Accurate identification of members of the Burkholderia cepacia complex in cystic fibrosis sputum.

UNLABELLED: The Burkholderia cepacia complex (BCC) is a group of closely related species which includes opportunistic pathogens causing chronic respiratory infections in immunocompromised patients, or individuals affected by cystic fibrosis (CF). Other Burkholderia species causing infection in the CF population are Burkholderia gladioli and Burkholderia pseudomallei. Traditional phenotypic analyses have been demonstrated to be inadequate for reliable identifications of isolates of BCC and B. gladioli. A pan-genomic analysis approach was used to design species-specific probes for Burkholderia cenocepacia, B. cepacia, Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia dolosa, Burkholderia pyrrocinia and B. gladioli. Multiplex real-time PCR assay was developed and tested using sputum specimens collected from CF patients spiked with Burkholderia species. The assay exhibited 100% sensitivity for all eight target species and detected 10(2) to 10(3)  CFU ml(-1) when applied to spiked sputum. Our PCR assay resulted highly specific for each of the Burkholderia species tested, allowing discrimination among Burkholderia and non-Burkholderia pathogens. Analysis carried out on 200 sputa positive for the presence of Burkholderia revealed that PCR assay and recA sequencing were fully comparable for identification of Burkholderia at the level of species. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia cepacia complex (BCC) has a complex taxonomic organization and its identification is a challenge for microbiology laboratories. Nonidentification or misidentification of BCC isolates represent a problem in epidemiology and treatment of cystic fibrosis patients. The high specificity and sensitivity of the multiplex Real-time PCR assay developed in this study indicates its potential to be a rapid and reliable method for the detection of Burkholderia at the level of species from sputum samples of cystic fibrosis patients.

Toll-like receptor kinetics in septic shock patients: a preliminary study.

The aim of this study is to evaluate some inflammatory parameter changes in septic shock patients and their possible correlation with clinical outcome, in particular when continuous veno-venous hemofiltration (CVVH) treatment is required. Considering the objective difficulty in enrolling this kind of patient, a preliminary study was initiated on seventeen septic shock patients admitted to a medical and surgical ICU. The mRNA expression of Toll-like receptor (TLR)-1, TLR-2, TLR-4, TLR-5, TLR-9, TNFα, IL-8 and IL-1ß was assessed, the plasmatic concentrations of IL-18, IL-2, IL-10 and TNFα were measured on the day of sepsis diagnosis and after 72 h. In those patients who developed acute renal failure unresponsive to medical treatment and who underwent CVVH treatment the same parameters were measured every 24 h during CVVH and after completion of the treatment. On sepsis diagnosis, gene expression of TLRs was up-regulated compared to the housekeeping gene in all the patients. After 72 h, in 35% of the patients a down-regulation of these genes was found compared to day 1, but it was not associated with a reduction of cytokine serum levels or improved clinical signs, better outcome or reduced mortality. After high volume hemofiltration treatment, cytokine serum levels and TLR expression were not significantly modified. In conclusion, considering the not numerous number of cases, from our preliminary study, we cannot certainly correlate TLR over-expression in septic shock patients with severity or outcome scores.

MALDI-TOF mass spectrometry and microsatellite markers to evaluate Candida parapsilosis transmission in neonatal intensive care units.

Recent studies on outbreaks of Candida showed an increased incidence of bloodstream infections in neonatal intensive care units (NICUs) caused by C. parapsilosis species, highlighting the need for the proper identification and epidemiology of these species. Several systems are available for molecular epidemiological and taxonomic studies of fungal infections: pulsed-field gel electrophoresis (PFGE) represents the gold standard for typing, but is also one of the most lengthy and expensive, while simple sequence repeats (SSRs) is based on polymerase chain reaction (PCR) amplification and is, therefore, faster. Only recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify and type microorganisms involved in nosocomial outbreaks. In our study, 19 strains of C. parapsilosis isolated from the blood cultures of neonates admitted to the University Hospital Federico II were genotyped by the amplification of eight SSR markers and by MALDI-TOF MS. Electrophoretic and spectrometric profile results were compared in order to identify similarities among the isolates and to study microevolutionary changes in the C. parapsilosis population. The discriminatory power and the unweighted pair group method with arithmetic mean (UPGMA) dendrograms generated were compared in order to evaluate the correlation of the groups established by the analysis of the clusters by both methods. Both methods were rapid and effective in highlighting identical strains and studying microevolutionary changes in the population. Our study evidenced that mass spectroscopy is a useful technique not only for the identification but also for monitoring the spread of strains, which is critical to control nosocomial infections.

Achromobacter xylosoxidans respiratory tract infection in cystic fibrosis patients.

The aims of this study were to evaluate the frequency of Achromobacter xylosoxidans infection in a cohort of cystic fibrosis patients, to investigate antimicrobial sensitivity, to establish possible clonal likeness among strains, and to address the clinical impact of this infection or colonization on the general outcome of these patients. The study was undertaken between January 2004 and December 2008 on 300 patients receiving care at the Regional Cystic Fibrosis Center of the Naples University "Federico II". Sputum samples were checked for bacterial identification. For DNA fingerprinting, pulsed-field gel electrophoresis (PFGE) was carried out. Fifty-three patients (17.6%) had at least one positive culture for A. xylosoxidans; of these, 6/53 (11.3%) patients were defined as chronically infected and all were co-colonized by Pseudomonas aeruginosa. Of the patients, 18.8% persistently carried multidrug-resistant isolates. Macrorestriction analysis showed the presence of seven major clusters. DNA fingerprinting also showed a genetic relationship among strains isolated from the same patients at different times. The results of DNA fingerprinting indicate evidence of bacterial clonal likeness among the enrolled infected patients. We found no significant differences in the forced expiratory volume in 1 s (FEV(1)) and body mass index (BMI) when comparing the case group of A. xylosoxidans chronically infected patients with the control group of P. aeruginosa chronically infected patients.

Candida albicans identification: comparison among nine phenotypic systems and a multiplex PCR.

BACKGROUND: Candida albicans is the most common fungal pathogen isolated from clinical samples and is also the most common yeast species carried as a commensal by healthy individuals although some non-C. albicans species account for an important number of infections. OBJECTIVES: To compare nine phenotypic systems for C. albicans identification [API 20C AUX; RapID Yeast Identification panel (RYIP); Vitek2 ID-YST system; chromogenic media, CHRO-Magar, Oxoid Chromogenic Candida Agar (OCCA), Candida ID2, Candida Identification Agar, CandiSelect 4, and Chromalbicans Agar] with multiplex PCR. PATIENTS/METHODS: A collection of 390 yeast strains was obtained by routine isolation from oral and vaginal swabs. All of the yeasts isolated were tested for germ tube formation, and then submitted to a multiplex PCR protocol tested in previous studies, and to nine phenotypical commercial methods, together with the reference ATCC strains. Comparison was limited to the ability of the tests to identify C. albicans. RESULTS: 253 isolates were provisionally identified as C. albicans by germ tube, and their identities were further confirmed with the multiplex PCR. Sensitivity of phenotypical systems ranged from 81.9% (Vitek2) to 87.7% (Candida ID2 e CHROMagar). For specificity, the highest value was 96.8% for Candida ID2, and the lowest value (75.1%) was for Chromalbicans Agar. CONCLUSIONS: Although with differences in discriminatory power, the methods tested showed overall acceptable levels of sensitivity and specificity respect to the multiplex PCR; therefore, all could be useful for C. albicans identification where molecular differentiation is not available.

A strain of Salmonella enterica serovar Virchow isolated in Turkey and carrying a CTX-M-3 extended-spectrum beta-lactamase.

A multiply resistant strain of Salmonella enterica subsp. enterica serovar Virchow was isolated in November 2002 from a catheterized patient admitted to the SSK Training Hospital in Ankara, Turkey. This isolate showed an antimicrobial susceptibility pattern compatible with the presence of a CTX-M-type ESBL, namely resistance to cefotaxime, aztreonam and cefepime, and intermediate susceptibility to ceftazidime. On checking for the presence of the bla(TEM), bla(SHV), and bla(CTX-M )resistance genes by PCR, negative results were obtained with the primers specific for SHV and TEM genes, while positive results were obtained with those specific for CTX-M-type genes. After sequencing, the beta-lactamase was identified as CTX-M-3. This is the first report of this enzyme in Salmonella Virchow and represents a further disquieting threat to the therapy of infections caused by Salmonella isolates.

Antibacterial activity of propolis and its active principles alone and in combination with macrolides, beta-lactams and fluoroquinolones against microorganisms responsible for respiratory infections.

Propolis is produced by bees and is reported to have several pharmaceutical properties. Its antibacterial activity against strains causing upper respiratory tract infections is particularly important: propolis might be used as a therapeutic agent to prevent the bacterial infections that sometimes overlap viral infections. In this study the in vitro activity of both an alcoholic solution and a hydroglyceric extract of propolis, as well as its active principles, was tested against bacteria responsible for respiratory infections (Streptococcus pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis and Streptococcus pyogenes). We also evaluated the in vitro activity of a combination of propolis and its active principles and some beta-lactams, macrolides and fluoroquinolones. Our results, though not demonstrating a clearly synergistic activity between antibiotics and propolis and its constituents, show the possibility of using natural preparations, due to their antimicrobial and anti-inflammatory properties, to enhance antibacterial therapy.

Distribution of mef(A)-containing genetic elements in erythromycin-resistant isolates of Streptococcus pyogenes from Italy.

In total, 124 Streptococcus pyogenes isolates were obtained from throat cultures of different symptomatic patients. All isolates showed M-phenotype macrolide resistance and contained the macrolide efflux gene mef(A). The isolates were screened for the presence and insertion site of mef(A)-containing genetic elements. In 25.8% of the isolates, mef(A) was found to be carried by elements belonging to the Tn1207.3/Phi10394.4 family inserted in the comEC gene, while 74.2% contained chimeric elements with a different genetic structure and chromosomal location, probably associated with the recently described 60-kb tet(O)-mef(A) element.

T serotyping and genomic profile of erythromycin-resistant or -sensitive Streptococcus pyogenes isolated in Campania Region, Italy.

Streptococcus pyogenes causes mild infections, such as pharyngitis, and severe infections, such as necrotizing fascitis. In recent years, erythromycin-resistant strains of S. pyogenes have been reported in many countries. In some areas of Italy, increased rates of erythromycin resistance were first observed in the mid-1990s. Here, we report epidemiological T serotyping, invasiveness, erythromycin resistance, and PFGE patterns of 99 S. pyogenes strains isolated at the Laboratory of Clinical Microbiology of the Second University of Naples, Italy. Regarding T serotyping, 26 of 99 strains were W+, 16 strains were U+, 16 were X+, and 14 were agglutinated by anti T serum. A low percentage revealed Y+. Twelve strains were not T serotyped. PFGE patterns showed species polymorphism; however, inside the various serotypes, we demonstrated a fair homogeneity. No correlation among invasiveness and T serotype or PFGE pattern has been shown. Twenty-two of 99 strains were erythromycin-resistant.

Commonly used antibiotics induce expression of Hsp 27 and Hsp 60 and protect human lymphocytes from apoptosis.

Heat shock proteins (Hsps) are abundant molecular chaperones participating in the cytoprotection. The kinetics of synthesis of Hsps closely correlates with the kinetics of development of resistance to cell death. In this study, we analysed the probable involvement of Hsp 27 and Hsp 60 in the protection of cells undergoing apoptosis. Human lymphocytes cultured in the presence of ampicillin or ceftriaxone produced Hsp 60 and Hsp 27, estimated by immunoblotting in a time-dependent manner and the increased levels of Hsp 60 and Hsp 27 correlated with enhanced resistance of the lymphocytes to apoptosis, as determined by flow cytometry. Cultures treated with ampicillin or ceftriaxone also exhibited smaller numbers of apoptotic cells than untreated cultures when exposed to the apoptosis-inducing agent staurosporine (1 mM). In contrast, cloramphenicol induced the production of only small amounts of Hsp 60, and no resistance apoptosis. Further studies are needed to clarify the potential role of Hsp 27 and Hsp 60 in the resistance of human cells to apoptosis and the effects of antibiotics on this phenomenon.

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in antiretroviral-naive HIV patients in Campania, Italy.

The aim of our study was to determine the prevalence of genotypic resistance to nucleoside analogues and protease inhibitors before and after 1997, the year of introduction of Highly Active Antiretroviral Therapy (HAART) in Campania (Italy). Forty-eight plasma HIV-RNA positive patients who had not been previously treated for HIV infection (naïve) were enrolled in two Divisions of Infectious Diseases. The main demographic characteristics were collected for each subject and the primary mutant genotypes were sought only in HIV-RNA positive patients with viral loads higher than 10,000 copies/ml. The diagnosis of HIV infection dated back to before 1996 for 21 out of 48 patients and to after 2000 for the other 27. INNO-Line Probe Assay (LiPA) HIV-RT and INNO-LiPA HIV protease (Innogenetics, Italy) were used to detect mutations conferring resistance to zidovudine, didanosine, zalcitabine, lamivudine, stavudine, saquinavir, indinavir, rotonavir, nelfinavir and amprenavir. No mutations associated with primary resistance to nucleoside analogues and protease inhibitors were detected in the 21 patients who had acquired HIV infection before 1996, whereas one or more mutations were seen in three of the 27 (11.1%) patients with HIV infection diagnosed after 2000. This study confirms that LiPA is a suitable tool for epidemiological surveys of HIV genotypic primary resistance. Drug-resistant HIV-1 genotypes, resistant both to nucleoside analogues and protease inhibitors, were detected only in subjects who had acquired HIV infection after 2000, most of whom had zidovudine-resistant mutants. These data suggest that the introduction of HAART has brought about the circulation of drug-resistant HIV genotypes.

Chlamydia pneumoniae infections prevent the programmed cell death on THP-1 cell line.

Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in chronic inflammatory disease and atherosclerosis. Here we show that infection with C. pneumoniae protects THP-1 cells against the apoptosis which spontaneously occurs in macrophages in the absence of an activation signal. Analysis by flow cytometry at different post-infection times revealed that 50+/-7% of THP-1 cells were apoptotic at 48 h after onset of the experiments, whereas C. pneumoniae-infected cultures (multiplicity of infection, MOI=30) displayed only 18+/-4% of cells in apoptosis. At MOI=20 and MOI=10 the cells susceptible to apoptosis at 48 h were 28+/-5% and 35+/-6% respectively. Moreover, the results show that heat-inactivated bacteria do not give significant protection against apoptosis, even at higher MOI (MOI=30), while UV-treated Chlamydia did provide a degree of protection against apoptosis. These data suggest that the anti-apoptotic effect of C. pneumoniae requires a heat-labile component released during infection, and that the effect is not lipopolysaccharide-dependent.

p53 and anti-p53 antibodies as possible markers of a switch towards a neoplastic phenotype in patients infected by Helicobacter pylori.

Helicobacter pylori is a definite carcinogen whose mechanism of action is still unknown. The aim of this work was (1) to determine the presence of p53 protein and related antibodies in patients affected by various gastric pathologies and chronically infected with H. pylori, and (2) to try to discover a test to be used as a marker of a possible switch towards a neoplastic phenotype.

Secondary metabolites of Aspergillus exert immunobiological effects on human monocytes.

This project focused on the effects of aflatoxin B1 (AFB1), a food-contaminating mycotoxin produced by fungi, genus Aspergillus, on the release and genetic expression of some important cytokines, i.e., (interleukin-1 alpha (IL-1 alpha), IL-6, tumor necrosis factor-alpha (TNF alpha)) by human monocytes. Monocytes, preincubated for different time periods with concentrations of AFB1 ranging from 0.01 to 1.0 pg/mL, were then activated with bacterial lipopolysaccharide. Cytokine levels were measured by immunoassay and mRNA by cDNA amplification. Pretreatment of monocytes with AFB1 resulted in a decrease in IL-1, IL-6 and TNF alpha release already at a concentration of 0.05 pg/mL. The gene expression of the cytokines considered was drastically affected by treatment with AFB1. In fact, AFB1 completely blocked the transcription of IL-1 alpha, IL-6 and TNF alpha mRNAs, while it did not affect beta-actin mRNA at the concentrations used. It therefore appears that AFB1 exerts its effect on cytokine release through selective inhibition of specific mRNA, without affecting general protein synthesis.