Isolation of KPC 3-producing Enterobacter aerogenes in a patient colonized by MDR Klebsiella pneumoniae.

We describe the interspecies transmission of the plasmid-mediated blaKPC-3 gene, which confers carbapenem resistance, between clinically relevant gram-negative bacteria in a single patient. A KPC-3 producing Enterobacter aerogenes was isolated from a hospitalized patient previously colonized and then infected by a Klebsiella pneumoniae ST101 carrying the blaKPC-3 gene. The strains showed identical plasmids. Since intense horizontal exchanges among bacteria can occur in the gut, clinicians should be aware that patients colonized by carbapenem-resistant K. pneumoniae could become carriers of other carbapenem-resistant Enterobacteriaceae.

Clostridium difficile infection in Italian urban hospitals: data from 2006 through 2011.

BACKGROUND: In developed countries, Clostridium difficile infection (CDI) represents an emerging threat in terms of morbidity and mortality rates. In our country limited CDI epidemiological data can be found. METHODS: Stool samples tested for C. difficile toxins from January 2006 to December 2011 in 5 large hospitals in Rome, Italy, were considered in the analysis. Repeated samples taken ≤ 2 months after a positive result were excluded. RESULTS: A total of 402 CDI episodes were identified. The incidence of CDI episodes progressively increased from 0.3 in 2006 to 2.3 per 10,000 patient-days in 2011. CDI episodes mostly occurred in patients > 60 years of age (77%). The >80 year-old age class reported the highest percentage of CDI episodes on tested samples (16%). Eighty percent (80%) of CDI episodes occurred in medical wards followed by surgery (10.2%) and intensive care units (9.8%). CONCLUSIONS: A significant increasing incidence of CDI episodes over the study period was observed during the years (p<.001), particularly in the older age groups. Medical wards experienced the highest number of CDI episodes as compared to intensive care and surgical wards. The increasing rate of CDI episodes over the last six years in our country, is alarming; urgent improvements in the surveillance systems and control programs are advisable.

Giardia intestinalis escapes oxidative stress by colonizing the small intestine: A molecular hypothesis.

Giardia intestinalis is the microaerophilic protozoon causing giardiasis, a common infectious intestinal disease. Giardia possesses an O(2) -scavenging activity likely essential for survival in the host. We report that Giardia trophozoites express the O(2) -detoxifying flavodiiron protein (FDP), detected by immunoblotting, and are able to reduce O(2) to H(2) O rapidly (∼3 µM O(2) × min × 10(6) cells at 37 °C) and with high affinity (C(50) = 3.4 ± 0.7 µM O(2)). Following a short-term (minutes) exposure to H(2) O(2) ≥ 100 µM, the O(2) consumption by the parasites is irreversibly impaired, and the FDP undergoes a degradation, prevented by the proteasome-inhibitor MG132. Instead, H(2) O(2) does not cause degradation or inactivation of the isolated FDP. On the basis of the elevated susceptibility of Giardia to oxidative stress, we hypothesize that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress.

Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis.

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN=116+/-10s(-1) at 1microM NO, T=37 degrees C). The activity is [O(2)]-dependent and characterized by an apparent K(M,O2)=22+/-7microM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with 5mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.

Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25-28S rRNA gene.

Clinically relevant yeasts are conventionally identified by a combination of phenotypic tests, which occasionally provide ambiguous results for atypical isolates or uncommon species. In this study, we evaluate a direct polymerase chain reaction-sequencing method, which exploits sequence divergence in the hypervariable D2 region of the large subunit of the 25-28S ribosomal RNA (rRNA) gene for identification of facultative pathogenic asco- and basidiomycota. A panel of 53 yeasts, including 40 clinical isolates and 13 reference strains representative of some clinically relevant taxa, was investigated by combining standard phenotypic tests with commercial identification systems (RapID, API 20C AUX), and results were compared with the taxonomic allocations inferred by D2 sequence analysis. Species-level resolution was achieved for almost all (52/53) strains by combining internet-based D2 sequence homology (BLAST and FASTA) searches in free-access synchronised databases with phylogenetic analysis. The phylogenetic information carried by the short D2 sequence substantiates a pattern of molecular evolution, which is similar to that inferred from analysis of the larger D1/D2 region, and consistent with previously published 25-28S rRNA phylogenetic architectures of facultative pathogenic yeast, including recently identified species. Inconsistency between conventional and molecular identification results was observed for 11/53 strains, likely on account of the ambiguous interpretation of phenotypic tests.

Ceftaroline Plus Ampicillin Against Gram-Positive Organisms: Results from E-Test Synergy Assays.

In an era of increasing drug resistance and limited numbers of antimicrobials in the drug production pipeline, healthcare-associated infections represent a growing public health threat. When therapeutic options are limited, clinicians often resort to using antimicrobial combinations that produce a synergistic effect on the target pathogen. Novel antibiotics are therefore welcome in the daily practice of medicine. For example, ceftaroline is a broad-spectrum cephalosporin active against a variety of bacteria, including methicillin-resistant Staphylococcus aureus, but with limited activity against enterococci, particularly Enterococcus faecium. In this study, we tested the efficacy of ceftaroline against clinical isolates of gram-positive bacteria (S. aureus, Enterococcus faecalis, and E. faecium) by the broth microdilution and E-test assays, and then evaluated the synergistic effect of ceftaroline and ampicillin using the E-test method. The time-kill assay was used to confirm the data on selected strains. This drug combination has been recently shown to be effective against E. faecalis and could offer the advantage of cost-effectiveness (compared to other synergistic associations) as well as good tolerability. The E-test was chosen because of its relative simplicity of use that makes it suitable for routine clinical laboratories as a quick tool to guide clinicians when confronted with difficult-to-treat infections that may require an empirical approach. Our results indicate the presence of a synergistic effect of ceftaroline and ampicillin on most of the strains used, especially E. faecium and E. faecalis. The fact that two of those Enterococcus strains were vancomycin resistant suggests that the possible use of this combination for combating the spread of vancomycin-resistant enterococci should be explored.

Evaluation of the inactivation effect of Triton X-100 on Ebola virus infectivity.

BACKGROUND: The recent Ebola virus disease outbreak occurred in West Africa since December 2013 highlighted the need of appropriate virus inactivation procedures to be set up to allow the necessary processing of specimens outside BSL-4 facilities and to perform laboratory tests without affecting clinical decisions. For this purpose, international guidelines suggest the pre-treatment of the samples with Triton X-100. OBJECTIVES: Due to the limited scientific evidence about the efficacy of Triton X-100 on enveloped-viruses, the aim of this work was to evaluate the effect of Triton X-100 on the virus infectivity and to establish the optimal conditions for its use. STUDY DESIGN: We evaluated the effect of Triton X-100 on the infectivity of enveloped-viruses such as West Nile virus (WNV) and Ebola virus (EBOV) at different experimental conditions. The residual virus infectivity was measured by limiting dilution assay on Vero E6 cells. Repeated experiments were performed, as specified, and for the titration of residual infectivity each dilution was tested in triplicate. RESULTS: Results obtained with WNV showed that infectivity was reduced by 6 Logs, after 1h of treatment with different concentrations of Triton X-100 (ranging from 0.5% to 0.05%). This effect was not time-dependent using 0.1% Triton X-100. Subsequently, we applied the method on EBOV and one hour exposure to 0.1% Triton X-100 strongly affected EBOV infectivity (4 Logs of infectivity reduction). CONCLUSIONS: We report that Triton X-100, when used alone, is able to strongly reduce the infectivity of a classical enveloped virus such as WNV and we provide, for the first time, scientific evidence that 0.1% Triton X-100 efficaciously affect Ebola virus infectivity. Even though a complete virus inactivation is not achieved, Triton X-100 certainly can contribute to mitigate the risk for the workers of accidental infection and improve the overall safety of the laboratory procedures. Further studies must be performed to deeply investigate alternative solutions able to balance higher level of safety and good performance in clinical chemistry and hematology parameters analysis, necessary for the appropriate and effective management of EVD patients.

Invasive Candidiasis due to Candida Norvegensis in a Liver Transplant Patient: Case Report and Literature Review.

Candida norvegensis is an emerging fluconazole-resistant pathogen isolated in most cases from skin and mucous membranes of immunocompromized patients. Documented invasive candidiasis (IC) due to C. norvegensis has been rarely reported, thus the clinical features of patients at risk for this pathogen are poorly defined. We report a liver transplant patient who developed IC due to C. norvegensis and review other cases of C. norvegensis IC published in the literature.

In vitro activity of doripenem in combination with various antimicrobials against multidrug-resistant Acinetobacter baumannii: possible options for the treatment of complicated infection.

AIMS: The aim of this study was to evaluate the in vitro activity of doripenem (DOR) alone and in combination with a variety of commonly used anti-Acinetobacter chemotherapeutic agents against 22 primary multidrug-resistant (MDR) Acinetobacter baumannii isolates (including 17 isolates that were resistant to DOR) from Intensive Care Unit patients. Antibiotic interactions were evaluated using the chequerboard method and the time-kill assay. RESULTS: Considering all antimicrobials in combination with DOR, chequerboard analysis showed synergy in 13 A. baumannii strains (54.2%). Seven strains (29.2%) showed ≥2 synergistic interactions. DOR showed synergy in combination with tigecycline (TIG) (eight strains), colistin (COL) (eight strains), amikacin (AMK) (four strains), ampicillin/sulbactam (two strains), and rifampicin (one strain). Remarkably, synergistic effects were detected only in DOR nonsusceptible strains. Time-kill assays confirmed synergy in eight isolates (giving 10 synergistic interactions) for DOR in combination with TIG (n=4), COL (n=5), and AMK (n=1). No antagonistic interactions were observed with both methods. CONCLUSIONS: This study demonstrates the in vitro synergistic activity of DOR in combination with TIG, COL, and AMK against DOR-resistant A. baumannii strains, opening the way to in vivo assessment of novel combination therapies for treatment of infections caused by MDR A. baumannii.

Cystic echinococcosis in a single tertiary care center in Rome, Italy.

BACKGROUND: Cystic echinococcosis (CE) is a chronic, clinically complex, and neglected disease. Its prevalence in Italy, a country of medium to high endemicity, remains poorly defined, as notification has long ceased to be mandatory. METHODS: We set up a retrospective cohort study involving all CE patients followed at our institute between January 2005 and December 2012. Demographical and clinical features were recorded and analyzed. RESULTS: CE was found in 28 patients (64.3%), mostly Italians from the central regions (50%), followed by subjects from the islands (33.3%) and Southern Italy (16.7%). Their median age was 45 years (IQR: 38.5-66.5), with Eastern Europeans being significantly younger (28 years, IQR: 19-39) than other patients (P ≤ 0.0001). A total of 149 cysts, mostly with hepatic localization (96%), were described. Based on the WHO classification, the cysts were mainly small (80.5%) and active (CE1 (73.8%); CE2 (7.4%)). Active cysts were more common in Eastern Europeans (85.7%) than Italians (66.7%). CONCLUSION: Our data confirm CE occurrence in Italy. We emphasize the importance to have a national CE registry, opportunely recently introduced. This is essential to assess CE prevalence in this country, implement appropriate control measures, and improve patient management.

Salmonella enterica ssp. arizonae infection in a 43-year-old Italian man with hypoglobulinemia: a case report and review of the literature.

INTRODUCTION: Salmonella enterica ssp. arizonae is an uncommon human pathogen with serious infections reported in immunocompromised hosts. In Europe, only a few cases have been described. Patients with this infection usually have a history of contact with reptiles or travel abroad. We present a case report of infection in a patient with hypoglobulinemia and a literature review. CASE PRESENTATION: We describe the case of a 43-year-old Caucasian Italian man with hypoglobulinemia who presented to our hospital with sepsis and diarrhea. A stool culture yielded S. enterica ssp. arizonae. Our patient was treated with oral ciprofloxacin and made a full recovery. We also present a review of the cases of S. enterica ssp. arizonae infections previously reported in Europe. CONCLUSIONS: The majority of infections from S. enterica ssp. arizonae occur in patients who are immunocompromised. Data from the literature suggests that it may be difficult to eradicate the bacteria and thus, prolonged antibiotic courses are often used. It would be advisable for clinicians to investigate for pre-existing immune dysfunction if S. enterica ssp. arizonae is isolated. In Italy, although there have only been a few cases, the likely route of transmission remains unclear and requires further surveillance.

The superoxide reductase from the early diverging eukaryote Giardia intestinalis.

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(•-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(•-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

HIV-related pneumococcal lung disease: does highly active antiretroviral therapy or bacteremia modify radiologic appearance?

We reviewed chest radiographs of 57 HIV-infected patients with pulmonary diseases in whom Streptococcus pneumoniae was the sole respiratory pathogen isolated to evaluate whether highly active antiretroviral therapy (HAART) or bacteremia modify radiographic appearance. Pneumococcal lung disease presented as lobar pneumonia in 40% of the cases, 54% of whom were on HAART; as bronchopneumonia in 42%, 58% on HAART; as interstitial infiltrates in 17%, 60% on HAART. Bacteremia was observed 38 times in 23 patients with CD4 less than 200/mm(3), and in 15 with CD4 greater than 200/mm(3) (p > 0.05). HAART does not significantly influences radiographic appearances of lung disease caused by Streptococcus pneumoniae (p > 0.05). Immunosuppression induced by HIV infection was a major risk factor for development of pneumococcal lung disease (p = 0.04) and influences radiographic appearance; bronchopneumonia (p = 0.006), in particular multifocal (p = 0.008), which was more frequent in subjects with CD4 less than 200/mm(3). Bacteremia influences radiographic appearance of pneumococcal lung disease; lobar pneumonia was more frequent (p = 0.003), and considering CD4 cell count, was more frequent if CD4 cell count was above 200/mm(3). An original finding of this study was the frequency of interstitial changes. This pattern of pneumonia, found in 17% of our patients, could represent a difference between HIV-seropositive and -seronegative subject in displaying pneumococcal lung disease.