Isolation of Campylobacter spp. from a pig slaughterhouse and analysis of cross-contamination.

The purpose of this study was to establish the prevalence and possible contamination routes of Campylobacter spp. in a pig slaughterhouse. Swab samples were taken from the last part of rectum, from the carcasses surface before meat inspection and from slaughter line surface from 4 different pig herds during slaughtering. Identification of Campylobacter isolates was determined by the use of phase-contrast microscopy, hippurate hydrolysis, indoxyl acetate hydrolysis tests and PCR based restriction fragment length polymorphism method (PCR-RFLP). Pulsed-field gel electrophoresis (PFGE) typing using two macro-restriction enzymes SmaI and SalI was applied to in-slaughterhouse contamination analysis of pig carcasses. The study showed that 28 (63.6%) of the 44 samples collected at slaughterhouse were contaminated by Campylobacter spp. Up to 5 different colonies were obtained from each swab sample and a total of 120 different isolates were collected. 23.4% (28 of 120) isolates were identified as C. jejuni (19 from carcasses and 9 from slaughter line surfaces) and 76.6% (92 of 120) isolates as C. coli (28 from faeces, 47 from carcasses and 17 from slaughter line surfaces). The typing results showed identity between isolates from successive flocks, different carcasses, and places in the slaughterhouse in contact with carcasses. The results suggest that cross-contamination originated in the gastro-intestinal tract of the slaughtered pigs and that cross-contamination happened during the slaughter process.

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania.

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type (M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38(T)) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles.Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1(T)) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.

Typing of Pseudomonas aeruginosa strains in Norwegian cystic fibrosis patients.

OBJECTIVES: Typing of Pseudomonas aeruginosa isolates from Norwegian cystic fibrosis (CF) patients with chronic Pseudomonas lung infection in order to see whether cross-infection might have occurred. METHODS: Isolates from 60 patients were collected during the years 1994-98, and typed by pulsed field gel electrophoresis. RESULTS: Seventy-one strains were identified. One large cluster of identical strains included 27 patients, and 13 smaller clusters of 2-4 patients were found (26 patients). Seven patients had a strain not shared by other patients (private strains). Harboring the main cluster strain was significantly associated with participation in summer camps and training courses (P = 0.004, chi-squared test). There were no associations with regular admissions to hospital (intravenous antibiotic courses) or smaller social gatherings of short duration. Small clusters and private strains were not associated with any of the risk factors. All strains were sensitive to colistin. The minimal inhibitory concentrations were generally lower in Norwegian P. aeruginosa strains compared with isolates from Danish patients. CONCLUSIONS: Our results indicate that cross-infection with P. aeruginosa between cystic fibrosis patients has occurred.

Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods.

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.

Diversity of Listeria monocytogenes isolates from cold-smoked salmon produced in different smokehouses as assessed by Random Amplified Polymorphic DNA analyses.

One hundred and forty-eight Listeria monocytogenes isolates originating from vacuum packed cold-smoked salmon produced in 10 different Danish smokehouses were compared by Random Amplified Polymorphic DNA (RAPD) profiling. A total of 16 different reproducible RAPD profiles were obtained using a standardised RAPD analysis by four primers separately. The grouping of the 148 strains was exactly the same for the four primers used. For a sub-set of 20 strains typed by Pulsed Field Gel Electrophoresis (PFGE), only one strain was allocated into a different group as compared to the grouping by RAPD typing. Different RAPD types dominated in products from different smokehouses. Some identical RAPD types were isolated in several smokehouses. In each of four smokehouses, one particular RAPD type could be repeatedly isolated from products. Each smokehouse/product carried its own specific RAPD type and this may indicate a possible persistence of closely related strains of L. monocytogenes in smokehouses.

Molecular epidemiology of contagious bovine pleuropneumonia in Tanzania based on amplified fragment length polymorphism and pulsed-field gel electrophoresis analysis.

The genetic diversity of 60 field strains of Mycoplasma mycoides ssp. mycoides, small colony type (M. mycoides SC), comprising 56 isolates from cattle in Tanzania, one from Kenya, two from Botswana and one from Portugal, as well as the type (PG1T) and vaccine (T1-SR49) strains, was investigated. The strains were analyzed for variations in the EcoRI and Csp6I restriction sites in the genomic DNA using the amplified fragment length polymorphism (AFLP) technique, and variations in the BamHI restriction sites using pulsed-field gel electrophoresis (PFGE). Six AFLP types were detected among the analysed strains. The AFLP profiles of the type and vaccine strains were indistinguishable from each other. Indistinguishable AFLP profiles were found for 55 Tanzanian field strains, one of them isolated in 1990 and the other 54 isolated in 1998/1999, although one strain isolated in 1999 showed a different profile. Strains from different countries revealed different AFLP profiles. Six PFGE types were detected among the analysed strains, with all the 56 Tanzanian field strains displaying indistinguishable PFGE profiles. Strains from different countries revealed different PFGE profiles, and so did the type and vaccine strains. The strong genomic homogeneity among M. mycoides SC strains associated with outbreaks of contagious bovine pleuropneumonia in different regions of Tanzania suggests that the outbreaks of the disease in the 1990-99 period might have been caused by a single epidemic clone. Moreover, this study has demonstrated that AFLP and PFGE are potential tools for molecular epidemiological studies of M. mycoides SC infections.

Comparative investigations of Listeria monocytogenes isolated from a turkey processing plant, turkey products, and from human cases of listeriosis in Denmark.

Listeria monocytogenes was isolated from critical control points in a Danish turkey processing plant, from turkey products and from cases of human listeriosis. During processing in the plant the prevalence of L. monocytogenes ranged from 25.9 to 41.4%. Cleaning and disinfection decreased the prevalence to 6.4%. Isolates of L. monocytogenes were characterized by pulsed-field gel electrophoresis (PFGE) using restriction endonuclease ApaI. Identical DNA types were obtained from turkey products and the processing line even after cleaning and disinfection. Two identical DNA types were demonstrated among isolates from turkey products and human cases of listeriosis. The prevalence of L. monocytogenes in turkey products ranged from 7.3 to 17.4% for ready-to-eat products and raw products, respectively. Since none of the 27 flocks examined before slaughter sampled positive for L. monocytogenes and the prevalence increased during processing, the potential risk from turkey meat was apparently due to factory hygiene rather than intrinsic contamination of the turkeys.

Genetic variations among Mycoplasma bovis strains isolated from Danish cattle.

The genetic heterogeneity of Mycoplasma bovis strains isolated in Denmark over a 17-year period was investigated. Forty-two field strains isolated from different geographic locations and specimens, including strains from 21 herds involved in two outbreaks of M. bovis-induced mastitis, and the type strain of M. bovis (PG45(T)) were assayed for variations in the BglII and MfeI restriction sites in the chromosomal DNA by using the amplified fragment length polymorphism (AFLP) fingerprinting technique. The obtained genomic fingerprints consisted of 62-68 AFLP fragments in the size range of 50-500 bp. Among the analyzed strains, 18 different AFLP profiles were detected. The similarity between individual fingerprints, calculated by Dice similarity coefficient, ranged from 0.9 to 1.0. Twenty-five strains, including 23 which were isolated during two outbreaks of M. bovis-induced mastitis which occurred 2 years apart, showed indistinguishable AFLP patterns. More genetic diversity was observed among the recent strains. The similarity of the genotypes of the field strains to that of the M. bovis type strain (PG45(T)) was 97.7%. The results of this study have demonstrated a remarkable genomic homogeneity of Danish strains of M. bovis that were probably epidemiologically related and which have remained stable for a considerable length of time. Furthermore, this study has demonstrated that AFLP can be used for genomic fingerprinting and discrimination of M. bovis strains.

Increasing prevalence of Mycoplasma bovis in Danish cattle.

A study on the prevalence of mycoplasmas in pneumonic bovine lungs was performed on material submitted for diagnostic purposes at the Danish Veterinary Laboratory, Copenhagen. Among the 50 examined cases 43 (86.0%) were found to be infected with mycoplasmas. The predominant mycoplasmas were Ureaplasma spp. (72.0%), M. dispar (48.0%) and M. bovis (24.0%). Other mycoplasmas were M. bovirhinis (20.0%) and M. bovigenitalium (6.0%). Among the infected lungs multiple species infections were predominant (76.7%) over single species infections (23.3%) with M. dispar-Ureaplasma (25.6%), M. bovis-Ureaplasma (18.6%) and M. dispar-M. bovirhinis-Ureaplasma (11.6%) infections being the most frequently encountered combinations. There appears to be an increasing prevalence of M. bovis (24.0%) as compared to earlier reports (0.6-2.0%), thus calling for special attention upon this mycoplasma. Pulsed field gel electrophoresis (PFGE) analysis of 11 field isolates of M. bovis from 9 different farms revealed different profiles except for 2 isolates which were recovered from the same farm. Because mycoplasmas belonging to the 'M. mycoides cluster' were not encountered during this study; it appears that the Danish cattle population is still free from this group of mycoplasma in spite of their presence in some other European countries.

Pseudomonas aeruginosa cross-infection among patients with cystic fibrosis during a winter camp.

Twenty-seven patients with cystic fibrosis from our Danish Cystic Fibrosis Center went to a winter camp for 1 week in November of 1990. This study is based on 22 of these patients. Prior to attending camp, 17 out of 22 patients harbored Pseudomonas aeruginosa in their sputum, but 5 patients did not. After returning from camp, all 22 patients harbored P. aeruginosa in the sputum, including the 5 patients whose sputum was free of P. aeruginosa before they went. Epidemiological typing used pulsed-field gel electrophoresis of the P. aeruginosa isolates was performed. The typing results showed that the 5 cystic fibrosis patients who were free of P. aeruginosa in their sputum prior to the winter camp had acquired P. aeruginosa isolates identical to the P. aeruginosa strains isolated from the other 17 cystic fibrosis patients. This constitutes a cross-colonization rate of 100%, the highest rate ever detected among patients with cystic fibrosis. We conclude that separate holiday camps based on the infection status of the patients with cystic fibrosis are necessary to avoid cross-infection of patients not infected with P. aeruginosa.

Mycoplasmas isolated from the respiratory tract of cattle and goats in Tanzania.

A microbiological study of the mycoplasma flora in the respiratory tracts of cattle and goats in selected regions of Tanzania is described. In the examination of cattle, mycoplasmas were isolated from 60 (17.8%) of the 338 examined lung samples, 8 (47.1%) of the 17 lymph nodes, 4 (13.3%) of the 30 pleural fluid samples and 4 (3.9%) of the 103 nasal swabs examined. All the isolates were identified as Mycoplasma mycoides subsp. mycoides, Small Colony type except for one isolate from pleural fluid which was identified as Mycoplasma arginini. M. mycoides subsp. mycoides, Small Colony type was isolated from samples originating from Dodoma, Iringa, Mbeya, Morogoro and Shinyanga regions where outbreaks of contagious bovine pleuropneumonia had been reported. In the examination of goats, mycoplasmas were isolated from 54 (34.0%) of the 159 examined lung samples, 41 (18.1%) of the 226 nasal swabs and 4 (40.0%) of the 10 pleural fluid samples. The species demonstrated were Mycoplasma capricolum subsp. capripneumoniae, M. mycoides subsp. mycoides, Small Colony type Mycoplasma ovipneumoniae and M. Capricolum subsp. arginini. The isolation of M. capripneumoniae in the Coast and Morogoro regions confirmed the presence of contagious caprine pleuropneumonia in the regions.

Epidemiology of Pseudomonas aeruginosa in cystic fibrosis and the possible role of contamination by dental equipment.

Cystic fibrosis (CF) patients often suffer from Pseudomonas aeruginosa lung infection yet the source of this organism is not known. In order to determine whether CF patients might be contaminated with P. aeruginosa from dental equipment, a total of 103 water samples from 25 dental sessions in Frederiksberg Municipal Oral Health Care Service were examined. Three samples (2.9%) were positive for P. aeruginosa. Three hundred and twenty-seven water samples from 82 dental sessions from various other Municipal Oral Health Services in Denmark, attended by CF patients, were also examined. Eighteen of 327 samples (5.5%) from nine sessions (11%) were positive for P. aeruginosa. In one case, genotypically identical (RFLP, pulsed-field gel electrophoresis) P. aeruginosa strains were found both in water from the dental equipment and in the CF patients sputum. This indicates a small risk for acquiring P. aeruginosa from dental sessions, which is however equal to the yearly 'natural background' incidence (1-2%) of acquisition of P. aeruginosa in our CF centre.

Genomic fingerprinting of 80 strains from the WHO multicenter international typing study of listeria monocytogenes via pulsed-field gel electrophoresis (PFGE).

An international multicenter typing study of Listeria monocytogenes was initiated by the World Health Organization (Food Safety Unit, Geneva) in order to evaluate the usefulness of various phenotypic and genotypic typing methods for L. monocytogenes, to select and standardize the most appropriate methods to define common nomenclature of varieties and to select specific reference strains. Pulsed-field gel electrophoresis was used in four laboratories for molecular characterization of a set of 80 'coded' strains distributed to all participating laboratories. The endonucleases ApaI and SmaI, used in all four laboratories, yielded between 21 and 28 restriction endonuclease digestion profiles (REDP). AscI was used, in addition, in laboratory A and displayed 21 REDP. The combination of ApaI, SmaI or AscI REDP established 25 to 33 genomic groups. depending on the laboratory and the number of viable strains. Agreement of typing data among the four laboratories ranged from 79 to 90%. Forty-nine (69%) of the 71 strains viable in all four laboratories were placed into exactly the same genomic groups in all four laboratories. The epidemiological relevance of the strains became known after decoding and it was shown that most of the epidemiologically related strains were correctly identified by the four groups of investigators. i.e., most related strains were placed into the same genomic groups by all four laboratories. Similar results were obtained when 11 duplicate cultures were analyzed-on average 84% of the duplicates were identified. Comparison of REDP obtained by laboratory A with REDP from previously analyzed set of 176 L. monocytogenes strains allowed the prediction of the serovar-groups of the 80 strains. These predictions of serovar-groups were later confirmed by serotyping results obtained by laboratories involved in the WHO multicenter typing study of L. monocytogenes. In general this study reconfirmed that PFGE is a very accurate and reproducible method for fine structure comparison and molecular typing of L. monocytogenes.

Lack of evidence of nosocomial cross-infection by Burkholderia cepacia among Danish cystic fibrosis patients.

Burkholderia cepacia isolates from nine of the ten Danish cystic fibrosis (CF) patients known between 1975 and the present day to carry this organism were investigated. Eight distinct genotypes were found with polymerase chain reaction ribotyping and pulsed-field gel electrophoresis. The results indicate that there is little patient-to-patient cross-infection with Burkholderia cepacia within the Danish CF population, even though the majority of patients attend the same CF clinic on a regular basis.

Listeria monocytogenes in poultry and poultry products: epidemiological investigations in seven Danish abattoirs.

Listeria monocytogenes was isolated from 11/236 (4 x 7%) caecal samples from parent flocks, providing broilers to the abattoirs investigated. Caecal samples from 2078 broilers representing 90 randomly selected broiler flocks were negative for L. monocytogenes. A total of 3080 samples from seven abattoirs including poultry processing line samples, and final products were also examined for L. monocytogenes. Listeria monocytogenes was isolated in 0 x 3% to 18 x 7% of the samples collected in the different abattoirs. Epidemiological typing of 247 L. monocytogenes isolates, including serotyping, phage typing, pulsed-field gel electrophoresis and ribotyping revealed 62 different clones. Based upon typability and discriminatory power, DNA typing methods used were found equally suitable as epidemiological markers. Serotyping and phage typing were not found useful as epidemiological markers for poultry isolates of L. monocytogenes since only 120/247 (48 x 6%) isolates were typable by phage typing and 230/247 (93 x 1%) L. monocytogenes belonged to serotype 01 while 6/247 (2 x 4%) belonged to 04. The discovery of a few dominating clones in each abattoir might indicate an endemic occurrence of L. monocytogenes. It is concluded that L. monocytogenes in the broiler production is primarily localized to the abattoirs. The incidence of L. monocytogenes may be reduced by improving the hygiene.

Comparison of ribotyping and genome fingerprinting of Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Forty Pseudomonas aeruginosa strains, previously characterized by pulsed-field gel electrophoresis, were ribotyped with EcoRI, BamHI, ClaI, and PvuII. Ribotyping with PvuII proved to be as discriminatory as pulsed-field gel electrophoresis with XbaI or DraI while EcoRI and BamHI were not. ClaI contributed further ribotypes, some of which might be due to a transposable element.

Epidemiology of Pseudomonas aeruginosa infection and the role of contamination of the environment in the Danish Cystic Fibrosis Centre.

In order to identify the possible reservoirs and routes of cross-infection with Pseudomonas aeruginosa, samples were collected during a six-week period in autumn 1992 from patients, their visiting parents, staff and the inanimate environment of the Danish Cystic Fibrosis (CF) Centre and from a control ward with common paediatric diseases. All the P. aeruginosa strains were phage typed and serotyped. From 240 CF patients, 310 strains of P. aeruginosa were isolated, and of these 283 (91.3%) belonged to the polyagglutinable phenotype, most often with a short phage type (31/188 or 109). P. aeruginosa was isolated from only six (0.6%) of 1000 swabs taken from the environment. These six environmental strains and 20 P. aeruginosa strains from CF patients with identical serotype and phage type were examined with pulsed field gel electrophoresis. None of the patients harboured strains similar to the environmental strains, indicating the present isolation procedure and hygienic precautions were effective in our CF centre, and prevented contamination of the environment with P. aeruginosa.

Detection of fimbrial and toxin genes in Escherichia coli and their prevalence in piglets with diarrhoea. The application of colony hybridization assay, polymerase chain reaction and phenotypic assays.

Two different genotypic methods, colony hybridization and polymerase chain reaction (PCR), were applied to detect enterotoxin, verotoxin and fimbrial genes in 708 Escherichia coli (E. coli) strains from piglets with diarrhoea. The results were compared with those obtained by phenotypic methods. DNA fragments specific for each of the enterotoxins LT, STa and STb, the verotoxins VT1, VT2 and VT2v, and for each of the fimbrial genes K88 (F4), K99 (F5), 987P (F6), F41 and F107, respectively, were used as probes in a colony hybridization assay of the E. coli strains. A PCR assay was used as genotypic test for the verotoxin gene. An Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to test for the presence of K88 and K99 fimbriae, and a Vero cell test was performed to test for verotoxin production. Toxin detection kits were applied to detect the E. coli heat-labile enterotoxin (LT) and the heat-stable (STa) enterotoxin. The correlation between the results obtained by genotypic and phenotypic methods was 97.7-100%. The prevalence of the different fimbrial and toxin genes in E. coli strains from piglets with neonatal and postweaning diarrhoea were recorded. The verotoxin and the fimbrial F107 genes were found to be more frequent in postweaning E. coli strains than in neonatal E. coli strains.

Polyagglutinable Pseudomonas aeruginosa from cystic fibrosis patients. A survey.

Chronic Pseudomonas aeruginosa lung infection is responsible for most of the mortallity and morbility observed in cystic fibrosis patients. During the course of the disease, the bacteria change from being O-serogroup typable (monoagglutinable), non-mucoid, resistant to normal human serum and motile, to become O-serogroup non-typable (polyagglutinable), serum-sensitive and non-motile. In spite of high levels of antibodies produced by the patient, and intensive antibiotic therapy it is not possible to eradiate the polyagglutinable bacteria from the lungs of the patients. The bacteria will reappear and it is not fully understood if it is the same strain which reappears, or it is a super-infection with a new strain which takes place. A reliable and stable typing method is needed to clarify this question. In the present study, the conventional typing methods, such as serotyping, phage typing and pyocin typing were compared with the newer DNA typing methods, such as, restriction fragment length polymorphism (RFLP) in combination with pulsed field gel electrophoresis (PFGE) and typing with a specific DNA probe. Typability, reproducibility and discriminatory power using the different typing methods were investigated. The conventional typing methods have proved to be adequate when typing P. aeruginosa isolates from non cystic fibrosis sources, but because the majority of cystic fibrosis P. aeruginosa isolates are polyagglutinable or non typable, serotyping is not useful. Phage typing also lacks discriminatory power as it lumps up to 40% of the isolates in the same phage group. Pyocin typing has the disadvantage of low reproducibility. Most of the conventional typing methods are based on receptors on the bacterial surface, which on exposure to the environmental conditions in the lung, are likely to provoke phenotypic changes of the bacteria. The obvious advantage of the newer DNA typing methods is that these methods are based on internal properties of the bacteria, as part of the bacterial genome is investigated. The present study has revealed that for the time being, restriction fragment length polymorphism (RFLP) and pulsed field gel electrophoresis (PFGE) in combination with phage typing is the best method of typing P. aeruginosa isolates from cystic fibrosis patients for epidemiological purposes.

Comparison of genome fingerprinting with conventional typing methods used on Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Two hundred Pseudomonas aeruginosa serial isolates from 61 cystic fibrosis patients were examined using restriction fragment length polymorphism in connection with pulsed field gel electrophoresis. A comparison was made of results obtained by genome fingerprinting and by conventional typing methods. It was possible to subdivide the majority of the genome types with the conventional typing methods, indicating the likelihood of bacterial phenotypes occurring in the lung of the cystic fibrosis patient. Two clusters of strains were observed among the 200 P. aeruginosa isolates from the 61 patients. Strains belonging to one cluster were present in 26 (42.6%) of the 61 patients. Strains belonging to the other cluster were present in 11 (18.0%) of the 61 patients. The occurrence of these clusters indicates that cross-infection has taken place among CF patients attending the Danish Cystic Fibrosis Centre. Conventional typing methods are based on the presence of specific bacterial surface structures. Therefore, conventional typing methods may sometimes lead to wrong classification of isolates from cystic fibrosis patients, especially if applied alone. A combination of two to six methods decreased the reproducibility of the typing results. The best combination was genome fingerprinting and phage typing, which yielded a reproducibility of 82.5%. Each time a method is added, the reproducibility decreases by an average of 14.4%.