Ubiquitous Environmental Bacterium Research Articles

Nontuberculous mycobacterial pulmonary disease (NTM PD) incidence trends in the United States, 2010–2019

BackgroundNontuberculous mycobacteria (NTM) are ubiquitous environmental bacteria that cause chronic lung disease. Rates of NTM pulmonary disease (NTM PD) have increased over the last several decades, yet national estimates in the United States (US) have not been assessed since 2015.MethodsWe used a nationally representative population of Medicare beneficiaries aged ≥ 65 years to assess rates of NTM PD in a high-risk population from 2010 to 2019. Poisson generalized linear models were used to assess the annual percent change in incidence in the overall population and among key demographic groups such as sex, geography, and race/ethnicity. We evaluated the relative prevalence of various comorbid conditions previously found to be associated with NTM PD.ResultsWe identified 59,724 cases of incident NTM PD from 2010 to 2019 from an annual mean population of 29,687,097 beneficiaries, with an average annual incidence of 20.1 per 100,000 population. NTM PD incidence was overall highest in the South and among women, Asian individuals, and persons aged ≥ 80 years relative to other studied demographic groups. The annual percent change in NTM PD incidence was highest in the Northeast, at 6.5%, and Midwest, at 5.9%, and among women, at 6.5%. Several comorbid conditions were highly associated with concurrent NTM diagnosis, including allergic bronchopulmonary aspergillosis, bronchiectasis, and cystic fibrosis.ConclusionsHere we provide current estimates of NTM PD incidence and prevalence and describe increasing trends in the US from 2010 to 2019. Our study suggests a need for improved healthcare planning to handle an increased future caseload, as well as improved diagnostics and therapeutics to better detect and treat NTM PD in populations aged ≥ 65 years.

Acquired resistance of Stenotrophomonas maltophilia to antimicrobials induced by herbicide paraquat dichloride.

Stenotrophomonas maltophilia, a ubiquitous environmental bacterium, is an important cause of nosocomial infections. Although banned in some countries, paraquat (PQ) is commonly used to control weeds. In this study, we investigated the effects of increasing concentrations of PQ on S. maltophilia and its antimicrobial resistance. The sequential exposure of S. maltophilia K279a to increasing concentrations of PQ induces the formation of strains with increased resistance to PQ. Among the 400 PQ-resistant isolates tested, 70 clones were resistant to 16 μg/ml ciprofloxacin (CIP), and around 18% of the PQ/CIP-resistant isolates showed increased resistance to all the tested antimicrobials including, the aminoglycosides, quinolones, cephalosporin, chloramphenicol, and co-trimoxazole. The results of the expression analysis of the antimicrobial resistance genes in the five selected PQ/CIP-resistant isolates demonstrated the high expression of genes encoding efflux pumps (smeYZ, smaAB, smaCDEF, smeDEF, smeVWX, and smtcrA) and the enzymes aph(3')-IIc, blaL1, and blaL2. However, expression of the genes known for PQ resistance (i.e., mfsA and sod) were not altered relative to the wild-type levels. Whole genome sequence analysis identified gene mutations that could account for the antimicrobial resistance, namely, smeT (TetR family regulatory protein), rplA (ribosomal protein L1), and acnA (aconitase A). Ectopic expression of wild-type AcnA partially complemented the fluoroquinolone-resistant phenotype of the mutant with mutated acnA, which suggests the role of aconitase A in antimicrobial susceptibility. Exposure of S. maltophilia to PQ thus induces the development of strains that increase resistance to multiple antimicrobials.

Glycerol metabolism impacts biofilm phenotypes and virulence in Pseudomonas aeruginosa via the Entner-Doudoroff pathway.

Pseudomonas aeruginosa is a ubiquitous bacterium and a notorious opportunistic pathogen that forms biofilm structures in response to many environmental cues. Biofilm formation includes attachment to surfaces and the production of the exopolysaccharide Pel, which is present in both the PAO1 and PA14 laboratory strains of P. aeruginosa. Biofilms help protect bacterial cells from host defenses and antibiotics and abet infection. The carbon source used by the cells also influences biofilm, but these effects have not been deeply studied. We show here that glycerol, which can be liberated from host surfactants during infection, encourages surface attachment and magnifies colony morphology differences. We find that glycerol kinase is important but not essential for glycerol utilization and relatively unimportant for biofilm behaviors. Among downstream enzymes predicted to take part in glycerol utilization, Edd stood out as being important for glycerol utilization and for enhanced biofilm phenotypes in the presence of glycerol. Thus, gluconeogenesis and catabolism of anabolically produced glucose appear to impact not only the utilization of glycerol but also glycerol-stimulated biofilm phenotypes. Finally, waxworm moth larvae and nematode infection models reveal that interruption of the Entner-Doudoroff pathway, but not abrogation of glycerol phosphorylation, unexpectedly increases P. aeruginosa lethality in both acute and chronic infections, even while stimulating a stronger immune response by Caenorhabditis elegans.IMPORTANCEPseudomonas aeruginosa, the ubiquitous environmental bacterium and human pathogen, forms multicellular communities known as biofilms in response to various stimuli. We find that glycerol, a common carbon source that bacteria can use for energy and biosynthesis, encourages biofilm behaviors such as surface attachment and colony wrinkling by P. aeruginosa. Glycerol can be derived from surfactants that are present in the human lungs, a common infection site. Glycerol-stimulated biofilm phenotypes do not depend on phosphorylation of glycerol but are surprisingly impacted by a glucose breakdown pathway, suggesting that it is glycerol utilization, and not its mere presence or cellular import, that stimulates biofilm phenotypes. Moreover, the same mutations that block glycerol-stimulated biofilm phenotypes also impact P. aeruginosa virulence in both acute and chronic animal models. Notably, a glucose-breakdown mutant (Δedd) counteracts biofilm phenotypes but shows enhanced virulence and stimulates a stronger immune response in Caenorhabditis elegans.

LISTERIA MONOCYTOGENES INFECTION IN FREE-RANGING RED FOXES (VULPES VULPES) AND EURASIAN LYNX (LYNX LYNX) IN SWITZERLAND.

Listeria monocytogenes is an ubiquitous environmental saprophytic bacterium causing listeriosis in domestic animals, humans, and occasionally wildlife. In animals, this foodborne zoonotic disease mainly occurs in ruminants and it is rare in carnivores. Seven red foxes (Vulpes vulpes) and one Eurasian lynx (Lynx lynx) were diagnosed with listeriosis between 2010 and 2021 at the Institute for Fish and Wildlife Health, Bern, Switzerland. Necropsy and histopathology revealed meningitis (six of seven red foxes), hepatitis (six of seven red foxes), pneumonia (five of seven red foxes), splenitis (two of seven red foxes) and splenomegaly (the Eurasian lynx, two of seven red foxes). Listeria monocytogenes was isolated from either lung, spleen, liver, or kidney of all animals. Serotyping detected L. monocytogenes serotype 1/2a in five red foxes and the Eurasian lynx and serotype 4b in two red foxes. Six red foxes were positive for canine distemper virus (CDV) by polymerase chain reaction, whereas the Eurasian lynx and one red fox were negative. One red fox that was positive for CDV and listeriosis was also diagnosed with salmonellosis. The identified L. monocytogenes serotypes are among the three most frequently isolated serotypes (1/2a, 1/2b, and 4b) from food or the food production environment and those that cause most listeriosis cases in humans and animals. Coinfection with CDV in six red foxes questions the role of CDV as potential predisposing factor for septicemic listeriosis. The detection of listeriosis in the regionally endangered Eurasian lynx and in carnivores highly abundant in urban settings, such as red foxes, reinforces the importance of wildlife health surveillance in a One Health context and adds the Eurasian lynx to the list of carnivores susceptible to the disease. Further investigations are required to assess the prevalence and epidemiology of L. monocytogenes in free-ranging carnivores and its interaction with CDV.

DEVELOPMENT AND PILOT VALIDATION OF A NOVEL PCR-BASED REPLICON TYPING SCHEME FOR PLASMID FAMILIES ASSOCIATED WITH ANTIBIOTIC RESISTANCE IN PSEUDOMONAS SPP.

Background. Pseudomonas species are ubiquitous environmental Gram-negative bacteria increasingly associated with difficult to treat healthcare-associated infections. Along with their substantial intrinsic antimicrobial resistance, the ability to acquire additional resistance and pathogenicity determinants contributes to increased morbidity and mortality. Plasmids represent the major vehicles of gene transfer among hospital strains. Accumulation and dissemination of resistance genes through horizontal gene transfer is exceptionally problematic since it leads to the emergence of multi-resistant and stable phenotypes highlighting the importance of novel tools for studying plasmid epidemiology. Materials and Methods. In this study we introduce a novel PCR-based replicon typing (PBRT) scheme for differentiation of various Pseudomonas spp. plasmid families requiring only two multiplex PCR (mPCR) assays. mPCR 1 is composed of previously published primer sets for IncP-1, IncP-7, IncP-9, IncQ, A/C, N, W, IncU. Primers for multiplex PCR 2 were designed after an in-depth in-silico bioinformatic analysis of the repA gene of more than 50 reference IncP-2, IncP-6, IncP-10, pKLC102-like and pMOS94-like plasmids some of which studied for the first time as a group. Results. The scheme was tested on a set of 90 previously genotyped multi-resistant clinical Pseudomonas spp. isolates. The detection rate of the target plasmid families was low in our strain collection. Replicons were registered in only 3/90 isolates from the IncP-7 (n=1), IncP-10 (n=1), and pMOS94-like (n=1) families. This pilot study demonstrates a novel PBRT scheme applicable to Pseudomonas spp. targeting plasmids of incompatibility groups known to harbour genes associated with antibiotic resistance.

Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection.

Legionella organisms are ubiquitous environmental bacteria that are responsible for human Legionnaires' disease, a fatal form of severe pneumonia. These bacteria replicate intracellularly in a wide spectrum of host cells within a distinct compartment termed the Legionella-containing vacuole (LCV). Effector proteins translocated by the Dot/Icm apparatus extensively modulate host cellular functions to aid in the biogenesis of the LCV and intracellular proliferation. RavZ is an L. pneumophila effector that functions as a cysteine protease to hydrolyze lipidated LC3, thereby compromising the host autophagic response to bacterial infection. In this study, we characterized the RavZ (RavZLP) ortholog in L. longbeachae (RavZLLO), the second leading cause of Legionella infections in the world. RavZLLO and RavZLP share approximately 60% sequence identity and a conserved His-Asp-Cys catalytic triad. RavZLLO is recognized by the Dot/Icm systems of both L. pneumophila and L. longbeachae. Upon translocation into the host, it suppresses autophagy signaling in cells challenged with both species, indicating the functional redundancy of RavZLLO and RavZLP. Additionally, ectopic expression of RavZLLO but not RavZLP in mammalian cells reduces the levels of cellular polyubiquitinated and polyneddylated proteins. Consistent with this process, RavZLLO regulates the accumulation of polyubiquitinated species on the LCV during L. longbeachae infection.

Detecting clusters of high nontuberculous mycobacteria infection risk for persons with cystic fibrosis – An analysis of U.S. counties

Nontuberculous mycobacteria are ubiquitous environmental bacteria that frequently cause disease in persons with cystic fibrosis (pwCF). The risks for NTM infection vary geographically. Detection of high-risk areas is important for focusing prevention efforts. In this study, we apply five cluster detection methods to identify counties with high NTM infection risk. Four clusters were detected by at least three of the five methods, including twenty-five counties in five states. The geographic area and number of counties in each cluster depended upon the detection method used. Identifying these clusters supports future studies of environmental predictors of infection and will inform control and prevention efforts.

The sbiTRS Operon Contributes to Stenobactin-Mediated Iron Utilization in Stenotrophomonas maltophilia.

Iron is an essential micronutrient for various bacterial cellular processes. Fur is a global transcriptional regulator participating in iron homeostasis. Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has emerged as an opportunistic pathogen. To elucidate the novel regulatory mechanism behind iron homeostasis in S. maltophilia, wild-type KJ and KJΔFur, a fur mutant, were subjected to transcriptome assay. A five-gene cluster, sbiBA-sbiTRS, was significantly upregulated in KJΔFur. SbiAB is an ATP type efflux pump, SbiT is an inner membrane protein, and SbiSR is a two-component regulatory system (TCS). The sbiTRS operon organization was verified by reverse transcription-PCR (RT-PCR). Localization prediction and bacterial two-hybrid studies revealed that SbiT resided in the inner membrane and had an intramembrane interaction with SbiS. In iron-replete conditions, SbiT interacted with SbiS and maintained SbiSR TCS in a resting state. In response to iron depletion stress, SbiT no longer interacted with SbiS, leading to SbiSR TCS activation. The iron source utilization assay demonstrated the contribution of SbiSR TCS to stenobactin-mediated ferric iron utilization but notto the utilization of hemin and ferric citrate. Furthermore, SmeDEF and SbiAB pumps, known stenobactin secretion outlets, were members of the SbiSR regulon. Collectively, in an iron-depleted condition, SbiSR activation is regulated by Fur at the transcriptional level and by SbiT at the posttranslational level. Activated SbiSR contributes to stenobactin-mediated ferric iron utilization by upregulating the smeDEF and sbiAB operons. SbiSR is the first TCS found to be involved in iron homeostasis in S. maltophilia. IMPORTANCE Therapeutic options for Stenotrophomonas maltophilia infections are limited because S. maltophilia is intrinsically resistant to several antibiotics. Iron is an essential element for viability, but iron overload is a lethal threat to bacteria. Therefore, disruption of iron homeostasis can be an alternative strategy to cope with S. maltophilia infection. The intricate regulatory networks involved in iron hemostasis have been reported in various pathogens; however, little is known about S. maltophilia. Herein, a novel sbiTRS operon, a member of Fur regulon, was characterized. SbiT, an inner membrane protein, negatively modulated the SbiSR two-component regulatory system by intramembrane protein-protein interaction with SbiS. In response to iron-depleted stress, SbiSR was activated via the regulation of Fur and SbiT. Activated SbiSR upregulated smeDEF and sbiAB, which contributed to stenobactin-mediated ferric iron utilization. A novel fur-sbiT-sbiSR-smeDEF/sbiAB regulatory circuit in S. maltophilia was revealed.

Nontuberculous Mycobacteria Lung Disease (NTM-LD): Current Recommendations on Diagnosis, Treatment, and Patient Management.

Nontuberculous mycobacteria (NTM) are a group of ubiquitous environmental bacteria that can be found in soil, dust, and water. Mycobacterium avium complex (MAC) is the most common pathogen and the one most associated with chronic pulmonary disease. In recent years, the prevalence of Mycobacterium avium complex-related pulmonary disease (MAC-PD) has increased and is an emerging public health concern. This is due to a combination of environmental and geographic factors, dynamic changes in organism virulence and antimicrobial susceptibility, and evolving host susceptibility. Given the dynamic nature of the disease, management of NTM pulmonary disease (NTM-PD) often includes a multimodal approach including antimicrobial therapy, airway clearance techniques, limiting environmental exposures, and reducing susceptibility to NTM through prevention of reflux and maintenance of body weight. This review will explore the most recent concepts in the diagnosis, treatment, and management of individuals with NTM pulmonary infection.

Characterization of Stenotrophomonas maltophilia phage AXL1 as a member of the genus Pamexvirus encoding resistance to trimethoprim–sulfamethoxazole

Stenotrophomonas maltophilia is a ubiquitous environmental bacterium capable of causing disease in humans. Antibiotics are largely ineffective against this pathogen due to numerous chromosomally encoded antibiotic resistance mechanisms. An alternative treatment option is phage therapy, the use of bacteriophages to selectively kill target bacteria that are causing infection. To this aim, we isolated the Siphoviridae bacteriophage AXL1 (vB_SmaS-AXL_1) from soil and herein describe its characterization. Host range analysis on a panel of 30 clinical S. maltophilia strains reveals a moderate tropism that includes cross-species infection of Xanthomonas, with AXL1 using the type IV pilus as its host surface receptor for infection. Complete genome sequencing and analysis revealed a 63,962 bp genome encoding 83 putative proteins. Comparative genomics place AXL1 in the genus Pamexvirus, along with seven other phages that infect one of Stenotrophomonas, Pseudomonas or Xanthomonas species. Functional genomic analyses identified an AXL1-encoded dihydrofolate reductase enzyme that provides additional resistance to the antibiotic combination trimethoprim–sulfamethoxazole, the current recommended treatment option for S. maltophilia infections. This research characterizes the sixth type IV pilus-binding phage of S. maltophilia and is an example of phage-encoded antibiotic resistance.

The fciTABC and feoABI systems contribute to ferric citrate acquisition in Stenotrophomonas maltophilia

BackgroundStenotrophomonas maltophilia, a member of γ-proteobacteria, is a ubiquitous environmental bacterium that is recognized as an opportunistic nosocomial pathogen. FecABCD system contributes to ferric citrate acquisition in Escherichia coli. FeoABC system, consisting of an inner membrane transporter (FeoB) and two cytoplasmic proteins (FeoA and FeoC), is a well-known ferrous iron transporter system in γ-proteobacteria. As revealed by the sequenced genome, S. maltophilia appears to be equipped with several iron acquisition systems; however, the understanding of these systems is limited. In this study, we aimed to elucidate the ferric citrate acquisition system of S. maltophilia.MethodsCandidate genes searching and function validation are the strategy for elucidating the genes involved in ferric citrate acquisition. The candidate genes responsible for ferric citrate acquisition were firstly selected using FecABCD of E. coli as a reference, and then revealed by transcriptome analysis of S. maltophilia KJ with and without 2,2′-dipyridyl (DIP) treatment. Function validation was carried out by deletion mutant construction and ferric citrate utilization assay. The bacterial adenylate cyclase two-hybrid system was used to verify intra-membrane protein–protein interaction.ResultsSmlt2858 and Smlt2356, the homologues of FecA and FecC/D of E. coli, were first considered; however, deletion mutant construction and functional validation ruled out their involvement in ferric citrate acquisition. FciA (Smlt1148), revealed by its upregulation in DIP-treated KJ cells, was the outer membrane receptor for ferric citrate uptake. The fciA gene is a member of the fciTABC operon, in which fciT, fciA, and fciC participated in ferric citrate acquisition. Uniquely, the Feo system of S. maltophilia is composed of a cytoplasmic protein FeoA, an inner membrane transporter FeoB, and a predicted inner membrane protein FeoI. The intra-membrane protein–protein interaction between FeoB and FeoI may extend the substrate profile of FeoB to ferric citrate. FeoABI system functioned as an inner membrane transporter of ferric citrate.ConclusionsThe FciTABC and FeoABI systems contribute to ferric citrate acquisition in S. maltophilia.

Nontuberculous Mycobacteria Infection Risk and Trace Metals in Surface Water: A Population-based Ecologic Epidemiologic Study in Oregon.

Rationale: Nontuberculous mycobacteria (NTM) are ubiquitous environmental bacteria, and some pathogenic species cause lung disease. Environmental factors contribute to increased NTM abundance, with higher potential for exposure and infection. Objectives: To identify water-quality constituents that influence the risk of NTM infection in Oregon. Methods: We conducted a population-based cohort study using patient incidence data from the Oregon statewide NTM laboratory data collected as part of a public health surveillance project from 2007 through 2012. To estimate the risk of NTM pulmonary infection (PI) from exposure to water constituents, we extracted water-quality data from the Water Quality Portal and associated these data with corresponding patient county of residence. Using generalized linear models, we modeled two outcomes: Mycobacterium avium complex species PI and Mycobacterium abscessus group species PI. Results: For every 1-unit increase in the log concentration of vanadium in surface water, infection risk increased by 49% among persons with Mycobacterium avium complex PI. Among those with Mycobacterium abscessus PI, we observed that for every 1-unit increase in the log concentration of molybdenum in surface water, infection risk increased by 41%. The highest risk of infection due to Mycobacterium abscessus group infection was concentrated in counties within the Northwestern region of Oregon. High infection risk associated with Mycobacterium avium complex species did not show any geographic pattern. Conclusions: Concentrations of the trace metals molybdenum and vanadium in surface water sources were associated with NTM infection in Oregon. These findings may help identify regions at higher risk of NTM infection to guide risk reduction strategies.

PqsR‐independent quorum‐sensing response of Pseudomonas aeruginosa ATCC 9027 outlier‐strain reveals new insights on the PqsE effect on RhlR activity

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum-sensing response regulates the expression of several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame-shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE-dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR-dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE-dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains. HIGHLIGHTS: Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR. This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE. Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE. The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction. The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation. Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.

The Zinc Transporter ZnuABC Is Critical for the Virulence of Chromobacterium violaceum and Contributes to Diverse Zinc-Dependent Physiological Processes.

Chromobacterium violaceum is a ubiquitous environmental bacterium that causes sporadic life-threatening infections in humans. How C. violaceum acquires zinc to colonize environmental and host niches is unknown. In this work, we demonstrated that C. violaceum employs the zinc uptake system ZnuABC to overcome zinc limitation in the host, ensuring the zinc supply for several physiological demands. Our data indicated that the C. violaceum ZnuABC transporter is encoded in a zur-CV_RS15045-CV_RS15040-znuCBA operon. This operon was repressed by the zinc uptake regulator Zur and derepressed in the presence of the host protein calprotectin (CP) and the synthetic metal chelator EDTA. A ΔznuCBA mutant strain showed impaired growth under these zinc-chelated conditions. Moreover, the deletion of znuCBA provoked reductions in violacein production, swimming motility, biofilm formation, and bacterial competition. Remarkably, the ΔznuCBA mutant strain was highly attenuated for virulence in an in vivo mouse infection model and showed low capacities to colonize the liver, grow in the presence of CP, and resist neutrophil killing. Overall, our findings demonstrate that ZnuABC is essential for C. violaceum virulence, contributing to subversion of zinc-based host nutritional immunity.

Nontuberculous mycobacterial infection and environmental molybdenum in persons with cystic fibrosis: a case\u2013control study in Colorado

RationaleNontuberculous mycobacteria (NTM) are ubiquitous environmental bacteria that may cause chronic lung disease and are one of the most difficult-to-treat infections among persons with cystic fibrosis (pwCF). Environmental factors likely contribute to increased NTM densities, with higher potential for exposure and infection.ObjectiveTo identify water-quality constituents that influence odds of NTM infection among pwCF in Colorado.MethodsWe conducted a population-based nested case–control study using patient data from the Colorado CF Center NTM database. We associated data from pwCF and water-quality data extracted from the Water Quality Portal to estimate odds of NTM infection. Using Bayesian generalized linear models with binomial-distributed discrete responses, we modeled three separate outcomes; any NTM infection, infections due to Mycobacterium avium complex species, and infections due to M. abscessus group species.ResultsWe observed a consistent association with molybdenum in the source water and M. abscessus group species infection among pwCF in all models. For every 1-unit increase in the log concentration of molybdenum in surface water, the odds of infection for those with M. abscessus group species compared to those who were NTM culture-negative increased by 79%. The odds of M. abscessus group infection varied by county; the counties with the highest probability of infection are located along the major rivers.ConclusionsWe have identified molybdenum in the source water as the most predictive factor of M. abscessus group infection among pwCF in Colorado. This finding will help inform patients at risk for NTM of their relative risks in residing within specific regions.

Enhanced spoVF operon increases host attachment and biocontrol ability of Bacillus subtilis for the management of Ceratocystis fimbriata in sweet potato

Bacillus subtilis is a ubiquitous environmental bacterium commonly used for the biological control of fungal plant diseases. The ability to form surface-attached microbial communities is essential for B. subtilis to colonize and protect plants from pathogen infection. In this work, it was found that enhanced spoVF operon altered the carbon metabolic pathways of B. subtilis 168, accelerating cell growth and glucose consumption, and inducing the production of antifungal metabolite dipicolinic acid (DPA). These metabolic effects significantly increased swimming motility and biofilm formation, enhancing the colonization ability and host attachment of B. subtilis. Mutant B. subtilis showed higher inhibitory activity in vivo against sweet potato pathogen Ceratocystis fimbriata in comparison with the wild-type strain. Collectively, novel insights on the key genes and pathways involved in biofilm formation and attachment of B. subtilis were revealed in this work.

Mining Public Mass Spectrometry Data to Characterize the Diversity and Ubiquity of P. aeruginosa Specialized Metabolites.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that causes chronic infections of burn wounds and in the lungs of cystic fibrosis (CF) patients. Vital to its infection is a myriad of specialized metabolites that serve a variety of biological roles including quorum sensing, metal chelation and inhibition of other competing bacteria. This study employed newly available algorithms for searching individual tandem mass (MS/MS) spectra against the publicly available Global Natural Product Social Molecular Networking (GNPS) database to identify the chemical diversity of these compounds and their presence in environmental, laboratory and clinical samples. For initial characterization, the metabolomes of eight clinical isolates of P. aeruginosa were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and uploaded to GNPS for spectral searching. Quinolones, rhamnolipids, phenazines and siderophores were identified and characterized; including the discovery of modified forms of the iron chelator pyochelin. Quinolones were highly diverse with the three base forms Pseudomonas quinolone signal 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), 4-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-4-quinolone-N-oxide (HQNO) having extensive variation in the length of their acyl chain from as small as 3 carbons to as large as 17. Rhamnolipids were limited to either one or two sugars with a limited set of fatty acyl chains, but the base lipid form without the rhamnose was also detected. These specialized metabolites were identified from diverse sources including ant-fungal mutualist dens, soil, plants, human teeth, feces, various lung mucus samples and cultured laboratory isolates. Their prevalence in fecal samples was particularly notable as P. aeruginosa is not known as a common colonizer of the human gut. The chemical diversity of the compounds identified, particularly the quinolones, demonstrates a broad spectrum of chemical properties within these the metabolite groups with likely significant impacts on their biological functions. Mining public data with GNPS enables a new approach to characterize the chemical diversity of biological organisms, which includes enabling the discovery of new chemistry from pathogenic bacteria.

Effect of hydrothermal hot-compression method on the antimicrobial performance of green building materials from heterogeneous cellulose wastes

Microbiomes in the built environment is an emerging topic of high-relevance, as biodeterioration and biodegradation of porous and polymer materials may be major public health risk factors. Epidemiological studies have confirmed indoor airborne microorganisms and their metabolites cause significant toxic immune reactions, with clinical effects associated with more than 60% of occupants’ chronic infections. The present study reports the potential inhibitory effect on microbial and fungal colonization of hydrothermal hot-compression for the manufacture of a series of novel cellulose-based panels engineered for insulating building applications. This method was examined as it is a green manufacturing technique which simultaneously self-bonds the cellulose fibres, as well as potentially having an anti-microbial effect. The technique renders the novel materials non-toxic, additive-free, biodegradation resistant, as well as increasing the recyclability potential of the materials at their end-of-life. Prototypes were formulated from three cellulosic materials sourced from complex post-consumer coffee industry heterogeneous waste stream ─jute textile, paper cups, and coffee grounds─ holding initially high-levels of microbiological contamination. The raw materials were characterized with Infrared Spectroscopy (FTIR) and Scanning Electronic Microscopy (SEM). Microbial species were identified using culture-dependent and culture-independent DNA quantitative methods for the raw materials, as well as the processed panels. The experimental results confirmed a high-level of bacterial and fungal contamination with ubiquitous environmental bacteria such as Bacillus, Burkholderia, and Stenotrophomonas species for the Jute textile and Paper cup fibres. However, the microbes were minimized or non-detectable for all three processed panel samples. This promising result demonstrates that complex cellulosic waste materials can be converted to low-microbial biomass potential industrial feedstock for the green manufacturing of building materials.

Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets.

Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection.IMPORTANCELegionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing.

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin-laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.IMPORTANCEPseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner.