Interaction of Legionella pneumophila and Helicobacter pylori with bacterial species isolated from drinking water biofilms.

BACKGROUND: It is well established that Legionella pneumophila is a waterborne pathogen; by contrast, the mode of Helicobacter pylori transmission remains unknown but water seems to play an important role. This work aims to study the influence of five microorganisms isolated from drinking water biofilms on the survival and integration of both of these pathogens into biofilms. RESULTS: Firstly, both pathogens were studied for auto- and co-aggregation with the species isolated from drinking water; subsequently the formation of mono and dual-species biofilms by L. pneumophila or H. pylori with the same microorganisms was investigated. Neither auto- nor co-aggregation was observed between the microorganisms tested. For biofilm studies, sessile cells were quantified in terms of total cells by SYTO 9 staining, viable L. pneumophila or H. pylori cells were quantified using 16 S rRNA-specific peptide nucleic acid (PNA) probes and cultivable cells by standard culture techniques. Acidovorax sp. and Sphingomonas sp. appeared to have an antagonistic effect on L. pneumophila cultivability but not on the viability (as assessed by rRNA content using the PNA probe), possibly leading to the formation of viable but noncultivable (VBNC) cells, whereas Mycobacterium chelonae increased the cultivability of this pathogen. The results obtained for H. pylori showed that M. chelonae and Sphingomonas sp. help this pathogen to maintain cultivability for at least 24 hours. CONCLUSIONS: It appears that M. chelonae may have an important role in the survival of both pathogens in drinking water. This work also suggests that the presence of some microorganisms can decrease the cultivability of L. pneumophila but not the viability which indicates that the presence of autochthonous microorganisms can lead to misleading results when the safety of water is assessed by cultivable methods alone.

The use of quaternary ammonium disinfectants selects for persisters at high frequency from some species of non-tuberculous mycobacteria and may be associated with outbreaks of soft tissue infections.

BACKGROUND: Non-tuberculous mycobacteria (NTM) are increasingly important as opportunistic infections after major and minor surgical procedures, likely because they are ubiquitous and not effectively killed by many commonly used disinfectants. Outbreaks of soft tissue infections with NTM appeared related to the use of commercial disinfectants based on quaternary ammonium compounds (QACs). METHODS: We studied the survival of clinical and environmental isolates of Mycobacterium abscessus, Mycobacterium massiliense, Mycobacterium chelonae and Mycobacterium fortuitum after 20 min, 60 min or 24 h exposures to different QACs, and the surviving bacteria were then re-exposed to QACs to see if the percentage of surviving bacteria had increased. The bacteria were labelled with a dnaA-gfp fusion and their level of QAC resistance monitored as increasing fluorescence. The QAC-resistant bacteria were then serially restreaked onto non-selective medium and retested for QAC survival. RESULTS: The frequency of survivors was <1 in 10(5) bacteria with Mycobacterium smegmatis, but >1 in 100 with the other mycobacteria studied. Different environmental and clinical isolates had similar QAC MICs, but QAC survivors of each strain were resistant. The QAC-surviving strains reverted to the original, non-resistant phenotype after several passages on non-selective medium. CONCLUSIONS: QACs should not be used in settings where even minimally invasive procedures are performed, as they select for a non-genetically determined reversible resistant phenotype that appears at high frequency with several rapidly growing mycobacterial species associated with healthcare-related infections. M. smegmatis behaves differently and is not an adequate model for testing the activity of disinfectants against NTM.

Screening for sterilizing activity of antibiotic combinations in an acid model of rapidly growing mycobacteria during the stationary phase of growth.

BACKGROUND: In view of the problems of correlation between the data provided by classical microbiological studies and clinical response, we designed an in vitro method to screen for the sterilizing activity of various antibiotics, individually or in combinations, against clinical isolates of various rapidly growing mycobacteria in the stationary phase. MATERIAL AND METHODS: We screened a large number of antibiotic combinations (4-16 microg/ml) for their sterilizing capacity in 26 Mycobacterium fortuitum clinical isolates, 7 Mycobacterium chelonae and 2 Mycobacterium abscessus clinical isolates (10(5) CFU). RESULTS: The best results against M. fortuitum were obtained with moxifloxacin, both on its own (13/26 strains) and in combination. This drug is also very active against M. chelonae (3/7 strains), and in combination with clarithromycin and amikacin exhibits sterilizing activity against all the strains studied. Combinations of clarithromycin with moxifloxacin or linezolid at high doses (16 microg/ml) exhibit activity against M. abscessus. CONCLUSIONS: The most relevant finding of our study is the good activity of moxifloxacin against these microorganisms in the stationary phase. This indicates the need to confirm these data in animal models or clinical trials in order to determine their true clinical importance.

Non-pigmented rapidly growing mycobacteria smooth and rough colony phenotypes pathogenicity evaluated using in vitro and experimental models.

Non-pigmented rapidly growing mycobacteria (NPRGM) are widely distributed in water, soil and animals. It has been observed an increasing importance of NPRGM related-infections, particularly due to the high antimicrobial resistance. NPRGM have rough and smooth colony phenotypes, and several studies have showed that rough colony variants are more virulent than smooth ones. However, other studies have failed to validate this observation. In this study, we have performed two models, invitro and in vivo, in order to assess the different pathogenicity of these two phenotypes. We used collection and clinical strains of Mycobacteriumabscessus, Mycobacterium fortuitum and Mycobacteriumchelonae. On the invitro model (macrophages), phagocytosis was higher for M. abscessus and M. fortuitum rough colony variant strains when compared to smooth colony variants. However, we did not find differences with colonial variants of M. chelonae. Survival of Galleriamellonella larvae in the experimental model was lower for M. abscessus and M. fortuitum rough colony variants when compared with larvae infected with smooth colony variants. We did not find differences in larvae infected with M. chelonae.Results of our in vivo study correlated well with the experimental model. This fact could have implications on the interpretation of the clinical significance of the NPRGM isolate colonial variants.

Treatment of water-based metalworking fluids to prevent hypersensitivity pneumonitis associated with Mycobacterium spp.

AIM: To prevent further outbreaks of hypersensitivity pneumonitis (HP), biocides are required which are capable of protecting water-based coolants from proliferating mycobacteria. The aim of this study was therefore, to test different biocide preparations on their mycobactericidal activity. METHODS AND RESULTS: Minimal inhibitory concentration values were determined for Mycobacterium chelonae and Mycobacterium immunogenum for triazine-based, methyloxazolidine-based, N/O-formal-based biocidal formulations. All biocides were effective already at a low dosage (<0.05%) irrespective of the presence or absence of organic soiling, except for one N/O-formal-based formulation containing Kathon 886 (CMI). Quenching of CMI in the presence of organic soiling was found to account for loss in efficacy as determined by high-performance liquid chromatography measurement. Preservation tests were carried out to investigate the efficacy of the biocidal preparations under practical conditions. CONCLUSIONS: Results indicate that methyloxazolidine-based biocidal preparations were most effective to prevent coolants from microbial contamination including rapidly growing mycobacteria. Furthermore, it could be demonstrated that common dipslides can be used to easily monitor coolants for contamination by mycobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data does not support the hypothesis that mycobacterial proliferation is enhanced by the reduction of competitive microbial population by biocides such as triazines as described earlier but rather suggests a protective effect of biocides regarding mycobacteria in the presence of competitive microbial flora, thereby preventing further outbreaks of HP.

Disseminated Mycobacterium chelonae infection in a patient with T-cell lymphoma.

Infections with rapidly growing mycobacteria are rare and most often seen in immunocompromised patients. We herein present the case of a 69-year-old man with a T-cell lymphoma treated by chemotherapy and mogamulizumab with a 6-month history of febrile episodes and subcutaneous nodules in both arms and arthritis of metacarpophalangeal joints. Blood cultures and DNA sequencing results demonstrated the growth of Mycobacterium chelonae. The patient was successfully treated with clarithromycin, moxifloxacin, and tobramycin, but died shortly after due to lymphoma progression.

The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome-cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.

In vitro antimicrobial susceptibility testing of rapidly growing mycobacteria using the tablet diffusion method: resistance pattern of Norwegian Mycobacterium fortuitum and Mycobacterium chelonae isolates.

Thirty-one Norwegian clinical isolates of rapidly growing mycobacteria classified as Runyon's group IV, including 20 Mycobacterium fortuitum and 11 Mycobacterium chelonae strains, were found resistant to a majority of tuberculostatic agents. Minimal inhibitory concentration (MIC) was determined for twelve other antimicrobial agents: amikacin, tobramycin, streptomycin, cefoxitin, imipenem, norfloxacin, ciprofloxacin, doxycycline, erythromycin, fusidic acid, co-trimoxazole and capreomycin. The agar plate dilution method was employed and compared with the agar tablet diffusion method. Regression lines were established correlating MIC values and inhibition zones. The agar tablet diffusion method was found to be a simple and useful method for testing antimicrobial susceptibilities of M. fortuitum and M. chelonae, and a good correlation between MIC values and zone sizes with twelve antimicrobial agents was revealed. Correlation coefficients for most of these antimicrobial agents were around -0.90. M. chelonae was generally more resistant than M. fortuitum. Four antimicrobial agents, capreomycin, norfloxacin, ciprofloxacin and amikacin, showed differences between M. fortuitum and M. chelonae large enough to allow the zone diameter to be used diagnostically.

Comparison of liquid chemical sterilization with peracetic acid and ethylene oxide sterilization for long narrow lumens.

The aim of this study was to determine how well peracetic acid liquid chemical sterilization (LCPAS) killed test organisms in the presence of 10% fetal bovine serum and 0.65% salt challenge (RPMI-S) compared with a 100% ethylene oxide (ETO) sterilizer and an ETO hydrochlorofluorocarbon (ETO-HCFC) sterilization method with long (125 cm), narrow (3-mm internal diameter) flexible lumens as the test carrier. The inoculated lumens were dried overnight before processing. The test organisms included Mycobacterium chelonei, Enterococcus faecalis, and Bacillus subtilis. For all 3 organisms tested, the LCPAS process resulted in a 6 log10 reduction in bacterial load compared with a 2.5 log10 to 6 log10 reduction for the 100% ETO and ETO-HCFC sterilizers. Sterilization was achieved for 100%, 61%, and 67% of the lumen test carriers for the LCPAS, 100% ETO, and ETO-HCFC sterilizers, respectively. The data indicate that of the sterilization methods evaluated, LCPAS was the most effective for sterilizing narrow flexible lumens in the presence of residual inorganic and organic soil. This effectiveness was achieved through a combination of organism wash-off and peracetic acid sterilant killing of organisms. Salt was the major compounding factor for effective ETO gas sterilization, because carriers inoculated with organisms in 10% fetal bovine serum alone all were sterilized by both 100% ETO and ETO-HCFC sterilization methods. Our data support the critical need to ensure adequate precleaning of narrow flexible lumen endoscopes before any sterilization method.

Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species.

DNA from several species of fast growing mycobacteria displayed a characteristic restriction fragment length polymorphism (RFLP) pattern when hybridizated to a Mycobacterium fortuitum 16S rRNA gene fragment. The resulting patterns were identical when comparing different strains belonging to the same species. The RFLP results were consistent with those obtained by DNA-DNA hybridization studies. Using this approach, we have been able to identify the number of copies for 16S rRNA genes in several fast-growing mycobacteria.

Rapidly growing mycobacterial infections after pedicures.

BACKGROUND: Rapidly growing mycobacteria (RGM) can cause a variety of cutaneous and systemic diseases. The causative organisms are typically Mycobacterium fortuitum or Mycobacterium chelonae (also known as Mycobacterium abscessus). Primary cutaneous lesions may develop after a variable latent period, from weeks to several months, and usually result from direct inoculation after trauma, from injections, or during surgery via contaminated medical instruments. Recently, investigators from the Centers for Disease Control and Prevention, Atlanta, Ga, and the California Department of Health Services, Berkeley, documented a large, unprecedented outbreak of community-acquired RGM infection, during which more than 100 patrons of a northern California nail salon contracted furunculosis in their legs as a result of exposure to whirlpool footbaths that were contaminated with M fortuitum. OBSERVATIONS: We report the clinical and epidemiological findings in 3 cases of lower extremity RGM infections that occurred after similar whirlpool footbath exposure at several different nail salons in southern California. These infections typically presented as recurrent furunculosis, causing considerable morbidity as a result of scarring, delayed diagnosis, and the need for long-term polymicrobial therapy. CONCLUSIONS: Rapidly growing mycobacterial infections related to pedicures may continue to occur in a sporadic fashion. Clinicians should consider the possibility of RGM infection and inquire about recent pedicures in a patient with recurrent lower extremity furunculosis and abscesses that are unresponsive to conventional antibiotic therapy.

Mycobacterium fortuitum and Mycobacterium chelonae biofilm formation under high and low nutrient conditions.

The rapidly growing mycobacteria (RGM) are broadly disbursed in the environment. They have been recovered from freshwater, seawater, wastewater and even potable water samples and are increasingly associated with non-tuberculous mycobacterial disease. There is scant evidence that non-tuberculous mycobacteria (NTM) and RGM form biofilms. Therefore, an experimental system was designed to assess the ability of RGM to form biofilms under controlled laboratory conditions. A flat plate reactor flow cell was attached to either a high or low nutrient reservoir and monitored by image analysis over time. Two surfaces were chosen for assessment of biofilm growth: silastic which is commonly used in medical settings and high density polyethylene (HDPE) which is prevalent in water distribution systems. The results show that Mycobacterium fortuitum and M. chelonae formed biofilms under both high and low nutrient conditions on both surfaces studied. These results suggest that RGM may form biofilms under a variety of conditions in industrial and medical environments.

Clinicopathologic study of satellite lesions in nontuberculous mycobacterial keratitis.

Multifocal stromal infiltrates or "satellite lesions" have been considered a characteristic feature of fungal keratitis. We examined two patients with nontuberculous mycobacterial keratitis who clinically presented with satellite lesions. The keratitis consisted of multifocal stromal infiltrates with indistinct white and fluffy margins. Both patients received topical fortified amikacin therapy with poor response. Lamellar keratectomy or penetrating keratoplasty was performed, respectively, in the two patients because of progressive stromal thinning and enlarging satellite lesions. Histopathologically, the main lesions consisted of dense infiltration of inflammatory cells with numerous acid-fast bacilli, while the satellite lesions were composed chiefly of inflammatory cells with fewer mycobacteria. Besides fungal keratitis, nontuberculous mycobacterial keratitis should also be considered when satellite lesions are present.

[Postoperative infection outbreak of M. chelonae subsp. abscessus].

OBJECTIVE: To study the pathogen of postoperative infection outbreak in a hospital in Shenzhen. METHODS: According to the Laboratory Science Procedure of Diagnostic Bacteriology in Tuberculosis published by Chinese Antituberculosis Association, Bergey's Manual of Determinative Bacteriology (ninth edition), and the Clinical Bacteriology Examination & Development, 97 wound exudates were cultivated by the traditional bacteriologic method. The strains were identified with three standard strains of M. chelonae subsp. chelonae, M. chelonae subsp. abscessus and M. fortuitum as controls. RESULTS: 26 fast-acid positive and fast growth nontuberculous mycobacterium were isolated from 97 exudates, then 41 strains including other 15 strains which were collected from the hospital were identified as M. chelonae subsp. abscessus. CONCLUSION: The outbreak of the postoperative infection happened in the hospital was caused by M. chelonae subsp. abscessus.

Gene replacement in Mycobacterium chelonae: application to the construction of porin knock-out mutants.

Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.

Localized cutaneous infections in immunocompetent individuals due to rapidly growing mycobacteria.

Rapidly growing mycobacteria (RGM) cause skin infections that are refractory to standard antibiotic regimens. Although typically associated with disseminated cutaneous or other systemic infections in immunocompromised patients, RGM sometimes cause localized cutaneous infections in immunocompetent hosts. These infections are almost always associated with precedent skin trauma and inoculation, and therefore have been implicated in outbreaks involving contaminated tattoo ink and inadequately sterilized acupuncture needles. Histologic features often include suppurative granulomatous inflammation, and microorganisms are rarely visualized with stains for acid-fast bacilli. The differential diagnosis includes granulomatous fungal and non-RGM bacterial infections as well as noninfectious suppurative or sarcoidlike conditions. Because no pathognomonic histologic features exist for cutaneous RGM infections, clinical suspicion and appropriate workup are essential to reach an accurate and timely diagnosis. Most localized cutaneous RGM infections in immunocompetent individuals respond well to either clarithromycin or amikacin, in combination with surgical debridement.

A pseudoepidemic of Mycobacterium chelonae: contamination of a bronchoscope and autocleaner.

BACKGROUND: At fibreoptic bronchoscopy the potential exists for contamination of bronchoscopes and microbiological specimens. Patients may also be cross infected with acid fast bacilli (AFB). During a five month period, 12 bronchial wash specimens of 65 patients undergoing bronchoscopy, one bronchoscope and an autocleaner, were contaminated with the AFB, Mycobacterium chelonae (MCH). AIM: To eradicate AFB contamination of bronchoscopy specimens by identifying sources of contamination and modifying disinfection procedures. METHODS: To identify the source of contamination, samples for AFB culture were taken from three bronchoscopes, the autocleaner and water taps. To eradicate MCH contamination, the bronchoscopes were soaked in 2% glutaraldehyde overnight and flushed with 70% alcohol. Disinfection procedures were altered by using sterile water and containers in cleaning. Autocleaner use was discontinued. RESULTS: The autocleaner, one bronchoscope and 12 bronchial wash specimens were contaminated with MCH. All contaminants had similar electrophoretic banding on probing of their DNA fragments, suggesting a common clone of origin. After the alterations in disinfection procedures and despite prolonged soaking in 2% glutaraldehyde, three further contaminated wash specimens were isolated from one bronchoscope. Only after ethylene oxide sterilisation of this bronchoscope was the contamination overcome. Since then no further MCH contamination has occurred. No patient required treatment and there has been no clinical evidence of mycobacterial disease. CONCLUSION: To avoid contamination of bronchoscopy specimens with MCH, use of autoclearners should be discouraged and sterile water and containers used in cleaning procedures. If MCH contamination occurs in this setting, the bronchoscope and dismantled valve mechanism should undergo ethylene oxide sterilisation.

Comparison of in vitro activities of gatifloxacin and ciprofloxacin against four taxa of rapidly growing mycobacteria.

By using current NCCLS broth microdilution methods, we found that gatifloxacin inhibited 90% of the isolates of the Mycobacterium fortuitum group at < or =0.12 micro g/ml and 90% of the Mycobacterium chelonae isolates at < or =4 micro g/ml. Gatifloxacin was generally fourfold more active than ciprofloxacin. We recommend that both gatifloxacin and ciprofloxacin be tested routinely against rapidly growing mycobacteria.

Comparison of the in vitro activity of the glycylcycline tigecycline (formerly GAR-936) with those of tetracycline, minocycline, and doxycycline against isolates of nontuberculous mycobacteria.

We compared the in vitro activity of the glycylcycline tigecycline (formerly GAR-936) with those of tetracycline, doxycycline, and minocycline by broth microdilution against 76 isolates belonging to seven species of rapidly growing mycobacteria (RGM) and 45 isolates belonging to five species of slowly growing nontuberculous mycobacteria (NTM). By using a resistance breakpoint of >4 micro g/ml for tigecycline and >8 micro g/ml for tetracycline, all RGM were highly susceptible to tigecycline, with inhibition of 50% of isolates at < or =0.12 micro g/ml and inhibition of 90% of isolates at 0.25 micro g/ml for Mycobacterium abscessus and inhibition of both 50 and 90% of isolates at < or =0.12 micro g/ml for M. chelonae and the M. fortuitum group. The MICs of tigecycline were the same for tetracycline-resistant and -susceptible strains, and RGM isolates were 4- to 11-fold more susceptible to tigecycline than to the tetracyclines. In contrast, no slowly growing NTM were susceptible to tigecycline, and isolates of M. marinum and M. kansasii were less susceptible to this agent than to minocycline. This new antimicrobial offers exciting therapeutic potential for the RGM, especially for isolates of the M. chelonae-M. abscessus group, against which the activities of the currently available drugs are limited.