Laboratory Isolates Research Articles

Multiple drug resistance in hookworms infecting greyhound dogs in the USA

Ancylostoma caninum is the most prevalent nematode parasite of dogs. We confirmed multiple-drug resistance (MDR) in several A. caninum isolates to all anthelmintic drug classes approved for the treatment of hookworms in dogs in the USA. Cases of MDR hookworms appear to be highly overrepresented in greyhounds. The aims of this study were to evaluate the drug-resistant phenotypes and genotypes of the A. caninum infecting greyhounds. Fecal samples from greyhounds of the USA were acquired from two greyhound adoption kennels, one active greyhound racing kennel, and three veterinary practices. Fecal egg counts (FECs) were performed on fecal samples from 219 greyhounds, and despite treatment with anthelmintics, the mean FEC was 822.4 eggs per gram (EPG). Resistance to benzimidazoles and macrocyclic lactones were measured using the egg hatch assay (EHA) and the larval development assay (LDA), respectively. We performed 23 EHA and 22 LDA on either individual or pooled feces, representing 54 animals. Mean and median IC50 and IC95 values for the EHA were 5.3 μM, 3.6 μM, and 24.5 μM, 23.4 μM, respectively. For the LDA, the median IC50 value was >1000 nM. These values ranged 62–81 times higher than our susceptible laboratory isolate. Only post-treatment samples were available. For samples collected <10 days post-treatment with albendazole, moxidectin, or a combination of febantel-pyrantel-moxidectin, the mean FEC were 349, 333, and 835 EPG, respectively. We obtained DNA from hookworm eggs isolated from 70 fecal samples, comprised of 60 individual dogs and 10 pools. Deep sequencing of the isotype 1 β-tubulin gene only revealed the presence of the F167Y (TTC>TAC) resistance polymorphism in 99% of these samples. These clinical, in vitro, and genetic data provide strong evidence that greyhound dogs in the USA are infected with MDR A. caninum at very high levels in prevalence and infection intensity.

ZnO Nanoparticles-Modified Dressings to Inhibit Wound Pathogens.

Zinc oxide (ZnO) nanoparticles (NPs) have been investigated for various skin therapies in recent years. These NPs can improve the healing and modulate inflammation in the wounds, but the mechanisms involved in such changes are yet to be known. In this study, we have designed a facile ZnO nano-coated dressing with improved antimicrobial efficiency against typical wound pathogens involved in biofilm and chronic infections. ZnO NPs were obtained by hydrothermal method and characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy. Antibacterial and antibiofilm effects were evaluated against laboratory and clinical isolates of significant Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) opportunistic pathogens, by quantitative methods. Our results have shown that the developed dressings have a high antibacterial efficiency after 6–24 h of contact when containing 0.6 and 0.9% ZnO NPs and this effect is similar against reference and clinical isolates. Moreover, biofilm development is significantly impaired for up to three days of contact, depending on the NPs load and microbial species. These results show that ZnO-coated dressings prevent biofilm development of main wound pathogens and represent efficient candidates for developing bioactive dressings to fight chronic wounds.

Mycobacterium Abscessus HelR Interacts with RNA Polymerase to Confer Intrinsic Rifamycin Resistance

Bacterial RNA polymerase is a target of the potent and broad-spectrum rifamycin group of antibiotics. Mutations within rpoB and inactivation by a diverse group of enzymes constitute the most widespread mechanisms of resistance. Rifampicin (RIF) constitutes the frontline therapy against M. tuberculosis as well as most slow-growing non-tuberculous mycobacteria (NTM) but is completely ineffective against M. abscessus. This high level of RIF resistance is despite the absence of mutations in the rifampicin resistance determining region of Mab_rpoB and is partially attributed to the presence of an ADP-ribosyltransferase (Arr) activity that inactivates RIF. Rifabutin (RBT), a close analogue of RIF, has recently been shown to be effective against M. abscessus in vitro and in a mouse model and comprises a promising therapeutic against M. abscessus infections. Using RNA sequencing we show that exposure of M. abscessus to sublethal doses of RIF and RBT results in ~25-fold upregulation of Mab_ helR in laboratory and clinical isolates; an isogenic deletion mutant of Mab_ helR is hypersensitive to RIF and RBT, and over-expression of Mab_ helR confers RIF tolerance in M. tuberculosis implying that Mab_helR constitutes a significant determinant of inducible RIF and RBT resistance. We demonstrate a preferential association of MabHelR with RNA polymerase in vivo in bacteria exposed to RIF and show that purified MabHelR can rescue transcription inhibition in the presence of RIF in in vitro transcription assays. Furthermore, MabHelR can dissociate RNAP from RIF-stalled initiation complexes in vitro, a species we envisage accumulates upon RIF exposure. Lastly, we show that the tip of the PCh-loop of Mab_ helR, in particular residues E496 and D497 that are in proximity to RIF, is critical for conferring RIF resistance without being required for dissociation of antibiotic-stalled RNAP complexes. This suggests that HelR may be additionally involved in displacing RIF bound to RNAP and function as an RNAP protection protein.

Development and validation of a reverse transcription real-time PCR assay for specific detection of PRRSGard vaccine-like virus.

Increasing use of modified live virus (MLV) vaccines presents challenges to interpret positive results of porcine reproductive and respiratory syndrome virus (PRRSV) screening PCR that can detect both wild-type and vaccine strains. Instead, vaccine-specific PCR provides a convenient tool to detect vaccine-like virus from a sample. Here we report the development and validation of a real-time RT-PCR specific for PRRSGard® , a newly available commercial PRRSV-2 MLV vaccine. Analytical specificity, sensitivity and diagnostic performance of PRRSGard PCR were evaluated and compared to a commercial PRRSV screening PCR (reference PCR). PRRSGard and reference PCRs did not cross-react with any of the 27 non-PRRSV swine pathogens. PRRSGard PCR did not cross-react with other PRRSV-2 vaccine viruses and 31 laboratory and field PRRSV-2 isolates representing various genetic lineages of PRRSV-2. PRRSGard and reference PCRs consistently detected up to 10-6 and 10-5 dilutions of PRRSGard vaccine virus, respectively. Based on testing serial dilutions of in vitro transcribed RNA, the 95% limit of detection of PRRSGard PCR was 16 genomic copies/reaction with CT cut-off value of 36 and 7 genomic copies/reaction with CT cut-off value of 37. Diagnostic performance of PRRSGard PCR was evaluated using 846 clinical samples (684 serum and 162 oral fluid samples). Compared to the reference screening PCR, diagnostic sensitivity, specificity and agreement of PRRSGard PCR were 95.34%, 98.85% and 97.52% with cut-off CT value of 36 and 98.14%, 96.56% and 97.16% with cut-off CT value of 37. In addition, PRRSGard PCR was able to detect PRRSGard vaccine virus in a sample even with the co-presence of another PRRSV strain. In summary, in contrast to a reference screening PCR that detects both vaccine and field PRRSV strains, PRRSGard PCR provides a convenient tool to specifically detect PRRSGard vaccine-like virus and to inform PRRSV vaccination protocols.

Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus

Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens that are associated with deterioration of lung function, e.g., in persons with cystic fibrosis (CF). There is evidence that co-infections with these pathogens cause airway inflammation and aggravate pathology in CF lungs. Intermicrobial competition of P. aeruginosa and A. fumigatus has been described, but it is unknown how anti-fungal therapy is affected. The anti-fungal azole voriconazole (VCZ), supernatants of P. aeruginosa laboratory isolates PA14 or PAO1, or clinical isolate Pa10 independently inhibited biofilm metabolism of A. fumigatus isolates 10AF and AF13073. When VCZ and supernatants were combined at their IC50s, synergistic effects on A. fumigatus were found. Synergistic effects were no longer observed when P. aeruginosa supernatants were prepared in the presence of iron, or when P. aeruginosa mutants were lacking the ability to produce pyoverdine and pyochelin. Combination of pure P. aeruginosa products pyoverdine, pyochelin, and pyocyanin with VCZ showed synergistic anti-fungal effects. Combining VCZ with P. aeruginosa supernatants also improved its MIC and MFC against planktonic A. fumigatus. In summary, in the case of P. aeruginosa–A. fumigatus co-infections, it appeared that the P. aeruginosa co-infection facilitated therapy of the Aspergillus; lower concentrations of VCZ might be sufficient to control fungal growth.

Synthesis of nZVI/PVP nanoparticles for bioremediation applications

The objective of this investigation is to synthesize and investigate zero-valent iron (ZVI) nanoparticles (NPs) for bioremediation applications. The ZVI-NPs were fabricated by chemical reduction using a ferrous salt solution with poly(N-vinylpyrrolidone) (PVP), used as a stabilizer. The synthesis was conducted with and without ultrasonic treatment. The ZVI NPs were fabricated and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD) analysis, and Fourier Transform Infrared Spectroscopy (FTIR). Experimental observations demonstrate that depending on synthesis conditions and coordination of stabilizers, NPs with different morphologies are formed. Colloidal solutions of the synthesized NPs were used in antimicrobial activity tests and biofilm formation assays for nine different control microorganisms: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 15692), Enterococcus faecalis (ATCC 29122), Klebsiella pneumoniae (laboratory isolates), Proteus vulgaris (laboratory isolates), Staphylococcus aureus (ATCC 29213), Bacillus cereus (DSMZ 4312), Bacillus subtilis (ATCC 6633), and Candida albicans (ATCC 10231). All control strains did not show antibacterial effect against PVP-stabilized ZVI NPs synthesized without ultrasonic treatment. However, biofilm results show that the highest absorbance values of the micro-organisms were tested in control wells. Although B. subtilis, E. coli, and K. pneumoniae were observed during biofilm formation, B. cereus, S. aureus, and P. aeruginosa biofilm formation reduced noticeably by Fe0/PVP-US (A1) NPs. For control strains, such as E. faecalis and C. albicans, no biofilm formation was observed. For Fe0/PVP (A2) NPs, biofilm formation of B. subtilis, E. faecalis, E. coli, K. pneumoniae, P. vulgaris, and C. albicans demonstrated positive effect, and B. cereus, S. aureus, P. aeruginosa showed negative effect. A strategic utilization of nZVI-PVP nanoparticles showed a great potential for effective, efficient, and sustainable bioremediation applications.

Microbial genetic and transcriptional contributions to oxalate degradation by the gut microbiota in health and disease.

Over-accumulation of oxalate in humans may lead to nephrolithiasis and nephrocalcinosis. Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbes can degrade oxalate using multiple ODPs and protect against its absorption. The exact oxalate-degrading taxa in the human microbiota and their ODP have not been described. We leverage multi-omics data (>3000 samples from >1000 subjects) to show that the human microbiota primarily uses the type II ODP, rather than type I. Furthermore, among the diverse ODP-encoding microbes, an oxalate autotroph, Oxalobacter formigenes, dominates this function transcriptionally. Patients with inflammatory bowel disease (IBD) frequently suffer from disrupted oxalate homeostasis and calcium oxalate nephrolithiasis. We show that the enteric oxalate level is elevated in IBD patients, with highest levels in Crohn's disease (CD) patients with both ileal and colonic involvement consistent with known nephrolithiasis risk. We show that the microbiota ODP expression is reduced in IBD patients, which may contribute to the disrupted oxalate homeostasis. The specific changes in ODP expression by several important taxa suggest that they play distinct roles in IBD-induced nephrolithiasis risk. Lastly, we colonize mice that are maintained in the gnotobiotic facility with O. formigenes, using either a laboratory isolate or an isolate we cultured from human stools, and observed a significant reduction in host fecal and urine oxalate levels, supporting our in silico prediction of the importance of the microbiome, particularly O. formigenes in host oxalate homeostasis.

Evaluation of changes in drug susceptibility and population genetic structure in Haemonchus contortus following worm replacement as a means to reverse the impact of multiple-anthelmintic resistance on a sheep farm

A population of Haemonchus contortus that was highly resistant to benzimidazoles and avermectin/milbemycins with a subpopulation that was resistant to levamisole, was replaced with a susceptible laboratory isolate of H. contortus in a flock of sheep. The anthelmintic susceptibility and population genetics of the newly established population were evaluated for 3.5 years using in vivo, in vitro, and molecular methods. Successful replacement of the resistant population with a susceptible population was confirmed using phenotypic and genotypic measurements; larval development assay indicated full anthelmintic susceptibility; albendazole treatment yielded 98.7% fecal egg count reduction; pyrosequence genotyping of single nucleotide polymorphisms in positions 167 and 200 of the isotype-1 beta tubulin gene were present at 0.0 and 1.7%, respectively; microsatellite genotyping indicated the background haplotype was similar to the susceptible isolate; and haplotypes of the isotype-1 beta tubulin gene were similar to the susceptible isolate. To sustain the susceptibility of the new population, targeted selective treatment was implemented using albendazole. Surprisingly, within 1.5 years post-replacement, the population reverted to a resistant phenotype. Resistance to albendazole, ivermectin, and moxidectin was confirmed via fecal egg count reduction test, larval development assay, and pyrosequencing-based genotyping. Targeted selective treatment was then carried out using levamisole. However, within one year, resistance was detected to levamisole. Population genetics demonstrated a gradual change in the genetic structure of the population until the final population was similar to the initial resistant population. Genetic analyses showed a lack of diversity in the susceptible isolate, suggesting the susceptible isolate had reduced environmental fitness compared to the resistant population, providing a possible explanation for the rapid reversion to resistance. This work demonstrates the power of combining molecular, in vitro, and in vivo assays to study phenotypic and genotypic changes in a field population of nematodes, enabling improved insights into the epidemiology of anthelmintic resistance.

Impact of benzimidazole resistance on fitness parameters and fumonisin B1 production in Fusarium verticillioides (Sacc) Niremberg

Fusarium verticillioides laboratory isolates resistant to carbendazim were readily obtained after mutagenesis with UV-irradiation. Three resistant phenotypes that included highly (HR, Resistant factors>30), moderately (MR, Rf: 14–25) and low (LR, Rf: 2–5) resistant isolates to carbendazim were identified by fungitoxicity tests in vitro. Mutant isolates were simultaneously resistant to thiabendazole and benomyl but not to the also tubulin-blocking fungicides diethofencarb and zoxamide. No cross resistance relationship was found between carbendazim and fungicides with different modes of action such as the dicarboximide iprodione, the QoI pyraclostrobin, the imidazole prochloraz, the triazoles flusilazole and epoxiconazole. Mutation(s) for resistance to benzimidazoles did not significantly affect mycelial growth rate whereas sporulation and pathogenicity were adversely affected in most of the resistant mutants. Moreover, low temperatures suppressed the expression of carbendazim-resistance in all resistant phenotypes. Most mutant isolates produced Fumonisin B1 in similar or less quantities than the wild-type isolate both in vitro and in vivo in the absence of carbendazim with the exception of one HR isolate which had up to 3-fold increased FB1 production. Sub lethal doses of carbendazim in the growth medium in vitro resulted in a significant reduction in FB1 levels in all cases even though most mutant isolates produced higher FB1 quantities than the wild type. A significant correlation was found between F. verticillioides sporulation and FB1 production on maize seeds. The potential risk of food and feed contamination by FB1 producing F. verticillioides isolates resistant to benzimidazoles and implications on resistance management strategies are discussed.

Heterogeneous Klebsiella pneumoniae Co-infections Complicate Personalized Bacteriophage Therapy.

Multidrug-resistant (MDR) organisms have increased worldwide, posing a major challenge for the clinical management of infection. Bacteriophage is expected as potential effective therapeutic agents for difficult-to-treat infections. When performing bacteriophage therapy, the susceptibility of lytic bacteriophage to the target bacteria is selected by laboratory isolate from patients. The presence of a subpopulation in a main population of tested cells, coupled with the rapid development of phage-resistant populations, will make bacteriophage therapy ineffective. We aimed to treat a man with multifocal urinary tract infections of MDR Klebsiella pneumoniae by phage therapy. However, the presence of polyclonal co-infectious cells in his renal pelvis and bladder led to the failure of three consecutive phage therapies. After analysis, the patient was performed with percutaneous nephrostomy (PCN). A cocktail of bacteriophages was selected for activity against all 21 heterogeneous isolates and irrigated simultaneously via the kidney and bladder to eradicate multifocal colonization, combined with antibiotic treatment. Finally, the patient recovered with an obviously improved bladder. The success of this case provides valuable treatment ideas and solutions for phage treatment of complex infections.Clinical Trial Registration www.chictr.org.cn, identifier ChiCTR1900020989.

Prevalence of Pathogenic Microorganisms and Determination of Possible Presence of Antimicrobial Residues in Industrial and Medical Wastes

Waste materials discharged from any industries without any treatment may cause serious environmental problems which may consequently affect public health. Present study design to screen the proliferation of microorganisms in different waste and environmental samples near industrial areas along with the assessment of the presence of antibacterial substances. The existence of the total viable bacteria and fungi was estimated up to 10 7 cfu/g and 10 4 cfu/g, respectively, in solid wastes. For liquid wastes, the total viable bacteria were recovered up to 10 7 cfu/mL and fungi were observed up to 10 5 cfu/mL. Both types of samples were found to be contaminated with an array of pathogenic bacteria including Klebsiella spp ., Staphylococcus spp ., and Vibrio spp . Conversely, some medical and pharmaceutical waste samples were found to inhibit the growth of laboratory isolates tested which indicate the presence of antibiotics residues. Finally, the huge microbial load and the possible presence of antibiotic or other antimicrobial residues render the samples a major public health concern.

In vitro antibacterial activity of green tea (Camellia sinensis) extract against staphylococcus aureus and MRSA

Context: There is a worldwide emergence of multi drug resistant bacteria. Recently no new antibiotics have been approved for use, although some are in the pipeline, undergoing phase 2 or 3 clinical trials. There is need for alternate compounds for treatment; one such agent is green tea. Green tea has antimicrobial effects against a variety of Gram positive and Gram negative bacteria (e.g., Staphylococcus aureus, Enterococcus sp, Escherichia coli, Salmonella sp.) Aims: To determine the antibacterial activity of green tea extract against Methicillin resistant and sensitive Staphylococcus aureus (MRSA &MSSA) Settings and Design: Department of Microbiology, Cross sectional study Materials and Methods: A total of 15 consecutive laboratory isolates of S. aureus (MSSA) and MRSA each, isolated between August to September 2017 were inoculated in media containing Green tea extract [epigallocatechin gallate (EGCG)] in concentrations of 2.5mg/ml, 5mg/ml, 10mg/ml. After overnight incubation the bacterial broth was subcultured using standard calibrated loop (0.01ml) onto Mueller Hinton agar. The lowest concentration of EGCG in which no growth occurred was considered as the minimum bactericidal concentration (MBC). Results: EGCG had antimicrobial activity against S. aureus ATCC-25923, P. aeruginosa ATCC-27853, K.pneumoniae ATCC-BAA 1705 and E.coli ATCC-25922- MBC 99.9% was 5mg/ml. But for E.faecalis ATCC 29212 MBC 99.9% was 10mg/ml. EGCG was effective against clinical isolates of MSSA and MRSA with MBC of 5mg/ml except 2 MRSA isolates for which MBC was 10mg/ml. Conclusions: Green tea extract- EGCG has antibacterial activity not only against standard bacterial strains but also against clinical isolates of S.aureus and MRSA. Keywords: Camellia sinensis, S.aureus, Minimum bactericidal concentration.

The occurrence of drug-resistant bacteria and screening the possible presence of residual antibiotics in poultry feed samples

The use of antibiotics in the poultry and livestock industries for the treatment and prevention of infectious diseases, and as growth promoters in poultry feeds has increased worldwide. Such frequent employment of antibiotics may contribute to the development and dissemination of bacterial antibiotic resistance. The present study was an attempt to isolate drug-resistant bacteria and to screen the probability of having residual antibiotics in the poultry feed samples. Therefore, a total of 18 samples inclusive of starter, grower and finisher of two poultry feed brands of reputed Bangladeshi feed companies were collected and subjected to microbiological analysis, antibiogram and agar well diffusion assay. All the samples contained extended numbers of total viable bacteria and fungi in an average of 108 and 107 cfu/g, respectively. Klebsiellaspp., Pseudomonas spp. and Bacillus spp. were predominantly present in the tested samples. E. coli and Vibrio spp. were also found in most of the samples. Most isolates have been determined to be multidrug-resistant. All the isolates showed resistance against Cefuroxime. Penicillin resistance was found in most of the isolates in greater proportion. Higher rate of resistance was evident against Novobiocin, Cephradine and Rifampicin. However, the bacterial isolates showed sensitivity to Tobramycin, Nalidixic acid and Neomycin. The poultry feed samples, especially starter and finisher of both brands noticeably had significant antimicrobial activity against the laboratory isolates indicative of the probable presence of residual antibiotics which might be used as supplements in the poultry feed samples.&#x0D; Stamford Journal of Microbiology, Vol.10 (1) 2020: 30-34

The Effectiveness of Varying Combination Ratios of A. cordifolia and M. indica against Field and Laboratory Strains of P. falciparum In Vitro.

Background Drug resistance in malaria is a global problem, with reports of Plasmodium parasites resistant to the current first-line antimalarial drug, artemisinin, expanding from Southeast Asia to Africa. There is therefore an urgent need to identify new drug candidates that will be effective against the existing malaria parasites. Drug combination therapy presents a myriad of advantages over monotherapy including delayed onset of resistance, potentiation, and synergism. This present study explored the effectiveness of combinations of aqueous extracts of Alchornea cordifolia (A. cordifolia) and Mangifera indica (M. indica) at clearing both laboratory and field isolates of P. falciparum. Methods Synchronized ring stage cultures of field (FA08) and laboratory strains (NF54 and CamWT_C580Y) of P. falciparum were subjected to combinations of different concentrations and ratios of aqueous extracts of A. cordifolia and M. indica. The growth inhibition of the individual plant extracts and their combinatory effects were studied in vitro using SYBR Green I drug assay. Results The A. cordifolia extract exhibited 50% inhibitory concentration (IC50) of 2.71, 7.80, and 3.56 μg/mL against the NF54, CamWT_C580Y, and FA08 parasite strains, respectively. Mangifera indica exhibited IC50 of 18.11, 20.08, and 10.23 μg/mL against the NF54, CamWT_C580Y, and FA08 parasite strains, respectively. Additive, synergistic and antagonistic interactions were observed at different combinations of A. cordifolia and M. indica extracts. Conclusion A combination product containing A. cordifolia and M. indica has the potential to serve as an effective antimalarial as majority of the tested combinations of aqueous extracts of A. cordifolia and M. indica extracts exhibited synergistic effects in vitro against the NF54, CamWT_C580Y, and FA08 P. falciparum strains.

Interactions between Candida albicans and Enterococcus faecalis in an Organotypic Oral Epithelial Model.

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause oral and disseminated infections. Similar to C. albicans, Enterococcus faecalis is a major opportunistic pathogen, which is of critical concern in immunocompromised patients. There is increasing evidence that E. faecalis co-exists with C. albicans in the human body in disease samples. While the interactive profiles between these two organisms have been studied on abiotic substrates and mouse models, studies on their interactions on human oral mucosal surfaces are non-existent. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and P52S) on an organotypic oral mucosal model. Our results demonstrated that the dual species biofilms resulted in profound surface erosion and significantly increased microbial invasion into mucosal compartments, compared to either species alone. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. By contrast, E. faecalis genes involved in quorum sensing, biofilm formation, virulence, and mammalian cell invasion were downregulated. This study highlights the synergistic cross-kingdom interactions between E. faecalis and C. albicans in mucosal tissue invasion.

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.

Systems biology analysis of the Clostridioides difficile core-genome contextualizes microenvironmental evolutionary pressures leading to genotypic and phenotypic divergence

Hospital acquired Clostridioides (Clostridium) difficile infection is exacerbated by the continued evolution of C. difficile strains, a phenomenon studied by multiple laboratories using stock cultures specific to each laboratory. Intralaboratory evolution of strains contributes to interlaboratory variation in experimental results adding to the challenges of scientific rigor and reproducibility. To explore how microevolution of C. difficile within laboratories influences the metabolic capacity of an organism, three different laboratory stock isolates of the C. difficile 630 reference strain were whole-genome sequenced and profiled in over 180 nutrient environments using phenotypic microarrays. The results identified differences in growth dynamics for 32 carbon sources including trehalose, fructose, and mannose. An updated genome-scale model for C. difficile 630 was constructed and used to contextualize the 28 unique mutations observed between the stock cultures. The integration of phenotypic screens with model predictions identified pathways enabling catabolism of ethanolamine, salicin, arbutin, and N-acetyl-galactosamine that differentiated individual C. difficile 630 laboratory isolates. The reconstruction was used as a framework to analyze the core-genome of 415 publicly available C. difficile genomes and identify areas of metabolism prone to evolution within the species. Genes encoding enzymes and transporters involved in starch metabolism and iron acquisition were more variable while C. difficile distinct metabolic functions like Stickland fermentation were more consistent. A substitution in the trehalose PTS system was identified with potential implications in strain virulence. Thus, pairing genome-scale models with large-scale physiological and genomic data enables a mechanistic framework for studying the evolution of pathogens within microenvironments and will lead to predictive modeling to combat pathogen emergence.

Pathology of Angiostrongylus cantonensis infection in two model avian hosts.

Angiostrongylus cantonensis causes severe neurological disorders in a wide range of warm-blooded animals, including several avian species. A laboratory isolate of A. cantonensis originating from French Polynesia, genotyped as clade 2, was used to assess the effect of experimental infection in chicken and Japanese quail. Low dose groups of birds were infected orally by 100 L3 larvae, high dose groups by 1500 L3 larvae and the birds in the third group were fed three infected snails, mimicking a natural infection. Clinical signs during the first week after infection, haematology, biochemistry, gross lesions and histology findings were used to assess the pathology of the infection. Some of the infected birds showed peripheral eosinophilia, while mild neurological signs were seen in others. No larvae were observed in serial sections of the central nervous system of infected birds 1 week after infection and no major gross lesions were observed during necropsy; histopathology did not reveal lesions directly attributable to A. cantonensis infection. Our results suggest that galliform birds are not highly susceptible to A. cantonensis infection and open a question of the importance of Galliformes in endemic areas as natural pest control, lowering the number of hosts carrying the infective larvae.

Antimicrobial Synergism Toward Pseudomonas aeruginosa by Gallium(III) and Inorganic Nitrite.

The ubiquitous involvement of key iron-containing metalloenzymes in metabolism is reflected in the dependence of virtually all bacteria on iron for growth and, thereby, potentially provides multiple biomolecular targets for antimicrobial killing. We hypothesized that nitrosative stress, which induces damage to iron metalloproteins, would sensitize bacteria to the ferric iron mimic gallium(III) (Ga3+), potentially providing a novel therapeutic combination. Using both laboratory and clinical isolates of Pseudomonas aeruginosa, we herein demonstrate that Ga3+ and sodium nitrite synergistically inhibit bacterial growth under both aerobic and anaerobic conditions. Nitric oxide also potentiated the antimicrobial effect of Ga3+. Because many chronic pulmonary infections are found as biofilms and biofilms have very high antibiotic tolerance, we then tested the combination against biofilms grown on plastic surfaces, as well as the apical surface of airway epithelial cells. Ga3+ and sodium nitrite had synergistic antimicrobial activity against both biofilms grown on plastic and on airway epithelial cell. Both Ga3+ and various NO donors are (independently) in clinical development as potential antimicrobials, however, we now propose the combination to have some particular advantages, while anticipating it should ultimately prove similarly safe for translation to treatment of human disease.

Multi-year diagnosis of unpredictable fouling occurrences in a full-scale membrane bioreactor

The membrane bioreactor (MBR) at the Traverse City Regional Wastewater Treatment Plant has experienced sudden and unpredictable periods of substantial permeability decline since 2011. Early observations detected irregularly-shaped Gram-positive bacteria that correlated with plant upsets. Use of biomolecular techniques, such as DNA sequencing of laboratory isolates and the mixed liquor microbial community, and fluorescent in situ hybridization, identified the dispersed organisms as members of the genus Staphylococcus. However, Staphylococcus species were consistently present during normal operation and therefore were more likely to be an indicator of the upset, not the cause. The results suggest that these microorganisms are responding to specific influent wastewater constituents. We chemically analysed seven mixed liquor samples from periods of permeability decline in 2017 and 2018, and four samples from a period of normal operation. During upset conditions, the total carbohydrate content exceeded that of normal operation by 40%. Additionally, mixed liquor calcium concentrations were 65% above normal during the upset in 2017. It is hypothesized and supported through multivariate statistical analysis and estimation of specific resistance to filtration values that a calcium-intermediated polymer bridging mechanism with extracellular polymeric substance constituents is a major contributor to fouling and permeability disruptions in the Traverse City MBR.