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Cleansing and storage practices for reusable feeding tube stylets are varied and lack consensus guidelines. Almost 40% of critical care nurses do not cleanse reusable stylets. Our proof-of-concept study aimed to identify potential microbial contamination of stylets before and after cleansing with 70% isopropyl alcohol to establish practice standards.This prospective, exploratory pilot study sampled reusable feeding tube stylets using three different stylet sample sets. Set 1 included human participant stylets sampled for microbiome profile precleansing, and postcleansing and reinsertion into feeding tubes (n = 4). Sets 2 and 3 included stylets stored at the bedside. Set 2 included precleansed stylets for microbiome profiles (n = 5). Set 3 included precleansed and postcleansed stylets sampled for quantitative cultures (n = 5). Careful handling and storage protocols were used. Microbiome profiling used 16s ribosomal RNA gene amplicon sequencing.Bacterial species identified on stylets were primarily common microflora and opportunistic pathogens, including Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas fulva, Cutibacterium acnes, Prevotella melaninogenica, and Lactobacillus paracasei. Microbiological culturing of stylet samples (set 3) did not yield growth for 9/10 samples; Staphylococcus capitis was identified in one postcleansed sample. Mean bacterial species diversity (alpha diversity) decreased following alcohol cleansing (M = 2.54 pre, M = 1.5 post; P = 0.006).The abundance of several potentially opportunistic pathogens indicated plausible risk for gut contamination secondary to reinsertion of stylets into small-bore feeding tubes. Stylet cleansing with 70% isopropyl alcohol reduced bacterial burden on the stylets, although viability was unknown. Careful cleansing, handling, and storage protocols for reusable stylets are necessary to minimize contamination.

Background: Functional Abdominal Pain disorders (FAPDs) are a group of heterogeneous gastrointestinal disorders with unclear pathophysiology. In children, FAPDs are more common in the winter months than summer months. The possible influence of school stressors has been proposed. Previously, our group showed differences in bacterial relative abundances and alpha diversity in the gut microbiome and its relationship with stressors in a cross-sectional evaluation of children suffering from FAPDs compared to a healthy control group. We present longitudinal data to assess whether the gut microbiome changes over school terms in the control and FAPDs groups. Methods: The longitudinal study included children with FAPDs (n = 28) and healthy controls (n = 54). Gastrointestinal symptoms, as well as stool microbiome, were assessed in both groups. Stool samples were serially collected from all participants during both the school term and summer vacation. The stool samples were subjected to total genomic extraction, 16S rRNA amplicon sequencing, and bioinformatics analysis. The gut microbiome was compared at school and during vacation. Other metrics, alpha diversity, and beta diversity, were also compared between the two school terms in every group. Results: In the healthy group, there were differences in microbiome composition between school terms and summer vacation. Conversely, we found no differences in the FAPDs group between the two terms. The healthy control group revealed differences (p-value < 0.05) in 55 bacterial species between the school term and vacation. Several of the differentially abundant identified bacteria were involved in short-chain fatty acids production (SCFAs), inflammation reduction, and gut homeostasis. Alpha diversity metrics, such as the Shannon index, were different in the control group and remained unchanged in the FAPDs group. Conclusion: Although preliminary, our findings suggest that the gut microbiome is static in FAPDs. This compares with a more dynamic healthy gut microbiome. Further studies are warranted to corroborate this and understand the interplay between stress, symptoms, and a less diverse and static microbiome. Future studies will also account for different variables such as diet and other patient demographic criteria that were missing in the current study.

Background Patients experience endotracheal intubation in various settings with wide-ranging risks for postintubation complications such as aspiration and ventilator-associated conditions. Objectives To evaluate associations between intubation setting, presence of aspiration biomarkers, and clinical outcomes. Methods This study is a subanalysis of data from the NO-ASPIRATE single-blinded randomized clinical trial. Data were prospectively collected for 513 adult patients intubated within 24 hours of enrollment. Patients with documented aspiration events at intubation were excluded. In the NO-ASPIRATE trial, intervention patients received enhanced oropharyngeal suctioning every 4 hours and control patients received sham suctioning. Tracheal specimens for α-amylase and pepsin tests were collected upon enrollment. Primary outcomes were ventilator hours, lengths of stay, and rates of ventilator-associated conditions. Results Of the baseline tracheal specimens, 76.4% were positive for α-amylase and 33.1% were positive for pepsin. Proportions of positive tracheal α-amylase and pepsin tests did not differ significantly between intubation locations (study hospital, transfer from other hospital, or field intubation). No differences were found for ventilator hours or lengths of stay. Patients intubated at another hospital and transferred had significantly higher ventilator-associated condition rates than did those intubated at the study hospital (P = .02). Ventilator-associated condition rates did not differ significantly between patients intubated in the field and patients in other groups. Conclusions Higher ventilator-associated condition rates associated with interhospital transfer may be related to movement from bed, vehicle loading and unloading, and transport vehicle vibrations. Airway assessment and care may also be suboptimal in the transport environment.

Upon infection with SARS-CoV-2, the virus that causes COVID-19, most people will develop no or mild symptoms. However, a small percentage of the population will become severely ill, and some will succumb to death. The clinical severity of COVID-19 has a close connection to the dysregulation of the patient’s immune functions. We previously developed a simple, nanoparticle-enabled blood test that can determine the humoral immune status in animals. In this study, we applied this new test to analyze the immune function in relation to disease severity in COVID-19 patients. From the testing of 153 COVID-19 patient samples and 142 negative controls, we detected a drastic decrease of humoral immunity in COVID-19 patients who developed moderate to severe symptoms, but not in patients with no or mild symptoms. The new test may be potentially used to monitor the immunity change and predict the clinical risk of patients with COVID-19.

In this prospective longitudinal study, we enrolled 54 healthy pediatric controls and 28 functional abdominal pain disorders (FAPDs) pediatric patients (mean age was 11 ± 2.58 years old). Fecal samples and symptom questionnaires were obtained from all participants over the course of the year. Clinical data assessment showed that FAPDs patients were more symptomatic than the control group. Microbiome analysis revealed that Phylum Bacteroidetes was higher in FAPDs compared to the control group (p &lt, 0.05), while phylum Firmicutes was lower in FAPDs (p &lt, 0.05). In addition, Verrucomicrobiota was higher in the control group than the FAPDs (p &lt, 0.05). At the genus level the relative abundance of 72 bacterial taxa showed statistically significant differences between the two groups and at the school term levels. In the control group, Shannon diversity, Observed_species, and Simpson were higher than the FAPDs (p &lt, 0.05), and beta diversity showed differences between the two groups (PERMANOVA = 2.38, p = 0.002) as well. Using linear discriminant analysis effect size (LEfSe), Enterobacteriaceae family and Megaspherae showed increased abundances in vacation term (LDA score &gt, 2.0, LEfSe, p &lt, 0.05). In the FAPDs group, the severity of symptoms (T-scores) correlated with 11 different taxa bacterial relative abundances using Pearson′s correlation and linear regression analyses. Our data showed that gut microbiome is altered in FAPDs compared to the control. Differences in other metrics such as alpha- and beta diversity were also reported between the two groups. Correlation of the severity of the disease (T-scores) correlated with gut microbiome. Finally, our findings support the use of Faecalibacterium/Bacteroides ratio as a potential diagnostic biomarker for FAPDs.

OBJECTIVES There are no tests or patient factors to help predict the best treatment approach for a patient with eosinophilic esophagitis (EoE). The prevalence of PPI responsive EoE in children ranges from 30%-71% with multiple studies showing similar characteristics in responders and non-responders. Eosinophil-derived neurotoxin (EDN), an eosinophilic granule protein, measured in esophageal brushing has been shown to be a viable measure of disease activity in EoE. Our aim is to determine if EDN can help predict response to PPI in pediatric patients with EoE. METHODS We conducted a prospective cross-sectional study to compare EDN between PPI-responsive and PPI-non-responsive EoE subjects from 2018 through 2020. Enrolled patients with active EoE were treated with high-dose PPI and underwent repeat endoscopy to determine PPI-responsiveness. EDN was measured at baseline endoscopy, prior to any treatment, and at follow up endoscopy, after PPI therapy. Subjects were divided into PPI-responsive and nonresponsive groups. EDN, endoscopic reference score (EREFS), and peak eosinophilic count (PEC) were compared. RESULTS Fifteen out of the 36 enrolled subjects with EoE (age range 2-18 years, 73.3% male) were PPI-responsive and 21 (age range 2-19 years, 95.2% male) were PPI-nonresponsive. EDN concentration was significantly higher in the PPI-nonresponsive group than in the PPI-responsive group (219.1 ± 229 mcg/mL vs 75.7 ± 60 mcg/mL respectively, p = 0.036). There was no difference between the two groups in EREFS (p = 0.55) or PEC (p = 0.15). CONCLUSIONS EDN measured in esophageal epithelial samples obtained by brushing during endoscopy may predict PPI-responsiveness in children and young adults with EoE.

Upon infection with SARS-CoV-2, the virus that causes COVID-19, most people will develop no or mild symptoms. However, a small percentage of the population will become severely ill, and some will succumb to death. The clinical severity of COVID-19 has a close connection to the dysregulation of the patient’s immune functions. We previously developed a simple, nanoparticle-enabled blood test that can determine the humoral immune status in animals. In this study, we applied this new test to analyze the immune function in relation to disease severity in COVID-19 patients. From the testing of 153 COVID-19 patient samples and 142 negative controls, we detected a drastic decrease of humoral immunity in COVID-19 patients who developed moderate to severe symptoms, but not in patients with no or mild symptoms. The new test may be potentially used to monitor the immunity change and predict the clinical risk of patients with COVID-19.

Coronavirus disease 2019 (COVID-19) has brought enormous loss and interruption to human life and the global economy since the first outbreak reported in China between late 2019 to early 2020, and will likely remain a public health threat in the months and years to come. Upon infection with SARS-CoV-2, the virus that causes COVID-19, most people will develop no or mild symptoms, however, a small percentage of the population will become severely ill, require hospitalization, intensive care, and some succumb to death. The current knowledge of COVID-19 disease progression with worsening symptom complex implicates the critical importance of identifying patients with high clinical risk compared to those who would be at lower risk for disease control and patient management with better therapeutic output. Currently no clinical test is available that can predict risk factors and immune status change at different severity scales. The immune system plays a critical role in the defense against infectious diseases. Extensive research has found that COVID-19 patients with poor clinical outcomes differ significantly in their immune responses to the virus from those who exhibit milder symptoms. We previously developed a nanoparticle-enabled blood test that can detect the humoral immune status change in animals. In this study, we applied this new test to analyze the immune response in relation to disease severity in COVID-19 patients. From the testing of 153 COVID-19 patient samples and 142 negative controls, we detected statistically significant differences between COVID-19 patients with no or mild symptoms from those who developed moderate to severe symptoms. Mechanistic study suggests that these differences are associated with type 1 versus type 2 immune responses. We conclude that this new rapid test could potentially become a valuable clinical tool for COVID-19 patient risk stratification and management.

Objectives The gold standard diagnostic procedure for food protein-induced proctocolitis (FPIP) requires flexible sigmoidoscopy (FS). To date there is no validated, noninvasive test to confirm FPIP diagnosis. Eosinophil-derived neurotoxin (EDN), a product of eosinophil (EOS) degranulation, has been shown to correlate with eosinophil infiltration in other tissues. Our objective was to compare EDN concentrations in rectal epithelial samples from infants with FPIP with those from a control population. Methods Children who underwent routine FS at Arnold Palmer Hospital for Children were enrolled in an IRB-approved, prospective, open-label pilot study between July 2017 and May 2019. We obtained rectal epithelial samples via: rectal swab, cytology brushing through FS, and rectal biopsy through FS. We then measured EDN levels in the samples and compared levels found in infants with FPIP against levels found in the control group. FPIP was defined as more than 60 EOS per 10 high-power fields (HPF) in rectal epithelial tissue obtained via rectosigmoid biopsy. Results Twenty-four patients were enrolled. The control group (n = 13) included patients with normal histopathology (84% boys, mean age 19 months, SD 6 months) and the FPIP group (n = 11) included patients with FPIP confirmed via biopsy (45% boys, mean age 6.9 months, SD 9 months). EDN concentration was significantly higher in the FPIP group than in the control group, for 2 sampling methods: rectal biopsy (183.6 ± 114.6 vs 76.6 ± 71.0 μg/mL; P = 0.010) and rectal swab (66.2 ± 64.8 vs 20.4 ± 22.2 μg/mL; P = 0.025). Conclusions EDN concentrations measured from rectal swab and rectal biopsy samples is elevated and may be a useful tool to screen for FPIP in children.

OBJECTIVES The aim of the study was to determine prevalence and characterize sucrase-isomaltase (SI) gene variants of congenital sucrase-isomaltase deficiency in non-Hispanic white pediatric and young adult patients with functional gastrointestinal disorders (FGIDs), and abnormal sucrase activity on histologically normal duodenal biopsy. METHODS Clinical symptoms and disaccharidase activities data were collected for an abnormal (low) sucrase (≤25.8 U, n = 125) activity group, and 2 normal sucrase activity groups with moderate (≥25.8-≤55 U, n = 250) and high (>55 U, n = 250) sucrase activities. SI gene variants were detected by next-generation sequencing of DNA from formalin-fixed paraffin-embedded tissues of these patients. FGIDs symptoms based on Rome IV criteria and subsequent clinical management of abnormal sucrase activity cases with pathogenic SI gene variants were analyzed. RESULTS Thirteen SI gene variants were found to be significantly higher in abnormal sucrase cases with FGIDs symptoms (36/125, 29%; 71% did not have a pathogenic variant) compared to moderate normal (16/250, 6.4%, P

To evaluate a deep oropharyngeal suction intervention (NO-ASPIRATE) in intubated patients on microaspiration, ventilator-associated events and clinical outcomes.Prospective, two-group, single-blind, randomized clinical trial.The study was conducted between 2014 - 2017 in 513 participants enroled within 24 hr of intubation and randomized into NO-ASPIRATE or usual care groups. Standard oral care was provided to all participants every 4 hr and deep oropharyngeal suctioning was added to the NO-ASPIRATE group. Oral and tracheal specimens were obtained to quantify α-amylase as an aspiration biomarker.Data were analysed for 410 study completers enrolled at least 36 hr: NO-ASPIRATE (N = 206) and usual care (N = 204). Percent of tracheal specimens positive for α-amylase, mean tracheal α-amylase levels over time and ventilator-associated events were not different between groups. The NO-ASPIRATE group had a shorter hospital length of stay and a subgroup with moderate aspiration at baseline had significantly lower α-amylase levels across time.Hospital length of stay was shorter in the NO-ASPIRATE group and a subgroup of intervention participants had lower α-amylase across time. Delivery of standardized oral care to all participants may have been an intervention itself and possibly associated with the lack of significant findings for most outcomes.This trial compared usual care to oral care with a deep suctioning intervention on microaspiration and ventilator-associated events, as this has not been systematically studied. Further research on the usefulness of α-amylase as an aspiration biomarker and the role of oral suctioning, especially for certain populations, is indicated.ClinicalTrials.gov: NCT02284178.目的: 评估插管患者的深口咽吸干预(非吸入)对微量误吸、呼吸机事故以及临床结果的影响。 设计: 前瞻性、两组、单盲、随机临床试验。 方法: 这项研究在2014年至2017年期间进行,513名实验对象在插管24小时内进行了登记,并被随机分为非吸入组或常规护理组。 每4个小时为所有实验对象提供一次标准的口腔护理,并将深口咽吸加入非吸入组。用口腔和气管标本量化了α-淀粉酶作为误吸生物标本。 结果: 对410名至少登记超过36小时的实验对象的数据进行了分析:未抽吸(N = 206)和常规护理(N = 204)。气管标本阳性α淀粉酶的比例即随着时间推移的气管α-淀粉酶水平与呼吸机相关事故两组间并无差别。非吸入组的住院时间更短,且基线误吸更少的子群随着时间推移明显降低了α-淀粉酶水平。 结论: 非吸入组的住院时间更短,随着时间推移干预子群的实验对象α-淀粉酶水平明显降低。向所有参与者提供标准化口腔护理本身可能就是一种干预并可能与大部分结果缺乏重大发现有关。 影响: 由于尚未得到系统研究,本次试验就微量误吸的深咽干预和呼吸机相关事故将常规护理和口腔护理进行了对比。需要对α-淀粉酶作为误吸生物标本的有效性以及口腔吸入的作用,尤其是对特定群体而言的作用,进行进一步研究。 试验注册编号: 临床试验。政府:NCT00654321。.

Patients with eosinophilic esophagitis (EoE) require frequent evaluation of mucosal inflammation via endoscopy. Instead of endoscopy, mucosal evaluation in adults with esophageal cancer and candidiasis is achieved using a cytology brush inserted through a nasogastric tube (NGT). We conducted a prospective cross-sectional study in children and young adults scheduled for routine esophagogastroduodenoscopy (EGD) where in Phase 1, we performed esophageal brushing through the endoscope under direct visualization and in Phase 2, we inserted the brush through a Cortrak® NGT prior to endoscopy. Eosinophil-derived neurotoxin (EDN) measured by ELISA in the samples extracted from brushes was validated as the sensitive biomarker. We collected 209 esophageal brushing samples from 94 patients and we found that EDN in brushing samples collected via EGD or NGT was significantly higher in patients having active EoE (n = 81, mean EDN 381 mcg/mL) compared with patients having gastroesophageal reflux disease (n = 31, mean EDN 1.9 mcg/mL, P = 0.003), EoE in remission (n = 47, mean EDN 3.7 mcg/mL, P = 0.003), or no disease (n = 50, mean EDN 1.1 mcg/mL, P = 0.003). EDN at a concentration of ≥10 mcg/mL of brushing sample was found to accurately detect active EoE. NGT brushing did not cause any significant adverse effects. We concluded that blind esophageal brushing using an NGT is a fast, less invasive, safe, and well-tolerated technique compared with EGD to detect and monitor EoE inflammation using EDN as the sensitive biomarker.

Tuberculosis (TB) is responsible for death of nearly two million people in the world annually. Upon infection, Mycobacterium tuberculosis (Mtb) causes formation of granuloma where the pathogen goes into dormant state and can live for decades before resuscitation to develop active disease when the immune system of the host is weakened and/or suppressed. In an attempt to better understand host-pathogen interactions, several groups have been developing in vitro models of human tuberculosis granuloma. However, to date, an in vitro granuloma model in which Mtb goes into dormancy and can subsequently resuscitate under conditions that mimic weakening of the immune system has not been reported. We describe the development of a biomimetic in vitro model of human tuberculosis granuloma using human primary leukocytes, in which the Mtb exhibited characteristics of dormant mycobacteria as demonstrated by (1) loss of acid-fastness, (2) accumulation of lipid bodies (3) development of rifampicin-tolerance and (4) gene expression changes. Further, when these micro granulomas were treated with immunosuppressant anti-tumor necrosis factor-alpha monoclonal antibodies (anti-TNFα mAbs), resuscitation of Mtb was observed as has been found in humans. In this human in vitro granuloma model triacylglycerol synthase 1deletion mutant (Δtgs1) with impaired ability to accumulate triacylglycerides (TG), but not the complemented mutant, could not go into dormancy. Deletion mutant of lipY, with compromised ability to mobilize the stored TG, but not the complemented mutant, was unable to come out of dormancy upon treatment with anti-TNFα mAbs. In conclusion, we have developed an in vitro human tuberculosis granuloma model that largely exhibits functional features of dormancy and resuscitation observed in human tuberculosis.

Mycobacterium tuberculosis (Mtb) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative Mtb fatty acyl-CoA reductase genes (fcr) expressed in E. coli display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated Mtb gene-knockout mutants for each reductase. Metabolic incorporation of( 14)C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δfcr1 mutant and partially in the Δfcr2 mutant. WE synthesis was inhibited in both Δfcr mutants. The Δfcr mutants exhibited faster growth rates, an increased uptake of (14)C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δfcr mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the fcr1 and fcr2 gene products are involved in WE synthesis under in vitro dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the Mtb reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of Mtb to currently used antibiotics thereby enhancing clearance of the pathogen from patients.

Background: Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen. Methodology/Principal Findings: We developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO2 (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells. Conclusions/Significance: The new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb .A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C26 was a major fatty acid in this TG, indicating that the TGS1 gene product uses C26 fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C26 : 0-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K m of 7.57 mM and V max of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 °C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.

Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis . We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase ( tgs ) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O 2 and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.

Five facultative sulfur chemolithotrophs were isolated from soils to study the diversity of sulfur lithotrophy. Phenotypic characteristics, including sulfur lithotrophic properties and chemotaxonomic features of the isolates, were similar to those of the members of the colorless sulfur bacteria. 16S rDNA sequence analyses rendered placing the isolates to three distinct phylogenetic clusters of alpha-proteobacteria. Three isolates, AS001, AS002, and KCT002, were identified as members of the genus Paracoccus. The strains AS001 and AS002, having identical 16S-rDNA sequence, showed significant 16S rDNA sequence similarity (99.1%) to Paracoccus versutus. The strain KCT002 showed highest (98%) 16S rDNA sequence similarity to P. alcaliphilus and 96% similarity to the pair AS001 and AS002. Isolate KCT001 appeared to be closely related to Pseudaminobacter salicylatoxidans, although sulfur lithotrophy of P. salicylotoxidans is not known. The other isolate, TCK, showed almost identical 16S rDNA (99.9%) sequence with two recently described unclassified chemolithoautotrophic arsenite oxidizing strains. Physiological and chemotaxonomic characteristics and phylogenetic analyses of the five new strains emphasize the need of polyphasic bacterial taxonomy of sulfur lithotrophs.

We have isolated and characterized a double-stranded DNA bacteriophage (TPC-1) of Bosea thiooxidans, a facultative sulfur chemolithotrophic bacterium. The name 'thiophage' is introduced for phage(s) infecting sulfur chemolithotrophic bacteria. Electron micrographs showed the phage particle with an icosahedral head and a very short wedge-like tail. TPC-1 is classified as the C1 morphotype of the Podoviridae family. Restriction map and terminal ends detection by end fill labeling of the TPC-1 genomic DNA showed that the genome is linear with 5' protruding cohesive termini. Contour length mapping of the DNA genome also revealed it to be a linear fragment with size ( approximately 44 kb) corresponding with the size estimated from restriction fragment analyses and proved the non-redundant nature of the linear genome topology. In colorless sulfur chemolithotrophic microorganisms, TPC-1 is the first report of a generalized transducing thiophage.

The acidophilic heterotroph, Acidiphilium symbioticum KM2, is highly resistant to several metals and harbors three plasmids of 3.8, 7.1, and 56 kb in size. The bacterium becomes extremely sensitive to metals when it is cured of its plasmids. A mini-plasmid library was constructed by ligating the plasmid DNA fragments generated by MboI partial digestion into the BamHI site of pBluescriptII KS+. The Lac(-)Amp(r) transformants of Escherichia coli DH5alpha, isolated after transformation with the library, were counter-selected on Cu(2+), Cd(2+), Ni(2+), and Zn(2+)-containing plates. Only Cd(2+)- and Zn(2+)-resistant colonies were developed, and, after screening, four types of recombinant plasmids designated as pNM201 (7.2 kb), pNM206 (3.4 kb), pNM208 (4.5 kb), and pNM215 (4.9 kb) were obtained. The DNA insert in pNM206 hybridized strongly with the 3.8-kb plasmid and weakly with the 7.1-kb plasmid of Acidiphilium symbioticum KM2. The DNA insert in pNM215 hybridized only with the 7.1-kb plasmid. These results strongly suggested that resistance to cadmium and zinc in A. symbioticum KM2 is mediated by these plasmids. The smallest insert of 422 bp in pNM206 conferring metal resistance in E. coli has no sequence similarity with the reported metal-resistant genes. All the putative ORFs are significantly rich (up to 37%) in basic amino acids, mainly arginine.

Chemolithotrophy, discovered by Sergei N. Winogradsky in 1887, originated from the observation of sulfur droplets in the filaments of Beggiatoa growing in the presence of hydrogen sulfide (18, 48). Among the few inorganic substrates used by bacteria in the chemolithotrophic process, a comparatively larger variety of reduced inorganic sulfur species support lithotrophic growth of a large number of phylogenetically diverse groups of bacteria and archaea (14). However, chemolithotrophic growth on sulfur was thought to be a conserved genetic trait and was used as the key taxonomic characteristic for the genus Thiobacillus (16, 17). As a result, a variety of physiologically and genetically unrelated eubacteria were classified as Thiobacillus species (14). Phylogenetic analyses based on 5S or 16S ribosomal DNA sequences had shown that the sulfur lithotrophs including the Thiobacillus species belong to the α, β, and γ subclasses of Proteobacteria (21, 22, 32). The knowledge of the biochemistry and molecular biology of sulfur lithotrophy in microbes must be considered important in understanding the genetic relatedness within the members of Thiobacillus and the relationship of this genus with other sulfur lithotrophs. Extensive biochemical investigations of the oxidative dissimilatory metabolism of sulfur compounds were reported previously (6, 24, 25, 29, 34, 35, 42). Even so, the mechanism involving the specific enzymes, proteins, or accessory factors is rather poorly understood. The element sulfur enjoys a wide range of oxidation states, −2 to +6, and sulfur lithotrophs are not necessarily similar in using specific sulfur species in their lithotrophic processes. Consequently, distinct biochemical pathways have been proposed for different sulfur lithotrophs (14, 16, 36). Thiosulfate is the common oxidizable substrate that is most suitable for the investigations of sulfur lithotrophic processes. For Paracoccus versutus (formerly Thiobacillus versutus), a thiosulfate-oxidizing periplasmic multienzyme system comprising enzyme A, enzyme B, and multiheme cytochromes was characterized (24, 25). The proposed mechanism is designated the Paracoccus sulfur oxidation (PSO) pathway (18). The function of enzyme A or enzyme B was not demonstrated. In Paracoccus denitrificans, a DNA region essential for sulfur oxidation (Sox) was identified (31). The sequence analysis revealed a partial open reading frame (ORF), soxA, and five additional ORFs (soxBCDEF) downstream of soxA (45, 46). soxB seems essential in sulfur lithotrophy, and the product SoxB appears similar to enzyme B of P. versutus (45). soxC encodes a sulfite dehydrogenase, the requirement for which in thiosulfate-dependent lithotrophic growth in P. denitrificans was experimentally verified (46). The products of soxD and soxE were suggested to be c-type cytochromes. The partial sequence available for soxF exhibits significant similarity with the flavoprotein of Chromatium vinosum, a thiosulfate-oxidizing phototrophic chemolithoautotroph (7, 14). The thiobacilli and other sulfur lithotrophs, phylogenetically close to P. denitrificans or P. versutus (α-3 subgroup [22]), may have acquired this PSO pathway (14, 18, 31) in the sulfur lithotrophic process. Neither P. versutus nor P. denitrificans uses tetrathionate, an oxidizable substrate commonly used to support lithotrophic growth of many species of Thiobacillus (14, 16). An alternative mechanism of thiosulfate oxidation via the formation of tetrathionate, coupled with the electron transport at the level of cytochrome b instead of cytochrome c, was proposed for obligately lithoautotrophic, moderately thermophilic Thiobacillus tepidarius (26, 47). The tetrathionate-utilizing sulfur lithotrophs such as Thiobacillus thiooxidans or Thiobacillus ferrooxidans, belonging to the β subclass of Proteobacteria and closely related to T. tepidarius (21, 22), may follow this tetrathionate intermediate pathway (16, 18). Further, cleavage of thiosulfate to sulfite and sulfur by rhodanese was demonstrated to be the primary reaction in the process of lithotrophy of thiosulfate by Thiobacillus novellus (6, 14, 16, 34). However, this process of sulfur lithotrophy, apparently distinct from the PSO or tetrathionate intermediate pathway, is yet to be investigated for other sulfur lithotrophs. Several facultative sulfur lithotrophs, KCT001, KCT002, AS001, and AS002, have been recently isolated and characterized by this laboratory. KCT002, AS001, and AS002 are classified as strains of Paracoccus, and the strain KCT001 is phylogenetically distinct from known strains of Thiobacillus, other sulfur lithotrophs, and other bacterial species of the α subclass of Proteobacteria (unpublished observation; C. Deb, E. Stackebrandt, A. Saha, and P. Roy, unpublished data). In the present study, transposon Tn5-mob insertional mutagenesis in KCT001 was performed to generate mutants impaired in the oxidation of sulfur compounds. A soxA gene was identified from transposon-adjacent genomic DNA of a thiosulfate oxidation-negative (Sox−) mutant. Two primers designed from this soxA gene and one from Tn5 were used in a PCR-based method to walk down the genome of transposon insertion Sox− mutants. We have shown that six independent insertion mutations were mapped within a DNA region of 4.4 kb in the genome of KCT001.

Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains of Thiobacillus ferrooxidans. The 1.4-kb fragment hybridized with IS T2, an insertion sequence of T. ferrooxidans. The 1.2-kb fragment was cloned and sequenced. The sequence (IS T445 ), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively. It displays 54.4% identity in 967 nucleotides of overlap with IS AE1 of Alcaligenes eutrophus. The IS T445 contains three open reading frames which have codon usage almost similar to 56 different coding genes of T. ferrooxidans. In Southern blots of restricted genomic DNAs probed with IS T445, each of the several strains of T. ferrooxidans gives a distinctive fingerprint. IS T445 is present in the range of 10–20 copies per genome in the four strains studied.

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism., Author Summary Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Cure and possible eradication of tuberculosis are limited by the lack of availability of any drug that can kill dormant Mtb. Understanding of the processes critical for dormancy and a reliable dormancy model suitable for high throughput screening of chemicals will help to discover drugs that can kill dormant Mtb. Storage of lipids for utilization as energy source is critically needed for dormancy. In the human lung, Mtb-infected macrophages are sequestered inside the hypoxic environments of the physical enclosure called granuloma in which Mtb becomes dormant. None of the currently used cell culture models of Mtb infection mimic this situation. We developed a model that mimics the environment inside the human granuloma by incubating Mtb-infected macrophages under hypoxia. We found that, under these conditions, macrophages accumulate lipid droplets and Mtb within these macrophages acquire a dormancy phenotype. We report how the pathogen inside the macrophages utilizes the host lipids to store lipids within the pathogen and acquire the hallmark traits of dormant Mtb. Thus, our novel model of Mtb dormancy may enable better understanding of the metabolic processes vital for the dormant pathogen and help to discover drugs that can kill latent pathogens.