Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS).

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies' (ONT) cloud-based What's In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT's framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC's 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT's MinION portability, ease-of-use, and identification capability in remote locations.

Secretome analysis of diarrhea-inducing strains of Escherichia coli.

Secreted proteins constitute a major part of virulence factors that are responsible for pathogenesis caused by Gram-negative bacteria. Enterohemorrhagic Escherichia coli, O157:H7, is the major pathogen often causing outbreaks. However, studies have reported that the significant outbreaks caused by non-O157:H7 E. coli strains, also known as "Big-Six" serogroup strains, are increasing. There is no systematic study describing differential secreted proteins from these non-O157:H7 E. coli strains. In this study, we carried out MS-based differential secretome analysis using tandem mass tags labeling strategy of non-O157:H7 E. coli strains, O103, O111, O121, O145, O26, and O45. We identified 1241 proteins, of which 565 proteins were predicted to be secreted. We also found that 68 proteins were enriched in type III secretion system and several of them were differentially expressed across the strains. Additionally, we identified several strain-specific secreted proteins that could be used for developing potential markers for the identification and strain-level differentiation. To our knowledge, this study is the first comparative proteomic study on secretome of E. coli Big-Six serogroup and the several of these strain-specific secreted proteins can be further studied to develop potential markers for identification and strain-level differentiation. Moreover, the results of this study can be utilized in several applications, including food safety, diagnostics of E. coli outbreaks, and detection and identification of bio threats in biodefense.

Training to Improve Precision and Accuracy in the Measurement of Fiber Morphology.

An estimated $7.1 billion dollars a year is spent due to irreproducibility in pre-clinical data from errors in data analysis and reporting. Therefore, developing tools to improve measurement comparability is paramount. Recently, an open source tool, DiameterJ, has been deployed for the automated analysis of scanning electron micrographs of fibrous scaffolds designed for tissue engineering applications. DiameterJ performs hundreds to thousands of scaffold fiber diameter measurements from a single micrograph within a few seconds, along with a variety of other scaffold morphological features, which enables a more rigorous and thorough assessment of scaffold properties. Herein, an online, publicly available training module is introduced for educating DiameterJ users on how to effectively analyze scanning electron micrographs of fibers and the large volume of data that a DiameterJ analysis yields. The end goal of this training was to improve user data analysis and reporting to enhance reproducibility of analysis of nanofiber scaffolds. User performance was assessed before and after training to evaluate the effectiveness of the training modules. Users were asked to use DiameterJ to analyze reference micrographs of fibers that had known diameters. The results showed that training improved the accuracy and precision of measurements of fiber diameter in scanning electron micrographs. Training also improved the precision of measurements of pore area, porosity, intersection density, and characteristic fiber length between fiber intersections. These results demonstrate that the DiameterJ training module improves precision and accuracy in fiber morphology measurements, which will lead to enhanced data comparability.

Influence of Sterilization Technologies on Electrospun Poly(ester urea)s for Soft Tissue Repair.

Degradable poly(ester urea)s (PEU)s were electrospun into nanofiber sheets and assessed for their potential to be used in soft tissue repair. The level of residual solvent was measured and the effects of ethylene oxide and electron beam sterilization techniques on molecular mass, mass distribution, and morphology were quantified. Two PEU compositions that formed stable nanofiber sheets were advanced into a pilot study in vitro and in vivo as candidate materials for hernia repair. Cell viability, spreading, proliferation, and migration were examined in vitro. Nanofiber sheets were implanted subcutaneously into mice and analyzed via microangiography and histology for tissue incorporation. Nanofiber sheets performed similarly to decellularized extracellular matrix (ECM) in vitro, but the lack of sufficient pore structure inhibited cellular infiltration after 14 days of culture. The lack of microporous features in nanofiber sheets also contributed to low levels of cellular infiltration, angiogenesis, and matrix deposition in vivo. A preliminary study to increase pore size in nanofibers was performed using coaxial electrospinning resulting in significant improvement in tissue infiltration in vivo.

Radiopaque, iodine functionalized, phenylalanine-based poly(ester urea)s.

The synthesis and characterization of iodine-functionalized phenylalanine-based poly(ester urea)s (PEUs) are reported. 4-Iodo-L-phenylalanine and L-phenylalanine were separately reacted with 1,6-hexanediol to produce two monomers, bis-4-I-L-phenylalanine-1,6-hexanediol-diester (1-IPHE-6 monomer) and bis-L-phenylalanine-1,6-hexanediol-diester (1-PHE-6 monomer). By varying the feed ratio of the 1-IPHE-6 and 1-PHE-6 monomers, the copolymer composition was modulated resulting in a wide variation in thermal, mechanical and radiopacity properties. Microcomputed tomography (µ-CT) projections demonstrate that increasing iodine content results in greater X-ray contrast. Compression tests of dry and wet porous scaffolds indicate that the poly(1-IPHE-6)0.24-co-poly(1-PHE-6)0.76 material results in the highest compression modulus. MC3T3 cell viability and spreading studies show PEUs are nontoxic to cells. As most medical device procedures require placement verification via fluoroscopic imaging, materials that possess inherent X-ray contrast are valuable for a number of applications.

Post-Electrospinning "Triclick" Functionalization of Degradable Polymer Nanofibers.

4-Dibenzocyclooctynol (DIBO) was used as an initiator for the ring-opening copolymerization of ε-caprolactone and 1,4,8-trioxaspiro[4.6]-9-undecanone (TOSUO) resulting in a series of DIBO end-functionalized copolymers. Following deprotection of the ketone group, the polymers were derivatized with aminooxyl-containing compounds by oxime ligation. Mixtures of keto- and alkyne-derivatized polymers were co-electrospun into well-defined nanofibers containing three separate chemical handles. Strain-promoted azide alkyne cycloaddition (SPAAC), oxime ligation, and copper-catalyzed azide alkyne cycloaddition (CuAAC) were used to sequentially functionalize the nanofibers first with fluorescent reporters and then separately with bioactive Gly-Arg-Gly-Asp-Ser (GRGDS), BMP-2 peptide, and dopamine. This translationally relevant approach facilitates the straightforward derivatization of diverse bioactive molecules that can be controllably tethered to the surface of nanofibers.

Extracellular protein biomarkers for the characterization of enterohemorrhagic and enteroaggregative Escherichia coli strains.

The extracellular proteins (ECPs) of enterohemorrhagic Escherichia coli (EHEC) can cause hemorrhagic colitis which may cause life threatening hemolytic-uremic syndrome, while that of enteroaggregative E. coli (EAEC) can clump to intestinal membranes. Liquid chromatography-electrospray ionization-tandem mass spectrometry based proteomics is used to evaluate a preliminary study on the extracellular and whole cell protein extracts associated with E. coli strain pathogenicity. Proteomics analysis, which is independent of genomic sequencing, of EAEC O104:H4 (unsequenced genome) identified a number of proteins. Proteomics of EHEC O104:H4, causative agent of the Germany outbreak, showed a closest match with E. coli E55989, in agreement with genomic studies. Dendrogram analysis separated EHEC O157:H7 and EHEC/EAEC O104:H4. ECP analysis compared to that of whole cell processing entails few steps and convenient experimental extraction procedures. Bacterial characterization results are promising in exploring the impact of environmental conditions on E. coli ECP biomarkers with a few relatively straightforward protein extraction steps.

Resorbable, amino acid-based poly(ester urea)s crosslinked with osteogenic growth peptide with enhanced mechanical properties and bioactivity.

Materials currently used for the treatment of bone defects include ceramics, polymeric scaffolds and composites, which are often impregnated with recombinant growth factors and other bioactive substances. While these materials have seen instances of success, each has inherent shortcomings including prohibitive expense, poor protein stability, poorly defined growth factor release and less than desirable mechanical properties. We have developed a novel class of amino acid-based poly(ester urea)s (PEU) materials which are biodegradable in vivo and possess mechanical properties superior to conventionally used polyesters (<3.5 GPa) available currently to clinicians and medical providers. We report the use of a short peptide derived from osteogenic growth peptide (OGP) as a covalent crosslinker for the PEU materials. In addition to imparting specific bioactive signaling, our crosslinking studies show that the mechanical properties increase proportionally when 0.5% and 1.0% concentrations of the OGP crosslinker are added. Our results in vitro and in an in vivo subcutaneous rat model show the OGP-based crosslinkers, which are small fragments of growth factors that are normally soluble, exhibit enhanced proliferative activity, accelerated degradation properties and concentration dependent bioactivity when immobilized.

Pulsed UV-C disinfection of Escherichia coli with light-emitting diodes, emitted at various repetition rates and duty cycles.

A 2010 study exposed Staphylococcus aureus to ultraviolet (UV) radiation and thermal heating from pulsed xenon flash lamps. The results suggested that disinfection could be caused not only by photochemical changes from UV radiation, but also by photophysical stress damage caused by the disturbance from incoming pulses. The study called for more research in this area. The recent advances in light-emitting diode (LED) technology include the development of LEDs that emit in narrow bands in the ultraviolet-C (UV-C) range (100-280 nm), which is highly effective for UV disinfection of organisms. Further, LEDs would use less power, and allow more flexibility than other sources of UV energy in that the user may select various pulse repetition frequencies (PRFs), pulse irradiances, pulse widths, duty cycles and types of waveform output (e.g. square waves, sine waves, triangular waves, etc.). Our study exposed Escherichia coli samples to square pulses of 272 nm radiation at various PRFs and duty cycles. A statistically significant correlation was found between E. coli's disinfection sensitivity and these parameters. Although our sample size was small, these results show promise and are worthy of further investigation. Comparisons are also made with pulsed disinfection by LEDs emitting at 365 nm, and pulsed disinfection by xenon flash lamps.

Extensive surgical and comprehensive postoperative medical management for cystic fibrosis chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis has a major impact on the quality of life of patients with cystic fibrosis (CF) and may contribute to progression of chronic lung disease. Despite multiple sinus surgeries, maxillary sinus involvement is a recurrent problem. The modified endoscopic medial maxillectomy (MEMM) permits debridement in the clinic, improves mucus clearance with nasal irrigations, and increases access for topical delivery of therapeutics. However, clinical outcomes of aggressive sinus surgery with regimented postoperative medical treatment have not been systematically evaluated. METHODS: CF patients completed the 22-Item Sinonasal Outcome Test questionnaires before sinus surgery (and bilateral MEMM) and at sequential postoperative visits. Objective measures included Lund-Kennedy endoscopic score and pulmonary function tests (forced expiratory volume at 1 second percent [FEV(1)%] predicted). Culture-directed antibiotic therapy, prednisone, and topical irrigations were initiated postoperatively. RESULTS: Twenty-two patients (mean age, 26.5 years; 4.9 prior sinus operations) underwent MEMM and sinus surgery. Symptom scores were significantly reduced at 60 days (primary outcome, 64.7 ± 18.4 presurgery versus 27.5 ± 15.3 postsurgery; p < 0.0001) and up to a year postoperatively (27.6 ± 12.6; p < 0.0001). Endoscopic scores were also reduced after surgery (10.4 ± 1.1 presurgery versus 5.7 ± 2.4 [30 days], 5.7 ± 1.4 [60 days], 5.8 ± 1.3 [120 days], and 6.0 ± 1.1 [1 year]; p < 0.0001)]. There were no differences in FEV(1)% predicted up to 1 year postoperatively, but hospital admissions secondary to pulmonary exacerbations significantly decreased (2.0 ± 1.4 versus 3.2 ± 2.4, respectively; p < 0.05). CONCLUSION: Prospective evaluation indicates sinus surgery with MEMM is associated with marked improvement in sinus disease outcomes. Additional studies are necessary to confirm whether this treatment paradigm is associated with improved CF pulmonary disease.

Evaluation of handheld assays for the detection of ricin and staphylococcal enterotoxin B in disinfected waters.

Development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation. The present study sought to assess the effectiveness of handheld assays (HHAs) in detecting ricin and Staphylococcal Enterotoxin B (SEB) in water. Performance of HHAs was evaluated in formulated tap water with and without chlorine, reverse osmosis water (RO) with chlorine, and RO with bromine. Each matrix was prepared, spiked with ricin or SEB at multiple concentrations, and then loaded onto HHAs. HHAs were allowed to develop and then read visually. Limits of detection (LOD) were determined for all HHAs in each water type. Both ricin and SEB were detected by HHAs in formulated tap water at or below the suggested health effect levels of 455 ng/mL and 4.55 ng/mL, respectively. However, in brominated or chlorinated waters, LODs for SEB increased to approximately 2,500 ng/mL. LODs for ricin increased in chlorinated water, but still remained below the suggested health effect level. In brominated water, the LOD for ricin increased to approximately 2,500 ng/mL. In conclusion, the HHAs tested were less effective at detecting ricin and SEB in disinfected water, as currently configured.

Identification of Yersinia pestis and Escherichia coli strains by whole cell and outer membrane protein extracts with mass spectrometry-based proteomics.

Whole cell protein and outer membrane protein (OMP) extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest neighbor database organism strains. These extracts were isolated from pathogenic and nonpathogenic strains of Yersinia pestis and Escherichia coli using ultracentrifugation and a sarkosyl extraction method followed by protein digestion and analysis using liquid chromatography tandem mass spectrometry (MS). Whole cell protein extracts contain many different types of proteins resident in an organism at a given phase in its growth cycle. OMPs, however, are often associated with virulence in Gram-negative pathogens and could prove to be model biomarkers for strain differentiation among bacteria. The mass spectra of bacterial peptides were searched, using the SEQUEST algorithm, against a constructed proteome database of microorganisms in order to determine the identity and number of unique peptides for each bacterial sample. Data analysis was performed with the in-house BACid software. It calculated the probabilities that a peptide sequence assignment to a product ion mass spectrum was correct and used accepted spectrum-to-sequence matches to generate a sequence-to-bacterium (STB) binary matrix of assignments. Validated peptide sequences, either present or absent in various strains (STB matrices), were visualized as assignment bitmaps and analyzed by the BACid module that used phylogenetic relationships among bacterial species as part of a decision tree process. The bacterial classification and identification algorithm used assignments of organisms to taxonomic groups (phylogenetic classification) based on an organized scheme that begins at the phylum level and follows through the class, order, family, genus, and species to the strain level. For both Gram-negative organisms, the number of unique distinguishing proteins arrived at by the whole cell method was less than that of the OMP method. However, the degree of differentiation measured in linkage distance units on a dendrogram with the OMP extract showed similar or significantly better separation than the whole cell protein extract method between the sample and correct database match compared to the next nearest neighbor. The nonpathogenic Y. pestis A1122 strain used does not have its genome available, and thus, data analysis resulted in an equal similarity index to the nonpathogenic 91001 and pathogenic Antiqua and Nepal 516 strains for both extraction methods. Pathogenic and nonpathogenic strains of E. coli were correctly identified with both protein extraction methods, and the pathogenic Y. pestis CO92 strain was correctly identified with the OMP procedure. Overall, proteomic MS proved useful in the analysis of unique protein assignments for strain differentiation of E. coli and Y. pestis. The power of bacterial protein capture by the whole cell protein and OMP extraction methods was highlighted by the data analysis techniques and revealed differentiation and similarities between the two protein extraction approaches for bacterial delineation capability.

Double-blind characterization of non-genome-sequenced bacteria by mass spectrometry-based proteomics.

Due to the possibility of a biothreat attack on civilian or military installations, a need exists for technologies that can detect and accurately identify pathogens in a near-real-time approach. One technology potentially capable of meeting these needs is a high-throughput mass spectrometry (MS)-based proteomic approach. This approach utilizes the knowledge of amino acid sequences of peptides derived from the proteolysis of proteins as a basis for reliable bacterial identification. To evaluate this approach, the tryptic digest peptides generated from double-blind biological samples containing either a single bacterium or a mixture of bacteria were analyzed using liquid chromatography-tandem mass spectrometry. Bioinformatic tools that provide bacterial classification were used to evaluate the proteomic approach. Results showed that bacteria in all of the double-blind samples were accurately identified with no false-positive assignment. The MS proteomic approach showed strain-level discrimination for the various bacteria employed. The approach also characterized double-blind bacterial samples to the respective genus, species, and strain levels when the experimental organism was not in the database due to its genome not having been sequenced. One experimental sample did not have its genome sequenced, and the peptide experimental record was added to the virtual bacterial proteome database. A replicate analysis identified the sample to the peptide experimental record stored in the database. The MS proteomic approach proved capable of identifying and classifying organisms within a microbial mixture.

Survival of viral biowarfare agents in disinfected waters.

Protecting civilian and military water supplies has received more attention since the United States began its war on terror in 2001. Both chlorine and bromine are used by branches of the U.S. military for disinfecting water supplies; however, limited data exists as to the effectiveness of these additives when used against viral biowarfare agents. The present study sought to evaluate the survival of selected viral biothreat agents in disinfected water. Disinfected water samples were spiked with vaccinia virus strain WR and Venezuelan equine encephalitis (VEE) virus strain TC-83 each separately to a final concentration of approximately 1 × 10(6) PFU/mL, and survival was assessed by plaque assay. Both viruses were inactivated by 1 mg/L free available chlorine (FAC) and 2mg/L total bromine within one hour. In conclusion, these results demonstrate that both chlorine and bromine are effective disinfectants against vaccinia virus and VEE strain TC-83 at the concentrations tested.

Effects of weak acids, UV and proton motive force inhibitors on pyrazinamide activity against Mycobacterium tuberculosis in vitro.

BACKGROUND: Pyrazinamide is a paradoxical frontline tuberculosis drug characterized by high sterilizing in vivo activity but poor in vitro activity. Pyrazinamide is thought to act by the entrapment of pyrazinoic acid in the bacterial cell, leading to acidification and membrane damage. Consequently, the effects of weak acids and molecules affecting membranes added to pyrazinamide were studied. OBJECTIVES: To examine the effects of weak acids, UV, oxidative stress and additional energy inhibitors on pyrazinamide activity in vitro against Mycobacterium tuberculosis as well as the effect of pyrazinamide on Escherichia coli ampicillin persisters. METHODS: Drug exposure experiments followed by cfu counts were performed to determine the effects of the above various factors on pyrazinamide activity in vitro against M. tuberculosis. RESULTS: Some weak acids such as benzoic acid, sorbic acid and propyl hydroxybenzoic acid could enhance the activity of pyrazinamide in vitro against old tubercle bacilli but not young bacilli whereas other weak acids such as salicylic acid and lactic acid did not appear to enhance pyrazinamide activity. While energy inhibitors carbonylcyanide m-chlorophenylhydrazone and dinitrophenol enhanced pyrazinamide activity for old tubercle bacilli but not young bacilli, valinomycin and KCN increased pyrazinamide activity for both young and old bacilli. Oxidative stresses due to H2O2 and menadione did not have a significant effect on pyrazinamide activity. UV, which presumably damages the membrane, enhanced the activity of pyrazinamide. Pyrazinamide, which otherwise has no activity against actively growing E. coli bacteria, could kill non-growing starved E. coli and also ampicillin-tolerant persisters. CONCLUSIONS: Some weak acids, UV and various energy inhibitors were found to enhance the activity of pyrazinamide in vitro against M. tuberculosis. Pyrazinamide shows preferential activity against both M. tuberculosis and E. coli persisters over the growing forms.

Evaluation of training on the use of Graseby syringe drivers for rural nonspecialist nurses.

AIM: to assess the impact of a training programme on nurse confidence in: setting up the Graseby syringe driver (GSD); explaining the GSD to patient and family; setting the rate on the GSD; putting appropriate type and dose of drugs in the GSD. STUDY DESIGN: training programme with pre-training, post-training and follow-up questionnaires. SAMPLE AND SETTING: palliative care nurse consultants presented half-day training sessions to 270 non-specialist nurses throughout the rural Grampians Health Region of Victoria, Australia. Nurses were from rural acute and sub-acute care settings, aged care facilities, and district nursing and nurse education services. MEASUREMENTS: demographic details of participants, previous experience and training with GSDs, comparative analyses of the four confidence parameters and participants' assessment of interest, new knowledge and usefulness of the training programme. RESULTS: increases in confidence levels were found in participating nurses in relation to each of the four confidence parameters. A follow-up survey tested residual benefit three months after the training programme. Statistically significant variations were found in nurses' confidence levels in relation to frequency of use. CONCLUSIONS: regular use of, and/or refresher sessions about the GSD are recommended to maintain optimum confidence, effective and safe nursing use of the GSD in palliative care.

Comparisons between nulliparous heifers and cows as oocyte donors for embryo production in vitro.

The aims of the present study were to compare (1) Holstein-Friesian heifers versus early postpartum lactating cows, and (2) different age categories of crossbred beef heifers versus cows, in terms of oocyte yield, morphological quality and developmental competence. Four experiments were designed to test the associated hypotheses. In Experiment 1, ovum pick up was carried out twice weekly for a period of 5 weeks on Holstein-Friesian heifers (n = 8) and early postpartum cows (n = 8). Oocytes were submitted to in vitro maturation (IVM), fertilization and culture. Significantly more follicles were punctured on the ovaries of heifers than cows (10.4 versus 7.8, P < 0.001). This was reflected in a significantly higher number of total oocytes (4.7 versus 2.8, P < 0.001) and grade 1-2 oocytes recovered/animal from heifers than from cows (3.0 versus 1.8, P < 0.05). There was no significant difference in the percentage of oocytes cleaving after fertilization, or in the percentage reaching the blastocyst stage between heifers and cows. In Experiment 2, oocytes were obtained by manual aspiration from the ovaries of slaughtered crossbred beef heifers (under 30 months, n = 1241) and cows (over 4 years old, n = 1125), and processed in vitro as above. No significant difference was observed between the two groups in terms of the number of aspirated follicles or oocytes recovered. A significantly higher proportion (P < 0.01) of cow oocytes than heifer oocytes reached the blastocyst stage (Day 8: 46.5% versus 33.4%). In Experiment 3, ovaries were separated according to age of heifer into three groups: (1) 12-18 months, (2) 19-24 months and (3) 25-30 months, and compared with cow oocytes. There was no significant difference in the blastocyst yield between the different age groups of heifers. Irrespective of heifer age, the blastocyst yield on Day 8 was significantly lower than that from cow oocytes (35.0, 35.2, 36.5 and 48.3%, respectively, P < 0.05). In Experiment 4, a significantly higher proportion (P < 0.001) of presumptive zygotes derived from abattoir-derived cow oocytes reached the blastocyst stage following culture in vivo in the ewe oviduct than those derived from heifer oocytes (Day 8: 53.1% versus 25.2% for cow and heifer oocytes, respectively). In conclusion, the origin of the oocyte has a significant impact on its subsequent developmental potential. These results would suggest that in an in vitro production system, cow oocytes should be preferentially used over those from heifers in order to maximize blastocyst development.

Anaerobic incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an unconventional front line tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. This disparity in PZA activity may reflect differences between the in vivo tissue environment and in vitro culture conditions. This study examined the effect of anaerobic conditions, which exist in granulomatous lesions in vivo, on PZA activity in vitro. Low oxygen enhanced the activity of PZA against Mycobacterium tuberculosis, with anaerobic conditions resulting in greater enhancement than microaerobic conditions. ATPase and respiratory chain enzyme inhibitors enhanced PZA activity under normal atmospheric conditions, but not under anaerobic conditions. Furthermore, the inhibitors did not enhance isoniazid or rifampicin activity. Nitrate as an alternative electron acceptor antagonized PZA activity under anaerobic conditions. These findings provide further support for a proposed mechanism of action of PZA in which the active form of PZA (pyrazinoic acid) depletes the membrane energy reserve. They also provide another explanation for the higher sterilizing activity of PZA within in vivo lesions with low oxygen than under in vitro drug susceptibility testing conditions with ambient oxygen.