Genetic evolution of invasive emm28 Streptococcus pyogenes strains and significant association with puerperal infections in young women in Finland.

OBJECTIVES: Streptococcus pyogenes or group A streptococcus (GAS) is a human specific pathogen that annually infects over 700 million individuals. GAS strains of type emm28 are an abundant cause of invasive infections in Europe and North America. METHODS: We conducted a population-based study on bacteraemic emm28 GAS cases in Finland, from 1995 to 2015. Whole-genome sequencing (WGS) was used to genetically characterize the bacterial isolates. Bayesian analysis of the population structure was used to define genetic clades. Register-linkage analysis was performed to test for association of emm28 GAS with delivery- or postpartum-related infections. A genome-wide association study was used to search for DNA sequences associated with delivery or puerperal infections. RESULTS: Among 3060 bacteraemic cases reported during the study period, 714 were caused by emm28. Women comprised a majority of cases (59 %, 422/714), and were significantly over-represented (84.4 %, 162/192, p < 0.0001) among cases in the childbearing age group (20-40 years). Register-linkage analysis revealed strong association (p < 0.0001) of emm28 bacteraemias with delivery and puerperium. In this register-linkage analysis, 120 women with GAS bacteraemia were identified and linked to delivery, infections during delivery or puerperium time. Among these the proportion of cases caused by emm28 was significantly higher than any other emm type (55.8%, 67/120, p < 0.0001). Among the four genetic subclades identified, SC1B has dominated among the bacteraemic cases since 2000. Altogether 620 of 653 (94.9%) isolates belonged to SC1B. No specific sequence or genetic clade was found nonrandomly associated with delivery or puerperal infections. CONCLUSIONS: Women of childbearing age were significantly overrepresented among bacteraemic emm28 GAS cases, and in particular were strongly associated with delivery and puerperium cases over the 21 years studied. The molecular mechanisms behind these associations are unclear and warrant further investigation.

Haemagglutinin mutations and glycosylation changes shaped the 2012/13 influenza A(H3N2) epidemic, Houston, Texas.

While the early start and higher intensity of the 2012/13 influenza A virus (IAV) epidemic was not unprecedented, it was the first IAV epidemic season since the 2009 H1N1 influenza pandemic where the H3N2 subtype predominated. We directly sequenced the genomes of 154 H3N2 clinical specimens collected throughout the epidemic to better understand the evolution of H3N2 strains and to inform the H3N2 vaccine selection process. Phylogenetic analyses indicated that multiple co-circulating clades and continual antigenic drift in the haemagglutinin (HA) of clades 5, 3A, and 3C, with the evolution of a new 3C subgroup (3C-2012/13), were the driving causes of the epidemic. Drift variants contained HA substitutions and alterations in the potential N-linked glycosylation sites of HA. Antigenic analysis demonstrated that viruses in the emerging subclade 3C.3 and subgroup 3C-2012/13 were not well inhibited by antisera generated against the 3C.1 vaccine strains used for the 2012/13 (A/Victoria/361/2011) or 2013/14 (A/Texas/50/2012) seasons. Our data support updating the H3N2 vaccine strain to a clade 3C.2 or 3C.3-like strain or a subclade that has drifted further. They also underscore the challenges in vaccine strain selection, particularly regarding HA and neuraminidase substitutions derived during laboratory passage that may alter antigenic testing accuracy.

Mycobacterium tuberculosis bacteraemia: experience from a non-endemic urban centre.

The isolation of Mycobacterium tuberculosis from blood culture specimens has been associated with human immunodeficiency virus (HIV) co-infection with variable impact on tuberculosis (TB) mortality reported. The overwhelming majority of M. tuberculosis bacteraemia cases were described in developing countries. We present a nested case-control analysis of clinical, sociodemographic and behavioural risk factors in patients with positive M. tuberculosis blood cultures compared with patients with negative blood cultures from a 9-year population-based active TB surveillance study conducted in Houston, Texas. There were 42 patients with M. tuberculosis bacteraemia, 47 blood culture negative patients and 3573 patients for whom no mycobacterial blood culture was requested. HIV infection was more common in patients for whom a mycobacterial blood culture was requested (79.8% versus 15.1% p <0.001). Of the patients with M. tuberculosis bacteraemia, six were HIV negative or had no documentation of HIV status, including five with immunosuppressive conditions other than HIV. Patients with M. tuberculosis bacteraemia were more likely than patients with negative blood cultures to be deceased at diagnosis or to die while on TB therapy (50.0% versus 17.0%, p <0.01), to report men-who-have-sex-with-men behaviour (31.7% versus 13.0%, p 0.03), to have renal failure (28.6% versus 6.4%, p 0.01), and to have HIV RNA levels higher than 500 000 copies/mL (61.9% versus 17.2%, p ≤0.01). Requests for mycobacterial culture of blood specimens were more common in HIV-infected individuals, and the presence of M. tuberculosis bacteraemia was associated with a significant increase in mortality.

Pharmacokinetic differences in exposure to camphor after intraruminal dosing in selectively bred lines of goats.

A pharmacokinetic dosing study with camphor was used to determine whether selection lines of high-juniper-consuming goats (HJC, n = 12) and low-juniper-consuming goats (LJC, n = 12) differed in their respective disposition kinetics. Postdosing plasma camphor concentrations were used to examine whether a timed single blood sample collected after intraruminal administration of camphor would be a useful screening test to aid in the identification of HJC. Yearling female Boer x Spanish goats (n = 24) received a single intraruminal dose of monoterpene cocktail (0.270 g/kg of BW) containing 4 different monoterpenes that represented their composition previously reported for Ashe juniper (Juniperus ashei). Camphor, the predominant monoterpene in Ashe juniper, was 49.6% of the mix and was the monoterpene analyzed for this study. Blood samples were taken at 15 time points from 0 to 8 h after dosing. Concentrations of camphor were measured in plasma using solid phase extraction and gas chromatography/flame-ionization detection analysis. Maximal plasma concentration of camphor was greater for LJC than HJC (P = 0.01), and area under the curve extrapolated to infinity was greater for LJC than HJC (P < 0.01). Total systemic exposure (area under the curve) to camphor was 5 times less in HJC goats. We conclude that 1) HJC goats possess internal mechanisms to reduce the bioavailability of camphor, and 2) a blood sample taken at 45 min or at 60 min after intraruminal administration of camphor may be useful for identifying HJC individual animals from within large populations of goats.

Molecular characteristics of pharyngeal and invasive emm3 Streptococcus pyogenes strains from Norway, 1988-2003.

A major virulence factor of group A streptococci (GAS) is the M protein. Strains with the M3 type are more often associated with necrotizing fasciitis (NF) and streptococcal toxic shock syndrome, and have a higher case fatality rate than strains of other M types. To better understand the epidemiology of M3 GAS strains in Norway, we analyzed 59 invasive and 69 pharyngeal isolates with respect to prophage content, allelic variation in emm3, mtsR encoding the metal transporter of Streptococcus repressor (mtsR), and sclB coding for streptococcal collagen-like protein B. The Norwegian emm3 strains were very homogeneous, mainly harboring the emm allele 3.1 and prophage profile PhiG3.01. Other prophage profiles were transient. The mutation in mtsR known to truncate the protein and result in decreased capacity to cause NF was not found in our isolates. The sclB gene usually harbored five or eight contiguous repeats of a CAAAA pentanucleotide sequence and a highly modular and variable collagen structural motif (CSM) region with 9 and 12 amino acid M3-specific conserved motif repeats distributed across the entire CSM region. Strains with 5 CAAAA repeats emerged in 1993 and these strains were associated with the increase in invasive M3 cases in the period 1993-2003.

Reduced in vitro functional activity of human NRAMP1 (SLC11A1) allele that predisposes to increased risk of pediatric tuberculosis disease.

Polymorphic variants within the human natural resistance-associated macrophage protein-1 (NRAMP1, also known as SLC11A1) gene have been shown to impact on susceptibility to tuberculosis in different human populations. In the mouse, Nramp1 is expressed at the macrophage phagosomal membrane and its activity can be assayed by the relative acquisition of mannose 6-phosphate receptor (M6PR) in Salmonella-containing vacuoles. Based on this M6PR recruitment assay, we have now developed an assay in primary human macrophages to test the function of human NRAMP1 gene variants. First, we established that M6PR acquisition was significantly higher (P = 0.002) in human U-937 monocytic cell lines transfected with NRAMP1 as compared to untransfected U-937 cells. Second, the M6PR assay was shown to be highly reproducible for NRAMP1 activity in monocyte-derived macrophages (MDM) from healthy volunteers. Finally, the assay was investigated in MDM from pediatric tuberculosis patients and significantly lower NRAMP1 activity was detected in MDM from individuals homozygous for the NRAMP1-274 high-risk allele (CC genotype) in comparison to heterozygous individuals (CT genotype; P=0.013). The present study describes both an assay for human NRAMP1 functional activity and concomitant evidence for reduced NRAMP1 function in the common genetic variant shown to be associated with tuberculosis susceptibility in pediatric patients.

Measurement of the Michel parameter rho in muon decay.

The TWIST Collaboration has measured the Michel parameter rho in normal muon decay, mu(+)--> e(+)nu(e)nu (mu). In the standard model, rho = 3/4. Deviations from this value imply mixing of left- and right-handed muon and electron couplings. We find rho=0.750 80+/-0.000 32(stat) +/- 0.000 97(syst) +/- 0.000 23, where the last uncertainty represents the dependence of rho on the Michel parameter eta. This result sets new limits on the W(L)-W(R) mixing angle in left-right symmetric models.

Recurrent tuberculosis in Houston, Texas: a population-based study.

OBJECTIVE: To determine the predictors of recurrence of tuberculosis (TB), the drug resistance pattern of Mycobacterium tuberculosis strains recovered from recurrent TB patients, and the frequency of re-infection with a new M. tuberculosis strain among patients with recurrent disease. DESIGN: A population-based, retrospective case-control study using the Houston Tuberculosis Initiative database. RESULTS: We found that, among 100 patients with recurrent TB who completed adequate therapy for a first episode of TB, not receiving directly observed therapy, pulmonary disease, HIV/AIDS diagnosis, not having a family physician, being unemployed and using public transportation were predictors of recurrent disease. There was a significant increase in drug-resistant M. tuberculosis strains in the second episode of disease compared to the first episode (21.3% vs. 8.2%, P = 0.04). Exogenous re-infection with a new strain of M. tuberculosis was found to cause 24-31% of recurrent TB. CONCLUSION: Recurrent TB in Houston is associated with a significant increase in drug-resistant M. tuberculosis strains. Re-infection with a new M. tuberculosis strain causes a significant proportion of recurrent TB in an area of low TB incidence. Patients with HIV/AIDS constitute a population at increased risk of disease recurrence.

New measurement of the cosmic-ray positron fraction from 5 to 15 GeV.

We present a new measurement of the cosmic-ray positron fraction at energies between 5 and 15 GeV with the balloon-borne HEAT-pbar instrument in the spring of 2000. The data presented here are compatible with our previous measurements, obtained with a different instrument. The combined data from the three HEAT flights indicate a small positron flux of nonstandard origin above 5 GeV. We compare the new measurement with earlier data obtained with the HEAT-e(+/-) instrument, during the opposite epoch of the solar cycle, and conclude that our measurements do not support predictions of charge sign dependent solar modulation of the positron abundance at 5 GeV.

Extra-pulmonary manifestations in a large metropolitan area with a low incidence of tuberculosis.

BACKGROUND: The increases in extra-pulmonary tuberculosis (EPTB) have been largely due to human immunodeficiency virus co-infection. The rates of EPTB have remained constant despite the decline in pulmonary tuberculosis (PTB) cases. OBJECTIVE: To evaluate covariates associated with EPTB. METHODS: A 4-year cohort of EPTB patients was compared with PTB cases. Enrollees were assessed for TB risk, medical records were reviewed, and Mycobacterium tuberculosis isolates were fingerprinted. RESULTS: We identified 538 EPTB cases (28.6%) in a total of 1878 enrollees. The most common sites of infection were lymph nodes (43%) and pleura (23%). EPTB cases included 320 (59%) males, 382 (71%) patients were culture-positive, and 332 (86.9%) patient isolates were fingerprinted. Fewer EPTB than PTB patients belonged to clustered M. tuberculosis strains (58% vs. 65%; P = 0.02). A multivariate model identified an increased risk for EPTB among African Americans (OR = 1.9, P = 0.01), HIV-seropositive (OR = 3.1, P < 0.01), liver cirrhosis (OR = 2.3, P = 0.02), and age <18 years (OR = 2.0, P = 0.04). Patients with concomitant pulmonary and extra-pulmonary infections were more likely to die within 6 months of TB diagnosis (OR = 2.3, P < 0.01). CONCLUSIONS: African American ethnicity is an independent risk factor for EPTB. Mortality at 6 months is partly due to the dissemination of M. tuberculosis and the severity of the underlying co-morbidity.

Extrathoracic tuberculosis lymphadenitis in adult HIV seronegative patients: a population-based analysis in Houston, Texas, USA.

OBJECTIVE: To evaluate the covariates associated with extrathoracic tuberculosis lymphadenitis (ETBL) among adult HIV-seronegative patients. METHODS: Enrollees were interviewed for TB risk assessment, their medical records were reviewed, and their Mycobacterium tuberculosis isolates underwent molecular characterization. Between 1 October 1995 and 30 September 1999, HIV-negative patients with ETBL were compared with other HIV-negative TB patients. RESULTS: We identified 73 ETBL cases (5%) out of a total of 1371 adult HIV-negative enrollees. Significant variables predicting ETBL in the univariate analysis included age < 45 years, female sex, Asian ethnicity, foreign birth, BCG vaccination, and infection with a M. tuberculosis isolate identified in major genetic group 1. Further analysis by birth country revealed increased ETBL risk for persons from countries other than the Americas and with a TB incidence > 25 per 100 000 per year. The multivariate model demonstrated increased risk for ETBL for patients of female sex (OR = 2.6, P < 0.01) and birth in Africa or South-east Asia (OR = 4.8; P = 0.03 and OR = 33.6; P = 0.01, respectively). CONCLUSIONS: In adult HIV-negative patients, ETBL occurs more frequently in females and in immigrants from countries other than the Americas; persons from India, South-east Asia and the Eastern Mediterranean exhibited the highest risk among these regions.

5' dinucleotide repeat polymorphism of NRAMP1 and susceptibility to tuberculosis among Caucasian patients in Houston, Texas.

SETTING: Houston Tuberculosis Initiative (HTI) and Baylor College of Medicine, Houston, Texas. OBJECTIVE: To further explore the association between the polymorphisms of NRAMP1 and human susceptibility/resistance to tuberculosis (TB), specifically to determine whether the reported association shown for blacks and Asians holds true for Caucasian populations. DESIGN: In a case-control study, 135 adult Caucasian TB patients and 108 adult Caucasian HIV-seronegative non-TB controls were analyzed for the association between the polymorphisms in NRAMP1 gene and clinical TB. RESULTS: Heterozygote at 5'(GT)n, a dinucleotide repeat polymorphism in the promoter of NRAMP1, was observed at significantly higher frequencies among HIV-negative patients with pulmonary TB (41.6%; OR 2.02; 95%CI 1.11-3.64), extra-pulmonary TB (66.7%; OR 4.80; 95%CI 1.34-17.15), and HIV-seropositive TB patients (50%; OR 3.77; 95%CI 1.33-10.66) in comparison with the controls (27.8%). Homozygotes (GT)(10,10) were over-represented among HIV-positive TB patients (18.2%; OR 6.86; 95%CI 1.55-30.21) compared to the controls (5.5%). CONCLUSION: These findings suggest that the 5'(GT)n polymorphism of NRAMP1 modifies TB susceptibility in this Caucasian population, and could possibly be related to the site of infection among HIV-negative individuals and HIV-coinfected TB.

Observation of ultraslow and stored light pulses in a solid.

We report ultraslow group velocities of light in an optically dense crystal of Pr doped Y2SiO5. Light speeds as slow as 45 m/s were observed, corresponding to a group delay of 66 micros. Deceleration and "stopping" or trapping of the light pulse was also observed. These reductions of the group velocity are accomplished by using a sharp spectral feature in absorption and dispersion that is produced by resonance Raman excitation of a ground-state spin coherence.

Evasion of human innate and acquired immunity by a bacterial homolog of CD11b that inhibits opsonophagocytosis.

Microbial pathogens must evade the human immune system to survive, disseminate and cause disease. By proteome analysis of the bacterium Group A Streptococcus (GAS), we identified a secreted protein with homology to the alpha-subunit of Mac-1, a leukocyte beta2 integrin required for innate immunity to invading microbes. The GAS Mac-1-like protein (Mac) was secreted by most pathogenic strains, produced in log-phase and controlled by the covR-covS two-component gene regulatory system, which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific to Mac that correlated with the course of disease, demonstrating that Mac was produced in vivo. Mac bound to CD16 (FcgammaRIIIB) on the surface of human polymorphonuclear leukocytes and inhibited opsonophagocytosis and production of reactive oxygen species, which resulted in significantly decreased pathogen killing. Thus, by mimicking a host-cell receptor required for an innate immune response, the GAS Mac protein inhibits professional phagocyte function by a novel strategy that enhances pathogen survival, establishment of infection and dissemination.

Amino acid sequence determinants of extended spectrum cephalosporin hydrolysis by the class C P99 beta-lactamase.

Class C beta-lactamases are commonly encoded on the chromosome of Gram-negative bacterial species. Mutations leading to increased expression of these enzymes are a common cause of resistance to many cephalosporins including extended spectrum cephalosporins. Recent reports of plasmid- and integrin-encoded class C beta-lactamases are a cause for concern because these enzymes are likely to spread horizontally to susceptible strains. Because of their increasing clinical significance, it is critical to identify the determinants of catalysis and substrate specificity of these enzymes. For this purpose, the codons of a set of 21 amino acid residues that encompass the active site region of the P99 beta-lactamase were individually randomized to create libraries containing all possible amino acid substitutions. The amino acid sequence requirements for the hydrolysis of ceftazidime, an extended spectrum cephalosporin commonly used to treat serious infections, were determined by selecting resistant mutants from each of the 21 libraries. DNA sequencing identified the residue positions that are critical for ceftazidime hydrolysis. In addition, it was found that certain amino acid substitutions in the omega-loop region of the P99 enzyme result in increased ceftazidime hydrolysis suggesting the loop is an important determinant of substrate specificity.

Tissue disposition and depletion of penicillin G after oral administration with milk in unweaned dairy calves.

OBJECTIVE: To determine tissue depletion of penicillin G in calves after oral ingestion with milk replacer and estimate a withdrawal period. DESIGN: Longitudinal controlled trial. ANIMALS: 26 Holstein calves. PROCEDURE: Once daily, 24 calves were fed milk replacer containing procaine penicillin G (0.68 mg/kg [0.31 mg/lb] of body weight); 2 calves served as controls. After 1 feeding, 12 calves were euthanatized in groups of 3 each 4, 6.5, 9.5, and 13 hours after feeding. After 14 days, 12 calves were euthanatized in groups of 3 each 4, 6.5, 9.5, and 13 hours after the final feeding. Concentrations of penicillin G were determined in tissues, blood, and urine by use of high-performance liquid chromatography. RESULTS: Penicillin G was not detected in muscle samples of treated calves. The highest concentrations of penicillin G in plasma, kidney, and liver were 13 ng/ml, 92 ng/g, and 142 ng/g, respectively. Thirteen carcasses had violative drug residues; 12 had violative residues in the liver only, and 1 had violative residues in the liver and kidney. A 21-hour withdrawal period was estimated. CONCLUSIONS AND CLINICAL RELEVANCE: Liver had the highest concentration of penicillin G and was most likely to have violative residues. Feeding calves milk containing penicillin G has the potential to cause violative drug residues in tissues. It is recommended to observe an appropriate withdrawal time prior to slaughter if calves are fed milk from cows treated with penicillin G.

Global differential gene expression in response to growth temperature alteration in group A Streptococcus.

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A Streptococcus (GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29 degrees C and 37 degrees C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29 degrees C relative to 37 degrees C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription-PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40 degrees C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.

Bioavailability and disposition of sodium and procaine penicillin G (benzylpenicillin) administered orally with milk to calves.

Eighteen 1-week-old Holstein calves were randomly assigned to one of three groups: (a) sodium penicillin G administered intravenously, (b) sodium penicillin G administered orally, or (c) procaine penicillin G administered orally. All calves were dosed with penicillin G at 4.0 mg/kg BW. At 5 weeks of age, the calves were dosed again. Blood samples were taken serially for 24 h after both dosings. Plasma was assayed for penicillin G by high performance liquid chromatography (HPLC). For i.v. administration, the area under the concentration-time curve (AUC), 7456 and 5508 ng/mL h, and systemic clearance, 0.54 and 0.73 L/kg h, were significantly different (P < 0.05) at 1 and 5 weeks of age, respectively. There were no significant differences between orally administered sodium and procaine penicillin G within the same age groups. Following oral (p.o.) administration, there were significant differences (P < 0.01) at 1 and 5 weeks of age in the AUC, 760 and 409 ng/mL h, terminal half-life, 2.1 and 1.6 h, time of maximum concentration (TMAX), 3.0 and 2.3 h, and maximum plasma concentration (CMAX), 85 and 58 ng/mL, respectively. Bioavailability was 10.2 and 7.4% at 1 and 5 weeks, respectively.