Biocidal Activity of Formaldehyde and Nonformaldehyde Biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in Pure and Mixed Suspensions in Synthetic Metalworking Fluid and Saline

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.     

Differential biocide susceptibility of the multiple genotypes of <Emphasis Type="Italic">Mycobacterium immunogenum</Emphasis>

The non-tuberculous mycobacterium Mycobacterium immunogenum colonizes industrial metalworking fluids (MWFs) presumably due to its relative resistance to the currently practiced biocides and has been implicated in occupational respiratory hazards, particularly hypersensitivity pneumonitis. With an aim to understand its inherent biocide susceptibility profile and survival potential in MWF, five different genotypes of this organism, including a reference genotype (700506) and four novel test genotypes (MJY-3, MJY-4, MJY-10 and MJY-12) isolated in our recent study from diverse MWF operations were evaluated. For this, two commercial biocide formulations, Grotan (Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine) and Kathon (5-chloro-2-methyl-4-isothiazolin-3-one) currently practiced for the control of microorganisms, including mycobacteria, in MWF operations were tested. Effect of the fluid matrix on the biocide susceptibility was investigated for the synthetic (S) and semi-synthetic (SS) MWF matrices. In general, the minimum inhibitory concentration values were higher for the HCHO-releasing biocide Grotan than the isothiazolone biocide Kathon. All genotypes (except the reference genotype) showed lower susceptibility in SS as compared to S fluid matrix for Grotan. However, in case of Kathon, a greater susceptibility was observed in SS fluid for majority of the test genotypes (MJY-3, 4 and 10). The test genotypes were more resistant than the reference genotype to either biocide in both fluid types. Furthermore, the individual genotypes showed differential biocidal susceptibility, with MJY-10 being the most resistant. These observations emphasize the importance of using the resistant genotypes of M. immunogenum as the test strains for formulation or development and evaluation of existing and novel biocides, for industrial applications.     

Presence of a single genotype of the newly described species Mycobacterium immunogenum in industrial metalworking fluids associated with hypersensitivity pneumonitis

Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.     

Presence of a Single Genotype of the Newly Described Species Mycobacterium immunogenum in Industrial Metalworking Fluids Associated with Hypersensitivity Pneumonitis

Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.     

Expanding the mycobacterial diversity of metalworking fluids (MWFs): evidence showing MWF colonization by Mycobacterium abscessus

Nontuberculous mycobacteria (NTM) have been associated with hypersensitivity pneumonitis in machinists. Only two species of NTM, namely Mycobacterium immunogenum and Mycobacterium chelonae, have been reported thus far to have the ability to colonize contaminated metalworking fluids (MWFs). Here, we report, for the first time, the presence and characterization (phenotypic and genotypic) of a third species, Mycobacterium abscessus, colonizing these harsh alkaline machining fluids. Two Mycobacterium morphotypes, smooth (S) and rough (R), were isolated (two isolates each) from an in-use industrial MWFs. Biocide susceptibility analysis using triclosan as a model yielded the same minimal inhibitory concentration for the two morphotypes. PCR-restriction analysis-based speciation of the morphotypes confirmed their identity as M. abscessus. Genotyping based on partial DNA sequences corresponding to the variable regions of the hsp65 gene and 16S-23S rRNA operon internal transcribed spacer region and randomly amplified polymorphic DNA-PCR analysis showed that both morphotypes belong to a single genotype. In addition, we isolated and confirmed two novel mycobacterial genotypes, one each of M. immunogenum and M. chelonae from additional in-use MWF screening. Taken together, this study expands the known mycobacterial species- and strain-diversity colonizing MWF. Furthermore, the study emphasizes the need for including M. abscessus species in the existing mycobacterial screening of contaminated MWF.     

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 decribed earlier but rather suggests a protective effect of biocides regarding mycobacteria in the presence of competitive microbial flora, thereby preventing further outbreaks of HP.     

Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods

Aims: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co‐contaminants, including culturable and non‐culturable sub‐populations, in metalworking fluids (MWF). Methods and Results: Auramine‐O‐rhodamine (AR) staining and LIVE/DEAD BacLight? Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non‐viable) of the MWF‐associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml^?1 and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining‐based microscopy allowed differential quantification of viable vs non‐viable cells. In general, a 3‐log difference was observed between the L/D microscopy count and culture count accounting for the presence of non‐culturable fraction in the bacterial population in in‐use MWF. Conclusions: The optimized AR staining‐ and the L/D staining‐based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non‐viable) of MWF‐associated mycobacteria and co‐contaminants in field MWF. Significance and Impact of the study: Early detection of MWF mycobacteria by rapid, low‐cost, less‐skill intensive and culture‐independent fluorescence‐based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.     

Factors influencing the microbial composition of metalworking fluids and potential implications for machine operator's lung

Hypersensitivity pneumonitis, also known as "machine operator's lung" (MOL), has been related to microorganisms growing in metalworking fluids (MWFs), especially Mycobacterium immunogenum. We aimed to (i) describe the microbiological contamination of MWFs and (ii) look for chemical, physical, and environmental parameters associated with variations in microbiological profiles. We microbiologically analyzed 180 MWF samples from nonautomotive plants (e.g., screw-machining or metal-cutting plants) in the Franche-Comté region in eastern France and 165 samples from three French automotive plants in which cases of MOL had been proven. Our results revealed two types of microbial biomes: the first was from the nonautomotive industry, showed predominantly Gram-negative rods (GNR), and was associated with a low risk of MOL, and the second came from the automotive industry that was affected by cases of MOL and showed predominantly Gram-positive rods (GPR). Traces of M. immunogenum were sporadically detected in the first type, while it was highly prevalent in the automotive sector, with up to 38% of samples testing positive. The use of chromium, nickel, or iron was associated with growth of Gram-negative rods; conversely, growth of Gram-positive rods was associated with the absence of these metals. Synthetic MWFs were more frequently sterile than emulsions. Vegetable oil-based emulsions were associated with GNR, while mineral ones were associated with GPR. Our results suggest that metal types and the nature of MWF play a part in MWF contamination, and this work shall be followed by further in vitro simulation experiments on the kinetics of microbial populations, focusing on the phenomena of inhibition and synergy.     

Decreased biocide susceptibility of adherent Legionella pneumophila.

In a study of the in vitro effectiveness of biocides against Legionella pneumophila, some aspects of the cooling tower environment were replicated in the laboratory, paying particular attention to water hardness and pH. Pieces of Douglas fir and polyvinyl chloride were colonized in a recirculating system and the comparative efficacy of two biocides (Bronopol and Kathon) against the sessile and planktonic populations was examined. While the biocides were relatively effective against the planktonic L. pneumophila population over a short period of time (minimum 9-12 h), substantially longer periods of time (maximum greater than 48 h) were required to reduce the number of cultivable bacteria to below detectable levels in the adherent population. The results indicate that failure to monitor the sessile population of L. pneumophila in laboratory studies of biocides may result in the use of incorrect dosages and/or contact times in field trials with apparently reduced in situ efficacy.     

Ortho-phthalaldehyde: a possible alternative to glutaraldehyde for high level disinfection

Ortho-phthalaldehyde (OPA) was tested against a range of organisms including glutaraldehyde-resistant mycobacteria, Bacillus subtilis spores and coat-defective spores. Glutaraldehyde (GTA) and peracetic acid (PAA) were tested for comparative purposes. Both suspension and carrier tests were performed using a range of concentrations and exposure times. All three biocides were very effective (> or = 5 log reduction) against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in suspension tests. OPA and GTA (PAA was not tested) were also very effective against Staph. aureus and Ps. aeruginosa in carrier tests. OPA showed good activity against the mycobacteria tested including the two GTA-resistant strains, but 0.5% w/v OPA was found not to be sporicidal. However, limited activity was found with higher concentrations and pH values. Coat-defective spores were more susceptible to OPA, suggesting that the coat may be responsible for this resistance. The findings of this study suggest that OPA is effective against GTA-resistant mycobacteria and that it is a viable alternative to GTA for high level disinfection.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.     

Mechanisms of bacterial resistance to biocides

Bacterial resistance to biocides is basically of two types: (i) intrinsic, a natural chromosomally-controlled property of an organism, (ii) acquired, resulting from genetic changes in a cell and arising either by mutation or by the acquisition of genetic material. Both types of resistance are discussed together with the underlying biochemical mechanisms where known. Specific examples of organisms are provided by reference to bacterial spores, mycobacteria, other Gram-positive bacteria and Gram-negative bacteria. The stability of resistance to biocides is considered, as is the possible linkage between biocide and antibiotic resistance.     

Importance of porins for biocide efficacy against Mycobacterium smegmatis

Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N'-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides.     

Surviving within the amoebal exocyst: the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> complex paradigm

Background  

Most of environmental mycobacteria have been previously demonstrated to resist free-living amoeba with subsequent increased virulence and resistance to antibiotics and biocides. The Mycobacterium avium complex (MAC) comprises of environmental organisms that inhabit a wide variety of ecological niches and exhibit a significant degree of genetic variability. We herein studied the intra-ameobal location of all members of the MAC as model organisms for environmental mycobacteria.     

Decreased biocide susceptibility of adherent Legionella pneumophila

J.B. WRIGHT. I. RUSESKA AND J.W. COSTERTON. 1991. In a study of the in vitro effectiveness of biocides against Legionella pneumophila , some aspects of the cooling tower environment were replicated in the laboratory, paying particular attention to water hardness and pH. Pieces of Douglas fir and polyvinyl chloride were colonized in a recirculating system and the comparative efficacy of two biocides (Bronopol and Kathon) against the sessile and planktonic populations was examined. While the biocides were relatively effective against the planktonic L. pneumophila population over a short period of time (minimum 9-12 h), substantially longer periods of time (maximum > 48 h) were required to reducc the number of cultivable bacteria to below detectable levels in the adherent population. The results indicate that failure to monitor the sessile population of L. pneumophila in laboratory studies of biocides may result in the use of incorrect dosages and/or contact times in field trials with apparently reduced in situ efficacy.     

Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.     

Effect of some biocides on non-tuberculous mycobacteria

The incidence of disease and nosocomial infections produced by non tuberculosis mycobacteria (NTM) has increased in immunocompetent patients, but also and more frequently, in immunosuppressed patients. Several studies have disclosed that mycobacteria are more resistant to biocides than non-sporulating bacteria; in addition, some species are particularly resistant. The biocide action of sodium hypochloride and glutaraldehyde on Mycobacterium aviumintracellulare, Mycobacterium gordonae, Mycobacterium fortuitum and Mycobacterium chelonae was studied, using a modified Kelsey Maurer test. For the different species, both the minimal inhibitory concentration (MIC) and the minimal action time were determined. Effectiveness of sodium hypochloride and glutaraldehyde against the different mycobacterial species varied. The same was true for different isolates of the same species. Sodium hypochloride effective MIC and exposure time (killing of 99.99% of all NTM) were 0.2% and 5 minutes, respectively. In order to achieve 100% killing, 0.5% MIC and 15 minute exposure were needed. In the case of glutaraldehyde, 99.99% of the bacteria were killed with 1% MIC and a 15 minute exposure. An effectiveness of 100% was achieved with a 2% MIC of glutaraldehyde and a 15 minute exposure. Sodium hypochloride and glutaraldehyde are effective biocides for mycobacteria. The first biocide is cheap and effective at low concentrations, but its corrosive and oxidant nature makes it impossible for use in hospitals or with laboratory equipment. Glutaraldehyde (neither corrosive nor oxidant) is a safe alternative for disinfection of this type of equipment. However, it is important to bear in mind that these pathogens may develop resistance to biocides.     

Microbial growth and accumulation in industrial metal-working fluids

The dynamics of microbial growth in metal-working fluids (MWF) and the effect of the addition of biocides were studied in large fluid systems, in this case, one central tank which holds 150 m3. In this system, populations of Pseudomonas pseudoalcaligenes (greater than 10(8) CFU/ml) were sustained for a year, although large quantities of biocides were added. Quantitation of 3-OH lauric acid, a marker for many Pseudomonas spp., by gas chromatography indicated that the bacterial biomass exceeded the viable counts by approximately 15 times. Fungi were grown on several occasions, the dominating genera being Fusarium and Candida. Soon after the old MWF was removed and the tank was provided with fresh MWF, which consisted of an emulsion of mineral oil in water, there was a massive growth of P. pseudoalcaligenes that reached levels of greater than 10(8) bacteria per ml. Initially, only low concentrations of other species were found for some weeks. After this period, different enterobacteria and other gram-negative rods often appeared at high concentrations (10(7) and 10(8) bacteria per ml, respectively). Bacteria identified as P. pseudoalcaligenes showed great variation with respect to colony morphology and a certain heterogeneity with respect to biochemical characteristics. Certain bacterial species grew as microcolonies on metal strips immersed in the circulating MWF, but P. pseudoalcaligenes was not recovered from this habitat. The total bacterial count in the air surrounding the machines in the metal-working shop showed an inverse relation to increasing distance from the machine. The concentration of bacteria in the air varied because of the number of machines in use, temperature, and humidity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of respirometry as a short-term method to assess the efficacy of biocides

This study shows that a short-term respirometric measurement based on the rate of oxygen uptake needed to oxidize glucose is a reliable and fast method to assess biocide efficacy against P. fluorescens cells. Respiratory activity using oxygen consumption rate, the determination of viable and nonviable cells using Live/Dead BacLight kit and colony formation units (CFU), were compared as indicators of the biocidal efficacy of ortho-phthalaldehyde (OPA). The results showed that determining the effect of OPA against P. fluorescens using the different methods leads to different conclusions. The minimum bactericidal concentration (MBC) was 80 mgl(-1), 100 mgl(-1) and 65 mgl(-1) respectively, using respiratory activity, viability using BacLight counts and culturability. The plate count method was shown to underestimate the biocidal action of OPA, whilst data from respirometry and viability using Live/Dead BacLight kit correlated strongly and were not statistically different when yellow cells were considered nonviable. Respirometry therefore represents an expeditious, non-destructive and accurate method to determine the antimicrobial action of biocides against aerobic heterotrophic bacteria.