Search

Mortality rates associated with C. gattii infections are estimated to be between 13% and 33%, depending on an individual’s predisposition, and C. gattii has caused at least 39 deaths in the PNW region. There have been four other international travel cases reported in patients from Europe and Asia with travel history to the PNW, but this report describes the first North American traveler who acquired C. deuterogattii infection presenting within the United States and the first case of a C. deuterogattii outbreak infection associated with anti-GM-CSF autoantibodies. Early and accurate diagnoses are important for disease prevention and treatment and for control of infectious diseases. Continual reporting of C. deuterogattii infections is necessary to raise awareness of the ongoing outbreak in the PNW and to alert travelers and physicians to the areas of endemicity with potential risks., The region encompassing the Pacific Northwest (PNW), Vancouver Island, Oregon, and Washington has been the location of an ongoing Cryptococcus gattii outbreak since the 1990s, and there is evidence that the outbreak is expanding along the West Coast into California. Here we report a clinical case of a 69-year-old, HIV-negative man from North Carolina who was diagnosed with a fungal brain mass by magnetic resonance imaging (MRI) and pathology. He had traveled to Seattle and Vancouver 3 years earlier and to Costa Rica 4 months prior to presentation. Phenotypic evidence showed that the fungal mass isolated from the patient’s brain represented C. gattii. In agreement with the phenotypic results, multilocus sequence typing (MLST) provided genotypic evidence that assigned the infecting organism within the C. gattii species complex and to the C. deuterogattii VGIIa clade. Whole-genome sequencing revealed >99.99% identity with the C. deuterogattii reference strain R265, indicating that the infecting strain is derived from the highly clonal outbreak strains in the PNW. We conclude that the patient acquired the C. gattii infection during his travel to the region 3 years prior and that the infection was dormant for an extended period of time before causing disease. The patient tested positive for anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies, supporting earlier reports that implicate these autoantibodies as a risk factor associated with C. gattii infection.

Members of the Mycobacterium abscessus group (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducible erm (41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring a truncation or a T28C substitution in erm (41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncated erm (41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 of erm (41) and 23S rRNA rrl gene mutations at position 2058 and/or 2059. Truncated and full-size erm (41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showed rrl mutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an active erm (41), inactive erm (41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation.

The smooth-to-rough colony morphology shift in Mycobacterium abscessus has been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistent M. abscessus infection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level of fadD23 , a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed as M. abscessus subspecies abscessus and were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations in mps1 and mps2 , 13% displayed previously unreported mutations in mmpL4a and mmpS4 , the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus (AFB) culture methods. Our work highlights applications of MALDI-TOF MS beyond identification, focusing on mycobacterial lipids relevant in virulence and adaptation. Later isolates displayed accumulation of triacylglycerol and reduced expression of fadD23 , sometimes preceding rough colony onset. Our results indicate that clonal diversification and a shift in lipid metabolism, including the loss of GPL, occur during chronic lung infection with M. abscessus . GPL loss alone may not account for all traits associated with rough morphology.

Mycobacterium abscessus ( M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii , and Mycobacterium massiliense . We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus , M. massiliense , and M. bolletii . Based on the M. abscessus ATCC 19977 T genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus , 15 M. massiliense , and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm (41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm (41) PCR for straightforward identification of M. abscessus , M. massiliense , and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group.

The most common cause of invasive aspergillosis (IA) in patients with chronic granulomatous disease (CGD) is Aspergillus fumigatus followed by A. nidulans ; other aspergilli rarely cause the disease. Here we review two clinical cases of fatal IA in CGD patients and describe a new etiologic agent of IA refractory to antifungal therapy. Unlike typical IA caused by A. fumigatus , the disease caused by the new species was chronic and spread from the lung to multiple adjacent organs. Mycological characteristics and the phylogenetic relationship with other aspergilli based on the sequence analysis of Mcm7 , RPB2 , and Tsr1 indicated that the new species, which we named as A. tanneri , belongs to Aspergillus section Circumdati . The species has a higher amphotericin B, voriconazole, and itraconazole MIC and causes more chronic infection in CGD mice than A. fumigatus . This is the first report documenting IA in CGD patients caused by a species belonging to the Aspergillus section Circumdati that is inherently resistant to azoles and amphotericin B. Unlike the results seen with many members of Aspergillus section Circumdati , ochratoxin was not detected in filtrates of cultures grown in various media. Our phenotypic and genetic characterization of the new species and the case reports will assist future diagnosis of infection caused by A. tanneri and lead to more appropriate patient management.

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis , an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis , CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H 2 O 2 but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O 2 -independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.

The aminoglycosides streptomycin, amikacin, and kanamycin and the cyclic polypeptide capreomycin are all widely used in second-line therapy for patients who develop multidrug-resistant tuberculosis. We have characterized a set of 106 clinical isolates of Mycobacterium tuberculosis using phenotypic drug susceptibility testing (DST) to determine the extent of resistance to each agent and cross-resistance between agents. These results were compared with polymorphisms in the DNA sequences of ribosome-associated genes previously implicated in resistance and with the clinical outcomes of subjects from whom these isolates were obtained. Thirty-six (34%) of these isolates displayed resistance to one or more of these agents, and the majority of these (20 of 36) showed cross-resistance to one or more agents. Most (33 of 36) of the resistant isolates showed polymorphisms in the 16S ribosome components RpsL and rrs . Three resistant strains (3 of 36) were identified that had no known polymorphisms in ribosomal constituents. For kanamycin and streptomycin, molecular DST significantly outperformed phenotypic DST using the absolute concentration method for predicting 4-month sputum conversion (likelihood ratios of 4.0 and 2.0, respectively) and was equivalent to phenotypic DST using the National Committee for Clinical Laboratory Standards (NCCLS)-approved agar proportion method for estimating MIC (likelihood ratio, 4.0). These results offer insight into mechanisms of resistance and cross-resistance among these agents and suggest that the development of rapid molecular tests to distinguish polymorphisms would significantly enhance clinical utility of this important class of second-line antituberculosis drugs.

A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus . This prompted us to characterize N. udagawae in comparison to A. fumigatus . Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55°C but not at 10°C, N. udagawae is able to grow at 10°C but fails to grow at >42°C. Furthermore, compared to A. fumigatus , the conidia of N. udagawae require longer incubation periods to germinate at 37°C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91 phox − / − mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens , but not with Escherichia coli . In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1β. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.

Chronic granulomatous disease (CGD) is an inherited immune deficiency characterized by increased susceptibility to infection with Staphylococcus , certain gram-negative bacteria, and fungi. Granulibacter bethesdensis , a newly described genus and species within the family Acetobacteraceae , was recently isolated from four CGD patients residing in geographically distinct locales who presented with fever and lymphadenitis. We sequenced the genome of the reference strain of Granulibacter bethesdensis , which was isolated from lymph nodes of the original patient. The genome contains 2,708,355 base pairs in a single circular chromosome, in which 2,437 putative open reading frames (ORFs) were identified, 1,470 of which share sequence similarity with ORFs in the nonpathogenic but related Gluconobacter oxydans genome. Included in the 967 ORFs that are unique to G. bethesdensis are ORFs potentially important for virulence, adherence, DNA uptake, and methanol utilization. GC% values and best BLAST analysis suggested that some of these unique ORFs were recently acquired. Comparison of G. bethesdensis to other known CGD pathogens demonstrated conservation of some putative virulence factors, suggesting possible common mechanisms involved in pathogenesis in CGD. Genotyping of the four patient isolates by use of a custom microarray demonstrated genome-wide variations in regions encoding DNA uptake systems and transcriptional regulators and in hypothetical ORFs. G. bethesdensis is a genetically diverse emerging human pathogen that may have recently acquired virulence factors new to this family of organisms.

Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations oftkmutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSVtkmutants in animal neurons is impaired. Here we report the detection of mutated HSVtksequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1tkopen reading frames from ganglia were amplified by PCR, cloned, and sequenced.tkmutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1tkmutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSVtkmutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected withtkmutant virus alone. These data suggest that diversetkmutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.

Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent life-threatening bacterial and fungal infections. We characterized the effects of single and combination antifungal therapy on survival, histopathology, and laboratory markers of fungal burden in experimental aspergillosis in the p47 phox − / − knockout mouse model of CGD. CGD mice were highly susceptible to intratracheal Aspergillus fumigatus challenge, whereas wild-type mice were resistant. CGD mice were challenged intratracheally with a lethal inoculum (1.25 × 10 4 CFU/mouse) of A. fumigatus and received one of the following regimens daily from day 0 to 4 after challenge ( n = 19 to 20 per treatment group): (i) vehicle, (ii) amphotericin B (intraperitoneal; 1 mg/kg of body weight), (iii) micafungin (intravenous; 10 mg/kg), or (iv) amphotericin B plus micafungin. The rank order of therapeutic efficacy based on prolonged survival, from highest to lowest, was as follows: amphotericin B plus micafungin, amphotericin B alone, micafungin alone, and the vehicle. Lung histology showed pyogranulomatous lesions and invasive hyphae, but without hyphal angioinvasion or coagulative necrosis. Treatment with micafungin alone or combined with amphotericin B produced swelling of invasive hyphae that was not present in mice treated with the vehicle or amphotericin B alone. Assessment of lung fungal burden by quantitative PCR showed no significant difference between treatment groups. Serum galactomannan levels were at background despite documentation of invasive aspergillosis by histology. Our findings showed the superior efficacy of the amphotericin B and micafungin combination compared to either agent alone after A. fumigatus challenge and also demonstrated unique features of CGD mice as a model for experimental aspergillosis.

Urinary nitrite and F2-isoprostanes, an index of oxidant stress, were elevated during chlamydial genital infection of mice. Enhancement of urinary nitrite and F2-isoprostanes was observed in phagocyte oxidase-deficient mice. Inhibition of inducible nitric oxide synthase reduced isoprostane excretion. We conclude that nitrogen radicals induce F2-isoprostane production and excretion during murine chlamydial genital infection.

The molecular methodologies used in our laboratories have allowed us to define a group of Nocardia isolates from clinical samples which resemble the type strain of Nocardia veterana . Three patient isolates and the type strain of N. veterana gave identical and distinctive restriction fragment length polymorphisms (RFLPs) for an amplified portion of the 16S rRNA gene. These three isolates and the N. veterana type strain also gave identical RFLPs for an amplified portion of the 65-kDa heat shock protein gene, but this pattern was identical to that obtained for the Nocardia nova type strain. Sequence analysis of both a 1,359-bp region of the 16S rRNA gene and a 441-bp region of the heat shock protein gene of the patient isolates showed 100% identities with the same regions of the N. veterana type strain. DNA-DNA hybridization of the DNA of one of the patient isolates with the DNA of the N. veterana type strain showed a relative binding ratio of 82%, with 0% divergence, confirming that the isolate was N. veterana . Biochemical and susceptibility testing showed no significant differences among the patient isolates and the N. veterana type strain. Significantly, the results of antimicrobial susceptibility testing obtained for our isolates were similar to those obtained for N. nova , indicating that susceptibility testing alone cannot discriminate between these species. We present two case studies which show that N. veterana is a causative agent of pulmonary disease in immunocompromised patients residing in North America. We also describe difficulties encountered in using 16S rRNA gene sequences alone for discrimination of N. veterana from the related species Nocardia africana and N. nova because of the very high degree of 16S rRNA gene similarity among them.

Candida glabrata is second only to Candida albicans as a cause of candidemia in adults, accounting for ca. 16 to 24% of candidemia cases (4, 18). Septicemia, endocarditis, pyelonephritis, esophagitis, and other deep sites of infection may also occur (5, 14). Mortality in patients with C. glabrata sepsis is high, unadjusted for the underlying disease, and at least equivalent to mortality in patients with C. albicans sepsis (6). Amphotericin B is considered the drug of choice. Although only 5 to 6% of C. glabrata isolates are resistant (MIC ≥ 64 μg/ml) to fluconazole in vitro, the clinical efficacy of this drug against C. glabrata infections has been difficult to establish (18, 22). C. glabrata infection largely occurs in patients with severe underlying diseases, rendering drug efficacy more difficult to assess. Catheter-acquired infections are a major cause of candidemia and may respond to catheter removal alone (14, 16). A randomized trial comparing fluconazole with amphotericin B in candidemia of nonneutropenic patients, largely catheter acquired, found equivalent results in patients with C. glabrata sepsis who were treated with fluconazole or amphotericin B (20). Four of 11 patients failed to respond to fluconazole, including one with a mixed infection, whereas 2 of 13 failed to respond to amphotericin B (P > 0.05). The range of error with a sample size this small is such that fluconazole could have provided a proportion cured that was 50% less than amphotericin B and still the difference would have failed to be statistically significant (Fisher's exact test). Complicating any decision about the use of fluconazole against C. glabrata infection is the 30-fold-lower susceptibility of C. glabrata compared to C. albicans (18). Drug concentrations used to distinguish susceptible from resistant C. glabrata were obtained from experience with C. albicans and may not apply to C. glabrata (21). An animal model for C. glabrata might provide some insight into the interpretation of fluconazole susceptibility testing, as has been reported for C. albicans (2). However, C. glabrata has failed to cause a progressive infection in normal mice or rats, necessitating the use of organ colony counts to assess drug efficacy. Intravenous inoculation of 108 C. glabrata into normal rats (11) or mice (7) did not cause obvious illness, progressive increase in organ colony counts, or death despite the enormous inoculum. Fluconazole at doses of 20 or 40 mg/kg given once daily by gavage for 7 days to rats beginning 3 days after infection reduced kidney but not liver colony counts (11). Only three rats were used in the control group, raising questions as to the reproducibility of this test. The fluconazole MIC for the C. glabrata strain used was 100 μg/ml. There have been two reports of the use of fluconazole in C. glabrata-infected mice rendered neutropenic by fluorouracil or irradiation (3, 13). Mice were given a very large intravenous inoculum (1 × 108 to 1.4 × 108 CFU) of isolates with fluconazole MICs ranging from 2 to >64 μg/ml. Fluconazole at 100 mg/kg twice daily by gavage for 5 days caused a significant reduction in the kidney CFU count in one of two experiments with the most susceptible isolate but no reduction in the splenic CFU count (3). In four experiments with other isolates and the same fluconazole regimen, the drug had no effect on the CFU in the spleen or other organs (3). In another report, mice infected with an isolate having an MIC of 16 μg/ml had a fourfold reduction in kidney but not in the splenic CFU counts after 7 days of fluconazole at 10 mg/kg (13). The wide scatter of organ CFU and the marginal evidence of efficacy left the therapeutic effect of fluconazole unclear in this model. Other drugs were effective in this same model. Intraperitoneal amphotericin B at 3 mg/kg daily caused roughly a 104-fold reduction in the colony counts of both the kidney and the spleen (3). Caspofungin, given intraperitoneally to normal (1) or neutropenic (13) mice inoculated with C. glabrata, reduced colony counts in the kidney (1, 13) but not in the spleen (13). We have explored the utility of a knockout (KO) strain of mice to determine efficacy of antifungal drugs against C. glabrata in vivo. This model avoids the confounding effects of cytotoxic drugs and neutropenia. C57BL/6 mice with disruption of the p47phox gene have deficient production of phagocyte oxidative products and are susceptible to infection with Aspergillus nidulans (8). Isolates of C. glabrata with a large range of fluconazole susceptibility were obtained from bone marrow transplant recipients or human immunodeficiency virus-infected patients and used to infect p47phox−/− KO mice. The mice developed a progressively lethal infection and proved useful for evaluating the relationship between in vivo efficacy and in vitro susceptibility. Caspofungin proved to be effective, as did amphotericin B, but fluconazole was not effective at 80 mg/kg daily, irrespective of in vitro fluconazole susceptibility.

Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide and downstream microbicidal reactive oxidants, leading to recurrent life-threatening bacterial and fungal infections. Xanthine oxidase (XO) is another enzyme known to produce superoxide in many tissues. Using the p47phox−/−mouse model of CGD, we evaluated the residual antibacterial activity of XO. Clearance ofBurkholderia cepacia, a major pathogen in CGD, was reduced in p47phox−/−mice compared to that in wild-type mice and was further inhibited in p47phox−/−mice by pretreatment with the specific XO inhibitor allopurinol. HepaticB. cepaciaburden was similar in the two genotypes, but allopurinol significantly reduced net hepatic killing and killing efficiency only in p47phox−/−mice. Clearance and killing of intravenousEscherichia coliwas intact in p47phox−/−mice and was unaffected by pretreatment with allopurinol. In CGD, XO may contribute to host defense against a subset of reactive oxidant-sensitive pathogens.

Molecular methods are increasingly used to identify pathogens that are difficult to cultivate. We report a case of disseminated infection with “ Mycobacterium tilburgii ,” a proposed species that has never been cultivated. The case illustrates the diagnostic utility of sequence analysis of the 16S rRNA gene directly from clinical specimens.

Chronic granulomatous disease (CGD) is a genetic disorder of NADPH oxidase in which phagocytes are defective in generating reactive oxidants. CGD patients suffer from recurrent infections and exuberant and persistent tissue granuloma formation. We hypothesized that abnormal granulomata in CGD may result from aberrant T-cell-mediated cytokine responses. To assess Th-1-type cytokine responses and granulomata, we challenged p47 phox −/− and wild-type mice with avirulent (SmD) or virulent (SmT) variants of Mycobacterium avium 2-151. To assess Th-2-type cytokine responses and granulomata, we used Schistosoma mansoni eggs (SME). Mononuclear cells were harvested, and cytokine responses were determined by enzyme-linked immunosorbent assay or reverse transcriptase PCR. Following SmD or SmT challenge, splenocytes from p47 phox −/− and wild-type mice generated similar polar Th-1 responses (increased levels of gamma interferon and basal levels of interleukin 4 [IL-4] and IL-5). By 8 weeks after SmT challenge, exuberant splenic granulomata developed in p47 phox −/− and wild-type mice. After SME challenge, thoracic lymph node mononuclear cells from p47 phox −/− and wild-type mice generated similar mixed Th-1 and Th-2 cytokine responses to SME antigen and concanavalin A. Peak lung granuloma sizes and rates of regression were similar in p47 phox −/− and wild-type mice. These results suggest that exuberant granulomatous inflammation in CGD is probably not the result of skewing of T-cell responses toward the Th-1 or Th-2 pole. Appropriate regression of established tissue granulomata in p47 phox −/− mice challenged with SME suggests that abnormal granuloma formation in CGD is stimulus dependent and is not an invariant feature of the disease.

strain J2315 (6), BC7 was isolated from a patient with “cepaciasyndrome” and K56-2 is less antibiotic resistant, making it moreamenable to genetic manipulation.Genomic DNA was prepared with the DNeasy blood andtissue kit (Qiagen), according to the manufacturer’s instruc-tions. A combination of the Roche 454 GS-FLX Titanium 8-kbmate-pair libraries (~12 coverage) and 100-bp Illumina frag-ment reads (50 coverage) were used for sequence determina-tions. All reads were used to generate hybrid assemblies withCelera Assembler version 7.0 (CA7.0) (7). Almost all of theintrascaffold breaks in these assemblies were estimated at 20bases, suggesting that they are ambiguities likely resulting fromthe high content of short tandem repeats in the G C-rich re-gions of the genome. Thus, we sought additional PacBio readsto obtain a better consensus of the K56-2 genome; short- andlong-length PacBio reads ( 2,000 and 2,000, respectively)were used to generate highly accurate, long, preassembledreads using the HGAP algorithm (Pacific Biosciences), whichwere combined with 454 reads and input into the CA7.0. K56-2assembled into 14 scaffolds containing 19 contigs. The finalcontig sequences were then run through the consensus polisherprogram, Quiver (PacBio), resulting in 3,516 base changes and624 base insertions or deletions.The BC7 assembly was initially in 7 scaffolds and 785 contigs.Since additional sequencing was not pursued for this genome, areference-guided gap closure pipeline was employed to resolvehundreds of gaps found throughout the assembly. The gap se-quences were predicted from the closed-genome reference(J2315) and used to recruit and locally assemble reads into thegaps to merge the adjacent contigs. The resulting assembly is 296contigs in 7 scaffolds.As in J2315, each genome has 3 chromosomes and 1 plasmid(6).ThechromosomesinBC7andK56-2haveverysimilarsizestothose reported in J2315 (3.83 Mb, 3.19 Mb, and 0.88 Mb), exceptfor chromosome 1 in K56-2, which has an estimated size of3.67 Mb, due to the absence of the large duplication in J2315. Thepresence of the plasmid was previously detected in K56-2, BC7,and J2315 (8). Sequence data confirm the presence of the plasmidin BC7 and K56-2, with practically no differences between thethree strains except for the presence of an additional copy of aninsertion element in the J2315 plasmid, pBCJ2315 (6). The ge-nomes were annotated using the annotation pipeline of the J.Craig Venter Institute (JCVI) (http://www.jcvi.org) and submit-ted to GenBank. Sequence data indicate that K56-2 and BC7 havesimilar gene contents to that of J2315, with 7,714 and 7,930 openreading frames (ORFs), respectively.

Mycobacterium massiliense ( Mycobacterium abscessus group) is an emerging pathogen causing pulmonary disease and skin and soft tissue infections. We report the genome sequence of the type strain CCUG 48898.

A strain of Streptococcus pyogenes resistant to multiple fluoroquinolones was isolated from the blood of an immunocompromised patient. Resistance to fluoroquinolones in S. pyogenes has not been previously studied. Compared to 10 sensitive strains of S. pyogenes , the fluoroquinolone-resistant clinical isolate of S. pyogenes presented point mutations in gyrA , predicting that serine-81 was changed to phenylalanine and that methionine-99 was changed to leucine, and in parC , predicting that serine-79 was changed to tyrosine. The mechanism of fluoroquinolone resistance in this isolate of S. pyogenes appears to be analogous to previously reported mechanisms for Streptococcus pneumoniae .

Reactive oxygen species (ROS) are required in a number of critical cellular signaling events, including those underlying hippocampal synaptic plasticity and hippocampus-dependent memory; however, the source of ROS is unknown. We previously have shown that NADPH oxidase is required for N-methyl-D-aspartate (NMDA) receptor-dependent signal transduction in the hippocampus, suggesting that NADPH oxidase may be required for NMDA receptor-dependent long-term potentiation (LTP) and hippocampus-dependent memory. Herein we present the first evidence that NADPH oxidase is involved in hippocampal synaptic plasticity and memory. We have found that pharmacological inhibitors of NADPH oxidase block LTP. Moreover, mice that lack the NADPH oxidase proteins gp91(phox) and p47(phox), both of which are mouse models of human chronic granulomatous disease (CGD), also lack LTP. We also found that the gp91(phox) and p47(phox) mutant mice have mild impairments in hippocampus-dependent memory. The gp91(phox) mutant mice exhibited a spatial memory deficit in the Morris water maze, and the p47(phox) mutant mice exhibited impaired context-dependent fear memory. Taken together, our results are consistent with NADPH oxidase being required for hippocampal synaptic plasticity and memory and are consistent with reports of cognitive dysfunction in patients with CGD.

Molecular methodologies have become useful techniques for the identification of pathogenic Nocardia species and for the recognition of novel species that are capable of causing human disease. Two isolates recovered from immunocompromised patients were characterized as Nocardia nova by biochemical and susceptibility testing results. The restriction fragment length polymorphism (RFLP) patterns obtained by restriction endonuclease analysis (REA) of an amplified portion of the heat shock protein gene were identical to those obtained with the type strain of N. nova . REA of an amplified portion of the 16S rRNA gene showed RFLP patterns that were unlike those obtained for the type strain of N. nova but that were similar to those obtained for the type strains of N. africana and N. veterana . Subsequent sequencing of a portion of the 16S rRNA gene produced identical results for the two patient isolates. Sequence analysis of 1,352-bp portions of the 16S rRNA gene indicated that these isolates were 99.8% similar to the recently described species N. veterana but were only 99.3, 98.1, and 98.1% similar to the type strains of N. africana , N. nova , and N. vaccinii , respectively. DNA-DNA hybridization studies confirmed that the two patient isolates belonged to the same species but were not closely related to N. africana , N. nova , N. vaccinii , or N. veterana . The patient isolates have been designated N. kruczakiae sp. nov. Because N. africana , N. veterana , and the new species are not readily differentiated from N. nova by phenotypic methods alone, the designation “ N. nova complex” can be used to designate isolates such as these that phenotypically resemble N. nova but that have not been definitively characterized by 16S rRNA gene sequencing or DNA-DNA hybridization.

It has been previously reported that although inducible nitric oxide synthase (iNOS) gene knockout (NOS2−/−) mice resolveChlamydia trachomatisgenital infection, the production of reactive nitrogen species (RNS) via iNOS protects a significant proportion of mice from hydrosalpinx formation and infertility. We now report that higher in vivo RNS production correlates with mouse strain-related innate resistance to hydrosalpinx formation. We also show that mice with a deletion of a key component of phagocyte NADPH oxidase (p47phox−/−) resolve infection, produce greater amounts of RNS in vivo, and sustain lower rates of hydrosalpinx formation than both wild-type (WT)NOS2+/+andNOS2−/−controls. When we induced an in vivo chemical block in iNOS activity in p47phox−/−mice usingNG-monomethyl-l-arginine (L-NMMA), a large proportion of these mice eventually succumbed to opportunistic infections, but not before they resolved their chlamydial infections. Interestingly, when compared to WT and untreated p47phox−/−controls, L-NMMA-treated p47phox−/−mice resolved their infections more rapidly. However, L-NMMA-treated p47phox−/−mice lost resistance to chronic chlamydial disease, as evidenced by an increased rate of hydrosalpinx formation that was comparable to that forNOS2−/−mice. We conclude that phagocyte oxidase-derived reactive oxygen species (ROS) regulate RNS during chlamydial urogenital infection in the mouse. We further conclude that while neither phagocyte oxidase-derived ROS nor iNOS-derived RNS are essential for resolution of infection, RNS protect from chronic chlamydial disease in this model.

Mycobacterium tuberculosisis an important respiratory pathogen the growth of which is controlled primarily by cytokine-activated macrophages. One of the principal mediators of this control is nitric oxide; however, superoxide has recently been shown to be protective in systemic mycobacterial infections. To determine whether superoxide is important in controllingM. tuberculosisduring primary pulmonary infection, mice lacking the cytosolic p47phoxgene (which is essential for effective superoxide production by the NADPH oxidase) were infected aerogenically. The lack of superoxide during an aerosol infection withM. tuberculosisresulted in a significant increase in bacterial growth over the early period of infection. Once antigen-specific gamma interferon-producing lymphocytes were detected in the draining lymph nodes, however, bacterial growth in the lung stopped. One interesting consequence of the lack of superoxide was an increase in neutrophilic infiltrates within the granuloma. This may be a consequence of increased tissue damage due to more rapid bacterial growth or may reflect a role for superoxide in controlling inflammation.

Isolates of the Mycobacterium avium complex were examined for hemolysin expression. Only invasive isolates of M. avium were observed to be hemolytic ( P < 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus, M. avium hemolysin appears to represent a virulence factor necessary for invasive disease.