Applied and Environmental Microbiology, September 2004, p . 5667-5671, Vol . 70, No . 9

Electrophoretic Mobility of Mycobacterium avium Complex Organisms

Darren Lytle,^1* Christy Frietch,¹ and Terry Covert²

National Risk Management Research Laboratory,¹ National Exposure Research Laboratory, U.S . Environmental Protection Agency, Cincinnati, Ohio²

Received 22 January 2004/ Accepted 20 April 2004

ABSTRACT

The electrophoretic mobilities (EPMs) of 30 Mycobacterium avium complex organisms were measured . The EPMs of 15 clinical isolates ranged from –1.9 to –5.0 µm cm V^–1 s^–1, and the EPMs of 15 environmental isolates ranged from –1.9 to –4.6 µm cm V^–1 s^–1 at pH 7 .

Nontuberculous mycobacteria (NTM) include Mycobacterium species that are not members of the Mycobacterium tuberculosis complex . Mycobacterium avium, Mycobacterium intracellulare, and MX organisms are NTM, and are included in the Mycobacterium avium complex (MAC) . MX organisms are MAC probe positive by the Accuprobe (Gen-Probe, Inc., San Diego, Calif.) or synthetic nucleic acid probe SNAP (Syngene, San Diego, Calif.) nucleic acid probe identification system but M . avium and M . intracellulare probe negative . MAC organisms are considered human pathogens and in recent years have emerged as a major cause of opportunistic infection in AIDS patients and other immune-compromised hosts (3, 6) . The most common disseminated bacterial infection in AIDS patients is from MAC organisms and is second only to the AIDS wasting syndrome as the most common cause of death (11) .

Many of the NTM are free-living saprophytes that have been isolated from numerous environments, such as water, soil, food, and animals . NTM are able to grow, persist, survive, and colonize in drinking water sources and distribution systems (2, 6) . One survey of U.S . public drinking water supplies found that NTM were detected in 35% of the samples, suggesting that drinking water may be an important source of human exposure to these organisms (2) . MAC organisms have been isolated worldwide from drinking water distribution systems and are believed to be a source of the M . avium organisms infecting immune-compromised hosts (6, 9) . In 1999, the U.S . Environmental Protection Agency (USEPA) published the "Drinking Water Contaminant Candidate List" (CCL), which included MAC organisms due to their clinical significance and their occurrence in drinking water (16) . The CCL lists chemical and microbial contaminants that will be considered for future regulatory action .

The surface properties of Mycobacterium have been studied to provide more information about the composition of the cell surface and catalase heterogeneity . In a collaborative study, the electrophoretic mobilities (EPMs) of catalases of eight BCG mycobacterial strains and three Mycobacterium phlei and five Mycobacterium fortuitum strains and an M . tuberculosis H37Rv strain were determined by polyacrylamide disk electrophoresis (15) . The EPMs of cells of Mycobacterium bovis BCG, M . phlei, Mycobacterium smegmatis, and Mycobacterium microti were compared in acetate-barbiturate solutions from pH 2.5 to 9 (10) . EPMs of all cells were identical, despite differences in growth medium, cell age, and chemical treatments . The similarities were attributed to common features of the bacterial cell wall structure . The EPMs of M . bovis BCG Tice cells were measured in phosphate-buffered water (ionic strengths of 0.005 to 0.1 M) with a variety of treatments (12) . An isoelectric point of 3.4 to 3.7 was determined, and the authors concluded from chemical treatment tests that the negative surface charge was due to carboxylic acids, phosphoesters, and strong acidic groups, possibly sulfates . The EPMs of representative strains of M . avium-M . intracellulare and Mycobacterium scrofulaceum were measured in buffers of ionic strength 0.05 M (7) . The isoelectric point varied from 3.5 to 4.5 . The negative charge of the bacterial cell wall was believed to be associated with amino groups and carboxyl and phosphate groups based on chemical and enzymatic tests .

The electrokinetic properties of MAC organisms in water have not been reported . Such information would be useful in predicting the ease of their removal during water treatment and the fate of these organisms in drinking water distribution systems . The objective of this study was to measure and compare the EPMs of MAC organisms in aqueous suspensions . The EPMs of MAC organisms were compared to the EPMs of other microbiological pathogens measured under similar experimental conditions . The effect of pH on the EPMs of MAC microorganisms was also examined .

Thirty MAC isolates (M . avium, M . intracellulare, and MX) from the USEPA's culture collection were used in the study (Table 1) . The isolates were obtained from studies by Glover et al . (9), Covert et al . (2), and Yoder et al . (17) . The isolates were identified by PCR amplification and sequencing regions of the 16S ribosomal gene, AccuProbe, SNAP, or by PCR-restriction fragment polymorphism, as previously described in these studies . The isolates were grown for 21 days (mid-log phase) at 37°C with 10% CO₂ in 20 ml of Middlebrook 7H9 broth with ADC enrichment (Difco Laboratories, Inc., Detroit, Mich.) . The cells were concentrated by centrifugation (12,857 x g) (Eppendorf centrifuge 5810R) and washed three times in 9.15 mM KH₂PO₄ buffered deionized water . The washed pellets were resuspended in 9.15 mM KH₂PO₄ buffer to an estimated 10⁶ CFU ml^–1 (McFarland Standard, BioMerieux, Durham, N.C.) and were used for subsequent EPM analyses .

 
EPM was measured with a Malvern Zetasizer 4 (Malvern Instruments, Ltd., Malvern, England) . This system measures EPM in micrometers centimeters per volt per second by laser doppler electrophoresis . Negative EPM values represent bacterial movement toward a positive electrode and a net negative surface charge . The instrument operation was checked daily with a MIN-U-SIL (high-purity, high-quality natural crystalline silica ground powder) solution with known EPM . Samples were injected directly into the 1.5-ml quartz capillary electrophoresis measurement cell with 10-ml disposable syringes . Prior to the measurement of each new sample, at least 20 ml of deionized water, followed by 10 ml of the sample to be measured, was rinsed through the capillary cell . Subsequent replicate measurements (typically four or five, depending on the sample volume) were made in 4-ml increments . These measurements were made at 25 ± 2°C . Acid/base titrations with 0.5 M each HCl and NaOH were used to adjust the sample pH to 7 . Statistical comparisons between means of groups were made with unpaired Students' t tests when data sets passed normality tests or the Mann-Whitney rank sum test when data sets failed normality test ( = 0.05) (4, 8) . Normality was tested with the Kolmogorov-Smirnov test . All statistical calculations were made with Sigmastat (version 2.0) (SPSS, Inc.) .

The EPMs of 15 clinical strains and 15 environmental MAC organisms suspended in 9.15 mM KH₂PO₄ buffered deionized water at neutral pH were measured (Table 1) . The EPMs of the clinical strains ranged from –1.94 to –4.95 µm cm V^–1 s^–1, with a mean (± standard deviation) of –3.19 ± 0.83 µm cm V^–1 s^–1 . The EPMs of the environmental strains ranged from –1.87 to –4.58 µm cm V^–1 s^–1, with a mean of –3.31 ± 1.11 µm cm V^–1 s^–1 . There was no significant statistical difference between the mean of clinical and environmental MAC isolates (P = 0.911) . The EPMs of the clinical and environmental groups were widely distributed, suggesting differences in the surface properties of strains do exist . The EPMs of MAC organisms were grouped according to species (M . avium, M . intracellulare, and MX) and source (Table 2) . Statistical comparisons between MAC groups (Table 2) were performed with the Mann-Whitney rank sum test . Significant statistical differences between clinical M . avium and clinical M . intracellulare and clinical M . avium and MX organisms existed . Statistical differences between environmental M . avium and MX and M . intracellulare and environmental MX organisms also existed, and statistical differences between clinical and environmental M . avium and clinical and environmental MX organisms were identified (Table 3) .

 
The EPMs of microbial surfaces are sensitive to suspension pH since the pH affects the dissociation of chemical groups on the cell wall . The pH of drinking waters varies from 6.5 to 10 depending upon the source water, treatment process, and corrosion control program . This study examined the effect of pH on the EPMs of MAC isolates . The effect of suspension pH (9.15 mM KH₂PO₄ buffered deionized water) on the EPMs of two environmental M . intracellulare strains is shown in Fig . 1 . The EPMs of both strains increased in the negative direction with increasing pH . The greatest EPM change occurred between pH 2 and 7, and both strains had an isoelectric point (zero point of charge) at a pH of approximately 2 . Figure 2 shows the impact of pH on the EPMs of two environmental M . avium strains . The EPMs dropped rapidly with decreasing pH . There were differences in EPMs between the two strains throughout the entire pH range . The isoelectric point of both strains was also near 2 . The impact of pH on the EPM of a MX strain is shown in Fig . 3 . The results were similar to those for the other species .

 
The EPMs of other environmentally important microorganisms suspended in 9.15 mM KH₂PO₄ buffer at near-neutral pH are shown in Table 4 (13) . The majority of the organisms are human pathogens and have been associated with drinking water contamination and waterborne disease outbreaks . Bacillus subtilis endospores have been measured through water treatment trains to determine the overall effectiveness of the water treatment processes (14) . Escherichia coli O157:H7 bacteria had the lowest negative EPM, –0.31 µm cm V^–1 s^–1, and spores of Encephalitozoon intestinalis had the greatest negative EPM, –3.1 µm cm V^–1 s^–1 . The EPMs of 14 clinical and environmental MAC strains measured in this study were more negative than those of all of the organisms listed in Table 4 . The EPMs of 10 MAC strains were more than 0.5 µm cm V^–1 s^–1 more negative than the EPMs of spores of E . intestinalis . These analyses showed the diversity in compositions of microbial cell walls, since EPM reflects the chemical composition of the cell walls . This study shows that the composition of the cell wall of MAC organisms is unique and very different from those of many waterborne pathogens .

 
The relatively large negative EPM measured for M . avium and M . intracellulare reflects the chemistry of the cell wall . Phosphate groups of phosphodiester linkages between the peptidoglycan and the arabinogalactan have been identified as the main components of the cell wall structure of mycobacteria (10) . Phosphate groups are highly ionized at pH values measured in drinking waters and could impart a strong negative charge .

The large negative charge of NTM may have potential practical water treatment implications . Since the majority of particles and surfaces in natural waters are negatively charged, more negatively charged microorganisms would become theoretically more stable as a result of stronger electrostatic repulsive interactions between surfaces (1) . If charge repulsion is the primary force responsible for the stability of microorganisms in water, the results of the study would suggest MAC organisms in aqueous environments would be very stable . Destabilization of MAC organisms during the commonly used drinking water treatment practice of chemical coagulation in a pH region, where charge neutralization is employed, would be more difficult and would require a greater coagulant concentration to achieve charge neutrality .

Hydrophobicity of microbial surfaces is also an important factor to consider when predicting the stability of microorganisms in aqueous environments and their ability to be removed during water treatment . Charged hydrophilic microorganisms tend to remain stable in water even after charge neutralization by salt addition . As the charge of hydrophobic microorganisms approaches neutrality, the ability of a microorganism to approach another hydrophobic surface increases . This increases the strength of attractive hydrophobic and van der Waals bonds, making it more likely for the microorganism to aggregate and adsorb to hydrophobic materials . Environmental opportunistic mycobacteria are reported to be among the most hydrophobic cells (5) . This contributes to their ability to be aerosolized, form biofilms, and resist disinfection (5) .

Additional studies are needed to examine the hydrophobicity of MAC organisms in water . Pilot plant studies are needed to determine how well conventional drinking water treatment processes remove these important, opportunistic pathogens .

ACKNOWLEDGMENTS

We thank Ian Laseke from the U.S . Environmental Protection Agency for assistance in laboratory work .

Any opinions expressed in this paper are those of the author(s) and do not necessarily reflect the official positions and policies of the USEPA . Any mention of products or trade names does not constitute recommendation for use by the USEPA .

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