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The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient’s antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient’s serum. Finally, we characterize one strain that was poorly recognized by the patient’s antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence. [ABSTRACT FROM AUTHOR]

The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient’s antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient’s serum. Finally, we characterize one strain that was poorly recognized by the patient’s antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is an emerging human pathogen and a significant cause of nosocomial infections among hospital patients worldwide. The enormous increase in multidrug resistance among hospital isolates and the recent emergence of pandrug-resistant strains underscores the urgency to understand how A. baumannii evolves in hospital environments. To this end, we undertook a genomic study of a polyclonal outbreak of multid rugresistant A. baumannii at the research-based National Institutes of Health Clinical Center. Comparing the complete genome sequences of the three dominant outbreak strain types enabled us to conclude that, despite all belonging to the same epidemic lineage, the three strains diverged before their arrival at the National Institutes of Health. The simultaneous presence of three divergent strains from this lineage supports its increasing prevalence in international hospitals and suggests an ongoing adaptation to the hospital environment. Further genomic comparisons uncovered that much of the diversification that occurred since the divergence of the three outbreak strains was mediated by homologous recombination across 20% of their genomes. Inspection of recombinant regions revealed that several regions were associated with either the loss or swapping out of genes encoding proteins that are exposed to the cell surface or that synthesize cell-surface molecules. Extending our analysis to a larger set of international clinical isolates revealed a previously unappreciated ability of A. baumannii to vary surface molecules through horizontal gene transfer, with subsequent intraspecies dissemination by homologous recombination. These findings have immediate implications in surveillance, prevention, and treatment of A. baumannii infections. [ABSTRACT FROM AUTHOR]

Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91phox −/− (X-linked) and p47 phox −/− (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patient's original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Koch's postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis. [ABSTRACT FROM AUTHOR]

Abstract: We describe the first case of a man diagnosed with laryngeal cancer presenting with nontypeable Haemophilus influenzae bacteremia and dissemination to a gouty joint and review the pertinent literature. [Copyright &y& Elsevier]

Bacterial whole-genome sequencing (WGS) of human pathogens has provided unprecedented insights into the evolution of antibiotic resistance. Most studies have focused on identification of resistance mutations, leaving one to speculate on the fate of these mutants once the antibiotic selective pressure is removed. We performed WGS on longitudinal isolates of Acinetobacter baumannii from patients undergoing colistin treatment, and upon subsequent drug withdrawal. In each of the four patients, colistin resistance evolved via mutations at the pmr locus. Upon colistin withdrawal, an ancestral susceptible strain outcompeted resistant isolates in three of the four cases. In the final case, resistance was also lost, but by a compensatory inactivating mutation in the transcriptional regulator of the pmr locus. Notably, this inactivating mutation reduced the probability of reacquiring colistin resistance when subsequently challenged in vitro. On face value, these results supported an in vivo fitness cost preventing the evolution of stable colistin resistance. However, more careful analysis of WGS data identified genomic evidence for stable colistin resistance undetected by clinical microbiological assays. Transcriptional studies validated this genomic hypothesis, showing increased pmr expression of the initial isolate. Moreover, altering the environmental growth conditions of the clinical assay recapitulated the classification as colistin resistant. Additional targeted sequencing revealed that this isolate evolved undetected in a patient undergoing colistin treatment, and was then transmitted to other hospitalized patients, further demonstrating its stability in the absence of colistin. This study provides a unique window into mutational pathways taken in response to antibiotic pressure in vivo, and demonstrates the potential for genome sequence data to predict resistance phenotypes. [ABSTRACT FROM AUTHOR]

We report the first cases of invasive mycosis by G. argillacea in chronic granulomatous disease, CGD (an inherited disorder of the NADPH oxidase), patients. G. argillacea is a previously underappreciated and frequently misidentified pathogen in CGD that should be excluded when P. variotii is identified morphologically.Background. Chronic granulomatous disease (CGD) is an inherited disorder of the nicotinamide adenine dinucleotide phosphate oxidase that leads to defective production of microbicidal superoxide and other oxidative radicals, resulting in increased susceptibility to invasive infections, especially those due to fungi.Methods. Geosmithia argillacea was identified from cultured isolates by genomic sequencing of the internal transcribed spacer region. Isolates previously identified as Paecilomyces variotii, a filamentous fungus closely resembling G. argillacea, were also examined.Results. We identified G. argillacea as the cause of invasive mycosis in 7 CGD patients. In 5 cases, the fungus had been previously identified morphologically as P. variotii. All patients had pulmonary lesions; 1 had disseminated lesions following inhalational pneumonia. Infections involved the chest wall and contiguous ribs in 2 patients and disseminated to the brain in 1 patient. Four patients with pneumonia underwent surgical intervention. All patients responded poorly to medical treatment, and 3 died.Conclusions. We report the first cases of invasive mycosis caused by G. argillacea in CGD patients. G. argillacea infections in CGD are often refractory and severe with a high fatality rate. Surgical intervention has been effective in some cases. G. argillacea is a previously underappreciated and frequently misidentified pathogen in CGD that should be excluded when P. variotii is identified morphologically. [ABSTRACT FROM AUTHOR]

Background Results Conclusions Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centers’ respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF center outbreak in which patient 2B was the index case.Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR occurred in isolates from both CF patients.Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains.We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts. [ABSTRACT FROM AUTHOR]

• Mesa Accula is not impacted by 28881 GGG > AAC mutations, despite FDA warning alert. • Mesa Accula can detect new viral variants B.1.1.7, R.1, P. 2, B.1.526 and B. 1. 351. • Continuous evaluation of SARS-CoV-2 variants' impact on diagnostic tests is needed. [ABSTRACT FROM AUTHOR]

Nasopharyngeal flocked swabs placed in viral transport media (VTM) are the preferred collection methodology for respiratory virus testing. Due to the rapid depletion of available reagents and swabs, we have validated an alternative swab placed in phosphate-buffered saline (PBS) for use in respiratory virus testing in a SARS-CoV-2 real-time polymerase chain reaction assay and a multiplexed respiratory virus panel. We collected nasopharyngeal (NP) swabs and oropharyngeal (OP) swabs from 10 healthy volunteers. Flocked swabs were placed in VTM and alternative swabs in PBS. In this feasibility study, we show that NP collection is better for detection of human material than OP collection, as measured by significantly lower RNase P gene cycle threshold values, and that a Dacron polyester swab in PBS shows equivalent detection of SARS-CoV-2 and RSV to a flocked swab in VTM in contrived specimens. Diluted SARS-CoV-2–positive patient specimens are detectable for up to 72 h at 4 °C. [ABSTRACT FROM AUTHOR]

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem). M. abscessus produces a β-lactamase (Bla Mab ) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a cystic fibrosis center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC > 512 µg/mL). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased bla Mab mRNA level. Concurrent increase in transcription of the preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 µg/mL). A frameshift mutation in msp2 responsible for the rough colony morphology and a SNP in ATP-dependent helicase hrpA cooccurred with the second increase in MIC (from 32 to 256 µg/mL). Increased Bla Mab expression and enzymatic activity may have been due to altered regulation of the ppiA-bla Mab operon by the mutated HrpA alone or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating M. abscessus , particularly imipenem-resistant strains., Competing Interests: The authors declare no conflict of interest.

The draft genome of Mucor velutinosus NIH1002, a 2011 isolate from a case of disseminated disease, was sequenced using PacBio long-read and HiSeq short-read technologies. The genome has 43 contigs, an N50 of 2.65 Mb, and 13,295 protein-coding genes. It is the most complete M. velutinosus genome to date., Competing Interests: The authors declare no conflict of interest.

Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem, meropenem). M. abscessus produces a β-lactamase (Bla Mab ) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 μg/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased bla Mab mRNA level. Concurrent increase in transcription of preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 μg/ml). A frameshift mutation in msp2 responsible for the rough colony morphology, and a SNP in ATP-dependent helicase hrpA co-occurred with the second increase in MIC (from 32 to 256 μg/ml). Increased Bla Mab expression and enzymatic activity may have been due to altered regulation of the ppiA-bla Mab operon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating M. abscessus , particularly imipenem resistant strains.

Rationale: The prevalence of nontuberculous mycobacterial (NTM) pulmonary disease varies geographically in the United States. Previous studies indicate that the presence of certain water-quality constituents in source water increases NTM infection risk., Objective: To identify water-quality constituents that influence the risk of NTM pulmonary infection in persons with cystic fibrosis in the United States., Methods: We conducted a population-based case-control study using NTM incidence data collected from the Cystic Fibrosis Foundation Patient Registry during 2010-2019. We linked patient zip code to the county and associated patient county of residence with surface water data extracted from the Water Quality Portal. We used logistic regression models to estimate the odds of NTM infection as a function of water-quality constituents. We modeled two outcomes: pulmonary infection due to Mycobacterium avium complex (MAC) and Mycobacterium abscessus species., Results: We identified 484 MAC cases, 222 M. abscessus cases and 2816 NTM-negative cystic fibrosis controls resident in 11 states. In multivariable models, we found that for every 1-standardized unit increase in the log concentration of sulfate and vanadium in surface water at the county level, the odds of infection increased by 39% and 21%, respectively, among persons with cystic fibrosis with MAC compared with cystic fibrosis-NTM-negative controls. When modeling M. abscessus as the dependent variable, every 1-standardized unit increase in the log concentration of molybdenum increased the odds of infection by 36%., Conclusions: These findings suggest that naturally occurring and anthropogenic water-quality constituents may influence the NTM abundance in water sources that supply municipal water systems, thereby increasing MAC and M. abscessus infection risk., Competing Interests: The authors declare that they have no conflicts of interest with regard to the content of this report. E.M.L., R.A.M., J.M., A.M.Z., and D.R.P. were supported by the Division of Intramural Research, NIAID. J.F. was supported by NSF award [1915277]. M.S. was supported by NSF award [1743597]. Authors receive funding to support open access publishing. This study was classified as nonhuman subjects research by the National Institutes of Health, Office of Human Subjects Research Protection because data were de-identified and investigators could not link back to identifiable data. The water-quality dataset and the R code for the analysis are available from the first author. The patient dataset cannot be made publicly available due to unique identifiers in the data.

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, β-glucan-binding receptor, Dectin-1. The patient's PBMCs failed to produce TNF-α and IL-1β in response to β-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1β and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1β-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.

Mycobacterium abscessus is an emerging nontuberculous mycobacterium (NTM) pathogen infecting susceptible people with cystic fibrosis (CF) and non-CF bronchiectasis. Here, we demonstrated the activity of an FDA-approved drug, disulfiram, against drug-susceptible and drug-resistant M. abscessus strains utilizing in vitro and intracellular macrophage assays and a zebrafish embryo infection model. These data demonstrate effective antimicrobial activity of disulfiram against M. abscessus infection in vivo and strongly support further study of disulfiram in human NTM infections.

Antibiotics are a modifiable iatrogenic risk factor for the most common human nosocomial fungal infection, invasive candidiasis, yet the underlying mechanisms remain elusive. We found that antibiotics enhanced the susceptibility to murine invasive candidiasis due to impaired lymphocyte-dependent IL-17A- and GM-CSF-mediated antifungal immunity within the gut. This led to non-inflammatory bacterial escape and systemic bacterial co-infection, which could be ameliorated by IL-17A or GM-CSF immunotherapy. Vancomycin alone similarly enhanced the susceptibility to invasive fungal infection and systemic bacterial co-infection. Mechanistically, vancomycin reduced the frequency of gut Th17 cells associated with impaired proliferation and RORγt expression. Vancomycin's effects on Th17 cells were indirect, manifesting only in vivo in the presence of dysbiosis. In humans, antibiotics were associated with an increased risk of invasive candidiasis and death after invasive candidiasis. Our work highlights the importance of antibiotic stewardship in protecting vulnerable patients from life-threatening infections and provides mechanistic insights into a controllable iatrogenic risk factor for invasive candidiasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Clarithromycin resistance in Mycobacterium abscessus subsp. abscessus , massiliense , and bolletii occurs through induction of erm (41) or mutations in rrl (23S rRNA) genes. Phenotypic detection of clarithromycin resistance is hindered by the need for extended incubation as well as co-occurrence of mixed populations of M. abscessus with different susceptibility profiles. We developed a quantitative EvaGreen-based droplet digital PCR (ddPCR) scheme for rapid detection of full-length or truncated erm (41) and a probe based ddPCR screening assay for assessment of 23S rRNA rrl mutational resistance. We tested 100 M. abscessus strains, synthetic mixes with different susceptibility profiles, and 13 positive MGIT samples. Truncated and full-length erm (41) genes were detected in 27/100 and 73/100 strains and 4/13 and 9/13 MGIT samples, respectively yielding a sensitivity and specificity of 100%. Clarithromycin resistance mutations in rrl were detected in 26/100 isolates, i.e., A2058G (18/100), A2058C (7/100), and A2059G (1/100), and in 3/13 MGIT samples, i.e., A2058G (2/13) and A2059G (1/13). A screening assay of rrl ddPCR (A2058A/A2058G probes) showed 100% sensitivity in detecting the wild type or A2058G mutation as well as identifying samples requiring further testing. Upon inclusion of additional ddPCR assays, we were able to detect A2058C and A2059G clarithromycin resistance-conferring mutations in the rrl gene. Our ddPCR scheme can differentiate between full-length and truncated erm (41) and identify clarithromycin resistance-conferring mutations in the rrl gene from clinical isolates and positive MGIT samples as well as deconvolute and quantitate mixed populations of M. abscessus with different clarithromycin resistance traits.

Mycobacterium abscessus (MAB) comprise rapidly growing, often multidrug-resistant (MDR), nontuberculous mycobacteria responsible for pulmonary and other infections in susceptible hosts. Antimicrobial peptides (APs) are natural and synthetic antimicrobials active against a range of microorganisms including mycobacteria. We evaluated APs activity against MAB reference and clinical strains. We observed minimal inhibitory concentrations of 1.6 to >50 μg/mL. Further work with the most active AP demonstrated protection of Acanthamoeba castellanii (AC) from killing by ingested MAB including MDR MAB strains. Antimicrobial peptides offer an attractive potential option for treatment of drug resistant treatment-refractory MAB., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Published by Elsevier Masson SAS.)

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry is a quick and accurate method for mycobacterial identification from protein extracts. Our new one-step extraction method successfully reduced routine multistep extraction procedure time from over 60 min to under 10 min and used only 1 μL loopful of mycobacteria while providing clinically acceptable identification scores (≥1.8). Overall, 86.8% and 4.4% of mycobacteria isolates (n = 68) were identified to the species/complex and genus levels, respectively, by one-step loop extraction method, comparable to the routine extraction method. Viability studies confirmed killing of mycobacterial isolates after 5 min in the extraction solution replacing lengthy heat killing step. Retrospective 7-month data analysis showed 100% of rapidly and slowly growing mycobacterial isolates were identified to the species/complex level by rapid extraction methods. Our rapid extraction methods substantially reduced processing time and microbial biomass required for testing without sacrificing quality and accuracy of mycobacterial identification., (Published by Elsevier Inc.)

Granulibacter bethesdensis is a pathogen reported to cause recurrent lymphadenitis exclusively in persons with chronic granulomatous disease. We report a case of fatal meningitis caused by a highly virulent G. bethesdensis strain in an adolescent in Europe who had chronic granulomatous disease.

Purpose: Mendelian suceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency predisposing to severe disease caused by mycobacteria and other intracellular pathogens. Delay in diagnosis can have an impact on the patient's prognosis., Methods: We evaluated the IFN-γ circuit by studying IFN-γ production after mycobacterial challenge as well as IL-12Rβ1 expression and STAT4 phosphorylation in response to IL-12p70 stimulation in whole blood of a 6-year-old Peruvian girl with disseminated recurrent mycobacterial infection diagnosed as multidrug-resistant tuberculosis. Genetic studies with Sanger sequencing were used to identify the causative mutation. Microbiological studies based on PCR reactions were used to diagnose the specific mycobacterial species., Results: We identified a homozygous mutation in the IL12RB1 gene (p. Arg211*) causing abolished expression of IL-12Rβ1 and IL-12 response. MSMD diagnosis led to a microbiological reevaluation of the patient, revealing a BCG vaccine-related infection instead of tuberculosis. Treatment was then adjusted, with good response., Conclusions: We report the first Peruvian patient with IL-12Rβ1 deficiency. Specific mycobacterial species diagnosis within Mycobacterium tuberculosis complex is still challenging in countries with limited access to PCR-based microbiological diagnostic techniques. Awareness of MSMD warning signs and accurate microbiological diagnosis of mycobacterial infections are of the utmost importance for optimal diagnosis and management of affected patients.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized fungal identification. Previously, we developed a MALDI-TOF MS mold extraction procedure and comprehensive database. While MALDI-TOF MS has become routine in a few laboratories, it has not yet become widespread. A major obstacle is the lack of a simple, reproducible and uniform protein extraction procedure. In this study, we developed and validated a rapid one-step protein extraction protocol for filamentous fungi. Excised molds were placed into tubes containing zirconia-silica beads and extraction solution without washing or ethanol inactivation steps. Extraction solutions containing different ratios of acetonitrile and formic acid were evaluated. Samples were then processed using a PowerLyzer high power bead based homogenizer and supernatants spotted for MALDI-TOF MS. The rapid method was evaluated prospectively and in parallel to our current mold extraction protocol for 3 months. Analysis of 106 clinical mold isolates resulted in an improved performance and a decrease in extraction time by 30 minutes to a total of 5 minutes of hands-on time. Acceptable identification scores (≥ 2.00) were achieved for up to 63.0% of mold isolates by the rapid method compared with 52.8% of isolates by the current routine protocol. Score comparisons between duplicate spots showed higher reproducibility of the rapid method as compared to the routine method. The rapid extraction method allows efficient analysis of clinical mold isolates both in scheduled batch runs and on an in-demand basis while providing a simple starting platform for laboratories adopting MALDI-TOF MS for mold identification., (Copyright © 2018. Published by Elsevier Inc.)

The incidence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing, but host responses in respiratory epithelium infected with NTM are not fully understood. In this work, we aimed to identify infection-relevant gene expression signatures of NTM infection of the respiratory epithelium. We infected air-liquid interface (ALI) primary respiratory epithelial cell cultures with Mycobacterium avium subsp. avium (MAC) or Mycobacterium abscessus subsp. abscessus (MAB). We used cells from four different donors to obtain generalizable data. Differentiated respiratory epithelial cells at the ALI were infected with MAC or MAB at a multiplicity of infection of 100:1 or 1,000:1, and RNA sequencing was performed at Days 1 and 3 after infection. In response to infection, we found down-regulation of ciliary genes but upregulation of genes associated with cytokines/chemokines, such as IL-32, and cholesterol biosynthesis. Inflammatory response genes tended to be more upregulated by MAB than by MAC infection. Primary respiratory epithelial cell infection with NTM at the ALI identified ciliary function, cholesterol biosynthesis, and cytokine/chemokine production as major host responses to infection. Some of these pathways may be amenable to therapeutic manipulation.