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ABSTRACTMycobacterium chimaerais an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellularecomplex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaerainfections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaeraclone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaerais a phylogenetically coherent group. In silicocomparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaerawith a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitroscreening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera. Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaeraqPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Tuberculosis (TB) represents a serious public health problem in Mozambique, with an estimated incidence rate of 548 cases per 100,000 population in 2011. Information on the molecular epidemiology of Mycobacterium tuberculosis(MTB) strains circulating in Mozambique is limited. This study provides the first description of the genetic diversity of MTB strains circulating in Beira city, the second largest town in Mozambique.

ABSTRACTBuruli ulcer (BU) is a necrotizing infection of skin and soft tissue caused by Mycobacterium ulcerans. In Australia, most cases of BU are linked to temperate, coastal Victoria and tropical, northern Queensland, and strains from these regions are distinguishable by variable-number tandem repeat (VNTR) typing. We present an epidemiological investigation of five patients found to have been infected during interstate travel and describe two nucleotide polymorphisms that differentiate M. ulceransstrains from northern Australia.

ABSTRACTTreponema pallidumis the causative agent of syphilis, a sexually transmitted infection of significant public health importance. Since 2000 there has been a marked increase in the number of cases of syphilis infections notified in Victoria, Australia, with the majority of cases occurring in men who have sex with men (MSM) and the highest incidence being in HIV-infected MSM. The molecular subtyping method described by Pillay et al. (A. Pillay et al., Sex. Transm. Dis. 25:408–414, 1998) has been used in this study to determine the diversity of T. pallidumsubtypes circulating locally and to look for any relationship between T. pallidumsubtypes and HIV status over a 6-year period (2004 to 2009). Treponema pallidumDNA was detected in 303 patient specimens (n= 3,652), and full subtyping profiles were obtained from 90 of these (from 88 patients). A total of 11 T. pallidumsubtypes were identified: types 14e (28, 31.1%), 14d (15, 16.7%), 14k (13, 14.4%), 14p (12, 13.3%), 14i (7, 7.8%) 14b (6, 6.7%), 14l (5, 5.6%), and 12i, 13b, 13i, and 13e (1 each, 1.1%). This study showed a similar level of variation among circulating T. pallidumstrains compared with that in other studies using the same methodology. A different mix of strains and different predominating strains have been found at each geographical study location, with type 14e emerging as the predominant local strain in Victoria. There was no detectable trend between T. pallidumsubtypes and the specimen collection site or stage of syphilis (where known), nor was there any relationship between particular strains and HIV status.

ABSTRACTThis work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulceransDNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.

ABSTRACTThe incidence of infectious syphilis in men who have sex with men and human immunodeficiency virus-infected patients has increased steadily in Victoria, Australia, since 2002. A TaqMan real-time PCR assay targeting the polAgene of Treponema pallidum(TpPCR) was developed. The analytical sensitivity of the assay was estimated to be 1.75 target copies per reaction. Initially, the assay was used to test a variety of specimens (excluding blood) from 598 patients. Of the 660 tests performed, positive PCR results were obtained for 55 patients. TpPCR results were compared with serology results for 301 patients being investigated for early syphilis. Of these patients, 41 were positive by both TpPCR and serology, 246 were negative by both TpPCR and serology, 4 were TpPCR positive but negative by serology, and 10 were TpPCR negative but showed evidence of recent or active infection by serology. Directly compared with serology, TpPCR showed 95% agreement, with a sensitivity of 80.39% and a specificity of 98.40%. Potential factors leading to the discrepant results are discussed. Concurrent serology on 21 patients with TpPCR-positive primary syphilitic lesions demonstrated that a panel of current syphilis serological tests has high sensitivity for the detection of early syphilis. We found that TpPCR is a useful addition to serology for the diagnosis of infectious syphilis. Direct comparison with other T. pallidumPCR assays will be required to fully assess the limitations of the assay.

ABSTRACTThe molecular basis for isoniazid resistance in Mycobacterium tuberculosisis complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosisisolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhAwere strongly associated with isoniazid resistance, while kasAmutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhApromoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhAwere associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpCor inhAand between mutations in kasAand mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

ABSTRACTElucidation of the molecular basis of isoniazid (INH) resistance in Mycobacterium tuberculosishas led to the development of different genotypic approaches for the rapid detection of INH resistance in clinical isolates. Mutations in katG, in particular the S315T substitution, are responsible for INH resistance in a large proportion of tuberculosis cases. However, the frequency of the katGS315T substitution varies with population samples. In this study, 52 epidemiologically unrelated clinical INH-resistant M. tuberculosisisolates collected in Australia were screened for mutations at katGcodon 315 and the fabG1-inhAregulatory region. Importantly, 52 INH-sensitive isolates, selected to reflect the geographic and genotypic diversity of the isolates, were also included for comparison. The katGS315T substitution and fabG1-inhA−15 C-to-T mutation were identified in 34 and 13 of the 52 INH-resistant isolates, respectively, and none of the INH-sensitive isolates. Three novel katGmutations, D117A, M257I, and G491C, were identified in three INH-resistant strains with a wild-type katGcodon 315, fabG1-inhAregulatory region, and inhAstructural gene. When analyzed for possible associations between resistance mechanisms, resistance phenotype, and genotypic groups, it was found that neither the katGS315T nor fabG1-inhA−15 C-to-T mutation clustered with any one genotypic group, but that the −15 C-to-T substitution was associated with isolates with intermediate INH resistance and isolates coresistant to ethionamide. In total, 90.4% of unrelated INH-resistant isolates could be identified by analysis of just two loci: katG315and the fabG1-inhA regulatory region.

ABSTRACTMutations at position 306 of embB(embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosisisolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P< 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P< 0.001 for trend). We examined the association between embB306 mutations and IS6110clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P= 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P= 0.008) and katG315 (odds ratio, 1.99; P= 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosisisolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA, is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins. In this study we investigated the architecture of the HBV minichromosome in more detail. In contrast to cellular chromatin the nucleosomal spacing of the HBV minichromosome has been shown to be unusually reduced by approximately 10 %. A potential candidate responsible for an alteration in the chromatin structure of the HBV minichromosome is the HBV core protein. The HBV core protein has been implicated in the nuclear targeting process of the viral genome. The association of the HBV core protein with nuclear HBV replicative intermediates could strengthen this role. Our findings, confirmed by in vivo and in vitro experiments indicate that HBV core protein is a component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes by 10 %. From this model of the HBV minichromosome we propose that the HBV core protein may have an impact on the nuclear targeting of the HBV genome and be involved in viral transcription by regulating the nucleosomal arrangement of the HBV regulatory elements, probably in a positive manner.

An intact activator‐binding site upstream of the σ54promoter of the pilin‐encoding pilEgene of Neisseria gonorrhoeaesuggests gonococci produce a protein capable of binding this sequence. We cloned a chimeric gene, rsp, that has sequence similarity to both the pilSand pilRgenes of Pseudomonas aeruginosaencoding a two‐component regulatory system that controls piliation. This gene is transcribed in N. gonorrhoeaeand indirect evidence suggests that Rsp binds to the activator‐binding site of the pilEgene. Despite this, mutation of rsphas no effect on piliation in N. gonorrhoeae, suggesting that the remnants of this regulatory system have persisted in the genome, despite the loss of its original function.

This selection of history journals suitable for an American public library includes firstly, those of a general nature; secondly, those devoted to the history of single countries; and thirdly, those limited to particular subdisciplines, such as oral history.

ABSTRACTA case of fever, sepsis, and chest lesions evident on a computed tomography scan of an indigenous man in northern Australia following burns to the feet is described. Sputum PCR testing revealed Mycobacterium leprae, and a fine-needle aspirate of the chest lesions demonstrated Cryptococcuscoinfection.

An intact activator-binding site upstream of the σ54 promoter of the pilin-encoding pilE gene of Neisseria gonorrhoeae suggests gonococci produce a protein capable of binding this sequence. We cloned a chimeric gene, rsp, that has sequence similarity to both the pilS and pilR genes of Pseudomonas aeruginosa encoding a two-component regulatory system that controls piliation. This gene is transcribed in N. gonorrhoeae and indirect evidence suggests that Rsp binds to the activator-binding site of the pilE gene. Despite this, mutation of rsp has no effect on piliation in N. gonorrhoeae, suggesting that the remnants of this regulatory system have persisted in the genome, despite the loss of its original function.

The Pseudomonas aeruginosa antibiotic supersusceptible mutant Z61 was 50–400-fold more susceptible than its wild-type parent K799 to 5 hydrophobic antibiotics. The strain Z61 outer membrane also demonstrated enhanced permeability towards a hydrophobic fluorescent probe. Strain Z61 cells had an altered cell surface, as revealed by phase-partitioning experiments, a lower amount of Lipid A phosphate, and a reduction in the number of Mg2+ binding sites in Lipid A, as demonstrated by dansyl polymyxin competition experiments. An antibiotic permation pathway directly through the outer membrane bilayer, rather than through porin proteins, is proposed for strain Z61.

Mucoid, alginate-producing Pseudomonas aeruginosa isolated from patients with cystic fibrosis were studied to determine the extent of heterogeneity of the isolates within individual sputa. Considerable heterogeneity involving cultural requirements for mucoid colonial growth was observed, and sensitivity to the ß-lactam antibiotic carbenicillin was also variable. For determining if the presence of alginate increased pulmonary survival of the bacteria, groups of rats were infected by transtracheal instillations of equivalent numbers of mucoid or nonmucoid P. aeruginosa, and survival was measured as the percentage of inoculum colony-forming units cultured from lung homogenates. Increased pulmonary survival of mucoid P. aeruginosa was observed in animals killed 3 hr or 6 hr after infection with unwashed bacteria. No difference in survival between mucoid and nonmucoid cells was observed when bacteria were washed prior to instillation. It was concluded that a single mucoid colony isolated from a sputum does not fully represent the population of mucoid P. aeruginosa within the patient and that pulmonary killing of mucoid P. aeruginosa can be less efficient than that for nonmucoid strains.