K+ efflux. These genes may encode K+/H+ antiporters, an activity postulated to mediate K+ efflux and demonstrated to exist in E. coli and other bacteria.

Development of ligase-assisted spacer addition for the measurement of microsatellites.

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation detection. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capabilities as a genotyping method.

Isothermal amplification and multimerization of DNA by Bst DNA polymerase.

We have demonstrated the isothermal in vitro amplification and multimerization of several different linear DNA targets using only two primers and the strongly strand-displacing exonuclease-negative Bst DNA polymerase. This reaction has been termed linear target isothermal multimerization and amplification (LIMA). LIMA has been compared with cascade rolling-circle amplification and has been found to be less sensitive but to yield similar variable-length multimeric dsDNA molecules. Products from several different LIMA reactions were characterized by restriction analysis and partial sequence determination. They were found to be multimers of subsets of the target sequence and were not purely primer derived. The sensitivities with respect to target concentration of several different LIMA reactions were determined, and they varied from 0.01 amol to 1 fmol. The sensitivity and specificity of LIMA were further tested using E. coli genomic DNA, and the selective amplification of a transposon fragment was demonstrated. A successful strategy for reducing LIMA-dependent background DNA synthesis in rolling-circle amplification embodiments was devised. This entailed the affinity purification of circular DNA templates before amplification.

Interrogation of multimeric DNA amplification products by competitive primer extension using bst DNA polymerase (large fragment).

Linear dsDNA composed of tandem repeats may be exponentially amplified by the strongly strand-displacing Bst DNA polymerase (large fragment) and two primers specific for opposite strands. When the repetitive DNA is derivedfrom rolling circle replication of a circular template, the reaction is termed cascade rolling circle amplification (CRCA). We have developed a variant of CRCA in which one primer is attached to the surface of a microwell and the other is labeled, thus enabling detection of amplified material using an ELISA-like protocol. The circular template is derived by annealing and ligation of a padlock on target DNA. It was found that there was good correlation between the synthesis of amplified material and signal. The specificity of the reaction with respect to single-nucleotide polymorphisms was investigated, and it was found that Bst DNA polymerase is prone to extension from primers with mismatched 3' ends. Reliable single nucleotide specificity was only obtained when pre-synthesized amplified material was interrogated by competitive primer extension.

A method for the isolation of RNA from Streptococcus salivarius and its application to the transcriptional analysis of the gtfJK locus.

The glucosyltransferases from oral streptococci cleave sucrose and polymerize the glucose moieties. In Streptococcus salivarius ATCC 25975, two glucosyltransferase-encoding genes, gtfJ and gtfK, are closely linked and transcribed in the same direction. A procedure for the isolation of intact RNA from this organism was devised. The procedure incorporated a high-temperature mutanolysin treatment and selective precipitation by LiCl. The RNA was subject to Northern hybridization and RNase protection assays and it was concluded that the two genes are transcribed separately. A potential factor-independent transcription terminator was located in the intergenic region.

Definition of a fundamental repeating unit in streptococcal glucosyltransferase glucan-binding regions and related sequences.

The C-termini of the glucosyltransferases (Gtfs) of oral streptococci are responsible for glucan binding. These glucan-binding domains (GBDs) are composed of a series of repeated sequences that have been classified into four different classes (A-D) by virtue of sequence similarity and which, by inference, have been suggested to be of functional importance. In contrast, we propose that repeat sequences evolve in response to selection for an increase in the number of copies of a particular domain through multiple duplication events occurring at different times. According to this hypothesis, repeats should possess various degrees of similarity, especially if only key residues are of functional importance. Analysis of the GBDs of the Gtfs indicated that a common fundamental repeat, designated the "YG" repeat, could be discerned within the "A", "B", "C", and "D" repeats. Similar elements were also conserved in the ligand-binding repeats of the Clostridium difficile toxins and the lysins and the PspA protein of Streptococcus pneumoniae, suggesting that similar selective pressures had also been imposed on these sequences. Analysis of the "YG" repeats present in the GtfJ and GtfK of Streptococcus salivarius indicated that some of the "YG" repeats in the GBDs of these proteins had arisen as a result of duplication events involving a series of three sequential "YG" repeats.

Detection and characterisation of extended spectrum beta-lactamases in Klebsiella pneumoniae causing nosocomial infection.

One hundred and ninety-five multi-resistant strains of Klebsiella pneumoniae were isolated at Princess Alexandra Hospital (PAH) between December 1991 and June 1995. All these organisms produced extended spectrum beta-lactamases (ESBLs) as detected by the double disc synergy test (DDST). Between June 1994 and June 1995, a second population of 67 multi-resistant but DDST negative strains was isolated. Twenty multi-resistant Klebsiella pneumoniae strains (16 DDST positive and four DDST negative) and one susceptible strain were selected for further study. These were tested for production of ESBLs by two double disc synergy methods and agar dilution minimum inhibitory concentrations (MICs) with and without clavulanic acid. Detected ESBLs were further characterised by isoelectric focusing. The confirmed DDST positive K. pneumoniae strains all produced ESBLs that focused at an isoelectric point (pI) of 7.6, suggesting the presence of SHV-2, SHV-2a, SHV-6, SHV-7 or SHV-8 enzymes. The multi-resistant DDST negative strains showed no clavulanic acid synergy and thus no evidence of the presence of ESBLs.

Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci--ompAVD4, ompB, 16S rRNA, groESL spacer region.

Koalas are infected with two species of Chlamydia, C. pecorum and C. pneumoniae. While it is known that significant genetic diversity occurs in the C. pecorum strains infecting koalas, very little is known about the C. pneumoniae strains that infect this host. In the current study, 10 isolates of koala C. pneumoniae were analysed at four gene loci and found to be different to both the human and horse C. pneumoniae strains at all loci (biovar differences ranging from 0.3% at groESL up to 9.0% at ompAVD4). All koala biovar isolates studied were found to be 100% identical at ompAVD4 (all 10 isolates) and at ompB (all three isolates) gene. This lack of allelic polymorphisms at ompAVD4 has now been observed for koala C. pneumoniae, human C. pneumoniae, guinea pig inclusion conjuctivitis C. psittaci and feline conjuctivitis C. psittaci and may be correlated to a lack of antibody response to the chlamydial major outer membrane protein (MOMP) in these same strain/host combinations. This study also provides the first documented case of natural C. pneumoniae infection causing a severe and extended respiratory episode in a captive koala population. This captive episode is in contrast to most free-range observations in which koala C. pneumoniae is rarely documented as causing respiratory, ocular or urogenital tract disease.

Epizootiology of Chlamydia infections in two free-range koala populations.

The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that. while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.

Biochemical studies on LevJ, a fructanase from Actinomyces naeslundii T14V.

The Actinomyces naeslundii T14V gene levJ encodes a sucrase with fructanase activity and may be responsible for the fructanase activity observed bound to the surface of A. naeslundii T14V cells. A large proportion of LevJ expressed in Escherichia coli was translocated to the periplasm, and translocation and enzymatic activity were not affected by deletion of a putative cell-wall anchor sequence. The pH optimum of the enzyme was found to be between 5.5 and 6.5 whether the substrate was sucrose or inulin, although inulinase activity was more sensitive than sucrose activity to perturbation of the pH from the optimum. The relation between LevJ inulinase activity and pH was similar to that of A. naeslundii whole cells. LevJ exhibited standard saturation kinetics with sucrose, and the K(m) was calculated to be 89 mM, but it was not possible to calculate a K(m) for inulin. Evidence for inhibition of inulinase activity but not sucrase activity by high concentrations of inulin was obtained.

Sequences of multiple bacterial genomes and a Chlamydia trachomatis genotype from direct sequencing of DNA derived from a vaginal swab diagnostic specimen.

Ultra-deep Illumina sequencing was performed on whole genome amplified DNA derived from a Chlamydia trachomatis-positive vaginal swab. Alignment of reads with reference genomes allowed robust SNP identification from the C. trachomatis chromosome and plasmid. This revealed that the C. trachomatis in the specimen was very closely related to the sequenced urogenital, serovar F, clade T1 isolate F-SW4. In addition, high genome-wide coverage was obtained for Prevotella melaninogenica, Gardnerella vaginalis, Clostridiales genomosp. BVAB3 and Mycoplasma hominis. This illustrates the potential of metagenome data to provide high resolution bacterial typing data from multiple taxa in a diagnostic specimen.

Community-associated meticillin-resistant Staphylococcus aureus carriage in hospitalized patients in tropical northern Australia.

BACKGROUND: Community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in remote Australian Aboriginal communities. It is a prominent clinical pathogen in northern Australia with potential for transmission within the local hospital setting. AIM: To determine epidemiological characteristics of S. aureus carriage within the Royal Darwin Hospital. METHODS: We screened two patient groups: an 'admission group' recruited within 48 h of admission; and an 'inpatient group' recruited five or more days after admission. S. aureus isolates were characterized by antibiotic susceptibility testing and genotyped by a multi-locus sequence type-based high-resolution melting scheme. FINDINGS: S. aureus carriage on admission was 30.7% of 225 compared with 34.8% among 201 inpatients, with MRSA carriage of 2.2% and 18.9% respectively. We isolated CA-MRSA from 0.9% and 10.4%, and healthcare-associated (HCA)-MRSA from 1.3% and 9.0% of the admission and inpatient groups, respectively. Among the inpatient group, hospital-associated ST239 was the most common MRSA strain. CA-MRSA was represented by one clonal complex (CC) in the admission group (CC5) and seven CCs in the inpatient group (CC1, 93, 5, 6, 30, 75, 88). CONCLUSION: Inpatient carriage of multiple CA-MRSA lineages suggests selection for and transmission within the hospital of not only typical HCA-MRSA, but also diverse CA-MRSA strains.

Carriage of methicillin-resistant Staphylococcus aureus by veterinarians in Australia.

OBJECTIVE: To estimate the prevalence of carriage of methicillin-resistant Staphylococcus aureus (MRSA) among Australian veterinarians. METHODS: Individuals attending veterinary conferences in Australia in 2009 were recruited to provide nasal swabs and complete a questionnaire about their professional activities. Swabs were processed by standard methods for detecting MRSA and questionnaire responses were used to group veterinarians according to their areas of major work emphasis (species and practice type). Prevalence was estimated for each of these grouping and contingency tables and regression tree analysis used to explain the variation in MRSA carriage. RESULTS: Among the 771 respondents 'industry and government veterinarians' (controls) had the lowest prevalence of MRSA carriage at 0.9%. Veterinarians with horses as a major area of work emphasis had a prevalence of 11.8% (13-fold that of controls) and those whose only major emphasis was horses had a prevalence of 21.4% (23-fold that of controls). Veterinarians with dogs and cats as a major activity had a 4.9% prevalence (5-fold that of controls). Prevalence rates for other major activities (pigs, dairy and beef cattle, avian and wildlife) were also increased, but were estimated from smaller numbers of respondents. Regression tree analysis clearly isolated equine veterinarians and dog and cat practitioners as groups at increased risk of carriage of MRSA. CONCLUSION: Carriage of MRSA is a notable occupational health issue for veterinarians in clinical practice in Australia, particularly those who work with horses.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes.

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson's Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59-119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.