Clinical manifestations and outcome of nocardiosis and antimicrobial susceptibility of Nocardia species in southern Taiwan, 2011-2021.

BACKGROUND/PURPOSE: Nocardiosis is an uncommon infectious disease. This study aimed to assess the clinical outcome of patients with nocardiosis and examine the antimicrobial susceptibility profiles of Nocardia spp. isolated. METHODS: We retrospectively reviewed the medical records of all inpatients diagnosed with nocardiosis between 2011 and 2021. The identification of Nocardia spp. at the species level was performed with the use of MALDI-TOF and 16S rRNA assays. The antimicrobial susceptibility of Nocardia spp. was performed using the microbroth dilution method. Factors associated with 90-day all-cause mortality were identified in multivariate logistic regression analysis. RESULTS: Of 60 patients with nocardiosis in the 11-year study period, the lungs (55.0%) were the most common site of involvement, followed by the skin and soft tissue (45.0%). Twenty-two patients (36.7%) died within 90 days following the diagnosis. All of the Nocardia isolates were susceptible to trimethoprim-sulfamethoxazole, linezolid, and amikacin, whereas more than 70% of the isolates were not susceptible to ciprofloxacin, imipenem-cilastatin, moxifloxacin, cefepime, and clarithromycin. Nocardiosis involving the lungs (relative risk [RR], 9.99; 95% confidence interval [CI], 1.52-65.50; p = 0.02), nocardiosis involving the skin and soft tissue (RR, 0.15; 95% CI, 0.02-0.92; p = 0.04), and treatment with trimethoprim-sulfamethoxazole (RR, 0.14; 95% CI, 0.03-0.67; p = 0.01) were independently associated with 90-day all-cause mortality. CONCLUSIONS: Nocardia spp. identified between 2011 and 2021 remained fully susceptible to trimethoprim-sulfamethoxazole, linezolid, and amikacin. Nocardiosis of the lungs, skin and soft tissue infection, and treatment with trimethoprim-sulfamethoxazole were independently associated with 90-day all-cause mortality.

Epidemiology of Nocardia Species at a Tertiary Hospital in Southern Taiwan, 2012 to 2020: MLSA Phylogeny and Antimicrobial Susceptibility.

The identification and antimicrobial susceptibility of Nocardia spp. are essential for guiding antibiotic treatment. We investigated the species distribution and evaluated the antimicrobial susceptibility of Nocardia species collected in southern Taiwan from 2012 to 2020. A total of 77 Nocardia isolates were collected and identified to the species level using multi-locus sequence analysis (MLSA). The susceptibilities to 15 antibiotics for Nocardia isolates were determined by the broth microdilution method, and the MIC50 and MIC90 for each antibiotic against different species were analyzed. N. cyriacigeorgica was the leading isolate, accounting for 32.5% of all Nocardia isolates, and the prevalence of Nocardia isolates decreased in summer. All of the isolates were susceptible to trimethoprim/sulfamethoxazole, amikacin, and linezolid, whereas 90.9% were non-susceptible to cefepime and imipenem. The phylogenic tree by MLSA showed that the similarity between N. beijingensis and N. asiatica was as high as 99%, 73% between N. niigatensis and N. crassostreae, and 86% between N. cerradoensis and N. cyriacigeorgica. While trimethoprim/sulfamethoxazole, amikacin, and linezolid remained fully active against all of the Nocardia isolates tested, 90.9% of the isolates were non-susceptible to cefepime and imipenem.

Effect of antifungal agents, lysozyme and human antimicrobial peptide LL-37 on clinical Candida isolates with high biofilm production.

Introduction. Candida species can form biofilms on tissues and medical devices, making them less susceptible to antifungal agents.Hypothesis/Gap Statement. Antifungal combination may be an effective strategy to fight against Candida biofilm.Aim. In this study, we investigated the in vitro activity of fluconazole, caspofungin and amphotericin B, alone and in combination, against 17 clinical Candida tropicalis and 6 Candida parapsilosis isolates with high biofilm formation. We also tested LL-37 and lysozyme for anti-biofilm activity against a selected C. tropicalis isolate.Methodology. Candida biofilms were prepared using the 96-well plate-based method. The minimum biofilm eradication concentrations were determined for single and combined antifungal drugs. The activity of LL-37 and lysozyme was determined by visual reading for planktonic cells and using the XTT assay for biofilms.Results. Under biofilm conditions, fluconazole plus caspofungin showed synergistic effects against 60.9% (14 of 23) of the tested isolates, including 70.6% of C. tropicalis [fractional inhibitory concentration index (FICI), 0.26-1.03] and 33.3% of C. parapsilosis (FICI, 0.04-2.03) isolates. Using this combination, no antagonism was observed. Amphotericin B plus caspofungin showed no effects against 78.3% (18 of 23) of the tested isolates. Amphotericin B plus fluconazole showed no effects against 65.2% (15 of 23) of the tested isolates and may have led to antagonism against 2 C. tropicalis and 2 C. parapsilosis isolates. LL-37 and lysozyme had no effect on biofilms of the selected C. tropicalis isolate.Conclusions. We found that fluconazole plus caspofungin led to a synergistic effect against C. tropicalis and C. parapsilosis biofilms. The efficacy of the antifungal combination therapies of the proposed schemes against biofilm-associated Candida infections requires careful and constant evaluation.

The Surface Protein Fructose-1, 6 Bisphosphate Aldolase of Klebsiella pneumoniae Serotype K1: Role of Interaction with Neutrophils.

Hypermucoviscosity phenotypic Klebsiella pneumoniae (HV-Kp) serotype K1 is the predominant pathogen of a pyogenic liver abscess, an emerging infectious disease that often complicates septic metastatic syndrome in diabetic patients with poor sugar control. HV-Kpisolates were more resistant to neutrophil phagocytosis than non-HV-Kpisolates because of different pathogen-associated molecular patterns. The protein expression of HV-Kp after interaction with neutrophils is unclear. We studied KP-M1 (HV phenotype; serotype K1), DT-X (an acapsularmutant strain of KP-M1), and E. coli (ATCC 25922) with the model of Kp-infected neutrophils, using a comparative proteomic approach. One the identified protein, namely fructose-1, 6-bisphosphate aldolase (FBA), was found to be distributed in the KP-M1 after infecting neutrophils. Cell fractionation experiments showed that FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane-localized FBA was surface-accessible to FBA-specific antibody. The fba gene expression was enhanced in high glucose concentrations, which leads to increasing bacterial resistance to neutrophils phagocytosis and killing. The KP-M1 after FBA inhibitors and FBA-specific antibody treatment showed a significant reduction in bacterial resistance to neutrophils phagocytosis and killing, respectively, compared to KP-M1 without treatment. FBA is a highly conserved surface-exposed protein that is required for optimal interaction of HV-Kp to neutrophils.

Impact of biofilm production by Candida species and antifungal therapy on mortality of patients with candidemia.

BACKGROUND AND OBJECTIVES: Few studies have investigated the clinical outcomes of patients with candidemia caused by Candida species with different levels of biofilm formation. We aimed to investigate the impact of antifungal therapy on the outcome of candidemia caused by Candida species that were categorised as low biofilm formers (LBFs), moderate biofilm formers (MBFs), and high biofilm formers (HBFs). METHODS: Adults with candidemia caused by LBF and HBF/MBF Candida species that were susceptible to fluconazole and caspofungin were included to investigate the impact of treatment with fluconazole vs an echinocandin on 30-day crude mortality. RESULTS: In total, 215 patients with candidemia received fluconazole and 116 patients received an echinocandin. In multivariate analysis, Pittsburgh bacteremia score ≥ 4 (adjusted odds ratio [AOR] =2.42; 95% confidence interval [CI], 1.32-4.41), malignancy (AOR = 3.45; 95% CI, 1.83-6.51), not removing the central venous catheter within 48 hours of a positive blood culture (AOR = 4.69; 95% CI, 2.61-8.45), and treatment with fluconazole for candidemia due to HBF/MBF Candida spp. (AOR = 2.23; 95% CI, 1.22-4.06) were independent factors associated with 30-day mortality. Of the 165 patients infected by HBF/MBF Candida isolates, those who received azole therapy had a significantly higher sepsis-related mortality rate than those who received echinocandin therapy (44.9% [49/109] vs 26.8% [15/56], P = .03). CONCLUSIONS: There was a trend of an independent association between fluconazole treatment and poor outcomes in the patients infected by HBF/MBF Candida strains.

Aspirin use is associated with reduced risk for recurrence of pyogenic liver abscess: a propensity score analysis.

Aspirin possesses anti-bacterial activity that may prevent recurrence of Klebsiella pneumoniae pyogenic liver abscess (KP-PLA). In ex-vivo study, aspirin was administered before bactericidal assay against serotype K1 K. pneumoniae. We identified 5,912 patients with PLA who had no known pre-existing hepatobiliary diseases or malignancy in Taiwan from 1999 to 2013 from nationwide cohort study. Multivariate Cox proportional hazards regression models was used to estimate the hazard ratios [HR] for the association between aspirin use and recurrent PLA. The PLA recurrence rate in patients taking aspirin daily for 30 or more days, from 90 days before to 90 days after the first PLA episode (aspirin users), and aspirin non-users was 42.5 and 74.6 per 1,000 person-years of follow-up, respectively. The population-based study showed a HR for PLA recurrence in aspirin users of 0.50 (95% confidence interval, 0.35-0.69), relative to that in non-users, after adjustments for confounders. An ex-vivo study indicated that aspirin was able to significantly enhance bacterial killing by leukocytes, whether collected from diabetic patients with KP-PLA recurrence or from healthy volunteers. Our results suggest that aspirin is associated with reduced risk for PLA recurrence among Taiwanese with PLA who had no preexisting hepatobiliary diseases or malignancy.

The Impact of Biofilm Formation on the Persistence of Candidemia.

This study aimed to determine the predictors of persistent candidemia and examine the impact of biofilm formation by Candida isolates in adult patients with candidemia. Of the adult patients with candidemia in Kaohsiung Chang Gung Memorial Hospital between January 2007 and December 2012, 68 case patients with persistent candidemia (repeated candidemia after a 3-day systemic antifungal therapy) and 68 control patients with non-persistent candidemia (Candida clearance from the bloodstream after a 3-day systemic antifungal therapy) were included based on propensity score matching and matching for the Candida species isolated. Biofilm formation by the Candida species was assessed in vitro using standard biomass assays. Presence of central venous catheters (CVCs) at diagnosis (adjusted odd ratio [AOR], 3.77; 95% confidence interval [CI], 1.09-13.00, p = 0.04), infection with higher biofilm forming strains of Candida species (AOR, 8.03; 95% CI, 2.50-25.81; p < 0.01), and receipt of suboptimal fluconazole doses as initial therapy (AOR, 5.54; 95% CI, 1.53-20.10; p < 0.01) were independently associated with persistent candidemia. Biofilm formation by Candida albicans, C. tropicalis, and C. glabrata strains was significantly higher in the case patients than in the controls. There were no significant differences in the overall mortality and duration of hospitalization between the two groups. Our data suggest that, other than presence of retained CVCs and use of suboptimal doses of fluconazole, biofilm formation was highly associated with development of persistent candidemia.

Delay in Human Neutrophil Constitutive Apoptosis after Infection with Klebsiella pneumoniae Serotype K1.

Klebsiella pneumoniae serotype K1 is a major cause of invasive syndrome defined by liver abscess with metastatic infections at other body sites. This culprit is known to be resistant to neutrophil phagocytosis and bactericidal activity. We hypothesized that K. pneumoniae serotype K1 might regulate neutrophil apoptosis and enhance the survival of the infected neutrophils that might serve as a vector for dissemination of the bacteria. Two serotypes of K. pneumoniae, KP-M1 isolated from a patient with liver abscess and DT-X (an acapsular mutant strain of KP-M1), were used to infect human neutrophils. The infected neutrophils were examined for their cytotoxicity, annexin V staining, proteins, DNA fragmentation, cytokine production, and viability that are involved in apoptosis. We found that KP-M1 was not destroyed and the ingested bacteria survived within neutrophils. While the uninfected neutrophils became apoptotic within 10 h, the neutrophils infected with KP-M1 could survive up to 24 h post infection. Constitutive apoptosis of KP-M1-infected neutrophils was significantly delayed compared to that of DT-X-infected or uninfected neutrophils (p < 0.01). KP-M1 modulated the anti-apoptotic effects by down-regulating the ratio of Bax to Bcl-2 and Mcl-1, and then delayed caspase-3 activation in the neutrophils, which was accompanied by inducing the anti-apoptotic cytokine, IL-8. These data suggest that K. pneumoniae serotype K1 can prolong the lifespan of infected neutrophils by delaying constitutive apoptosis within the first several hours of infection.

Antifungal susceptibility of Candida species isolated from patients with candidemia in southern Taiwan, 2007-2012: impact of new antifungal breakpoints.

The Clinical and Laboratory Standard Institute (CLSI) revised the clinical breakpoints (CBPs) for the azoles and echinocandins against Candida species in 2012. We aimed to report the epidemiology of candidemia and antifungal susceptibility of Candida species and evaluate the impact of new CBPs on antifungal susceptibility in our region. All blood isolates of Candida species were obtained from 2007 to 2012. The minimum inhibitory concentrations of fluconazole, voriconazole, echinocandins and flucytosine against Candida isolates were determined by Sensititre YeastOne system. Differences in susceptibility rates between the CBPs of previous and revised versions of CLSI were examined. Of 709 Candida isolates, the fluconazole-susceptible rate was 96.5% in Candida albicans, 85.8% in Candida tropicalis and 92.1% in Candida parapsilosis by the revised CBPs. Compared with the susceptibility results by previous CBPs, the marked reductions in susceptibility of C. albicans, C. tropicalis and C. parapsilosis to fluconazole, that of C. tropicalis and C. parapsilosis to voriconazole, that of C. tropicalis and Candida glabrata to anidulafungin and that of C. tropicalis, C. glabrata and Candida krusei to caspofungin by revised CBPs were found. In conclusion, Candida albicans and C. parapsilosis remain highly susceptible to fluconazole. The non-susceptible rates of Candida species to azoles and echinocandins increase with interpretation by the revised CBPs.

Impact of glycemic control on capsular polysaccharide biosynthesis and opsonophagocytosis of Klebsiella pneumoniae: Implications for invasive syndrome in patients with diabetes mellitus.

Klebsiella pneumoniae (KP), with production of abundant capsular polysaccharide (CPS), is capable of causing invasive syndrome. Environmental glucose stimuli may increase CPS biosynthesis. We aimed to investigate the relationship between glycemic control and KP-mediated invasive syndrome in diabetic patients and the effect of glucose on CPS biosynthesis. Diabetic patients with community-acquired KP bacteremia were included to study the risk factors of invasive syndrome. KP-M1, a serotype-K1 KP clinical isolate, was used to examine the CPS biosynthesis and cps gene expression, and the effect of exogenous glucose on bacterial phagocytosis and killing. We found that invasive syndrome was significantly more common in diabetic patients who were infected with strains expressing the K1 serotype (adjusted odds ratio [AOR], 8.32; 95% confidence interval [CI], 1.56-44.24; p=0.01), and had poor glycemic control (HbA1c ≥9%; AOR, 5.66; 95% CI, 2.01-15.92; p<0.01). Pre-incubation of KP-M1 in media containing different gradient glucose concentrations enhanced CPS biosynthesis and cps gene expression in high glucose (0.5%) concentration, which leads to increasing bacterial resistance to phagocytosis and killing. High glucose levels reflected by poor glycemic control may stimulate CPS biosynthesis and cps gene expression of highly virulent KP, which increase resistance to phagocytosis and contribute to development of invasive syndrome.

Comparative effectiveness of flomoxef versus carbapenems in the treatment of bacteraemia due to extended-spectrum ß-lactamase-producing Escherichia coli or Klebsiella pneumoniae with emphasis on minimum inhibitory concentration of flomoxef: a retrospective study.

This study compared treatment outcomes of adult patients with bacteraemia due to extended-spectrum ß-lactamase-producing Escherichia coli or Klebsiella pneumoniae (ESBL-EK) receiving flomoxef versus those receiving a carbapenem as definitive therapy. In propensity score matching (PSM) analysis, case patients receiving flomoxef shown to be active in vitro against ESBL-EK were matched with controls who received a carbapenem. The primary endpoint was 30-day crude mortality. The flomoxef group had statistically significantly higher sepsis-related mortality (27.3% vs. 10.5%) and 30-day mortality (28.8% vs. 12.8%) than the carbapenem group. Of the bacteraemic episodes caused by isolates with a MICflomoxef of ≤1 mg/L, sepsis-related mortality rates were similar between the two treatment groups (8.7% vs. 6.4%; P=0.73). The sepsis-related mortality rate of the flomoxef group increased to 29.6% and 50.0% of episodes caused by isolates with a MICflomoxef of 2-4 mg/L and 8 mg/L, respectively, which was significantly higher than the carbapenem group (12.3%). In the PSM analysis of 86 case-control pairs infected with strains with a MICflomoxef of 2-8 mg/L, case patients had a significantly higher 30-day mortality rate (38.4% vs. 18.6%). Multivariate regression analysis revealed that flomoxef therapy for isolates with a MICflomoxef of 2-8 mg/L, concurrent pneumonia or urosepsis, and a Pitt bacteraemia score ≥4 were independently associated with 30-day mortality. Definitive flomoxef therapy appears to be inferior to carbapenems in treating ESBL-EK bacteraemia, particularly for isolates with a MICflomoxef of 2-8 mg/L, even though the currently suggested MIC breakpoint of flomoxef is ≤8 mg/L.

Increasing trend of fluconazole-non-susceptible Cryptococcus neoformans in patients with invasive cryptococcosis: a 12-year longitudinal study.

BACKGROUND: This study aimed to investigate the rate of fluconazole-non-susceptible Cryptococcus neoformans in Southern Taiwan for the period 2001-2012 and analyze the risk factors for acquiring it among patients with invasive cryptococcosis. METHODS: All enrolled strains were isolated from blood or cerebrospinal fluid samples of the included patients. If a patient had multiple positive results for C. neoformans, only the first instance was enrolled. Susceptibility testing was performed using the Clinical and Laboratory Standards Institutes M27-A3 broth micro-dilution method. The MIC interpretative criteria for susceptibility to fluconazole were ≤ 8 µg/ml. A total of 89 patients were included. Patients (n = 59) infected by fluconazole-susceptible strains were compared with those (n = 30) infected by non-susceptible strains. The patients' demographic and clinical characteristics were analyzed. RESULTS: The rate of fluconazole-non-susceptible C. neoformans in the study period significantly increased over time (p < 0.001). The C. neoformans isolated in 2011-2012 (odds ratio: 10.68; 95 % confidence interval: 2.87-39.74; p < 0.001) was an independent predictive factor for the acquisition of fluconazole-non-susceptible C. neoformans. CONCLUSIONS: The rate of fluconazole-non-susceptible C. neoformans has significantly increased recently. Continuous and large-scale anti-fungal susceptibility tests for C. neoformans are warranted to confirm this trend.

Correlation of anti-fungal susceptibility with clinical outcomes in patients with cryptococcal meningitis.

BACKGROUND: This study aimed to investigate the correlation of minimum inhibiting concentrations (MICs), obtained by broth micro-dilution, and clinical response in patients with cryptococcal meningitis. METHODS: Using retrospective analyses covering the period 2001-2010, factors affecting clinical therapeutic cure in patients with cryptococcal meningitis 10 weeks after the start of anti-fungal therapy were identified. Specific emphasis was placed on the role of anti-fungal susceptibility. RESULTS: Of 46 patients with cryptococcal meningitis identified, 21 were cured after 10 weeks of treatment. Overall, 12 strains (26.1%) were resistant to fluconazole (>8 µg/ml) and 8 (17.4%) had an MIC >1 µg/ml for amphotericin B. Twenty-three patients received combination amphotericin B and fluconazole as their initial antifungal therapy, 17 were given amphotericin B only, five received fluconazole only, and one received a combination of amphotericin B and flucytosine. After 2 weeks, all patients received fluconazole (400-600 mg daily for 8 weeks at least, then 200 mg daily thereafter). The presence of isolates resistant to fluconazole (MIC >8 µg/ml; 4.8% vs. 44%, p < 0.01) were statistically significant among patients who were cured. Anti-fungal susceptibility, reflected by fluconazole MIC >8 µg/ml, was an independent predictor of therapeutic cure at 10-week evaluation (OR = 15.7; 95% CI: 1.8-135.9; p = 0.01), but higher MIC of amphotericin B (>1 µg/ml) was not. CONCLUSIONS: The MICs of fluconazole, determined by the CLSI method, may be a potential predictor of therapeutic cure in patients with cryptococcal meningitis.