Search

At the end of 2022 and in the following months, an increase in the incidence of Streptococcus pyogenesinfections was observed in many European countries that was simultaneously accompanying to enhance of invasive infections (iGAS). We have showed a risen trend of S. pyogenesinfections among preschoolers after the pandemic event. A thorough epidemiological investigation of both paediatric and adult samples positive for S. pyogenesindicate a more complex scenario leading to need of important improvement in surveillance programs.

The pandemic marked the beginning of an era of dynamic and rapid changes in the diagnosis of respiratory infections. Herein we describe Legionnaires’ disease trend in the years 2016–2023 in a large Italian hospital showing how improvements in diagnostic algorithms impact on its detection.

Introduction: A serious complication associated with Central Venous Access Device (CVAD) is infection because of bacterial contamination, either by the extra-luminal or by the intra-luminal route. We evaluated the efficacy, the safety, and the cost-effectiveness of two strategies for non-inferiority in controlling bacterial colonization of the exit-site of Peripherally-Inserted Central Catheters (PICC).Methods: After PICC placement, a skin swab of the exit site was taken and cultured. In group A the exit site was sealed with N-butyl-cyanoacrylate glue, while in group B a chlorhexidine-releasing sponge dressing was applied. A second skin culture was taken at day 7.Results: A total of 51 patients were enrolled in each group. In 42 patients the second skin culture was not performed because of 20 patients were lost at follow-up or deceased and in 22 patients the dressing needed to be changed early, because of local bleeding (13 cases, in group B) or because of dressing detachment (four in group A and five in group B). The microbiological study was completed in 36 patients in group A and 24 in group B. No microorganisms were isolated in any patient.Conclusions: Both strategies were effective in controlling bacterial colonization. Glue was effective in reducing local bleeding, and it was more cost-effective than sponge dressing. During the first week, when local bleeding and bacterial colonization must be prevented, glue might be more appropriate than chlorhexidine-releasing dressing; after the first week chlorhexidine-releasing dressing might be preferable, considering that the safety of glue application on the skin for prolonged periods is still questionable.

Whether subglottic secretions (SS) culture during invasive mechanical ventilation may aid microbiological surveillance is unknown. We conducted a prospective study to assess SS cultures predictivity of endotracheal aspirate (ETA) and bronchoalveolar lavage (BAL) isolates.

Introduction: Urinary tract infections (UTIs) are among the most common bacterial infections, affecting 150 million people worldwide each year. Importantly, the incidence of UTI increases markedly with age. The increasing resistance to empirically prescribed antimicrobial agents complicates the management of this disease. This makes UTI an important issue in increasingly aging population and updated epidemiological investigation is advisable. To understand the epidemiological variation in UTI resistance patterns among differently aged populations, we conducted a retrospective study among patients presenting to the emergency department (ED) of a large tertiary-care hospital in Italy during January 2013 and June 2017. Methods: 1281 patients who presented with UTI symptoms to the ED, were stratified into four age categories: young adults (18-44 years old;), adults (45-64), the elderly (65-84), and the oldest old (≥ 85). Inclusion criteria were urine collected in ED patients with UTI symptoms and first positive culture from one given patient in a given year. Results: 362 (28.2%) patients had a urine culture with positive result, leading to a total of 459 germs isolated, stratified into four categories: young adults (58 isolates, 12.6%), adults (98, 21.4%), the elderly (174, 37.9%), and the oldest old (129, 28.1%). Escherichia coli represents the 60% of all monomicrobial infections, followed in frequency by Klebsiella pneumoniae (15%), and Enterococcus faecalis (5%). The other 20% of the infections are caused by various germs. The most common association of germs in polymicrobial is E. coli + E. faecalis, accounting for the 28% of all infections. Overall, we found a peak of susceptibility to amoxicillin (AMX) in the oldest old ( 81%), significantly higher compared to young adults (54%), adults (47%) and elderly (35%) (p<0,001). For ciprofloxacin (CIP) there is a greater susceptibility in the young adult (55.5%), but not so marked compared to the other three groups; for fosfomicin (FOS) the susceptibility was greater in the group of adults (60%) compared to young adults, elderly and the oldest old. Also for trimethoprim/ sulfamethoxazole (TMP-SMX) we found greater susceptibility in the adult group (60%), followed by the oldest old (57,6%), young adults (49%) and elderly (47%). Conclusion: Age-related differences in antimicrobial-resistant microorganisms were evident for adults with UTI, and could potentially contribute to the risk of inappropriate empirical therapy in elderly patients. Thus, different empirical antimicrobial regimens should be considered for distinct age groups.

To assess the accuracy of differential time to positivity (DTP) method for the diagnosis of catheter-related bloodstream infections (CRBSI) in the routine practice of our intensive care unit (ICU).

ABSTRACTSensititre YeastOne (SYO) is an affordable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method for antifungal susceptibility testing. In this study, the MICs of yeast isolates from 1,214 bloodstream infection episodes, generated by SYO during hospital laboratory activity (January 2005 to December 2013), were reanalyzed using current CLSI clinical breakpoints/epidemiological cutoff values to assign susceptibility (or the wild-type [WT] phenotype) to systemic antifungal agents. Excluding Candida albicans(57.4% of all isolates [n= 1,250]), the most predominant species were Candida parapsilosiscomplex (20.9%), Candida tropicalis(8.2%), Candida glabrata(6.4%), Candida guilliermondii(1.6%), and Candida krusei(1.3%). Among the non-Candidaspecies (1.9%), 7 were Cryptococcus neoformansand 17 were other species, mainly Rhodotorulaspecies. Over 97% of Candidaisolates were susceptible (WT phenotype) to amphotericin B and flucytosine. Rates of susceptibility (WT phenotype) to fluconazole, itraconazole, and voriconazole were 98.7% in C. albicans, 92.3% in the C. parapsilosiscomplex, 96.1% in C. tropicalis, 92.5% in C. glabrata, 100% in C. guilliermondii, and 100% (excluding fluconazole) in C. krusei. The fluconazole-resistant isolates consisted of 6 C. parapsilosiscomplex isolates, 3 C. glabrataisolates, 2 C. albicansisolates, 2 C. tropicalisisolates, and 1 Candidalusitaniaeisolate. Of the non-Candidaisolates, 2 C. neoformansisolates had the non-WT phenotype for susceptibility to fluconazole, whereas Rhodotorulaisolates had elevated azole MICs. Overall, 99.7% to 99.8% of Candidaisolates were susceptible (WT phenotype) to echinocandins, but 3 isolates were nonsusceptible (either intermediate or resistant) to caspofungin (C. albicans, C. guilliermondii, and C. krusei), anidulafungin (C. albicansand C. guilliermondii), and micafungin (C. albicans). However, when the intrinsically resistant non-Candidaisolates were included, the rate of echinocandin nonsusceptibility reached 1.8%. In summary, the SYO method proved to be able to detect yeast species showing antifungal resistance or reduced susceptibility.

ABSTRACTWe compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients =16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated =5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P= 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P= 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P= 0.003), Gram-positive cocci (P= 0.013), and Escherichia coli(P= 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P= 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h; P= 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h; P< 0.001). Similarly high percentages of microorganisms in BacT/Alert and Bactec cultures (93.8% and 93.3%, respectively) were identified by direct matrix-assisted laser desorption ionization–time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings.

ABSTRACTOur aims were to identify (i) risk factors associated with the acquisition of multidrug-resistant (MDR, to 3 or more classes of antimicrobials) Proteus mirabilisisolates responsible for bloodstream infections (BSIs) and (ii) the impact on mortality of such infections. Risk factors for acquiring MDR P. mirabilisBSIs were investigated in a case-case-control study; those associated with mortality were assessed by comparing survivors and nonsurvivors in a cohort study. The population consisted of 99 adult inpatients with P. mirabilisBSIs identified by our laboratory over an 11-year period (1999 to 2009), 36 (33.3%) of which were caused by MDR strains, and the overall 21-day mortality rate was 30.3%. Acquisition of an MDR strain was independently associated with admission from a long-term care facility (odds ratio [OR], 9.78; 95% confidence interval [CI], 1.94 to 49.16), previous therapy with fluoroquinolones (OR, 5.52; 95% CI, 1.30 to 23.43) or oxyimino-cephalosporins (OR, 4.72; 95% CI, 1.31 to 16.99), urinary catheterization (OR, 3.89; 95% CI, 1.50 to 10.09), and previous hospitalization (OR, 2.68; 95% CI, 10.4 to 6.89). Patients with MDR P. mirabilisBSIs received inadequate initial antimicrobial therapy (IIAT, i.e., treatment with drugs to which the isolate displayed in vitroresistance) more frequently than those with non-MDR infections; they also had increased mortality and (for survivors) longer post-BSI-onset hospital stays. In multivariate regression analysis, 21-day mortality was associated with septic shock at BSI onset (OR, 12.97; 95% CI, 32.2 to 52.23), P. mirabilisisolates that were MDR (OR, 6.62; 95% CI, 16.4 to 26.68), and IIAT (OR, 9.85; 95% CI, 26.7 to 36.25), the only modifiable risk factor of the 3. These findings can potentially improve clinicians' ability to identify P. mirabilisBSIs likely to be MDR, thereby reducing the risk of IIAT—a major risk factor for mortality in these cases—and facilitating the prompt implementation of appropriate infection control measures.

ABSTRACTWe evaluated the reliability of the Bruker Daltonik's MALDI Biotyper system in species-level identification of yeasts directly from blood culture bottles. Identification results were concordant with those of the conventional culture-based method for 95.9% of Candida albicans(187/195) and 86.5% of non-albicans Candidaspecies (128/148). Results were available in 30 min (median), suggesting that this approach is a reliable, time-saving tool for routine identification of Candidaspecies causing bloodstream infection.

We tested the activities of anidulafungin and other antifungal agents against clinical isolates of different fungal species. For Candida species, high sessile MIC90s (SMIC90s) were obtained for fluconazole, voriconazole, and amphotericin B, whereas the anidulafungin SMIC90s were very low, as were those for caspofungin. Comparatively, for Aspergillus species, higher SMIC90values were obtained not only for amphotericin B and voriconazole but also for the echinocandins.

ABSTRACTWe tested the activities of anidulafungin and other antifungal agents against clinical isolates of different fungal species. For Candidaspecies, high sessile MIC90s (SMIC90s) were obtained for fluconazole, voriconazole, and amphotericin B, whereas the anidulafungin SMIC90s were very low, as were those for caspofungin. Comparatively, for Aspergillusspecies, higher SMIC90values were obtained not only for amphotericin B and voriconazole but also for the echinocandins.

ABSTRACTThe Vitek 2 yeast susceptibility test was evaluated by testing 122 Candidaisolates against fluconazole and voriconazole. Excellent categorical agreement with the CLSI broth microdilution method was observed (97.5% for both the azoles). Moreover, the Vitek 2 system was able to identify all but 2 of 59 investigated fluconazole-resistant organisms.

ABSTRACTExtended-spectrum-β-lactamase (ESBL)-producing strains of Escherichia coliare a significant cause of bloodstream infections (BSI) in hospitalized and nonhospitalized patients. We previously showed that delaying effective antimicrobial therapy in BSI caused by ESBL producers significantly increases mortality. The aim of this retrospective 7-year analysis was to identify risk factors for inadequate initial antimicrobial therapy (IIAT) (i.e., empirical treatment based on a drug to which the isolate had displayed in vitro resistance) for inpatients with BSI caused by ESBL-producing E. coli. Of the 129 patients considered, 56 (43.4%) received IIAT for 48 to 120 h (mean, 72 h). Independent risk factors for IIAT include an unknown BSI source (odds ratios [OR], 4.86; 95% confidence interval [CI], 1.98 to 11.91; P= 0.001), isolate coresistance to ≥3 antimicrobials (OR, 3.73; 95% CI, 1.58 to 8.83; P= 0.003), hospitalization during the 12 months preceding BSI onset (OR, 3.33; 95% CI, 1.42 to 7.79; P= 0.005), and antimicrobial therapy during the 3 months preceding BSI onset (OR, 2.65; 95% CI, 1.11 to 6.29; P= 0.02). IIAT was the strongest risk factor for 21-day mortality and significantly increased the length of hospitalization after BSI onset. Our results underscore the need for a systematic approach to the management of patients with serious infections by ESBL-producing E. coli. Such an approach should be based on sound, updated knowledge of local infectious-disease epidemiology, detailed analysis of the patient's history with emphasis on recent contact with the health care system, and aggressive attempts to identify the infectious focus that has given rise to the BSI.

ABSTRACTBloodstream infections (BSI) caused by extended-spectrum β-lactamase (ESBL)-producing organisms markedly increase the rates of treatment failure and death. We conducted a retrospective cohort analysis to identify risk factors for mortality in adult in-patients with BSI caused by ESBL-producing Enterobacteriaceae(ESBL-BSI). Particular attention was focused on defining the impact on the mortality of inadequate initial antimicrobial therapy (defined as the initiation of treatment with active antimicrobial agents >72 h after collection of the first positive blood culture). A total of 186 patients with ESBL-BSI caused by Escherichia coli(n= 104), Klebsiella pneumoniae(n= 58), or Proteus mirabilis(n= 24) were identified by our microbiology laboratory from 1 January 1999 through 31 December 2004. The overall 21-day mortality rate was 38.2% (71 of 186). In multivariate analysis, significant predictors of mortality were inadequate initial antimicrobial therapy (odds ratio [OR] = 6.28; 95% confidence interval [CI] = 3.18 to 12.42; P< 0.001) and unidentified primary infection site (OR = 2.69; 95% CI = 1.38 to 5.27; P= 0.004). The inadequately treated patients (89 of 186 [47.8%]) had a threefold increase in mortality compared to the adequately treated group (59.5% versus 18.5%; OR = 2.38; 95% CI = 1.76 to 3.22; P< 0.001). The regimens most commonly classified as inadequate were based on oxyimino cephalosporin or fluoroquinolone therapy. Prompt initiation of effective antimicrobial treatment is essential in patients with ESBL-BSI, and empirical decisions must be based on a sound knowledge of the local distribution of pathogens and their susceptibility patterns.

ABSTRACTNosocomial Candidabloodstream infections rank among infections with highest mortality rates. A retrospective cohort analysis was conducted at Catholic University Hospital to estimate the risk factors for mortality of patients with candidemia. We reviewed records for patients with a Candidabloodstream infection over a 5-year period (January 2000 through December 2004). Two hundred ninety-four patients (42.1% male; mean age ± standard deviation, 65 ± 12 years) were studied. Patients most commonly were admitted with a surgical diagnosis (162 patients [55.1%]), had a central venous catheter (213 [72.4%]), cancer (118 [40.1%]), or diabetes (58 [19.7%]). One hundred fifty-four (52.3%) patients died within 30 days. Of 294 patients, 168 (57.1%) were infected by Candida albicans, 64 (21.7%) by Candida parapsilosis, 28 (9.5%) by Candida tropicalis, and 26 (8.8%) by Candida glabrata. When fungal isolates were tested for biofilm formation capacity, biofilm production was most commonly observed for isolates of C. tropicalis(20 of 28 patients [71.4%]), followed by C. glabrata(6 of 26 [23.1%]), C. albicans(38 of 168 [22.6%]), and C. parapsilosis(14 of 64 [21.8%]). Multivariable analysis identified inadequate antifungal therapy (odds ratio [OR], 2.35; 95% confidence interval [95% CI], 1.09 to 5.10; P= 0.03), infection with overall biofilm-forming Candidaspecies (OR, 2.33; 95% CI, 1.26 to 4.30; P= 0.007), and Acute Physiology and Chronic Health Evaluation III scores (OR, 1.03; 95% CI, 1.01 to 1.15; P< 0.001) as independent predictors of mortality. Notably, if mortality was analyzed according to the different biofilm-forming Candidaspecies studied, only infections caused by C. albicans(P< 0.001) and C. parapsilosis(P= 0.003) correlated with increased mortality. Together with well-established factors, Candidabiofilm production was therefore shown to be associated with greater mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.

Nosocomial Candida bloodstream infections rank among infections with highest mortality rates. A retrospective cohort analysis was conducted at Catholic University Hospital to estimate the risk factors for mortality of patients with candidemia. We reviewed records for patients with a Candida bloodstream infection over a 5-year period (January 2000 through December 2004). Two hundred ninety-four patients (42.1% male; mean age ± standard deviation, 65 ± 12 years) were studied. Patients most commonly were admitted with a surgical diagnosis (162 patients [55.1%]), had a central venous catheter (213 [72.4%]), cancer (118 [40.1%]), or diabetes (58 [19.7%]). One hundred fifty-four (52.3%) patients died within 30 days. Of 294 patients, 168 (57.1%) were infected by Candida albicans, 64 (21.7%) by Candida parapsilosis, 28 (9.5%) by Candida tropicalis, and 26 (8.8%) by Candida glabrata. When fungal isolates were tested for biofilm formation capacity, biofilm production was most commonly observed for isolates of C. tropicalis (20 of 28 patients [71.4%]), followed by C. glabrata (6 of 26 [23.1%]), C. albicans (38 of 168 [22.6%]), and C. parapsilosis (14 of 64 [21.8%]). Multivariable analysis identified inadequate antifungal therapy (odds ratio [OR], 2.35; 95% confidence interval [95% CI], 1.09 to 5.10; P = 0.03), infection with overall biofilm-forming Candida species (OR, 2.33; 95% CI, 1.26 to 4.30; P = 0.007), and Acute Physiology and Chronic Health Evaluation III scores (OR, 1.03; 95% CI, 1.01 to 1.15; P < 0.001) as independent predictors of mortality. Notably, if mortality was analyzed according to the different biofilm-forming Candida species studied, only infections caused by C. albicans (P < 0.001) and C. parapsilosis (P = 0.003) correlated with increased mortality. Together with well-established factors, Candida biofilm production was therefore shown to be associated with greater mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.

ABSTRACTExtended-spectrum beta-lactamases (ESBLs) are a large, rapidly evolving group of enzymes that confer resistance to oxyimino cephalosporins and monobactams and are inhibited by clavulanate. Rapid reliable detection of ESBL production is a prerequisite for successful infection management and for monitoring resistance trends and implementation of intervention strategies. We evaluated the performance of the new VITEK 2 ESBL test system (bioMe´rieux, Inc, Hazelwood, Mo.) in the identification of ESBL-producing Enterobacteriaceaeisolates. We examined a total of 1,129 clinically relevant Enterobacteriaceaeisolates (including 218 that had been previously characterized). The ESBL classification furnished by the VITEK 2 ESBL test system was concordant with that of the comparison method (molecular identification of beta-lactamase genes) for 1,121 (99.3%) of the 1,129 isolates evaluated. ESBL production was correctly detected in 306 of the 312 ESBL-producing organisms (sensitivity, 98.1%; positive predictive value, 99.3%). False-positive results emerged for 2 of the 817 ESBL-negative isolates (specificity, 99.7%; negative predictive value, 99.3%). VITEK 2 ESBL testing took 6 to 13 h (median, 7.5 h; mean ± SD, 8.2 ± 2.39 h). This automated short-incubation system appears to be a rapid and reliable tool for routine identification of ESBL-producing isolates of Enterobacteriaceae.

Objectives: Several studies have documented the potent in vitro activity of caspofungin against Candida spp. This is of special concern for Candida glabrata infections that are often resistant to many azole antifungal agents and, consequently, difficult to treat. The aim of the present study was to expand the data on the in vitro activity of caspofungin against azole-resistant isolates of C. glabrata.Methods: A total of 50 clinical isolates of C. glabrata were tested for susceptibility to caspofungin. The isolates were cross-resistant to multiple azoles, including fluconazole, itraconazole, ketoconazole and voriconazole. Expression of the resistance-related CgCDR1 and CgCDR2 genes was evaluated by quantitative RT–PCR analysis. The MICs of caspofungin were determined by using the National Committee for Clinical Laboratory Standards M27-A2 reference method.Results: C. glabrata isolates exhibited increased expression of the CDR efflux pump(s), and this was in accordance with their high-level azole resistance. In contrast, all the isolates were highly susceptible to caspofungin (100% of isolates were inhibited at ≤1 mg/L).Conclusions: Our results represent further evidence for the excellent antifungal potency of caspofungin, particularly against C. glabrata isolates expressing cross-resistance to azoles.

ABSTRACTWe have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene AFR1in Cryptococcus neoformansis involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of AFR1in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of C. neoformans, strain BPY22; an afr1mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an AFR1-overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the AFR1gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of C. neoformans.

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene AFR1 in Cryptococcus neoformans is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of AFR1 in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of C. neoformans, strain BPY22; an afr1 mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an AFR1-overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the AFR1 gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of C. neoformans.

ABSTRACTThe increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabratainfections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1and CgCDR2genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrataisolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2(which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1and CgCDR2exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11and analysis of its expression showed no mutation or upregulation in any C. glabrataisolate, suggesting that CgERG11is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

ABSTRACTProduction of extended-spectrum ß-lactamases (ESBLs) is an important mechanism of ß-lactam resistance in Enterobacteriaceae. Identification of ESBLs based on phenotypic tests is the strategy most commonly used in clinical microbiology laboratories. The Phoenix ESBL test (BD Diagnostic Systems, Sparks, Md.) is a recently developed automated system for detection of ESBL-producing gram-negative bacteria. An algorithm based on phenotypic responses to a panel of cephalosporins (ceftazidime plus clavulanic acid, ceftazidime, cefotaxime plus clavulanic acid, cefpodoxime, and ceftriaxone plus clavulanic acid) was used to test 510 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Providencia stuartii, Morganella morganii, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Citrobacter freundii, and Citrobacter koseri. Of these isolates, 319 were identified as ESBL producers, and the remaining 191 were identified as non-ESBL producers based on the results of current phenotypic tests. Combined use of isoelectric focusing, PCR, and/or DNA sequencing demonstrated that 288 isolates possessed blaTEM-1- and/or blaSHV-1-derived genes, and 28 had a blaCTX-Mgene. Among the 191 non-ESBL-producing isolates, 77 isolates produced an AmpC-type enzyme, 110 isolates possessed TEM-1, TEM-2, or SHV-1 ß-lactamases, and the remaining four isolates (all K. oxytocastrains) hyperproduced K1 chromosomal ß-lactamase. The Phoenix ESBL test system gave positive results for all the 319 ESBL-producing isolates and also for two of the four K1-hyperproducing isolates of K. oxytoca. Compared with the phenotypic tests and molecular analyses, the Phoenix system displayed 100% sensitivity and 98.9% specificity. These findings suggest that the Phoenix ESBL test can be a rapid and reliable method for laboratory detection of ESBL resistance in gram-negative bacteria.

ABSTRACTSpontaneous chylothorax is rare in adults. We present an unusual case that was complicated by Prevotella biviaempyema. Full recovery was achieved with chest tube drainage and prompt treatment with intravenous clindamycin.

ABSTRACTKPC-type carbapenemases are emerging in Klebsiella pneumoniaeand other Gram-negative pathogens worldwide. Rapid and sensitive detection of these resistance determinants has become relevant to clinical management and infection control. We evaluated the bioMérieux EasyQ real-time PCR assay for blaKPCdetection with 300 members of the Enterobacteriaceae, including 29 control strains producing known carbapenemases and 271 nonreplicate clinical isolates. The EasyQ assay correctly detected all of the 111 isolates harboring blaKPCgenes, with no false positives, and results were available within 2 h.

We directly tested 484 organisms from clinical (n= 310) and simulated (n= 174) positive blood cultures using the NG-Test Carba 5 assay for carbapenemase-producing Enterobacteralesdetection. The assay identified all but 4 of the KPC (170/171), OXA-48-like (22/22), VIM (19/21), and NDM (14/15) producers with no false positives.

Introduction Staphylococcus lugdunensis is an unusually virulent coagulase-negative staphylococcus that has rarely been implicated in central nervous system infections.Case presentation Two children hospitalized in the Neurosurgery Unit developed ventriculitis caused by methicillin-resistant Staphylococcus lugdunensis following placement of external ventriculostomy drains. The causative organisms were identified by molecular studies. The patients recovered without significant sequelae after high doses of intrathecal vancomycin.Conclusion Distinguishing Staphylococcus lugdunensis from other coagulase-negative staphylococcus species is crucial because it carries a substantial risk for severe central nervous system infections displayed by patients with implanted cerebrospinal fluid devices. Clinicians should not underestimate the importance of the isolation of this species from cerebrospinal fluid specimens.