A Culture Supernatant from an Actinomycete sp. Affects Biofilm Formation and Virulence Expression of Candida auris.

The multidrug-resistant pathogen Candida auris is characterized by its aggregation under certain conditions, which affects its biofilm formation, drug susceptibility, and pathogenicity. Although the innate tendency to aggregate depends on the strain, the mechanism regulating C. auris aggregation remains unclear. We found that the culture supernatant from one of the 95 Actinomyces strains isolated from a deep-sea environment (IMAs2016D-66) inhibited C. auris aggregation. The cells grown in the presence of IMAs2016D-66 exhibited reduced hydrophobicity, biofilm formation, and enhanced proteolytic activity. In addition, the efflux pump activity of the fluconazole-resistant C. auris strain LSEM 3673 was stimulated by IMAs2016D-66, whereas no significant change was observed in the fluconazole-susceptible strain LSEM 0643. As the relationship between aggregative tendency and virulence in C. auris is still unclear, IMAs2016D-66 can serve as a tool for investigating regulatory mechanisms of phenotype switching and virulence expression of C. auris. Understanding of phenotype switching may help us not only to understand the pathogenicity of C. auris, but also to design new drugs that target the molecules regulating virulence factors.

Rifampicin exerts anti-mucoviscous activity against hypervirulent Klebsiella pneumoniae via binding to the RNA polymerase ß subunit.

OBJECTIVES: In hypervirulent Klebsiella pneumoniae (hvKP), the hypermucoviscous capsule is known to be a major virulence determinant. We previously discovered that rifampicin (RFP), a bactericidal drug that binds to and inhibits the ß subunit of RNA polymerase (RpoB), elicits anti-mucoviscous activity against hvKP by suppressing rmpA, a regulator of capsule production. Here, we aimed to determine whether RFP exerts this effect at sub-growth-inhibitory concentrations via its binding to RpoB. METHODS: Five spontaneous RFP-resistant mutants (R1-R5) were prepared from an hvKP clinical isolate and subjected to whole genome sequencing and mucoviscosity analyses. Subsequently, a two-step allelic exchange procedure was used to create a rpoB mutant R6 and revertants with wild-type rpoB from R1-R5 (named R1'-R5'). Transcription levels of rmpA and the capsular polysaccharide polymerase gene magA and capsule thickness of R1-R5 and R1'-R5' grown without or with RFP were evaluated by quantitative reverse transcription polymerase chain reaction and microscopic observation using India ink staining. RESULTS: R1-R5 all had non-synonymous point mutations in rpoB and were highly resistant to the bactericidal effects and anti-mucoviscous activity of RFP. While the properties of R6 were similar to those of R1-R5, the responses of R1'-R5' to RFP were identical to those of the wild type. rmpA and magA transcription levels and capsule thickness correlated well with the mucoviscosity levels. CONCLUSIONS: RFP exerts anti-mucoviscous activity by binding to RpoB. The mechanism of how this causes rmpA suppression remains to be explored.

Siderophore production as a biomarker for Klebsiella pneumoniae strains that cause sepsis: A pilot study.

BACKGROUND/PURPOSE: Klebsiella pneumoniae bacteremia-induced sepsis is a clinically important condition with a high mortality rate and various known virulence factors. However, studies on the association of these virulence factors with the occurrence of K. pneumoniae bacteremia-induced sepsis are scarce. We aimed to investigate clinical variables and virulence factors in patients with K. pneumoniae bacteremia-induced sepsis. METHODS: We retrospectively reviewed the medical records of 76 patients with K. pneumoniae bacteremia between January 2012 and July 2017. Patients were divided into sepsis (n = 25) and non-sepsis (n = 51) groups. Patient background characteristics, antimicrobial regimens, and prognosis were evaluated. We assessed the distribution of virulence factors related to K. pneumoniae, such as mucoviscosity, capsular polysaccharide, and siderophores. Siderophore production levels were determined by measuring the orange halo zone on chrome azurol S agar plate assay. RESULTS: There were no intergroup differences in male-to-female ratio and age. Multivariable analysis revealed that siderophore production level (p < 0.01) was an independent predictor of K. pneumoniae bacteremia-induced sepsis. Furthermore, the optimal cut-off point of siderophore production to predict sepsis was 9.6 mm (sensitivity, 86%; specificity, 76%; AUC, 0.81). CONCLUSION: Siderophore production was an independent predictor of sepsis caused by K. pneumoniae bacteremia. The optimal cut-off point for siderophore production for sepsis occurrence prediction was 9.6 mm. To improve outcomes, patients with K. pneumoniae bacteremia-induced sepsis with high siderophore production levels should be managed prudently.

Genomic characterization of triple-carbapenemase-producing Acinetobacter baumannii.

OBJECTIVES: To characterize Acinetobacter baumannii OCU_Ac16a, a clinical isolate co-harbouring three acquired carbapenemase genes, bla NDM-1, bla TMB-1, and bla OXA-58, and assess the clinical significance of so-called multiple-carbapenemase producers. METHODS: OCU_Ac16a and its close relative, OCU_Ac16b, which lacks the bla NDM-1, were isolated from sputum cultures of a patient at Osaka City University Hospital. We subjected these strains to whole-genome analysis, particularly focusing on the genetic context of each carbapenemase gene. The transmissibility and functionality of each carbapenemase gene were analysed by conjugation and transformation experiments and antimicrobial susceptibility tests. RESULTS: bla TMB-1 was located in a class 1 integron on the chromosome, whereas bla NDM-1 and bla OXA-58 were found on plasmids named pOCU_Ac16a_2 and pOCU_Ac16a_3, respectively. pOCU_Ac16a_2 (which exhibited highly efficient self-transmissibility) and pOCU_Ac16a_3 (which did not show transmissibility but could be introduced into another A. baumannii strain via electroporation) could both confer carbapenem resistance (MICs ≥512 and ≥32 mg/L, respectively) on the recipient strain. The functionality of bla TMB-1 was evident from the high resistance of OCU_Ac16b to ceftazidime and cefepime (MICs ≥256 and 48 mg/L, respectively), and the high resistance of OCU_Ac16a to cefiderocol (MIC 32 mg/L) could be explained by the additive effect of bla NDM-1 and bla TMB-1. CONCLUSIONS: Our data revealed the genomic organization of OCU_Ac16a and demonstrated that all the carbapenemase genes are functional, each contributing to the extremely high broad-spectrum resistance of OCU_Ac16a to ß-lactams. As multiple-carbapenemase producers can be serious health threats as drug-resistant pathogens and disseminators of carbapenemase genes, close attention should be paid to their emergence.

Complete Genome Sequence of Acinetobacter pittii OCU_Ac17, Isolated from Human Venous Blood.

Acinetobacter pittii isolate OCU_Ac17 was obtained from the venous blood of a patient at a hospital in Japan. We present its complete 4.108-Mbp genome sequence (1 chromosome plus 3 plasmids), analyzed by combining long-read (Flongle) and short-read (MiniSeq) sequencing.

Complete Genome Sequence of a Clinical Isolate of Acinetobacter baumannii Harboring 11 Plasmids.

Here, we report the complete genome sequence of an Acinetobacter baumannii isolate harboring 11 plasmids, obtained at a hospital in Japan in 2016. The complete 4.07-Mbp genome sequence (1 chromosome and 11 plasmids) was analyzed by a combination of long-read (Flongle) and short-read (NovaSeq 6000) sequencing.

Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development.

Nutritional status contributes to the regulation of immune responses against pathogens, and malnutrition has been considered as a risk factor for tuberculosis (TB). Mycobacterium tuberculosis (Mtb), the causative agent of TB, can modulate host lipid metabolism and induce lipid accumulation in macrophages, where the bacilli adopt a dormant phenotype. In addition, serum lipid components play dual roles in the regulation of and protection from Mtb infection. We analyzed the relationship between nutritional status and the humoral immune response in TB patients. We found that serum HDL levels are positively correlated with the serum IgA specific for Mtb antigens. Analysis of the relationship between serum nutritional parameters and clinical parameters in TB patients showed that serum albumin and CRP levels were negatively correlated before treatment. We also observed reduced serum LDL levels in TB patients following treatment. These findings may provide insight into the role of serum lipids in host immune responses against Mtb infection. Furthermore, improving the nutritional status may enhance vaccination efficacy.

Complete Genome Sequence of Acinetobacter baumannii ATCC 19606T, a Model Strain of Pathogenic Bacteria Causing Nosocomial Infection.

Acinetobacter baumannii ATCC 19606T, which is often used in genetic studies as a routine model microorganism, belongs to sequence type 52 (ST52), showing beta-lactam resistance. We present the complete 3.996-Mbp genome sequence (1 chromosome plus 2 plasmids), generated by combining long-read (MinION) and short-read (MiniSeq) sequencing data.

Detection of Rhizopus-specific antigen in human and murine serum and bronchoalveolar lavage.

Mucormycosis is a deep-seated fungal infection that mainly develops in patients with severe immunodeficiencies such as those with malignant hematological diseases. Despite poor prognosis, there is no reliable and minimally invasive diagnostic method-such as serodiagnosis-for making a clinical decision regarding the condition. As early diagnosis and early treatment improve the prognosis of mucormycosis, the development of a sensitive early diagnostic method is important. We had previously identified a Rhizopus-specific antigen (RSA) by signal sequence trapping and retrovirus-mediated expression (SST-REX), and evaluated its utility as a diagnostic antigen by constructing a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect serum RSA levels in inoculated mice. In this study, we used the RSA-specific rabbit monoclonal antibodies generated by novel hybridoma technology to improve the sensitivity of the ELISA system. We observed an increase in serum and bronchoalveolar lavage fluid (BALF) levels of RSA in mouse model 1 day after inoculation, suggesting that this newly developed monoclonal antibody-based ELISA system may be useful for the diagnosis of mucormycosis in the early stages of infection. In addition, we measured RSA levels in human serum and BALF, and found that serum RSA level was higher in mucormycosis patients (15.1 ng/ml) than that in invasive pulmonary aspergillosis patients (0.53 ng/ml) and the negative control (0.49 ng/ml). Our results suggest that RSA may be a powerful tool for the diagnosis of pulmonary mucormycosis, and its differentiation from other deep-seated mycoses such as aspergillosis.

Clinical and virulence factors related to the 30-day mortality of Klebsiella pneumoniae bacteremia at a tertiary hospital: a case-control study.

Klebsiella pneumoniae bacteremia is a critical clinical presentation that is associated with high mortality. However, extremely few studies have investigated the virulence factors related to mortality of K. pneumoniae bacteremia in patients. The present study elucidated clinical and virulence factors associated with the 30-day mortality of K. pneumoniae bacteremia at a tertiary hospital. The medical records of 129 patients with K. pneumoniae bacteremia admitted to Osaka City University Hospital between January 2012 and December 2018 were retrospectively reviewed. Patient background characteristics, antimicrobial regimens, and prognosis were evaluated. Additionally, virulence factors were assessed using multiplex polymerase chain reaction to elucidate their association with K. pneumoniae. The 30-day mortality was 10.9% in patients with K. pneumoniae bacteremia. The male-to-female ratio, age, and underlying disease did not differ between the non-survivor and survivor groups. Multivariate analysis showed that sepsis (odds ratio (OR), 7.46; p = 0.005) and iutA (OR, 4.47; p = 0.046) were independent predictors associated with the 30-day mortality of K. pneumoniae bacteremia. Despite the relatively low 30-day mortality of patients with K. pneumoniae bacteremia, the treatment of those with sepsis and those infected with K. pneumoniae harboring iutA may require careful management for improving their outcomes.

Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia.

Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia

Abstract Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

Clinical characteristics of bacteremia caused by hypermucoviscous Klebsiella pneumoniae at a tertiary hospital.

This study aimed to assess the predictive factors of bacteremia due to hypermucoviscous Klebsiella pneumoniae (hvKP), as well as the mortality. The medical records of 114 patients with K. pneumoniae bacteremia who were divided into the hvKP (n = 24) and non-hvKP (n = 90) groups and were retrospectively reviewed. The male-to-female ratio, age, and underlying disease did not differ between the 2 groups. Mortality was higher among patients in the hvKP bacteremia group than in the non-hvKP bacteremia group (29.2% vs 6.7%). Multivariate analysis showed that the independent predictors associated with hvKP bacteremia were abscess (P = 0.01) and no antibiotic exposure (P = 0.02); thus, early assessment of these conditions is important. For patients with a history of abscess and no antibiotic exposure, it is necessary to administer treatment while keeping the risk of hvKP in mind.

Discovery of anti-mucoviscous activity of rifampicin and its potential as a candidate antivirulence agent against hypervirulent Klebsiella pneumoniae.

A recent increase in the incidence of hypervirulent Klebsiella pneumoniae (hvKP) infections, especially those caused by a sublineage of clonal group CG23 (CG23-I), is raising serious health concerns worldwide. The high virulence of hvKP is, at least in part, attributed to the overproduction of capsular polysaccharide (CPS), which is triggered by a positive regulator of capsular polysaccharide synthesis (cps) genes, named rmpA (regulator of mucoid phenotype A). Although extensive research has been conducted on the mechanisms of hvKP virulence, no study has focused on the development of antivirulence therapeutics. This study attempted to identify and validate an antimicrobial agent able to suppress hvKP hypermucoviscosity. A total of 18 commercially available antimicrobial agents, including ß-lactams, quinolones and aminoglycosides, were tested. Rifampicin (RFP) was found to have strong anti-mucoviscous activity against CG23-I hvKP even at subinhibitory concentrations. Polysaccharide extracts from hvKP showed substantially lowered viscosity when cells were grown with RFP. Moreover, microscopic observations demonstrated that RFP treatment results in a drastic reduction in the thickness of the CPS layer around hvKP cells. RFP treatment decreased transcript levels of rmpA and rmpA-regulated cps genes, indicating that RFP suppresses mucoviscosity of hvKP through inhibition of rmpA transcription. These data suggest that RFP may serve as a potential antivirulence agent for refractory hvKP infection.

Mortality caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae bacteremia; a case control study: alert to Enterobacteriaceae strains with high minimum inhibitory concentrations of piperacillin/tazobactam.

This study aimed to assess the prognostic factors of patients with bacteremia due to extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) as well as the antimicrobial susceptibility, particularly to piperacillin/tazobactam (PTZ), among ESBL-PE strains. The medical records of 65 patients with ESBL-PE bacteremia divided into the survivor group (n = 52) and nonsurvivor group (n = 13) were retrospectively reviewed. The male-to-female ratio, age, underlying disease, leukocyte count, C-reactive protein level, and treatment did not differ between the 2 groups. Multivariate analysis showed that the independent predictors associated with hospital mortality of ESBL-PE bacteremia were sepsis (P = 0.047) and febrile neutropenia (P = 0.008); thus, early assessment of these conditions is important. Further, the minimum inhibitory concentration values of ESBL-PE isolates in nonsurvivors tended to be higher than those in survivors. PTZ should be used with caution in cases of ESBL-PE strains with low susceptibility to the drug.

Genome-Based Epidemiological Analysis of 13 Acinetobacter Strains Isolated from Blood Cultures of Hospitalized Patients from a University Hospital in Japan.

The genus Acinetobacter comprises many species that can cause infectious diseases. Despite their importance as nosocomial pathogens, the clinical distributions of individual species or clones are not well understood. In this study, we aimed to characterize 13 Acinetobacter strains isolated from blood cultures from Osaka City University Hospital. We conducted whole-genome sequencing to reveal their genetic background. We also performed PCR-based open reading frame typing (POT) and compared the results with those of multilocus sequence typing (MLST) to confirm its reliability as a genotyping method. Although biochemical analysis suggested that most isolates were A. baumannii, genomic analysis revealed that the collection of Acinetobacter isolates comprised six different species, with non-baumannii Acinetobacter species representing the majority. All strains possessed an inherent ADC-type ß-lactamase gene, whereas the distribution of OXA-type enzymes was limited to A. baumannii, A. pittii, and A. colistiniresistens. While MLST properly discriminated four A. baumannii strains as different clones, POT failed to distinguish three of the four A. baumannii strains from each other, highlighting a potential pitfall that may be encountered when applying POT to non-epidemiological A. baumannii strains.

Longitudinal Evaluation of Humoral Immunity and Bacterial and Clinical Parameters Reveals That Antigen-Specific Antibodies Suppress Inflammatory Responses in Active Tuberculosis Patients.

A novel tuberculosis vaccine to replace BCG has long been desired. However, recent vaccine trials focused on cell-mediated immunity have failed to produce promising results. It is worth noting that most commercially available successful vaccines rely on humoral immunity. To establish a basic understanding of humoral immunity against tuberculosis, we analyzed and evaluated longitudinal levels and avidity of immunoglobulin to various tuberculosis antigens compared with bacterial and clinical parameters during treatment. We found that levels of IgG antibodies against HrpA and HBHA prior to treatment exhibited a positive correlation with bacterial burden. Analysis of changes in CRP during treatment revealed an association with high levels of specific IgG and IgA antibodies against mycobacterial antigens. Levels of CRP prior to treatment were negatively associated with IgG avidity to CFP-10 and MDP1 and IgA avidity to HrpA, while IgA avidity to MDP1 and Acr exhibited a negative correlation with CRP levels after 60 days of treatment. These results may provide insight for the development of a novel tuberculosis (TB) vaccine candidate to induce protective humoral immunity against tuberculosis.

High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages.

Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.

Successful Treatment of Intestinal Mycosis Caused by a Simultaneous Infection with Lichtheimia ramosa and Aspergillus calidoustus.

A 53-year-old woman was hospitalized due to septic shock after developing pneumococcal pneumonia after undergoing esophageal cancer surgery. Her transverse colon became perforated after receiving antimicrobial chemotherapy; therefore, emergency subtotal colectomy was performed. Fungi detected in both her colon tissue and a drainage sample indicated intestinal mucormycosis. Early intensive treatment with high-dose liposomal amphotericin B was successful, and she was subsequently discharged from the hospital. The fungal isolates were identified to be Lichtheimia ramosa and Aspergillus calidoustus via gene sequencing using panfungal primers as well as species-specific primers against elongation factor 1 and beta-tubulin for detecting Lichtheimia and Aspergillus, respectively.

Clinical Characteristics and Low Susceptibility to Daptomycin in Enterococcus faecium Bacteremia.

Enterococcus faecium has high levels of resistance to multiple antibiotics, and the mortality due to E. faecium bacteremia is high. Accordingly, E. faecium strains with low susceptibility to daptomycin are a concern in clinical practice. This study assessed the predictive factors and prognosis of patients with bacteremia due to E. faecium as well as the antimicrobial susceptibility, particularly to daptomycin, among E. faecium isolates. The medical records of patients admitted to Osaka City University Hospital with E. faecalis (n = 60) and E. faecium (n = 48) bacteremia between January 2011 and March 2016 were retrospectively reviewed. The E. faecalis group (mean age: 62.0 years) included 22 women, and the E. faecium group (mean age: 59.1 years) included 19 women. Predictive factors for infection, prognosis, and isolate antimicrobial susceptibilities were evaluated. The mean Sequential Organ Failure Assessment score and mortality rate did not differ between the two groups. The independent predictors of E. faecium bacteremia in multivariate analysis included quinolone use (p = 0.025), malignancy (p = 0.021), and prolonged hospitalization (p = 0.016). Cardiovascular disease was associated with a reduced risk of E. faecium bacteremia (p = 0.015). Notably, the percentage of E. faecium isolates with low daptomycin susceptibility was higher than that of E. faecalis (8.5% vs. 0%, p = 0.036). Thus, E. faecium should be considered when administering antibiotic therapy to patients with a history of these predictors. Furthermore, the use of daptomycin should be avoided in case of E. faecium with low susceptibility to daptomycin.