Marja-Leena Katila

Mycobacteria in water and loose deposits of drinking water distribution systems in Finland

ABSTRACT Drinking water distribution systems were analyzed for viable counts of mycobacteria by sampling water from waterworks and in different parts of the systems. In addition, loose deposits collected during mechanical cleaning of the main pipelines were similarly analyzed. The study covered 16 systems at eight localities in Finland. In an experimental study, mycobacterial colonization of biofilms on polyvinyl chloride tubes in a system was studied. The isolation frequency of mycobacteria increased from 35% at the waterworks to 80% in the system, and the number of mycobacteria in the positive samples increased from 15 to 140 CFU/liter, respectively. Mycobacteria were isolated from all 11 deposits with an accumulation time of tens of years and from all 4 deposits which had accumulated during a 1-year follow-up time. The numbers of mycobacteria were high in both old and young deposits (medians, 1.8 × 105 and 3.9 × 105 CFU/g [dry weight], respectively). Both water and deposit samples yielded the highest numbers of mycobacteria in the systems using surface water and applying ozonation as an intermediate treatment or posttreatment. The number and growth of mycobacteria in system waters correlated strongly with the concentration of assimilable organic carbon in the water leaving the waterworks. The densities of mycobacteria in the developing biofilms were highest at the distal sites of the systems. Over 90% of the mycobacteria isolated from water and deposits belonged to Mycobacterium lentiflavum, M. tusciae, M. gordonae, and a previously unclassified group of mycobacteria. Our results indicate that drinking water systems may be a source for recently discovered new mycobacterial species.

Susceptibility Testing with the Manual Mycobacteria Growth Indicator Tube (MGIT) and the MGIT 960 System Provides Rapid and Reliable Verification of Multidrug-Resistant Tuberculosis

ABSTRACT The objective of the study was to compare the manual Mycobacteria Growth Indicator Tube (MGIT) method and the BACTEC MGIT 960 system to the BACTEC 460 method for susceptibility testing of Mycobacterium tuberculosis. The evaluation was based on testing of 36 M. tuberculosis strains with various susceptibilities to isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM). In addition, five of the strains generating discrepant results in testing for EMB were analyzed for heteroresistance. For INH, the susceptibility test results obtained by the MGIT 960 and the manual MGIT systems agreed with the BACTEC 460 results in 94 and 97% of the cases, respectively. The results of susceptibility to RMP were all in agreement. For SM, 78 and 72% of the results obtained by the MGIT 960 and the manual MGIT systems, respectively, agreed with the BACTEC 460 results. In contrast, less than 80% of the results for susceptibility to EMB obtained by the two MGIT methods agreed with the BACTEC 460 results. All five strains analyzed for EMB heteroresistance were found to consist of resistant and susceptible subpopulations. The average turnaround times were 6.4 days for the MGIT 960 system, 6.5 for the manual MGIT system, and 8.7 days for the BACTEC 460 method. Both MGIT methods can be regarded as accurate and rapid alternatives to the BACTEC 460 method for detection of strains resistant to INH and RMP. However, more studies are needed for solving the problems associated with susceptibility testing to EMB and SM.

Osteitis after newborn vaccination with three different Bacillus Calmette-Guérin vaccines : twenty-nine years of experience

Newborns in Finland have been vaccinated with Bacillus Calmette-Guérin (BCG) since the 1950s. Until the end of 1970 the vaccine was made from BCG strain Gothenburg by the Swedish BCG laboratory in Gothenburg and from 1971 on from the same strain in Copenhagen, Denmark. It was replaced by the Glaxo vaccine in 1978. Complications caused by BCG vaccination have been under follow-up, and the data have been collected from nationwide registers. In this study we analyzed the incidence rates of BCG osteitis between the years 1960 and 1988. From 1960 to 1970 the incidence rate was from 2.7 to 13.0/100 000 BCG-vaccinated infants (mean, 7.3; median, 6.9). The incidence increased during the years 1971 to 1978 when it varied between 15.3 and 72.9/100 000 BCG-vaccinated infants (mean, 36.9; median, 30.4). Since 1978 the incidence has varied between 1.7 and 10.1/100 000 BCG-vaccinated infants (mean, 6.4; median, 7.2). In Britain no reports of BCG osteitis have been published despite the use of the same Glaxo vaccine. Our results indicate that the incidence of BCG osteitis in a given population depends on the BCG vaccine used. The follow-up of BCG complications is an essential part of BCG vaccination program.

Mycobacterium palustre sp. nov., a potentially pathogenic, slowly growing mycobacterium isolated from clinical and veterinary specimens and from Finnish stream waters

Taxonomic studies were performed on a phenotypically homogeneous group of 13 mycobacteria isolated from clinical, veterinary and stream-water samples. The methods applied included chromatographic analyses of bacterial lipids, biochemical tests and sequencing of the 16S rDNA and the internal transcribed spacer 1 (ITS1) region. Positive results in urease, Tween 80 hydrolysis and pyrazinamidase tests and a negative result in a semi-quantitative catalase test, combined with the ability to grow at 42 degrees C, distinguished this group among the yellow-pigmented, slowly growing mycobacteria. Unique fatty acid and mycolic acid profiles in chromatographic analyses and the results of gene sequencing indicated that the novel isolates represent a previously undescribed species, for which the name Mycobacterium palustre sp. nov. is proposed. The fatty acid profile obtained by GLC was characterized by the presence of several methyl-branched fatty acid markers. The most prominent markers were 2-methyleicosanoic, tetracosanoic and hexacosanoic acids. According to 16S rDNA sequencing, M. palustre is phylogenetically closest to Mycobacterium kubicae, a recently described species. M. palustre gives a false-positive result in a hybridization test with the AccuProbe Mycobacterium avium complex. One of the strains was isolated from a lymph-node biopsy from a child with cervical lymphadenitis. Thus, M. palustre should be listed among potential inducers of paediatric lymphadenitis. The veterinary isolates originated from the lymph nodes of slaughter pigs. The majority of the strains were recovered from natural waters, which highlights the role of the environment as a source of potentially pathogenic mycobacteria. The type strain of M. palustre is strain E846T (= DSM 44572T = ATCC BAA-377T).

Persistent Legionella pneumophila colonization of a hospital water supply: efficacy of control methods and a molecular epidemiological analysis

After a nosocomial outbreak caused by Legionella pneumophila serogroup 5, the hospital water distribution system, which was found to be colonized by L. pneumophila serogroups 5 and 6, was decontaminated by the superheat and flush method and by installing an additional heat‐shock unit in one of the hot water circuits. This unit exposed the recirculated water to a temperature of 80 °C. The efficacy of the decontamination measures was evaluated by monitoring the temperatures and legionella concentrations at different parts of the hot water distribution system. The genetic diversity of the colonizing legionella flora was examined using two genotyping methods: amplified fragment length polymorphism analysis (AFLP) and random amplified polymorphic DNA (RAPD) analysis. Selected serogroup 6 strains were also analyzed by sequence‐based typing (SBT). The results indicated that long‐term eradication of serogroup 5 strains was never achieved. Only one serogroup 6 strain was never isolated after the superheat and flush. In all, according to genetic fingerprints, the diversity of Legionella strains in a hospital water system remains stable over the years regardless of the use of recommended disinfection procedures.

Recurrence of urinary tract infections in adult patients with community-acquired pyelonephritis caused by E. coli : A 1-year follow-up

In a prospective study, 42 women were followed for recurrence of urinary tract infections (UTIs) for 1 y after an index episode of community-acquired pyelonephritis caused by Escherichia coli. Altogether, 26 repeat episodes were detected. Of these, 20 occurred at least 1 month after the index episode and were regarded as recurrences. In all, 40% (17 of 42) of the women had recurrences. An earlier history of UTI increased the risk of recurrence: 52% of the 29 women with previous UTI had at least 1 recurrence, compared with 15% of the 13 patients without previous UTI. E. coli caused the majority (73%) of the recurrences. Genotype comparisons by RAPD-PCR analysis between E. coli isolates from a patient showed that 75% of the original and recurrent strains were genetically non-identical. Of the 54 E. coli strains, 42 were carrying genes coding for G adhesins of P fimbriae: 40 isolates carried class II, 1 class III and 1 carried both class II and III G adhesin genes. Each of the virulence-associated factors (genes for G adhesins, MRHA, haemolysin, type 1C fimbriae, and O and K antigens) was evenly distributed among E. coli isolates of index episodes, independent of the recurrences. The index isolates, however, had more virulence-associated factors than did the isolates from the recurrences which were mainly due to lower UTIs.In a prospective study, 42 women were followed for recurrence of urinary tract infections (UTIs) for 1 y after an index episode of community-acquired pyelonephritis caused by Escherichia coli. Altogether, 26 repeat episodes were detected. Of these, 20 occurred at least 1 month after the index episode and were regarded as recurrences. In all, 40%, (17 of 42) of the women had recurrences. An earlier history of UTI increased the risk of recurrence: 52%, of the 29 women with previous UTI had at least 1 recurrence, compared with 15%, of the 13 patients without previous UTI. E. coli caused the majority (73%) of the recurrences. Genotype comparisons by RAPD-PCR analysis between E. coli isolates from a patient showed that 75%. of the original and recurrent strains were genetically non-identical. Of the 54 E. coli strains, 42 were carrying genes coding for G adhesins of P fimbriae: 40 isolates carried class II, I class III and 1 carried both class II and III G adhesin genes. Each of the virulence-associated factors (genes for G adhesins, MRHA, haemolysin, type 1C fimbriae, and O and K antigens) was evenly distributed among E. coli isolates of index episodes, independent of the recurrences. The index isolates, however, had more virulence-associated factors than did the isolates from the recurrences which were mainly due to lower UTIs.

COMPARISON OF MYCOBACTERIA-INDUCED CYTOTOXICITY AND INFLAMMATORY RESPONSES IN HUMAN AND MOUSE CELL LINES

Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factorα, TNF-α; interleukin 6, IL-6; and interleukinβ, IL-1β) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-α induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-α after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1β. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factor alpha, TNF-alpha; interleukin 6, IL-6; and interleukin beta, IL-1beta) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-alpha induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-alpha after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1beta. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.

Nosocomial Legionella pneumophila serogroup 5 outbreak associated with persistent colonization of a hospital water system.

An outbreak of infections caused by Legionella pneumophila serogroup 5 was detected in a university hospital, and nosocomial reservoirs of the legionella epidemic were examined. Clinical isolates from two patients who had been affected by the L. pneumophila serogroup 5 outbreak, and from another patient with a legionella infection caused by the same serogroup 3 years later, were compared to L. pneumophila serogroup 5 isolates from the hospital water supply by two molecular methods, amplified fragment length polymorphism (AFLP) analysis and random amplified polymorphic DNA analysis (RAPD). Genotyping confirmed the epidemiological linkage of the first two patients, and linked their infections with the hospital water supply. The third clinical strain, which was also linked to the hospital water, was very similar to the epidemic strain. Even though the water distribution system was sanitized (superheat and flush sanitation), the epidemic strain was shown to be persisting in the hospital water outlets several years after its initial discovery.

Intrauterine bacterial growth at birth and risk of asthma and allergic sensitization among offspring at the age of 15 to 17 years

BACKGROUND Microbial colonization of the airways and intestine during birth might have an effect on the risk of asthma and allergic diseases later in life. OBJECTIVE We sought to evaluate the association between intrauterine microbial growth at the time of delivery and the development of asthma and allergic sensitization among offspring. METHODS Intrauterine bacterial culture results were recorded at the time of cesarean delivery of 460 children who were born at Kuopio University Hospital during 1990-1992. When the children reached the age of 15 to 17 years, self-administered questionnaires were sent to the mothers, and 382 of the children were also examined by using skin prick tests. RESULTS Intrauterine growth of potential pathogenic anaerobic bacteria and Streptococcus species at birth was associated with an increased risk of doctor-diagnosed asthma ever (odds ratio [OR], 4.51 [95% CI, 1.56-13.0]; OR, 2.53 [95% CI, 1.19-5.38]) and doctor-diagnosed current asthma (OR, 7.34 [95% CI, 2.44-22.03]; OR, 3.37 [95% CI, 1.46-7.76]) at the age of 15 to 17 years compared with the risk seen in subjects with negative microbial cultures. These findings remained significant also after applying the Bonferroni correction. No significant association after the Bonferroni correction was detected between intrauterine microbial growth and allergic sensitization among offspring. CONCLUSION The results of this study indicated that specific intrauterine microbial growth at the time of birth might increase the risk of asthma among offspring through inflammatory mechanisms. These results indicate new potential targets for future studies on the effects of maternal vaginal microflora and intrauterine infection in the development of asthma among children.

Pulmonary Infection Caused by an Unusual, Slowly Growing Nontuberculous Mycobacterium

ABSTRACT Mycobacterium triplex, a recently described slowly growing nontuberculous mycobacterium, was isolated from a Finnish patient with pulmonary mycobacteriosis. The disease was successfully treated with antimycobacterial drugs. The strain isolated, which was similar to the type strain but differed slightly from the species description, was regarded as a variant of M. triplex sensu stricto. According to present knowledge this variant of the species has never been isolated before.