Maria Cristina Ghezzi

Serum-Mediated Enhancement of TNF-α Release by Human Monocytes Stimulated with the Yeast Form of Candida albicans

The role of serum in the production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes stimulated with yeast-form Candida albicans was studied. Pre-exposure of C. albicans to human pooled serum enhanced both TNF-alpha mRNA and cytokine secretion compared with C. albicans preincubated with medium only. Serum factors involved were >30 kDa, were efficiently inhibited by D-mannose, and recognized both Ca++-dependent and -independent pathways. Preincubation of yeasts with rabbit mannose-binding protein (MBP) resulted in dose-related enhancement of TNF-alpha secretion, through a Ca++-dependent pathway inhibited by D-mannose. TNF-alpha levels were similarly induced in C. albicans preincubated with vitronectin and with serum. Ca++ depletion did not affect cytokine release, while D-mannose supplementation displayed inhibition. The latter effect was abolished after Ca++ depletion. These data call for an involvement of both MBP and vitronectin in the serum-mediated enhancement of TNF-alpha release upon stimulation of monocytes with yeast forms of C. albicans.

Antimicrobial Susceptibility, Biochemical and Genetic Profiles of Staphylococcus haemolyticus Strains Isolated from the Bloodstream of Patients Hospitalized in Critical Care Units

Abstract Staphylococcus haemolyticus strains (n=20), responsible of blood stream infections, were consecutively isolated from patients hospitalized in two different wards at high risk of infection. Strains displayed high rate of resistance to oxacillin (90%). All strains but two with decreased susceptibility (MIC = 4 μg/mL), were sensitive to vancomycin. Ten strains were resistant to teicoplanin. Among the strains susceptible to glycopeptides, three displayed heteroresistance to vancomycin and seven to teicoplanin, when tested by Etest technique with 2 x McFarland inoculum. Biochemical reactions allowed to assign strains to eight biotypes, with 11 strains clustering under two main biotype A and biotype B. Pulsed-field-gel-electrophoresis (PFGE) identified 11 different PFGE-types. Seven strains grouping under the major PFGE-type 1 and three strains clustering in PFGE-type 2, closely correlated to biotype A and biotype B respectively. Seven teicoplanin-resistant isolates clustered in the PFGE-type 1, two in the PFGE-type 2 and one in PFGE-type 5. Therefore, teicoplanin-resistant strains were biochemically and genetically related and clonally distributed, despite different clones of S. haemolyticus circulated in the units during the study period.

Clostridium difficile Infection and Candida Colonization of the Gut: Is There a Correlation?

TO THE EDITOR—We read with interest the article of Manian and Bryant about the possible protective role of gut colonization by Candida species against Clostridium difficile infection (CDI) [1]. Although Candida has been involved as part of the normal intestinal microbiota [2], no recent data seem to confirm the authors’ results. Conversely, an opposite role for non-albicans Candida (NAC) colonization was suggested by Nerandzic et al in a trial on 548 patients with CDI [3]. Recent observations conducted in our 1300-bed teaching hospital Policlinico “Umberto I” in Rome showed that patients treated for severe CDI developed at least 1 episode of candidemia, thus hypothesizing a link between CDI and candidemia [4]. We designed a prospective casecontrol study in diarrheic patients with suspected CDI from November 2013 until June 2014 to investigate whether Candida colonization of the gut and CDI may be linked. Stool specimens (n = 140; Bristol stool chart 5–7 [5]) from consecutive patients were evaluated for glutamate dehydrogenase and CD toxin A/B by immunochromatography tests (C-Diff Quick Check Complete, Techlab); positive samples were screened for the C. difficile Pa-Loc gene and for 027 ribotype by reverse transcription polymerase chain reaction (RT-PCR) (GeneXpert, Cepheid, Sweden). Candida was revealed by plating 10 μL of all samples on Sabouraudchloramphenicol agar (Liofilchem, Italy), incubated for 24–48 hours at 37°C. Candida colonization was defined as the growth of ≥10 colony-forming units per milliliter of stool sample. The yeasts were typed through their biochemical profiles (Api ID 32 C, bioMérieux). Mantel-Haenszel test and Stata 11 software were used for statistical analysis; an α error <.05 was accepted. The 140 patients averaged 65 ± 22.98 years of age, and 52% were male. One hundred patients had negative results and 40 positive results for CDI, as revealed by both the immunochromatography reactivity and by the RT-PCR for the Pa-Loc gene presence, which showed that 10 patients were infected by the 027 ribotype. CDI was significantly associated with Candida colonization (83% CDI positive vs 67% CDI negative; χ = 3.91; P < .05). Candida albicans was the species more often implicated ( χ = 4.82; P = .02; Table 1). All except 1 of the ten 027 ribotype–infected patients were colonized by the yeast, 7 of which were C. albicans (χ = 0.37; P = .5). Our data provided evidence that Candida colonization and CDI are linked, thus suggesting a role for the yeast during CDI. The prevalence of yeasts observed in our study was higher than that reported by others [1, 3], probably due to differences in the cultural and typing methods, or to different study populations. Indeed, Manian and Bryant evoked a protective role for Candida in competing with C. difficile, as they observed a lower prevalence of Candida colonization in only 16.7% of the CDIpositive patients. Unfortunately, the authors did not perform any quantitative assessment of Candida growth, referring only to the overgrowth of the yeast covering >50% of an agar plate [1]. Similarly, the higher prevalence of NAC observed in Nerandzic et al’s study (68% NAC vs 32% of C. albicans) can be affected by the storage conditions used in their study [3]. Further studies are in progress to find out a correlation between CDI and candidemia and to reveal the pathogenic mechanisms underlying this association.

Analysis of methods commonly used for glycopeptide and oxazolidinone susceptibility testing in Enterococcus faecium isolates

The susceptibility to teicoplanin, vancomycin and linezolid of 30 clinical isolates of Enterococcus faecium was tested by Vitek 2, Phoenix, Etest, broth microdilution and disc diffusion tests. The vanA and vanB resistance genes and the 23S rRNA gene G2576T mutation were detected by PCR and PCR-RFLP, respectively. Resistance rates to teicoplanin ranged from 3% for Vitek 2 to 57.6% for the Phoenix test, and those to vancomycin ranged from 56.7% for Vitek 2 to 86.7% for the Phoenix test. Only two out of 25 strains carrying the vanA gene were univocally recognized as the VanA phenotype. The only strain with the G2576T mutation did not carry the vanA gene and showed resistance to linezolid by the disc diffusion, Vitek 2 and broth dilution methods (MIC>8 microg ml(-1)), but was susceptible when tested with the Phoenix test and Etest (MIC<or=4 microg ml(-1)). Therefore, the resistance to glycopeptides and linezolid was not univocally detected by the susceptibility testing methods used in this study.

Voriconazole treatment of Candida tropicalis meningitis: persistence of (1,3)-β-D-glucan in the cerebrospinal fluid is a marker of clinical and microbiological failure

Introduction: Infections are still the most common complications of cerebral shunt procedures. Even though fungal etiologies are considered to be rare, they are associated with significant morbidity and mortality. Due to their uncommonness, diagnostic procedures and optimal therapy are poorly defined. We report a case of Candida tropicalis infection of ventriculo-peritoneal cerebrospinal fluid (CSF) shunt in a 49-year-old immune competent male treated with voriconazole (VOR). Methods: Microbiological and CSF markers (1,3-b-D-glucan-BDG) of fungal infection, biofilm production capacity, sensitivity of serial isolates of the pathogen, and the concentration of the antifungal drug have been monitored and related to the clinical course of this infection. Results: Despite appropriate treatment with VOR, in terms of adequate achieved CSF drug concentrations and initial effective therapeutic response, loss of VOR susceptibility of the C tropicalis and treatment failure were observed. Conclusion: Biofilm production of the C. tropicalis isolate might have had a significant role in treatment failure. Of interest, clinical and microbiological unfavorable outcome was anticipated by persistence of BDG in CSF. Rising titers of this marker were associated with relapse of fungal infection.

Protective features of monoclonal antibodies to Escherichia coli during experimental infection of mice with homologous and heterologous serotypes of E. coli

Murine monoclonal antibodies (MAbs) MT1F and ARM1-4, recognising proteins on the surface of untreated Escherichia coli O6:K-, protected 100% of mice challenged intraperitoneally with 2 x LD50 of the same strain. MAb MT1F protected 70% of animals challenged with 2 x LD50 of E. coli O111:B4, whereas ARM1-4 gave complete protection. Lower survival was observed in mice given either MAb and challenged with E. coli O128:K-, with values ranging from 30 to 42%. However, the protection afforded against E. coli O111:B4 and E. coli O128:K- was significantly improved when the mice were pre-treated with a mixture of the two MAbs. Control mice, pre-treated with unrelated ascitic fluid and challenged with any of the E. coli serotypes, showed 100% mortality and organ histological lesions resembling those of the early stages of septic shock. The mice had high levels of circulating endotoxin and tumour necrosis factor-alpha (TNF-alpha) at 90 min after challenge. In contrast, mice treated with MAbs and surviving the infection displayed moderate histological lesions, enhanced bacterial clearance and lower serum levels of TNF-alpha, despite circulating endotoxin levels that were higher than in the control group. Protection by the MAbs was probably due to the prevention of the bacterial spread to organs and of the cascade of events leading to septic shock. This occurred in spite of the presence of high levels of circulating endotoxin.

The Correlation Between Biofilm Production and Catheter-Related Bloodstream Infections Sustained by Candida. A Case Control Study

Biofilm forming capacity of yeasts colonizing the intravenous devices is considered a key factor involved in the pathogenesis of Candida catheter-related bloodstream infections (CCRBSI). The biofilm production of strains of Candida spp. isolated both from the CVC and from the blood of patients with CCRBSI was compared to that of strains isolated from patients not having CCRBSI. Results, expressed in terms of Biofilm Index (BI), revealed that biofilm-producing strains were isolated in the CCRBSI group with a frequency significantly higher than in the non-CCRBSI group (χ2 = 4.25, p = 0.03). The species more frequently cultured was C. parapsilosis complex (including C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis). When this species was isolated from the CVC tip cultures of the CCRBSI group it showed BIs significantly (p = 0.05) higher than those found in the non-CCRBSI group. All the strains of C. tropicalis isolated from the CCRBSI group produced biofilm. Instead most of the isolates of C. glabrata were non-producers. The cumulative BI of non-albicans Candida strains isolated from CCRBSI patients was significantly higher than that of non-albicans strains cultured from patients non-CCRBSI (χ2 = 6.91; p = 0.008). C. albicans was a biofilm producer both in the CCRBSI and in the non-CCRBSI group. When isolated from the blood it showed enhanced biofilm production in the CCRBSI group only, while when colonizing the CVC it displayed high BIs both in the CCRBSI group and in non-CCRBSI group. Our data seem to indicate that the biofilm production capacity should be considered in the clinical management of CCRBSI.

Biotimer assay: A reliable and rapid method for the evaluation of central venous catheter microbial colonization

Adherent bacteria and biofilm frequently colonize central venous catheters (CVCs). CVC colonization is correlated to infections and particularly to bloodstream ones. The classical microbiological methods to determine of CVC colonization are not fully reliable and are time-consuming. BioTimer Assay (BTA) is a biological method already used to count bacteria adherent to abiotic surfaces and biofilm without sample manipulation. BTA employs specific reagents whose color changed according to bacterial metabolism. BTA is based on the principle that a metabolic reaction will be faster when more bacteria are present in the sample. Therefore, the time required for color changes of BTA reagents determines the number of bacteria present in the sample through a correlation line. Here, for the first time, we applied BTA and a specifically developed laboratory procedure to evaluate CVC colonization in comparison with the routine microbiological method (RMM). 125 CVCs removed from patients for suspected catheter-related bloodstream infection (CRBSI) or at hospital discharge were examined. BTA was reliable in assessing sterility and CVC colonization (100% agreement with RMM) and in recognizing the presence of fermenting or non-fermenting bacteria (97.1% agreement with RMM) shortening the analytical time by between 2- and 3-fold. Moreover, the reliability of BTA as early alert of CRBSI was evaluated. The sensitivity, specificity, positive, and negative predictive values for BTA as an early alert of CRBSI were 100, 40.0, 88.8 and 100%, respectively. In conclusion, BTA and the related laboratory procedure should be incorporated into routine microbiological methods since it can be considered a reliable tool to evaluate CVC colonization in a very short time and a rapid alert for CRBSIs.

Candidaemia in a tertiary care academic hospital in Italy. The impact of C. parapsilosis complex on the species distribution and antifungal susceptibility

Purpose. To analyse the species distribution and the susceptibility profiles to the major antifungal agents of Candida isolated from bloodstream infections (BSIs) in both intensive care units (ICUs) and non‐ICU wards in a tertiary care hospital in Italy from 2010 until 2015. Methodology. Episodes of Candida BSI were recorded in a retrospective observational cohort study. Yeasts were isolated from both blood and intravascuIar devices (IVDs) and their susceptibility to antifungal drugs was tested using the microdilution method. Results. 514 Candida BSIs were evidenced and 19% of these episodes were associated with the presence of an IVD. The trend of the general incidence increased significantly throughout the study period, ranging from 1.42 to 3.63 (mean 2.52) episodes/1000 admissions. The incidence of Candida BSIs and IVD‐associated candidaemia was significantly higher in ICUs relative to the other wards. The most frequently isolated species were C. albicans and C. parapsilosis complex, with the latter presenting a significant increased trend of isolation. C. parapsilosis complex was most frequently involved in IVD‐related candidaemia, coinfections and late recurrent infections. Furthermore, the MIC50s of C. parapsilosis complex were significantly enhanced for echinocandins compared to the MIC50s for the same drugs and the other yeasts, while the MIC50s of C. albicans for amphotericin B showed a significant increase during the study period, ranging from 0.1 to 0.5 &mgr;g ml−1. Conclusions. A progressively enhanced incidence of Candida BSIs, a relatively high impact of C. parapsilosis complex and changes in the susceptibility profiles of the isolated yeasts were evidenced during the observation period.

Successful conservative treatment of peripheral candidal thrombophlebitis: Case report

Martina Carnevalini, Federico Faccenna, Anna Paola Massetti, Giammarco Raponi, Maria Cristina Ghezzi, Cecilia Venditti and Mario Venditti Department of Infectious Diseases and Tropical Medicine, ‘‘La Sapienza’’ University of Rome, Rome, Italy,Department of Vascular Surgery, ‘‘La Sapienza’’ University of Rome, Rome, Italy and Department of Clinical Microbiology, ‘‘La Sapienza’’ University of Rome, Rome, Italy