HemU and TonB1 contribute to hemin acquisition in Stenotrophomonas maltophilia.

Introduction: The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of Stenotrophomonas maltophilia is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake. Methods: Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of S. maltophilia. Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction. Results: Smlt3896-hemU-exbB2-exbD2-tonB2 and tonB1-exbB1-exbD1a-exbD1b operons were selected as candidates for hemin acquisition. Compared with the parental strain, hemU and tonB1 mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the Smlt3896 and tonB2 mutants was comparable to that of the parental strain. HemA expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated hemA expression. Like hemA, hemU was repressed by Fur in iron-replete conditions; however, hemU was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike hemA and hemU, the TonB1-exbB1-exbD1a-exbD1b operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression. Conclusion: HemA, HemU, and TonB1 contribute to hemin acquisition in S. maltophilia. Fur represses the expression of hemA and hemU in iron-replete conditions. HemA expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. HemU expression is regulated by low iron and hemin levels in a hemP-dependent manner.

Risk factors for prolonged mechanical ventilation in critically ill patients with influenza-related acute respiratory distress syndrome.

BACKGROUND: Patients with influenza-related acute respiratory distress syndrome (ARDS) are critically ill and require mechanical ventilation (MV) support. Prolonged mechanical ventilation (PMV) is often seen in these cases and the optimal management strategy is not established. This study aimed to investigate risk factors for PMV and factors related to weaning failure in these patients. METHODS: This retrospective cohort study was conducted by eight medical centers in Taiwan. All patients in the intensive care unit with virology-proven influenza-related ARDS requiring invasive MV from January 1 to March 31, 2016, were included. Demographic data, critical illness data and clinical outcomes were collected and analyzed. PMV is defined as mechanical ventilation use for more than 21 days. RESULTS: There were 263 patients with influenza-related ARDS requiring invasive MV enrolled during the study period. Seventy-eight patients had PMV. The final weaning rate was 68.8% during 60 days of observation. The mortality rate in PMV group was 39.7%. Risk factors for PMV were body mass index (BMI) > 25 (kg/m2) [odds ratio (OR) 2.087; 95% confidence interval (CI) 1.006-4.329], extracorporeal membrane oxygenation (ECMO) use (OR 6.181; 95% CI 2.338-16.336), combined bacterial pneumonia (OR 4.115; 95% CI 2.002-8.456) and neuromuscular blockade use over 48 h (OR 2.8; 95% CI 1.334-5.879). In addition, risk factors for weaning failure in PMV patients were ECMO (OR 5.05; 95% CI 1.75-14.58) use and bacteremia (OR 3.91; 95% CI 1.20-12.69). CONCLUSIONS: Patients with influenza-related ARDS and PMV have a high mortality rate. Risk factors for PMV include BMI > 25, ECMO use, combined bacterial pneumonia and neuromuscular blockade use over 48 h. In addition, ECMO use and bacteremia predict unsuccessful weaning in PMV patients.

Emergence of OXA-48-producing hypervirulent Klebsiella pneumoniae strains in Taiwan.

The OXA-48-producing hypervirulent Klebsiella pneumoniae (hvKP) strains were rarely reported. In this study, we characterized three carbapenem-resistant hvKP strains (KP2185, NCRE61, and KP2683-1) isolated from renal abscess, scrotal abscess, and blood samples in a Taiwan hospital. The three strains belonged to two different clones: ST23 K1 (KP2683-1) and ST11 KL64 (KP2185 and NCRE61). KP2683-1 exhibited the highest virulence in an in vivo model. Whole-genome sequencing analysis showed that KP2185 and NCRE61 acquired IncFIB type plasmids containing a set of virulence genes (iroBCDN, iucABCD, rmpA, rmpA2, and iutA), while KP2683-1 acquired an IncL type plasmid harboring blaOXA-48.

Modulatory role of SmeQ in SmeYZ efflux pump-involved functions in Stenotrophomonas maltophilia.

BACKGROUND: SmeYZ is a constitutively expressed efflux pump in Stenotrophomonas maltophilia. Previous studies demonstrated that: (i) smeYZ inactivation causes compromised swimming, oxidative stress tolerance and aminoglycoside resistance; and (ii) the ΔsmeYZ-mediated pleiotropic defects, except aminoglycoside susceptibility, result from up-regulation of entSCEBB'FA and sbiAB operons, and decreased intracellular iron level. OBJECTIVES: To elucidate the modulatory role of SmeQ, a novel cytoplasmic protein, in ΔsmeYZ-mediated pleiotropic defects. METHODS: The presence of operons was verified using RT-PCR. The role of SmeQ in ΔsmeYZ-mediated pleiotropic defects was assessed using in-frame deletion mutants and functional assays. A bacterial adenylate cyclase two-hybrid assay was used to investigate the protein-protein interactions. Gene expression was quantified using quantitative RT-PCR (RT-qPCR). RESULTS: SmeYZ and the downstream smeQ formed an operon. SmeQ inactivation in the WT KJ decreased aminoglycoside resistance but did not affect swimming and tolerance to oxidative stress or iron depletion. However, smeQ inactivation in the smeYZ mutant rescued the ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. In the WT KJ, SmeQ positively modulated SmeYZ pump function by transcriptionally up-regulating the smeYZQ operon. Nevertheless, in the smeYZ mutant, SmeQ exerted its modulatory role by up-regulating entSCEBB'FA and sbiAB operons, decreasing intracellular iron levels, and causing ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. CONCLUSIONS: SmeQ is the first small protein identified to be involved in efflux pump function in S. maltophilia. It exerts modulatory effect by transcriptionally altering the expression of target genes, which are the smeYZQ operon in the WT KJ, and smeYZQ, entSCEBB'FA and sbiAB operons in smeYZ mutants.

Assuring safety of fecal microbiota transplantation in the COVID-19 era: A single-center experience.

Background and Aim: Fecal microbiota transplantation (FMT) is used to treat recurrent or refractory Clostridioides difficile infection (CDI). In the past, screening of fecal donors required surveillance of personal behavior, medical history, and diseases that could be transmitted by the blood or fecal-oral route. In addition, the exclusion of multidrug-resistant organisms (MDROs) has been recommended since 2018. This task has become more complicated in the era of the coronavirus disease-2019 (COVID-19) pandemic. To prevent fecal transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is crucial to commence screening for SARS-CoV-2, alongside other traditional tests. Our aim was to investigate whether hidden carriers of SARS-CoV-2 were enrolled for stool donation, and the status of the presence or incidence of MDRO during fecal donation in Taiwan. Methods: Fecal products collected from March 2019 to December 2022 were tested for MDRO and nucleic acid amplification tests for SARS-CoV-2 using the pooling method. The period of fecal product collection crossed the time before and during the COVID pandemic in Taiwan. Results: A total of 151 fecal samples were collected. The fecal products were tested using polymerase chain reaction (PCR) to detect SARS-CoV-2. The results were negative for all stocks. This was similar to the results of MDRO testing. The safety of FMT products has been guaranteed during the pandemic. Conclusion: Our FMT center produced MDRO-free and COVID-19-free products before and during the COVID-19 outbreak in Taiwan. Our protocol was effective for ensuring the safety of FMT products.

Interaction of multidrug-resistant hypervirulent Klebsiella pneumoniae with components of human innate host defense.

IMPORTANCE: Klebsiella pneumoniae strains with a combination of multidrug resistance and hypervirulence genotypes (MDR hvKp) have emerged as a cause of human infections. The ability of these microbes to avoid killing by the innate immune system remains to be tested fully. To that end, we compared the ability of a global collection of hvKp and MDR hvKp clinical isolates to survive in human blood and resist phagocytic killing by human neutrophils. The two MDR hvKp clinical isolates tested (ST11 and ST147) were killed in human blood and by human neutrophils in vitro, whereas phagocytic killing of hvKp clinical isolates (ST23 and ST86) required specific antisera. Although the data were varied and often isolate specific, they are an important first step toward gaining an enhanced understanding of host defense against MDR hvKp.

Prognostic factors for poor outcomes in patients with severe COVID-19 treated with remdesivir plus dexamethasone in Taiwan.

BACKGROUND: Coronavirus disease-2019 (COVID-19) remains a global public health concern, and remdesivir plus dexamethasone combination therapy is suggested for patients with severe disease. However, the factors associated with poor outcomes in these patients remain unclear. We identified the factors associated with poor outcomes in Taiwanese patients with severe COVID-19 treated with remdesivir plus dexamethasone. METHODS: Adults with severe COVID-19 (oxygen saturation <94% on room air or requiring supplemental oxygen) treated with remdesivir and dexamethasone were identified between 1 May and 31 July 2021. The main outcomes were 14-day non-recovery, 28-day mortality, and progression to respiratory failure requiring invasive mechanical ventilation or death in initially non-ventilated patients. The prognostic factors associated with poor outcomes were analyzed by multivariate logistic regression and Cox regression. RESULTS: Of the 110 patients treated with remdesivir and dexamethasone, 57 (51.8%) recovered within 14 days and 6 (5.5%) died within 28 days. Of the 89 initially non-ventilated patients, 12 (13.5%) progressed to respiratory failure or death. Charlson Comorbidity Index, SOFA score, and admission to remdesivir treatment interval were associated with 14-day non-recovery. C-reactive protein level was associated with 28-day mortality. Pneumonia Severity Index and admission to remdesivir treatment interval were associated with progression to respiratory failure requiring invasive mechanical ventilation or death in initially non-ventilated patients. CONCLUSION: High disease severity on admission and delayed initiation of remdesivir therapy were associated with poor outcomes in COVID-19 patients treated with remdesivir and dexamethasone.

High carriage rate of extended-spectrum ß-lactamase Enterobacterales and diarrheagenic Escherichia coli in healthy donor screening for fecal microbiota transplantation.

The safety of fecal microbiota transplantation (FMT) has been highlighted by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli bacteremia transmitted from donors and acquisition of diarrheagenic E. coli (Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC)) via FMT. The use of donor screening criteria to lower the risk of pathogen transmission via FMT is essential. This study aimed to demonstrate the outcomes of our strict donor screening program. This study was conducted at our FMT center between January 2019 and June 2022. Our donor screening program included an initial questionnaire and subsequent blood and stool testing. We further used selective culture for third-generation cephalosporin-resistant (3GCR) Enterobacterales and multiplex PCR to detect diarrheagenic E. coli in stools. The resistance mechanisms and sequence type of 3GCR Enterobacterales were determined. A total of 742 individuals were assessed, and 583 participants (78.6%) were excluded after questionnaire. Of the remaining 159 participants undergoing stool and blood tests, 37 participants were finally qualified (5.0%, 37/742). A high fecal carriage rate of ESBL-producing Enterobacterales (35.2%, 56/159), including E. coli (n=53) and Klebsiella pneumoniae (n=5), and diarrheagenic E. coli (31.4%, 50/159), including EPEC (n=41), enteroaggregative E. coli (n=11), enterotoxigenic E. coli (n=4), and STEC (n=1), was noted. CTX-M-79 and CTX-M-15 were dominant in E. coli and K. pneumoniae, respectively. The sequence types of the ESBL-producing strains were diverse. The screening for 3GCR Enterobacterales and diarrheagenic E. coli in stool is necessary. Our findings also support the effectiveness of multiplex PCR panels in FMT donor screening programs.

Implication of the σE Regulon Members OmpO and σN in the ΔompA299-356-Mediated Decrease of Oxidative Stress Tolerance in Stenotrophomonas maltophilia.

Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA299-356, an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by ΔompA299-356. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA299-356 mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJΔOmpA299-356. KJΔOmpA299-356 complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJΔOmpA299-356. Next, the involvement of the three σ factors in the ΔompA299-356-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the ΔompA299-356-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σE decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the ΔompA299-356-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation. IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal ß-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σP and σE expression levels. The outcomes of σP and σE activation are different and are linked to ß-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.

Mortality and ventilator dependence in critically ill patients with ventilator-associated pneumonia caused by carbapenem-resistant Acinetobacter baumannii.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a key pathogen associated with ventilator-associated pneumonia (VAP). Research on treatment outcomes, especially ventilator dependence, in patients with VAP caused by CRAB remains limited. METHODS: This retrospective multicenter study included ICU-admitted patients with VAP caused by CRAB. The original cohort was included as the mortality evaluation cohort. The ventilator dependence evaluation cohort included cases that survived more than 21 days after VAP and without prolonged ventilation before VAP onset. The mortality rate, ventilator dependence rate, clinical factors associated with treatment outcomes, and treatment outcome differences with various VAP onset times were investigated. RESULTS: In total, 401 patients with VAP caused by CRAB were analyzed. The 21-day all-cause mortality rate was 25.2%, and the 21-day ventilator dependence rate was 48.8%. Clinical factors associated with 21-day mortality included lower body mass index, higher sequential organ failure assessment score, vasopressors usage, CRAB persistence, and VAP onset time > seven days. Clinical factors associated with 21-day ventilator dependence included older age, vasopressors usage, and VAP onset time > seven days. CONCLUSIONS: ICU-admitted patients with CRAB-related VAP had high mortality and ventilator dependence rates. Older age, vasopressor usage, and longer VAP onset time were independent factors associated with ventilator dependence.

Association between microcirculation in spontaneous breathing trial and extubation success.

PURPOSE: This study assessed the association between changes in sublingual microcirculation after a spontaneous breathing trial (SBT) and successful extubation. MATERIALS AND METHODS: Sublingual microcirculation was assessed using an incident dark-field video microscope before and after each SBT and before extubation. Microcirculatory parameters before the SBT, at the end of the SBT, and before extubation were compared between the successful and failed extubation groups. RESULTS: Forty-seven patients were enrolled and analysed in this study (34 patients in the successful extubation group and 13 patients in the failed extubation group). At the end of the SBT, the weaning parameters did not differ between the two groups. However, the total small vessel density (21.2 [20.4-23.7] versus 24.9 [22.6-26.5] mm/mm2), perfused small vessel density (20.6 [18.5-21.8] versus 23.1 [20.9-25] mm/mm2), proportion of perfused small vessels (91 [87-96] versus 95 [93-98] %), and microvascular flow index (2.8 [2.7-2.9] versus 2.9 [2.9-3]) were significantly lower in the failed extubation group than in the successful extubation group. The weaning and microcirculatory parameters did not differ significantly between the two groups before the SBT. CONCLUSIONS: More patients are required to investigate the difference between baseline microcirculation before a successful SBT and the change in microcirculation at the end of the SBT between the successful and failed extubation groups. Better sublingual microcirculatory parameters at the end of SBT and before extubation are associated with successful extubation.

Safety and immunogenicity of homologous versus heterologous booster dose with AZD1222, mRNA-1273, or MVC-COV1901 SARS-CoV-2 vaccines in adults: An observer-blinded, multi-center, phase 2 randomized trial.

OBJECTIVES: To report the safety and immunogenicity profile of a protein subunit vaccine (MVC-COV1901) compared to AZD1222 and mRNA-1273 when given as a third (booster) dose to individuals who have completed different primary vaccine regimens. METHODS: Individuals were classified according to their primary vaccine regimens, including two-dose MVC-COV1901, AZD1222, or mRNA-1273. A third dose of either half-dose MVC-COV1901, full-dose MVC-COV1901, standard-dose AZD1222, half-dose mRNA-1273 was administered in a 1:1:1:1 treatment ratio to individuals with an interval range of 84-365 days after the second dose. Endpoints included safety, humoral immunogenicity, and cell-mediated immune response on trial days 15 and 29. Exploratory endpoint included testing against variants of concern (Omicron). RESULTS: Overall, 803 participants were randomized and boosted - 201 received half-dose MVC-COV1901, 196 received full-dose MVC-COV1901, 203 received AZD1222, and 203 received half-dose mRNA-1273. Reactogenicity was mild to moderate, and less in the MVC-COV1901 booster group. Heterologous boosting provided the best immunogenic response. Boosting with mRNA-1273 in MVC-COV1901 primed individuals induced the highest antibody titers, even against Omicron, and cell-mediated immune response. CONCLUSIONS: Overall, MVC-COV1901 as a booster showed the best safety profiles. MVC-COV1901 as a primary series, with either homologous or heterologous booster, elicited the highest immunogenic response. CLINICALTRIALS: gov registration NCT05197153.

Tenofovir use is associated with a decreased risk of hepatocellular carcinoma among men with HIV irrespective of coinfection status.

Background & Aims: Tenofovir is recommended as part of the first-line antiretroviral therapy (ART) to treat people living with HIV (PLWH) with HBV coinfection. However, the effects of tenofovir-containing ART on hepatocellular carcinoma (HCC) risk among PLWH with/without chronic hepatitis virus infections remain unclear. Methods: This study included 23,838 PLWH. All of them were males aged ≥20 years and followed prospectively during 2000-2017. Four major nationwide registries - the Human Immunodeficiency Virus surveillance database, Taiwan Cancer Registry, Death Certification System, and National Health Insurance Database - were applied to define ART and comorbidities and ascertain newly diagnosed HCC. Tenofovir-containing ART was identified through prescription records. Cox proportional hazards models were used to determine the association of tenofovir use with HCC incidence. Results: HCC incidence was lower among ever users of tenofovir than among never users (24.2 and 85.7 per 100,000 person-years, respectively). Ever users had significantly reduced HCC risk (adjusted hazard ratio 0.20, 95% CI 0.13-0.31). The effect of tenofovir use on reduced risk for HCC consistently favored never users across many prespecified subgroups, including HBV or HCV coinfection (p <0.05). The findings were consistent in subgroups of PLWH diagnosed with HIV before tenofovir's approval and in those born before the nationwide roll-out of neonatal HBV vaccination. Conclusions: Our findings underscore the need for randomized controlled trials of tenofovir in combination with long-acting injectable ART regimens to assess its safety and efficacy in PLWH, particularly in those with HBV or HCV coinfection. Impact and implications: Tenofovir's effect on the risk of hepatocellular carcinoma (HCC) among people living with HIV with hepatitis B or C coinfection remains under investigated. This nationwide prospective cohort study, comprising 23,838 men living with HIV, showed that tenofovir-containing antiretroviral therapy was associated with reduced risk of HCC (adjusted relative risk: 0.20, 95% CI 0.13-0.31), which was consistently observed across many prespecified subgroups. The effect of tenofovir use on HCC risk should be further investigated in PLWH, particularly following the development of long-acting injectable ART regimens.

Microbiota analysis in the hemodialysis population - Focusing on Enterobacteriaceae.

BACKGROUND: Infection is a recognized risk factor for mortality among hemodialysis (HD) population, including infection caused by Enterobacteriaceae. We aimed to investigate Enterobacteriaceae in gut microbiota among HD patients and to analyze associations between microbiota and clinical parameters. METHODS: This prospective study of microbiota analysis in HD patients was conducted in April-May 2019. A control group without recent antibiotic use or hospitalization was used for comparison. Stool samples underwent 16S rRNA sequencing, using Greengenes 16S rRNA database for microbiota analysis. RESULTS: Among 96 hemodialysis (HD) patients, mean age was 61.9 ± 0.8 years and mean duration of HD was 6.5 ± 0.7 years. No significant differences were found in alpha diversity between HD and control groups (HD group 949.5, controls 898; p = 0.16) although significant between-group differences were found in beta diversity (p < 0.001). At phylum level, HD group had a higher abundance of Firmicutes and Proteobacteria, but lower abundance of Bacteriodetes. At genus level, Escherichia-Shigella complex increased among HD patients who had hospitalization with 1 year (median 0.024 vs 0.004, p = 0.054) and Klebsiella was associated with emergency room visit within 1 year among HD patients (p = 0.002). CONCLUSIONS: Alpha diversity in HD patients is not lower than that in healthy controls but significant between-group differences are found in microbiota composition according to beta diversity, due to decreased Bacteriodetes and increased Firmicutes and Proteobacteria. Deeper microbiota analyses for Enterobacteriaceae are necessary. Whether change in dietary components can help to decrease mortality among dialysis population warrants further research.

COVID-19 associated mold infections: Review of COVID-19 associated pulmonary aspergillosis and mucormycosis.

COVID-19-associated mold infection (CAMI) is defined as development of mold infections in COVID-19 patients. Co-pathogenesis of viral and fungal infections include the disruption of tissue barrier following SARS CoV-2 infection with the damage in the alveolar space, respiratory epithelium and endothelium injury and overwhelming inflammation and immune dysregulation during severe COVID-19. Other predisposing risk factors permissive to fungal infections during COVID-19 include the administration of immune modulators such as corticosteroids and IL-6 antagonist. COVID-19-associated pulmonary aspergillosis (CAPA) and COVID-19-associated mucormycosis (CAM) is increasingly reported during the COVID-19 pandemic. CAPA usually developed within the first month of COVID infection, and CAM frequently arose 10-15 days post diagnosis of COVID-19. Diagnosis is challenging and often indistinguishable during the cytokine storm in COVID-19, and several diagnostic criteria have been proposed. Development of CAPA and CAM is associated with a high mortality despiteappropriate anti-mold therapy. Both isavuconazole and amphotericin B can be used for treatment of CAPA and CAM; voriconazole is the primary agent for CAPA and posaconazole is an alternative for CAM. Aggressive surgery is recommended for CAM to improve patient survival. A high index of suspicion and timely and appropriate treatment is crucial to improve patient outcome.

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

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

(Pro)renin receptor inhibition attenuated liver steatosis, inflammation, and fibrosis in mice with steatohepatitis.

The (Pro)renin receptor (PRR) is reportedly involved in hepatic lipid metabolism and hepatocyte PRR knockdown protects mice against hepatosteatosis. However, the impact of PRR inhibition on liver inflammation and fibrosis in nonalcoholic steatohepatitis (NASH) remains unclear. Herein, C57BL/6 mice were fed a normal chow diet or fast food diet (FFD) for 24 weeks. Lentivirus-mediated PRR short hairpin RNA (shRNA) or handle region peptide (HRP), a PRR blocker, was administered for PRR inhibition. Mouse primary hepatocytes were cultured with palmitic acid, prorenin, siRNA-targeted PRR, and HRP. In FFD-fed mice, PRR inhibition via lentivirus-mediated PRR knockdown or HRP significantly attenuated liver steatosis, inflammation, and fibrosis. Mechanistically, PRR knockdown or HRP decreased hepatic acetyl-CoA carboxylase (ACC) abundance and upregulated peroxisome proliferator-activated receptor-alpha (PPARα). HRP treatment also decreased hepatic PRR expression. In addition, intrahepatic oxidative stress, apoptosis and inflammatory cell recruitment were ameliorated by PRR knockdown or HRP treatment, along with suppression of proinflammatory cytokine expression. PRR inhibition downregulated the hepatic expression of profibrotic factors, as well as TGF-ß1/SMAD3 pathway. In primary mouse hepatocytes, PRR knockdown with siRNA or HRP downregulated cellular ACC and increased PPARα expression. In conclusion, our findings revealed that PRR inhibition attenuated hepatic steatosis, inflammation, and fibrosis in mice with NASH. Accordingly, targeting PRR signaling may serve as a potential treatment for NASH.

Decolonization of carbapenem-resistant Klebsiella pneumoniae from the intestinal microbiota of model mice by phages targeting two surface structures.

Background: Klebsiella pneumoniae is a normal component of the human gastrointestinal tract microbiota. However, in some cases, it can cause disease. Over the past 20 years, the prevalence of antibiotic-resistant bacteria, such as carbapenem-resistant K. pneumoniae (CRKP), has been increasing. Materials and methods: We attempted to specifically eliminate CRKP from a mouse model with the human intestinal microbiota. To establish humanized microbiota-colonized mice, we administered K64 CRKP-containing human microbiota to germ-free mice by fecal microbiota transplantation. Then, we used two phages, one targeting the capsule (φK64-1) and one targeting O1 lipopolysaccharide (φKO1-1) of K64 K. pneumoniae, to eliminate CRKP. Results: In untreated control and φKO1-1-treated K64-colonized mice, no change in CRKP was observed, while in mice treated with φK64-1, a transient reduction was observed. In half of the mice treated with both φKO1-1 and φK64-1, CRKP was undetectable in feces by PCR and culture for 60 days. However, in the other 50% of the mice, K. pneumoniae was transiently reduced but recovered 35 days after treatment. Conclusion: Combination treatment with φK64-1 and φKO1-1 achieved long-term decolonization in 52.3% of mice carrying CRKP. Importantly, the composition of the intestinal microbiota was not altered after phage treatment. Therefore, this strategy may be useful not only for eradicating drug-resistant bacterial species from the intestinal microbiota but also for the treatment of other dysbiosis-associated diseases.

Clinical and Microbiological Characteristics of Bacteremic Pneumonia Caused by Klebsiella pneumoniae.

Klebsiella pneumoniae is a common pathogen of nosocomial pneumonia worldwide and community-acquired pneumonia (CAP) in Asia. Previous studies have shown that K. pneumoniae bacteremic CAP is associated with high mortality. We aimed to revisit K. pneumoniae bacteremic pneumonia in the current era and determine the risk factors associated with 28-day mortality. Between January 2014 and August 2020, adult patients with K. pneumoniae bacteremic pneumonia in a medical center in Taiwan were identified. Clinical and microbiological characteristics were compared between CAP and nosocomial pneumonia. Risk factors for 28-day mortality were analyzed using multivariate logistic regression. Among 150 patients with K. pneumoniae bacteremic pneumonia, 52 had CAP and 98 had nosocomial pneumonia. The 28-day mortality was 52% for all patients, 36.5% for CAP, and 60.2% for nosocomial pneumonia. Hypervirulent K. pneumoniae was more prevalent in CAP (61.5%) than in nosocomial pneumonia (16.3%). Carbapenem-resistant K. pneumoniae was more prevalent in nosocomial pneumonia (58.2%) than in CAP (5.8%). Nosocomial pneumonia, a higher Severe Organ Failure Assessment score, and not receiving appropriate definitive therapy were independent risk factors for 28-day mortality. In conclusion, revisiting K. pneumoniae bacteremic pneumonia in the current era showed a high mortality rate. Host factors, disease severity, and timely effective therapy affect the treatment outcomes of these patients.

Involvement of the hemP-hemA-smlt0796-smlt0797 Operon in Hemin Acquisition by Stenotrophomonas maltophilia.

The hemin acquisition system of Stenotrophomonas maltophilia was elucidated in this study. To identify the TonB-dependent outer membrane receptor for hemin in S. maltophilia, the hemin acquisition systems of Pseudomonas aeruginosa were referenced. PhuR, HasA, and HxuA are three known TonB-dependent outer membrane receptors involved in hemin acquisition by P. aeruginosa. Thus, HemA (Smlt0795) and Smlt2937, the orthologs of PhuR and HasA/HxuA in S. maltophilia, were first considered. KJΔEnt, a stenobactin-null strain, was used as the parental strain for the hemin utilization assay. Deletion of hemA, but not Smlt2937, of KJΔEnt impaired hemin acquisition under iron-depleted conditions, indicating that HemA is the TonB-dependent receptor for hemin uptake. The hemA gene is a member of the hemP-hemA-smlt0796-smlt0797 operon, whose expression was upregulated in a fur mutant and under iron-depleted conditions. The contribution of the hemP-hemA-smlt0796-smlt0797 operon to hemin acquisition was investigated by in-frame deletion mutant construction and hemin utilization assays. Inactivation of hemP, smlt0796, and smlt0797 of KJΔEnt insignificantly affected hemin acquisition under iron-depleted conditions. However, hemP deletion in a fur mutant increased hemin acquisition under iron-depleted conditions. Collectively, we revealed that (i) HemA likely functions as the outer membrane receptor for hemin uptake; (ii) HemP, a predicted transcriptional factor, apparently functions as a repressor of the expression of the hemA transcript; and (iii) in a fur mutant, HemP has a negative impact on hemin acquisition under iron-depleted conditions. IMPORTANCE Stenotrophomonas maltophilia is an emerging multidrug-resistant opportunistic pathogen, increasing the difficulty of treatment of this infection. Iron is a critical element for bacterial viability. Heme is the most abundant iron source in the human host; thus, heme is the major iron source for a pathogen in the infection niche. Blocking iron acquisition from heme can be an alternative strategy to control S. maltophilia infection. Although several hemin acquisition systems have been reported in various pathogens, very little is known about the hemin acquisition systems of S. maltophilia. By in-frame deletion mutant construction and hemin utilization assays, we demonstrated that HemA (Smlt0795) is the TonB-dependent outer membrane receptor for hemin uptake and that HemP (Smlt0794), a predicted transcriptional factor, had a negative impact on hemin acquisition in a fur mutant. The negative regulatory role of HemP in hemin acquisition is first reported.