Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.     

Molecular Characterization of Vancomycin-Resistant Enterococci from Hospitalized Patients and Poultry Products in The Netherlands

Vancomycin-resistant enterococci (VRE) pose an emerging health risk, but little is known about the precise epidemiology of the genes coding for vancomycin resistance. To determine whether the bacterial flora of consumer poultry serves as a gene reservoir, the level of contamination of poultry products with VRE was determined. VRE were genotyped by pulsed-field gel electrophoresis (PFGE), and transposon structure mapping was done by PCR. The vanX-vanY intergenic regions of several strains were further analyzed by sequencing. A total of 242 of 305 (79%) poultry products were found to be contaminated with VRE. Of these VRE, 142 (59%) were high-level-vancomycin-resistant Enterococcus faecium strains (VREF). PFGE revealed extensive VREF heterogeneity. Two genotypes were found nationwide on multiple occasions: type A (22 of 142 VREF [15%]) and type B (14 of 142 VREF [10%]). No PFGE-deduced genetic overlap was found when VREF from humans were compared with VREF from poultry. Two vanA transposon types were identified among poultry strains. In 59 of 142 (42%) of the poultry VREF, the size of the intergenic region between vanX and vanY was ~1,300 bp. This transposon type was not found in human VREF. In contrast, all human strains and 83 of 142 (58%) of the poultry VREF contained an intergenic region 543 bp in size. Sequencing of this 543-bp intergenic vanX-vanY region demonstrated full sequence conservation. Though preliminary, these data suggest that dissemination of the resistance genes carried on transposable elements may be of greater importance than clonal dissemination of resistant strains. This observation is important for developing strategies to control the spread of glycopeptide resistance.     

Phylogenetic Analysis of the DNA Polymerase Gene of a Novel Alphaherpesvirus Isolated from an Indian <Emphasis Type="Italic">Gyps</Emphasis> Vulture

The DNA polymerase gene of a novel herpesvirus, vulture herpesvirus (VHV), isolated from an Indian Gyps vulture was completely sequenced using primer walking and transposon insertion strategies. DNA sequencing analysis revealed a single open reading frame (ORF) of 3660 nucleotides (53% G-C content) able to encode 1219 amino acids. Identification was based on a nucleotide sequence identity of approximately 50% to other herpesvirus sequences found in Genbank. Nine motifs were identified that are conserved amongst all known herpesviruses and are found within the 3–5 exonuclease and DNA binding functional domains of the DNA polymerase enzyme. Phylogenetic analysis using Clustal W with neighbour-joining revealed VHV to group within the subfamily Alphaherpesvirinae, more closely related to the avian herpesviruses than to those of other species. Partial sequence data also revealed VHV to contain other genes fundamental to the structure and replication of all herpesvirus genomes. A Real Time PCR Taqman assay specific for the VHV DNA polymerase gene was designed to detect the presence of VHV genomic material in post mortem tissue samples from diseased birds. Positive tissues included the spleen, rectum, thymus, kidney and brain. A herpesvirus specific to vultures may pose a threat to the management of captive breeding programs being established to assist the survival of wild populations of Gyps vultures.     

Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus

CHROMagar MRSA was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus (MRSA). A well-defined collection consisting of 216 MRSA strains and 241 methicillin-susceptible Staphylococcus aureus isolates was used. The sensitivity of CHROMagar MRSA after 24 h of incubation was 95.4%, increasing to 100% after 48 h. The specificity was already 100% after 24 h.     

The Role of Sialic Acid in Opsonin-Dependent and Opsonin-Independent Adhesion of Listeria monocytogenes to Murine Peritoneal Macrophages

The adhesion of listeriae to host cells employs mechanisms which are complex and not well understood. Listeria monocytogenes is a facultative intracellular pathogen responsible for meningoencephalitis, septicemia, and abortion in susceptible and immunocompromised individuals. Subsequent to colonization and penetration of the gut epithelium, the organism attaches to resident macrophages and replicates intracellularly, thus evading the humoral immune system of the infected host. The focus of these studies was to investigate the attachment of the organism to murine peritoneal macrophages in an opsonin-dependent and opsonin-independent fashion. Assessment of competitive binding experiments by immunofluorescence and enzyme-linked immunosorbent assays showed that adhesion of the organism to macrophages in the presence or absence of opsonins was inhibited (90%) by N-acetylneuraminic acid (NAcNeu). In addition, the lectin from Maackia amurensis, with affinity for NAcNeu-α(2,3)galactose, blocked binding of L. monocytogenes to host cells. Oxidation of the surface carbohydrates on the organism by using sodium metaperiodate resulted in a dose-dependent reduction (up to 98%) in adherence to macrophages. Monoclonal antibody to complement receptor 3 did not prevent listeriae from binding to mouse macrophages or from replicating within the infected cells whether or not normal mouse serum was present. Based on our results, we propose the involvement of NAcNeu, a member of the sialic acid group, in the attachment of L. monocytogenes to permissive murine macrophages.     

Differing Patterns of P-Selectin Expression in Lung Injury

Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of ¹²⁵I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression was sustained for the next 7 hours, in striking contrast to the pattern of P-selectin expression in the cobra venom factor model, in which upregulation was very transient (within the 1st hour). In the immune complex model, injury and neutrophil accumulation were P-selectin dependent. Upregulation of P-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor α. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl sulfoxide sensitive but neutrophil independent. Different mechanisms that may explain why upregulation of lung vascular P-selectin is either transient or sustained are discussed.     

Spread of old and new clones of epidemic methicillin-resistant Staphylococcus aureus in Poland

Objective: To study the relatedness among methicillin-resistant Staphylococcus aureus (MRSA) isolates originating from two regions of Poland using different epidemiologic typing methods.
Methods: Forty-five MRSA isolates (19 from Warsaw and 26 from the Grajewo region) were collected between 1995 and 1996. For phenotypic epidemiologic analysis, antimicrobial susceptibility testing (AST) with a panel of 19 antibiotics was performed. For genotypic epidemiologic analysis, pulsed-field gel electrophoresis (PFGE) of Smal-digested chromosomal DNA, restriction endonuclease analysis of plasmid (REAP) DNA digested by Hin dIII, random amplification of polymorphic DNA (RAPD) and binary typing (BT) of genomic DNA by hybridization with five different RAPD-generated strain-specific DNA probes, were used.
Results: Six clusters of clonally related strains were found among the MRSA isolates analyzed. Three of these, identified in both regions, were related to previously described Polish epidemic clones, designated HeEMRSA-Pol1 (heterogeneously methicillin resistant—18 isolates) and HoEMRSA-Pol1 (homogeneously resistant—two clones, six isolates each). The remaining three clones, identified in the Grajewo region only, are previously undescribed. One of these, represented by 11 isolates, appears to be new epidemic heterogeneous MRSA clone (HeEMRSA-Pol2). Results of PFGE and BT in general showed good correlation, and, in some cases, RAPD using AP1 and AP7 primers could discriminate between isolates belonging to single PFGE or BT types. Broad AST and REAP can provide useful additional information concerning relatedness.
Conclusion: Evidence for the spread of previously recognized epidemic MRSA clones in Poland and the presence of a new epidemic heterogeneously resistant clone of MRSA in hospitals outside Warsaw is documented.     

Stress,coping, family conflict,and adolescent alcohol use

This study examined alcohol use among seventh graders in relation to life events, daily hassles, the supportive quality of the family environment, coping, and anxiety. Four hundred twenty-five students participated, 228 girls and 197 boys. Stepwise regression and discriminant function analyses indicated that the students reported more alcohol use if they also reported more life events, more daily hassles, and more conflict in the family. A stress-buffering effect of low family conflict on life events could not be substantiated for extent of alcohol use. The results are discussed in the context of the developmental transitions of adolescence.     

Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.     

Decreased hypocretin-1 (Orexin-A) levels in the cerebrospinal fluid of patients with myotonic dystrophy and excessive daytime sleepiness

STUDY OBJECTIVE: Myotonic dystrophy type 1 is a multisystem disorder with myotonia, muscle weakness, cataracts, endocrine dysfunction, and intellectual impairment. This disorder is caused by a CTG triplet expansion in the 3' untranslated region of the DMPK gene on 19q13. Myotonic dystrophy type 1 is frequently associated with excessive daytime sleepiness, sharing with narcolepsy a short sleep latency and the presence of sleep-onset rapid eye movement periods during the Multiple Sleep Latency Test. Since narcolepsy is characterized by a dysfunction of the hypothalamic hypocretin system, we investigated whether patients with myotonic dystrophy type 1 with excessive daytime sleepiness have abnormalities in the hypocretin system. DESIGN/PARTICIPANTS: Six patients with myotonic dystrophy type 1 complaining of excessive daytime sleepiness and 13 healthy controls without a sleep disorder were included. The patients with myotonic dystrophy type 1 were evaluated using clinical interviews, nocturnal polysomnograms, and Multiple Sleep Latency Tests. All patients had a confirmed genetic diagnosis for DM1 and were HLA typed. Cerebrospinal fluid hypocretin-1 levels were measured using a direct radioimmunoassay in patients and controls. Setting: University hospital sleep laboratory. INTERVENTIONS: N/A. MEASUREMENT AND RESULTS: The mean sleep latency on Multiple Sleep Latency Tests was abnormal in all patients (< 5 minutes in 2, < or = 8 in 4) and 2 sleep-onset rapid eye movement periods were observed in 2 subjects. All patients were HLA-DQB1*0602 negative. Hypocretin-1 levels were significantly lower in patients versus controls (p < 0.001); 1 case with 2 sleep-onset rapid eye movement periods had hypocretin-1 levels in the range generally observed in narcolepsy (< 110 pg/mL). Three cases had intermediate levels (110-200 pg/mL). Hypocretin-1 levels did not correlate clinically with disease severity or duration or with subjective or objective sleepiness reports. CONCLUSIONS: A dysfunction of the hypothalamic hypocretin system may mediate sleepiness and abnormal Multiple Sleep Latency Test results in patients with myotonic dystrophy type 1.