Antimicrobial activity of cuprous oxide-coated and cupric oxide-coated surfaces.

BACKGROUND: Disease can be spread through contact with contaminated surfaces (fomites). For example, fomites have been implicated in the spread of meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Antimicrobial surface treatments are a potential method of reducing disease transmission from fomites, and broad-spectrum activity is desirable. AIM: To test cuprous oxide (Cu2O) and cupric oxide (CuO) coatings for antimicrobial activity against 12 micro-organisms including bacteria and fungi. METHODS: We fabricated two surface coatings. The Cu2O coating was fabricated in a simple two-step process using polyurethane to bind the active copper oxide particles; CuO was prepared by heat treatment of Cu2O particles in air to produce cupric oxide (CuO) and to cause early-stage sintering to form a continuous coating. The antimicrobial activity was examined with 10 µL of microbial suspension droplets followed by counting cells as colony-forming units (cfu). FINDINGS: The coatings rapidly killed nine different micro-organisms, including Gram-negative and Gram-positive bacteria, mycobacteria and fungi. For example, the Cu2O/PU coating killed 99.9997% of P. aeruginosa and 99.9993% of S. aureus after 1 h. Efficacy was not reduced after weekly cleanings. The antimicrobial activity of the Cu2O coating was unchanged after abrasion treatment, and the coatings were not cytotoxic to human cells. CONCLUSION: The combination of broad-spectrum antimicrobial activity, abrasion resistance, and low toxicity of the Cu2O coating suggests potential use in healthcare settings.

Disinfection and cleaning of heater-cooler units: suspension- and biofilm-killing.

BACKGROUND: Non-tuberculous mycobacteria (NTM) infections in cardiac surgery patients, caused by Mycobacterium chimaera or Mycobacterium abscessus, have been traced to NTM-aerosols produced by heater-cooler units of cardiopulmonary bypass equipment. AIM: To develop a protocol to disinfect the water reservoir(s) of heater-coolers to reduce NTM numbers and thereby prevent potential NTM aerosolization; and to devise an approach to disrupt surface biofilms of heater-coolers to reduce reinoculation of the heater-cooler reservoir(s) after disinfection. METHODS: A laboratory-scale Centers for Disease Control and Prevention bioreactor and a heater-cooler were inoculated with M. chimaera or M. abscessus to measure the ability of different disinfection protocols to reduce NTM colony-forming units in water and biofilm samples and to delay the reappearance of NTM after disinfection. FINDINGS: The combination of an enzyme detergent cleaning agent and Clorox® were equivalent to Clorox alone in reducing M. chimaera cfu in heater-cooler water reservoir samples. However, reappearance of those bacteria was delayed by 12 weeks by the combination of enzyme detergent cleaning agent and Clorox exposure compared to Clorox disinfection alone. CONCLUSION: A combination of an enzyme detergent and Clorox was an effective disinfection treatment and significantly delayed the reappearance of M. chimaera in the heater-cooler reservoir.

Adherence and biofilm formation of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus to household plumbing materials.

AIMS: Measure adherence and biofilm formation by cells of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus on common household plumbing materials namely stainless steel, glass, zinc-galvanized steel, copper and polyvinyl chloride (PVC). METHODS AND RESULTS: Coupons in a CDC biofilm reactor were exposed to cell suspensions containing 10(5) NTM colony forming units (CFU) per ml and adherence measured for 6 h. Biofilm formation (increased numbers of adherent CFU) was measured weekly to 21 days in the absence of substantial numbers of suspended mycobacterial cells. Adherence was rapid and substantial with 2000-15 000 CFU cm(-2) adhering within 1-6 h at room temperature. Biofilm numbers reached as high as 10(7)  CFU cm(-2) . Biofilm-grown cells of Myco. avium were more adherent compared with suspension-grown cells. CONCLUSIONS: Mycobacterium avium, Myco. intracellulare and Myco. abscessus readily adhered and formed biofilms on all types of plumbing materials. Factors influencing adherence and biofilm formation were species, plumbing material and prior growth.

Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment.

A majority of the Mycobacterium species, called the nontuberculous mycobacteria (NTM), are natural inhabitants of natural waters, engineered water systems, and soils. As a consequence of their ubiquitous distribution, humans are surrounded by these opportunistic pathogens. A cardinal feature of mycobacterial cells is the presence of a hydrophobic, lipid-rich outer membrane. The hydrophobicity of NTM is a major determinant of aerosolization, surface adherence, biofilm-formation, and disinfectant- and antibiotic resistance. The NTM are oligotrophs, able to grow at low carbon levels [>50 microg assimilable organic carbon (AOC) l(-1)], making them effective competitors in low nutrient, and disinfected environments (drinking water). Biofilm formation and oligotrophy lead to survival, persistence, and growth in drinking water distribution systems. In addition to their role as human and animal pathogens, the widespread distribution of NTM in the environment, coupled with their ability to degrade and metabolize a variety of complex hydrocarbons including pollutants, suggests that NTM may be agents of nutrient cycling.

Microbial-mediated release of bisphenol A from polycarbonate vessels.

AIM: To identify the source of bisphenol A (BPA) [2,2'-bis(4-hydroxyphenyl) propane] in cultures of an antibiotic-producing Bacillus sp. strain grown in polycarbonate flasks. METHODS AND RESULTS: Although a culture of an antibiotic-producing Bacillus sp. strain grown in a new, rinsed polycarbonate flask yielded BPA, duplicate cultures grown in thoroughly washed polycarbonate flasks did not. Cells of Escherichia coli strain C were grown in new polycarbonate flasks rinsed three-times with 100 ml distilled H2O. BPA was only recovered from cultures grown in new polycarbonate flasks, but not from the autoclaved medium incubated in parallel. CONCLUSIONS: BPA was present in either Bacillus or E. coli cultures, probably due to its release from inadequately washed polycarbonate flasks. Standard autoclaving did not result in BPA appearance; microbial growth was required. Polycarbonate vessels for microbial cultures should be thoroughly washed to avoid the appearance of BPA in culture medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study rigorously demonstrates that the presence of BPA in culture medium was a consequence of microbial growth or metabolism in inadequately washed polycarbonate flasks. As BPA exhibits antimicrobial and oestrogenic activity, searches for novel drugs or production of recombinant chemotherapeutic agents could be derailed by the artefactual appearance of BPA.

Effect of different cell fractions of Mycobacterium avium and vaccination regimens on Mycobacterium avium infection.

Because of the availability of uniform genetic stocks and the ability to modulate stress levels, chickens were investigated as a host for the development of an antimycobacterial vaccine. The imposition and the timing of stress significantly influenced the outcome of Mycobacterium avium infection in chickens. Simple, whole cell or lysate vaccines and combinations of vaccine preparations were identified that led to high levels of protection. In addition, short-term stress at the time of vaccination significantly increased the protective efficacy of M. avium vaccine preparations. Post-infection vaccination of M. avium-infected chickens was also shown to significantly reduce the number of lesions and colony counts.

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth.

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.

A luciferase-based method for assessing chlorine-susceptibility of Mycobacterium avium.

A rapid and quantitative assay for the disinfection of the water-borne pathogen, Mycobacterium avium, was developed using firefly luciferase as a reporter gene. There was a correlation between the quantity of light produced and the number of colony-forming units. In chlorine-disinfection studies of a luciferase-carrying derivative of M. avium, there was a strong correlation (r2=0.96) between colony forming units and relative light units. It was discovered that chlorine was rapidly lost from suspensions containing 10(6) M. avium cells/ml. The luciferase-based test can be used to rapidly measure susceptibility of M. avium to different disinfectants used in water treatment.

Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems.

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter(-1). The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r(2) = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm(2)). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.

Rapid coliphage detection assay.

A rapid coliphage detection assay was developed, based on the phage-induced release of beta-galactosidase from cells of Escherichia coli. The assay could detect as few as five coliphage per sample without an overnight incubation period. The range of acceptable assay parameters was identified.

Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity.

A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa. Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.

Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium avium.

Environmental and patient isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five M. avium strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of M. avium in drinking water.

High rates of disseminated infection due to non-tuberculous mycobacteria among AIDS patients in Finland.

OBJECTIVE: to determine the rate of disseminated infection due to non-tuberculous mycobacteria (NTM) among Finnish AIDS patients, and to analyse the epidemiology of these infections. METHODS: in a prospective cohort study HIV-infected patients with CD4 counts < 200 x 10(6)/l were interviewed, and had mycobacterial blood cultures performed at baseline and at 6 months, then subsequently for clinical indications; autopsies were performed on patients who died. The cohort was followed at least for 24 months or to death. Water samples were collected from the homes of patients and from the environment and cultured for organisms of the Myobacterium avium complex (MAC). Environmental and clinical isolates were compared using pulsed field gel electrophoresis (PFGE). RESULTS: NTM infection occurred in 22 (43%) of 51, 19 isolates were Mycobacterium avium, two M. genavense and one M. intracellulare. Multivariate analysis identified urban residence (P=0.04) and eating raw fish (P=0.04) as independent risk factors. Molecular analysis revealed two clusters of related isolates (three M. avium, two M. genavense) among urban residents. CONCLUSION: AIDS patients in Finland have high rates of disseminated infection due to NTM. Clusters of identical organisms and association with urban residence suggests that these are newly acquired infections in advanced AIDS.

The international epidemiology of disseminated Mycobacterium avium complex infection in AIDS. International MAC Study Group.

OBJECTIVE: To determine rates of disseminated Mycobacterium avium complex (MAC) infection among AIDS patients in developed and developing countries, and to determine whether different rates reflect differences in exposure or immunity, or both. DESIGN: Prospective cohort study. SETTING: University hospitals and outpatient AIDS programs. METHODS: HIV-infected subjects with CD4 counts < 200 x 10(6)/l were interviewed and had CD4 lymphocyte counts, blood cultures for mycobacteria (baseline and at 6 months), and skin tests with purified protein derivative (PPD) and M. avium sensitin. RESULTS: Among 566 study patients rates of disseminated MAC were 10.5-21.6% in New Hampshire, Boston and Finland compared to 2.4-2.6% in Trinidad and Kenya (P < 0.001). PPD skin test reactions > or = 5 mm were present in 20% of patients from Kenya compared to 1% at other sites (P < 0.001). Among patients from the United States and Finland, multiple logistic regression indicated that occupational exposure to soil and water was associated with a decreased risk of disseminated MAC, whereas the following were associated with an increased risk of disseminated MAC: low CD4 count, swimming in an indoor pool, history of bronchoscopy, regular consumption of raw or partially cooked fish/shellfish and treatment with granulocyte colony-stimulating factor. CONCLUSIONS: Rates of disseminated MAC in AIDS are higher in developed than developing countries and are due to both differences in exposure and differences in immunity. These data provide a rationale for prevention of MAC through both active immunization and reduction in exposure to the organism.

Isolation and characteristics of Mycobacterium avium complex from water and soil samples in Uganda.

SETTING: Mycobacterium avium complex organisms have not been isolated from late stage AIDS patients in Uganda. This could possibly be due to the absence of M. avium complex in the Uganda environment. OBJECTIVE AND DESIGN: Determine whether M. avium complex organisms could be isolated from water and soils collected in the living environment of Ugandan AIDS patients. RESULTS: Representatives of the M avium complex were isolated from 3 of 7 (43%) water and 3 of 7 (43%) soil samples collected in Kampala, Uganda. The average number of colony-forming units per ml water was 3.3 and average colony-forming units per gram of soil was 7825. In terms of growth characteristics, antimicrobial susceptibility patterns, and the presence or absence of plasmids and IS901, Ugandan M. avium complex isolates were similar to those isolated from the US and European AIDS patients and their environment. CONCLUSIONS: M. avium complex organisms sharing genetic and physiological characteristics of M. avium complex isolates recovered from patients with AIDS can be isolated from water and soil samples in Uganda.

Recovery of Mycobacterium avium from cigarettes.

Mycobacterium avium was recovered from tobacco, cigarette paper, and cigarette filters. M. avium could also be recovered from cigarette filters after the cigarettes had been smoked.