Lucio Romano

A Scan of Chromosome 10 Identifies a Novel Locus Showing Strong Association with Late-Onset Alzheimer Disease

Strong evidence of linkage to late-onset Alzheimer disease (LOAD) has been observed on chromosome 10, which implicates a wide region and at least one disease-susceptibility locus. Although significant associations with several biological candidate genes on chromosome 10 have been reported, these findings have not been consistently replicated, and they remain controversial. We performed a chromosome 10–specific association study with 1,412 gene-based single-nucleotide polymorphisms (SNPs), to identify susceptibility genes for developing LOAD. The scan included SNPs in 677 of 1,270 known or predicted genes; each gene contained one or more markers, about half (48%) of which represented putative functional mutations. In general, the initial testing was performed in a white case-control sample from the St. Louis area, with 419 LOAD cases and 377 age-matched controls. Markers that showed significant association in the exploratory analysis were followed up in several other white case-control sample sets to confirm the initial association. Of the 1,397 markers tested in the exploratory sample, 69 reached significance (P

Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter‐encoding gene, CnAFR1, involved in the resistance to fluconazole

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ‐cal meningitis, the most frequently encountered life‐threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole‐resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole‐resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter‐encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock‐out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

In vitro susceptibility of Vibrio spp. isolated from the environment

Bacteria of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera and human intestinal diseases. Some of these species may show resistance to different antibiotics including cefotaxime, tetracycline and chloramphenicol. The susceptibility to different antibiotics was tested using 40 Vibrio alginolyticus, eight V. parahaemolyticus and six V. vulnificus strains isolated in the coastal waters of Northern Sardinia (Italy). The frequency of resistance to beta-lactams was unexpectedly high. More than 80% of Vibrio isolates were resistant to ampicillin and 2.5% of V. alginolyticus were resistant to ceftazidime and cefotetan. Forty percent of V. alginolyticus and three V. vulnificus isolates gave a positive nitrocefin test. PCR was also performed using selected primers chosen for having common sequences of bla(TEM) and bla(SHV) genes.

Characterization of clinical isolates of Enterobacteriaceae from Italy by the BD Phoenix extended-spectrum beta-lactamase detection method.

ABSTRACT Production of extended-spectrum β-lactamases (ESBLs) is an important mechanism of β-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-M gene. Among the 191 non-ESBL-producing isolates, 77 isolates produced an AmpC-type enzyme, 110 isolates possessed TEM-1, TEM-2, or SHV-1 β-lactamases, and the remaining four isolates (all K. oxytoca strains) hyperproduced K1 chromosomal β-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.

Recurrent Ventriculoperitoneal Shunt Infection Caused by Small-Colony Variants of Staphylococcus aureus

Phenotypic variants of Staphylococcus aureus may be misidentified by routine microbiological methods, and they may also respond poorly to antibacterial treatment. Using molecular methods, we identified small-colony variants of methicillin-resistant S. aureus (which were misidentified by 3 widely used automated identification systems as methicillin-susceptible coagulase-negative staphylococci) as the cause of recurrent ventriculoperitoneal shunt-related meningitis.

Commercial systems for fluconazole susceptibility testing of yeasts: comparison with the broth microdilution method.

Fluconazole susceptibility was tested in 100 clinical yeast isolates (65 Candida albicans, 13 C. glabrata, 8 C. tropicalis, 7 C. parapsilosis, 3 Saccharomyces cerevisiae, 1 each of C. krusei, C. lusitaniae, Cryptococcus neoformans, Rhodotorula glutinis) and two control strains (Candida krusei ATCC 6258, C. parapsilosis ATCC 22019) using broth microdilution (reference method), disk diffusion, Etest strips, Sensititre YeastOne, Candifast, Integral System Yeasts. Using M27-A breakpoints, isolates were classified as susceptible (81%), susceptible-dose dependent or Resistant with broth dilution. Rates of concordance with the reference method were good for Sensititre YeastOne, Etest and disc-diffusion (81.2%-94.7%) but very low for the Candifast (3.1%) and Integral System (16.6%), which classified most susceptible isolates as resistant. Lack of standardisation (inoculum, medium composition) and non-objective interpretation schemes may be the cause of their poor performance. Sensititre YeastOne, Etest and disc-diffusion are potentially useful for fluconazole antifungal susceptibility testing of yeasts in clinical laboratories.

Molecular Basis of Rifampin and Isoniazid Resistance in Mycobacterium bovis Strains Isolated in Sardinia, Italy

ABSTRACT Fourteen of 22 (68%) Mycobacterium bovis strains isolated from cattle in Sardinia were found to be resistant to rifampin and isoniazid. Analysis of the rpoB and the katG, oxyR-ahpC, and inhA gene regions of these strains was performed in order to investigate the molecular basis of rifampin and isoniazid resistance, respectively. The most frequent mutation, encountered in 6 of 10 strains (60%), was in the rpoBgene; it occurred, at codon position 521 and resulted in leucine changed to proline. This suggests that codon 521 may be important for the development of rifampin resistance in M. bovis. Resistance to isoniazid is associated in Mycobacterium tuberculosis with a variety of mutations affecting one or more genes. Our results confirm the difficulty of interpreting the sequence variations observed in clinical strains of M. bovis. M. bovis strains isolated from the same geographic area showed similar mutations within the genes responsible for rifampin and isoniazid resistance. Our results represent the first study to elucidate the molecular genetic basis of drug resistance in M. bovis isolated from cattle.

Tsukamurella tyrosinosolvens and Rhizobium radiobacter sepsis presenting with septic pulmonary emboli

Septic pulmonary embolism (SPE) is an uncommon, but life-threatening event that is usually associated with extrapulmonary infections. We report the first case of bilateral SPE secondary to a central venous catheter-related bloodstream infection involving pathogens commonly considered environmental contaminants: Tsukamurella tyrosinosolvens and Rhizobium radiobacter. Empirical levofloxacin treatment was confirmed by in vitro susceptibility data and produced prompt clinical improvement, but removal of the infected line proved indispensable for eradication of the infection. Laboratory personnel should be aware of the pathogenic potential of these environmental organisms, particularly in immunocompromised hosts with indwelling catheters.

Entamoeba dispar: A Rare Case of Enteritis in a Patient Living in a Nonendemic Area

Entamoeba dispar, a common noninvasive parasite, is indistinguishable in its cysts and trophozoite forms from Entamoeba histolytica, the cause of invasive amebiasis, by microscopy. To differentiate the two species seems to be a problem for laboratory diagnosis. Recent experimental studies showed that  E. dispar can be considered pathogenic too. We present a rare case of enteritis due to E. dispar.

Early Detection of Negative BACTEC MGIT 960 Cultures by PCR-Reverse Cross-Blot Hybridization Assay

ABSTRACT We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures. Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay. PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system. Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay. In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures.