Prevalence of Mycobacteria in Pools

7/29/2019 Prevalence of Mycobacteria in Pools

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684 E. LEONI ET AL.

people with different health conditions and are thus moresusceptible to infection from opportunistic bacteria. Thereis evidence that, in AIDS patients, the M. avium complexinfection is associated with swimming pool use (Von Reynet al. 1996).

The aim of this study was to assess the prevalence of non-tubercular mycobacteria in the swimming pool environment.

MATERIALS AND METHODS

The study took place in 12 public indoor swimming pools inthe city of Bologna (Emilia-Romagna region). They were allindoor pools open all year except for a short period of closureduring the summer to service the lters. The water leavingthe pool is partly discarded (1015%) and mixed with waterin a feed tank. It then undergoes preltration, ltration andchlorination. In three of the pools diatomaceous earth is usedfor the ltration, while in the other nine a coagulation/ltration system with rapid triple-media sand lters is used.

Water samples

In each swimming pool a chemical and microbiological exam-ination was carried out on the water in the pool itself (68samples) and at different phases of the treatment process (24

samples for each phase): water leaving the pool (outgoing),after ltration and after chlorination (incoming).Microbiological samples were taken in 1-l sterile bottles

containing 10% sodium thiosulphate (1 ml l 1 ). The poolwater was collected at a depth of 50 cm and at four differentpoints about 1 m away from the pool edge. At the momentof sampling, the temperature, pH (direct reading pH meter,Orion 701A, Cambridge, MA, USA) and free and total chlor-ine residual (DPD method, colourimeter Model DC 1100;La Motte, Chestertown, MD, USA) were noted. Parametersregarding conductivity, oxidability and ammonia were mea-sured in the laboratory according to the techniques given inStandard Methods for the Examination of Water and Wastewater (APHA, AWWA, WEF 1992).

Microbiological samples were analysed within 12 h of col-lection, using the standard plate method to determine thetotal heterotrophic counts per ml at 36 C (Plate Count Agar;Oxoid, Basingstoke, UK) and the standard MF technique todetermine Gram-negative bacteria (McConkey Agar; Difco,Detroit, MI, USA). The Gram-negative bacteria isolated onMcConkey Agar were then subcultured and identied withthe API 20E and API 20NE systems (Biomerieux, MarcyLe toule, France) for lactose-fermenting and non-lactose-fer-menting strains, respectively.

The mycobacteria were isolated by decontaminating 1 l

water with hexadecyl-pyridinium chloride (Fluka, Millwau-kee, WI, USA) at a nal concentration of 004%, in accord-ance with the technique proposed by Fischeder et al. (1991).

1999 The Society for Applied Microbiology, Journal of Applied Microbiology 87 , 683688

After a 30-min exposure at room temperature, differentamounts of water (5, 20, 200and500 ml)were ltered throughlter membranes (HAWG 045 mm; Millipore, Bedford, MA,USA) which were rinsed with 300 ml sterile water to removethe cetylperidinium chloride residual and then placed onplatesof Oleic AcidAlbuminAgar (Middlebrook7H10 med-ium; Biolife, Milan, Italy). The plates were incubated in thedark at 30C under 5% CO 2 and read for growth after 1, 2,4 and 8 weeks. Colonies with morphology, pigmentation andgrowth rate typical of mycobacteria were stained by the Ziehl-Neelsen method. Acid-fast rods were subcultured on Lo w-enstein-Jensen slants and examined for cultural and bio-chemical characteristics, including temperatures and growthtime, pigment production, growth on McConkey agar and5% NaCl, niacin production, hydrolysis of Tween 80, nitrateand tellurite reduction, catalase, arylsulphatase and ureaseactivities.

Surfaces

Microbiological contamination was measured on the surfacesof pool edges, shower oors and changing-room benches.Samples were taken with sterilized cotton swabs, which wererubbed across surface areas of 20 20 cm. Four samplingsites were selected for each side of the pool edge, four sites

for the oor of each shower and four for the changing-roombenches. Immediately after use the swabs were placed inwide-necked asks, containing a known volume of sterilebuffer solution (pH 72), which were shaken vigorously todilute the collected material. The suspension was then takento the laboratories, shaken again for 20 min and examined forheterotrophic bacteria, Gram-negative bacteria and myco-bacteria. Colony-forming units per volume unit were con-verted into colony-forming units per surface unit, taking intoaccount the relation between the sampling surface and thevolume of buffer solution used to dilute material from theswabs.

Statistical analysis

For the statistical analysis the bacteriological data were trans-formed into log 10 (x 1). Correlations between mycobacteriaand other physical, chemical and bacteriological variableswere studied using the analysis of linear regression. A pairedt -test was used to compare the differences between the bac-terial contamination of the various phases of the water cycle.

RESULTS

The frequencies of isolation and the variation intervals of mycobacteria from the pool water and the surfaces are shownin Table 1.


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Table 1 Mycobacteria contamination of pool water and surfaces

Water ( n 68) Pool edge oor (n 35) Shower oor (n 44) Positive samples Range Positive samples Range Positive samples Range(%) (cfu 100 ml 1 ) (%) (cfu 100 cm 2 ) (%) (cfu 100 cm 2 )

All the species 88 (2968) 100 (1694580) 100 (610850) Mycobacterium gordonae 74 (1840) 80 (1091700) 80 (310430) M. chelonei 38 (2360) 38 (61312) 49 (64140) M. fortuitum 35 (2250) 31 (6637) 43 (172334) M. avescens 6 (1078) 11 (1251375) 18 (8312) M. phlei 3 (20120) 6 (1593) 7 (25187) M. terrae 3 (213) 6 (410) 5 (1035)

M. marinum 0 0 5 (4575)

Water samples

Non-tubercular mycobacteria were found in 882% poolwater samples (60 of the 68 samples examined) at con-centrations between 2 and 968 cfu 100 ml 1 . The most fre-quent species was M. gordonae, isolated from 735% of thesamples, followed by fast-growing mycobacteria such as M.chelonei , isolated from 382% of the samples, and M. fortu-

itum, from 353%. The concentrations of positive samplesvaried from 1 to 840 cfu 100 ml 1 for M. gordonae, from 2 to360 cfu 100 ml 1 for M. chelonei and from 2 to 250 cfu100 ml 1 for M. fortuitum (Table 1). Sporadic recoveries weremade of M. avescens (59% of the samples), M. phlei (29%)and M. terrae (29%).

No statistically signicant correlation was found betweenthe concentrations of mycobacteria and water temperature(mean 287 C; S.D. 11C), free residual chlorine (mean052mg l 1 ; S.D. 016mg l 1 ) and ammonia (mean 086 mgl 1 ; S.D. 035 mg l 1 ). Nevertheless, mycobacteria were absentin all the samples with a level of available chlorine above07 mg l 1 . There was no correlation between the presenceof mycobacteria and other bacteriological parameters (totalheterotrophic count and Gram-negative bacteria). The mostcommonly found species of Gram-negative bacteria wasPseudomonas aeruginosa, isolated in 103% of the samples atconcentrations ranging from 1 to 40 cfu l 1 .

The circulation of micro-organisms in the pool environ-ment was calculated by measuring theconcentrationof micro-bes at the various phases of the water cycle: incoming, in thepool (samples taken when thepool was at itsbusiest), outgoingand after ltration. The mean values and standard deviations(log units) of the most signicant microbial indices, relativeto 24 measurements for each phase, are shown in Fig.1. A

progressive increase in the microbial counts can be noted inthe water from the pool and from the outlet point, withstatistically signicant differences (paired t -test; P 001).


Fig. 1 Variations in concentrations of bacteria at different phasesof the water cycle (mean values 2 1 S.D. )

The outgoing water was the most contaminated, with 667%

of samples having a count at 36 C of more than 200 cfu l 1

(the limit established by current Italian regulations). Pseudo-monas aeruginosa, which was always absent in the incoming


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686 E. LEONI ET AL.

water, was found in 25% of the pool water samples (540 cful 1 ) and in 667% of samples from outgoing water (10320cfu l 1 ). The distribution of mycobacteria was more uniformin the different phases of the water cycle, with 667% positivesamples of the incoming water (geometric mean 102 cfu100 ml 1 ), 833% of the pool water (248 cfu 100 ml 1 ),917% of the outgoing water (1164 cfu 100 ml 1 ) and 667%of the ltered water (295 cfu 100 ml 1 ).

Filtration improved the microbiological quality sig-nicantly (Fig.1), but the reduction obtained by this processin the different bacteriological parameters was onlysignicantfor Ps. aeruginosa (P 0001) and the total count ( P 001),but not for the mycobacteria. No statistically signicant dif-ferences in the percentage of decrease in microbial con-tamination were observed between the two ltration systems.Both methods show a high efciency in the elimination of Ps.aeruginosa, with mean decreases (calculated as a mean of thereductions obtained for each determination) of over 99%(Table 2). The ltering process brought about a much lowerreduction, however, in the mesophilic plate count, 616% forthe sand lters and 501% for the diatomaceous lters. Thepercentage mean reduction of mycobacteria was also quitelow, only 465% for the sand lters and 633% for the diato-maceous lters. On average, the water leaving the pool andundergoing ltration had a free chlorine concentration of

047mg l 1

(S.D.

014mg l 1

). The mycobacteria isolated inthe various phases of the cycle belonged to the same speciesas those found in the pool water, with a strong prevalenceof M. gordonae and fast-growing mycobacteria, which wererecovered at relative percentages similar to those of the poolwater.

Surfaces

Mycobacteria were found in all samples taken from the pooledges and shower oors (Table1). They were not found,

Table 2 Mean microbial concentration (log units) and percentage reduction obtained by ltration of outgoing waterFiltration system Total cfu at 36C (log cfu ml 1 ) Pseudomonas aeruginosa (log cfu l 1 ) Mycobacteria (log cfu 100 ml 1 ) Rapid sand ltersOutgoing water 228 096 203Filtered water 208 015 156Reduction (%) 616 994 465

Diatomaceous earth ltersOutgoing water 254 136 217Filtered water 230 03 142

Reduction (%) 501 998 633* Calculated as mean of the reductions obtained for each determination.


however, on the changing-room benches. The most frequentspecies was M. gordonae (in 80% of pool edge samples and796% of shower oor samples), followed by M. chelonei (382% and 486%, respectively) and M. fortuitum (314%and 432%). Sporadic recoveries were made of M. avescens(in 114% of pool edge samples and 182% of shower oorsamples), M. phlei (57% and 68%) and M. terrae (57%and 45%). Mycobacterium marinum was isolated on only twooccasions from shower oors (45% of samples) at con-centrations of 45 and 75 cfu 100 cm 2 .

DISCUSSION

The results of our study show that the swimming poolenvironment provides a suitable habitat for the survival andreproduction of mycobacteria. The widespread circulation of these species of micro-organisms throughout the water cycleand their considerable adaptability can be seen from the highpercentages of recovery in all types of samples examined:667% of samples of water entering the pool, 917% of sam-ples of water leaving the pool and in 100% of samples takenfrom the surfaces of the pool edge and shower oors. Of the various surfaces investigated, only the changing-roombenches were found to be free from mycobacteria. Thesendings suggest that mycobacteria may survive and grow on

the surfaces which are continuously perfused with water,contributing to biolm formation. Hall-Stoodleyand Lappin-Scott (1998) showed the ability of rapidly growing myco-bacteria to colonize surfaces and Schulze Ro bbecke and Fis-cheder (1989)) reported that biolms were an importanthabitat for the proliferation of mycobacteria and sites for themycobacterial contamination of water distribution systems.

The ltration and chlorine treatment of the water broughtabout a fairly low mean reduction in the mycobacteria count;the arithmetic mean of single reductions was only 521%after ltration and 603% after full treatment. No statistically


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signicant differences were found between the sand anddiatomaceous lters; the capacity of both systems to reducemycobacteria and heterotrophic plate count was quite low,probably as a result of the ideal habitat environmental bacteriafound in the ltering material, where concentrations of residual chlorine were lower. This nding would suggest thatthe chlorine residual needs to be controlled at all phases of recirculation in order to prevent the bacteria from breedingin the organic material contained in the lters.

Our study conrmed that the most common species of mycobacteria to be found in swimming pool environmentswas the saprophytic species M. gordonae (Runyons group II).Not only is this species able to survive, but it can also repro-duce in the water and on surfaces, taking advantage of thepresence of organic molecules (Havelaar et al. 1985). A moreserious matter is that of the isolationof pathogenic andoppor-tunistic mycobacteria. The only example of the former to beisolated was M. marinum (Runyons group I). Although thisspecies was never found in the water, it was recovered ontwo occasions from shower oors (45% of samples). Themycobacteria most commonly responsible for opportunisticinfections ( M. avium complex, M. xenopi and M. kansasii )were not found, but the high recovery percentage of M.chelonei and M. fortuitum from both water and surfaces,although in very low concentrations, gives cause for concern.

The presence of mycobacteria in the water is enhancedabove all by the rather high average temperatures of theswimming pools investigated, as well as by the concentrationsof chlorine, which were always lower than the 1 mg l 1 freechlorine considered necessary to control mycobacteria (Pel-letier et al. 1988). Unlike the study of Daillaoux etal . (1980)),no statistical correlation was found between mycobacteriadensities and free chlorine concentrations in pool water.Nevertheless, the high level of chlorine found in the samplesnegative for mycobacteria (070092 mg l 1 ) suggest that anincreased concentration of available chlorine is an importantfactor in controlling mycobacterial contamination.

Although infections from mycobacteria other than M. mar-inum have never been linked with any certainty to swimmingpool environments, the health risk in subjects with weakenedimmune systems should not be underestimated, given thewide diffusion of these micro-organisms and the direct con-tact pool users have with the water and the resulting aerosol.

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Prevalence of Mycobacteria in Pools