Marianne Nielsine Skov

Clonal lines of Salmonella enterica serotype enteritidis documented by IS200-, ribo-, pulsed-field gel electrophoresis and RFLP typing

Sixty-two selected strains of Salmonella serotype Enteritidis of 33 phage types (PTs), and one strain classified as RDNC, were characterised by four different chromosomally based typing methods to elucidate genetic relationships among strains of different phage types. Based on IS200-hybridisation patterns, two major groups, containing strains of the most commonly encountered phage types, and six minor groups (seven with the RDNC strain included) were observed. IS200 pattern was a stable epidemiological marker in strains of all phage types except PT 6a and 14b. Ribotyping separated strains of the phage types into one major and five minor groups; the pattern of the RDNC strain was not seen with other strains. More than one ribotype was observed among strains of Enteritidis PTs 6, 7, 14b and 21. By pulsed-field gel electrophoresis, strains of 21 of the 33 phage types formed one large cluster when bands > 125 kb were used as the criterion for separation. Among strains belonging to PTs 1, 6, 7 and 14b, more than one pattern was observed by this method. By probing with five random cloned fragments of the Enteritidis chromosome, strains from 27 of 31 phage types examined showed the same hybridisation pattern. With the combined use of four genotypic methods, two groups of strains, representing eight and seven of 33 Enteritidis phage types, were formed; these two groups may be considered as the main evolutionary lines of Enteritidis. Strains of the remaining phage types, and the RDNC strain, belonged to separate groups.

Verocytotoxin-Producing Escherichia coli in Wild Birds and Rodents in Close Proximity to Farms

ABSTRACT Wild animals living close to cattle and pig farms (four each) were examined for verocytotoxin-producing Escherichia coli (VTEC; also known as Shiga toxin-producing E. coli). The prevalence of VTEC among the 260 samples from wild animals was generally low. However, VTEC isolates from a starling (Sturnus vulgaris) and a Norway rat (Rattus norvegicus) were identical to cattle isolates from the corresponding farms with respect to serotype, virulence profile, and pulsed-field gel electrophoresis type. This study shows that wild birds and rodents may become infected from farm animals or vice versa, suggesting a possible role in VTEC transmission.

Species Identification of Clinical Isolates of Anaerobic Bacteria: a Comparison of Two Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems

ABSTRACT We compared two matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems (Shimadzu/SARAMIS and Bruker) on a collection of consecutive clinically important anaerobic bacteria (n = 290). The Bruker system had more correct identifications to the species level (67.2% versus 49.0%), but also more incorrect identifications (7.9% versus 1.4%). The system databases need to be optimized to increase identification levels. However, MALDI-TOF MS in its present version seems to be a fast and inexpensive method for identification of most clinically important anaerobic bacteria.

Colonisation and infection of the paranasal sinuses in cystic fibrosis patients is accompanied by a reduced PMN response

BACKGROUND We studied whether the sinuses might be foci for Pseudomonas aeruginosa lung infection. METHODS Endoscopic Sinus Surgery was performed in 78 CF patients; PFGE was used for bacterial genotyping. Material from sinuses and lungs were Gram-stained to detect biofilms. Immunoglobulins were measured in serum and saliva. RESULTS When P. aeruginosa was cultured simultaneously from the sinuses and the lungs they were genetically identical in 38 of the 40 patients (95%). In the sinuses, P. aeruginosa formed biofilms with minimal cellular inflammation, probably because of a significantly higher local production of secretory IgA compared with IgG (p<0.001). CONCLUSIONS We have shown that P. aeruginosa form biofilm in the sinuses, which constitute an important bacterial reservoir for subsequent lung infection. The high amount of IgA in the upper airways probably protects P. aeruginosa from the inflammatory immune system, and they can proceed unnoticed into a permanent infectious focus that cannot be eradicated with antibiotics.

Increased antigen-specific Th-2 response in allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis

The majority of patients with cystic fibrosis (CF) become colonized with Aspergillus fumigatus (A. fumigatus) in the lower respiratory tract, the prevalence being up to 60%. Between 1–11% of CF patients develop allergic bronchopulmonary aspergillosis (ABPA). Previous studies of ABPA in selected patients suffering from cystic fibrosis or atopic asthma have suggested a pathogenic role for antigen‐specific “Th2‐like” T lymphocytes. The aim of this study was to evaluate the quantitative importance of such Th2 cells, using improved techniques for measuring interleukin‐4 (IL‐4) and IL‐5 secretion in unseparated peripheral blood mononuclear cell (PBMC) suspensions from CF patients with ABPA and from a control group without ABPA. Thus, 20 patients with CF, heavily colonized with A. fumigatus in the lower respiratory tract, were studied: 10 patients with ABPA, and 10 without. Unseparated PBMC were stimulated in vitro by A. fumigatus antigen and by an unrelated antigen (tetanus toxoid) as a control. After 6 days of stimulation, IL‐4 and IL‐5 (markers for Th2 cell activity) and interferon‐gamma (IFN‐γ) (marker for Th1 cell activity) were quantified in the supernatants by enzyme‐linked immunosorbent assay.

16S rRNA Gene Sequencing in Routine Identification of Anaerobic Bacteria Isolated from Blood Cultures

ABSTRACT A comparison between conventional identification and 16S rRNA gene sequencing of anaerobic bacteria isolated from blood cultures in a routine setting was performed (n = 127). With sequencing, 89% were identified to the species level, versus 52% with conventional identification. The times for identification were 1.5 days and 2.8 days, respectively.

In Vivo Emergence of Aspergillus terreus with Reduced Azole Susceptibility and a Cyp51a M217I Alteration

Azole resistance in Aspergillus terreus isolates was explored. Twenty related (MB) and 6 unrelated A. terreus isolates were included. CYP51A sequencing and RAPD genotyping was performed. Five MB isolates were itraconazole susceptible, whereas the minimum inhibitory concentrations (MICs) for 15 MB isolates were elevated (1-2 mg/L). Voriconazole and posaconazole MICs were 0.5-4 and 0.06-0.5 mg/L, respectively, for MB isolates but 0.25-0.5 and <0.03-0.06 mg/L, respectively, for controls. Sequencing identified a Cyp51Ap M217I alteration in all 15 isolates with elevated itraconazole MICs. Genotyping showed that 18 of 20 MB isolates were identical and unique, suggesting endogenous origin. In conclusion, itraconazole resistance in A. terreus was linked to an M217I Cyp51A alteration.

Antimicrobial Drug Resistance of Salmonella Isolates from Meat and Humans, Denmark

We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue.

Genomic lineage of Salmonella enterica serotype Gallinarum

Forty-eight strains of Salmonella enterica serotype Gallinarum of biotypes Gallinarum and Pullorum were characterised by three chromosomally based typing methods. The patterns obtained were compared with those of strains of eight other serotypes of Salmonella of O serogroup D. The same PvuII and PstI IS200 patterns were commonly observed among strains of both biotypes and the three SmaI ribotypes of serotype Gallinarum strains differed in only one or two bands, supporting the view that members of these two biotypes are closely related. The same IS200 patterns were also commonly observed among strains of serotype Enteritidis, indicating its evolutionary relationship with serotype Gallinarum. NotI pulsed-field gel electrophoresis (PFGE) patterns divided strains into 24 types. Based on a similarity analysis, two clusters were formed. One contained the majority of biotype Gallinarum strains and two atypical strains of Pullorum; the other contained strains of biotype Pullorum and an otherwise typical strain of biotype Gallinarum. Two atypical strains of biotype Pullorum remained unclustered by PFGE analysis. The grouping of strains differed according to the typing method used, but the majority of strains within each of the biotypes Gallinarum and Pullorum were very similar by the chromosomal markers analysed.

An epidemiological study of Salmonella enterica serovar 4, 12:b:- in broiler chickens in Denmark

Epidemiological investigations of isolates of Salmonella enterica serovar 4, 12:b:- were carried out to establish particular molecular markers to assign isolates to a common origin. Plasmid profiling demonstrated that over 50% of 291 isolates, obtained between 1991 and 1996, were plasmid-free. The remaining isolates exhibited a common trend in plasmid content of 105 and 2kb. Although no specific correlation to any particular source within the poultry industry was discernible using plasmid analysis, there were indications of clonality with local divergence. Ribotyping with EcoRI demonstrated limited discriminative potential as 96% of the isolates expressed a common profile. Ribotyping with HindIII failed to further differentiate the isolates. IS200 (PstI) typing and PFGE (NotI and XbaI) afforded some degree of further discrimination with selected isolates. Each technique produced four profiles, but dominant profiles were also apparent. Eighteen variables were selected for multivariate logistic regression analysis in order to identify risk areas associated with broiler flocks within the industry. An increased risk for S. 4, 12:b:- infection was only associated with the feedmills used. Random effects at the house and/or farm level were also found to be statistically significant. Of the 16 feedmills associated with the isolation of 4, 12:b:-, six were deemed to be significant risk factors.