Effect of Amplicon Size on PCR Detection of Bacteria Exposed to Chlorine

Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Shawn C. McCarty and Ronald M. Atlas

Department of Biology, University of Louisville, Louisville, Kentucky 40292

The effect of amplicon size on the Several methods for the detection of larly during chlorine disinfection--the PCR detection of Legionella pneumo- microorganisms in environmental sam- most common treatment for the destruc- phila after chlorine inactivation was ples using PCR have been developed. (1) tion of pathogens in drinking water and investigated. Two amplicons specific Roche Molecular Systems and the Per- a process demonstrated to produce chro- to the L. pneumophila mip gene were kin-Elmer Corporation have developed a mosomal lesions(6)--longer amplicons used for the PCR analyses: a 6SO-bp commercial PCR kit for the detection of may be more reflective of inactivation amplicon and smaller 168-bp ampli- the water-associated pathogen Legionella than smaller amplicons. Treatment with con within the 6SO-bp amplicon; a pneumophila, the EnviroAmp Legionella bleach (chlorine) of glassware, equip- 108-bp amplicon specific to species kit, which allows the semiquantitative ment, and benchtops is routinely recom- rRNA coding sequence also was used. detection of the genus Legionella and mended for laboratories performing PCR After exposure to chlorine, viable species L. pneumophila in under 6 hr. (2) to prevent contamination with DNA that agar grown cells were not detected The amplicon for Legionella detection is may give unwanted amplification; ade- by plate counts or direct counts with a 108-bp region of a 5S rRNA coding quate chlorine treatment eliminates p-iodonitrotetrazolium (INT) after 1 gene, and the amplicon for L. pneumo- DNA that can be amplified by PCR. In rain for treatment at 10 mg/I, after 2 phila detection is a 168-bp region of the this study we determined the effect of rain for treatment at S rag~I, and af- macrophage infectivity potentiator (mip) amplicon length on the PCR detection of ter 4 rain for treatment at 2.S mg/I; gene. bacteria after chlorine disinfection of viable water grown cells were The public health significance of a water; chlorination is the method most present at least 4 rain after biocide PCR positive result rests with distin- commonly used for disinfection of pota- addition even with a chlorine dose of guishing viable from nonviable organ- ble waters and has been used to treat S mg/I. At the lO-mg/I dosage, PCR isms, because PCR procedures can detect cooling to decontaminate them from Le- products from the 168-bp amplicon DNA trapped inside dead or even pre- gionella species. were detected on agarose gels up to served cells. (3'4) PCR positive results may 16 rain after chlorination; even after require confirmation using conven- 24 hr of PCR the 168-bp products tional procedures like plate counts or an- were detectable using a capture imal infectivity studies, unless it can be MATERIALS AND METHODS probe hybridization assay. However, demonstrated that a particular PCR pro- Growth and Maintenance of L. the 6SO-bp target was not detected cedure detects only viable cells. Studies pneumophila Cultures after 4 min chlorine contact time at performed using histological samples the same biocide dosage using agar- preserved for PCR examination have The L. pneumophila strain used for this ose gels, and PCR products could not shown that the signals from longer am- investigation was isolated from a cooling be detected by hybridization after 32 plicons are lost more rapidly than tower and confirmed using a fluorescent rain. At lower chlorine concentra- smaller amplicons. (4) A PCR-based assay antibody test (Genetic Systems Corp., Se- tions, a similar pattern was seen with to assess DNA damage induced by the attle, WA). This strain was maintained the 168-bp amplicon detectable anticancer drug cisplatin has been devel- on BCYE agar (BDMS, Inc., Cincinnati, longer after biocide addition than oped on the basis of loss of signal from OH). For chlorination experiments, 10 the 6SO-bp mip amplification target. long amplicons (750 and 2000 bp) after BCYE agar plates were inoculated and in- On the basis of these data, larger am- drug treatment. (s) In the same assay a cubated at 37~ for 5 days. The surface of plicons appear to correlate better smaller amplicon (150 bp) showed no the plates was then flooded with dechlo- with viability of L. pneumophila in wa- appreciable loss of signal following cis- rinated filter-sterilized (DCFS) tap water ter samples. platin treatment. If the likelihood of and placed on an orbital shaker for 20 DNA damage in any defined sequence is min at 200 rpm to loosen the cells. DCFS proportional to the length, a relation- tap water was prepared using powered ship between amplicon length and per- activated carbon, filtered through a 0.2- sistence in dead cells may exist. Particu- p,M membrane, and then autoclaved; the

3:181-1859 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $5.00 PCR Methods and Applications 181 Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

effectiveness of elimination of residual Assessment of L. pneumophila counts were performed by placing 9 ml chlorine was confirmed by chemical Viability of the sample into a well of a six-position analysis. The cells were then transferred microtiter plate; 1 ml of a 1% (wt/vol) to a sterile centrifuge tube and pelleted Viability was assessed using plate counts aqueous INT solution was added, and by centrifugation at 1500g for 30 min. and direct counts using p-iodonitrotetra- the microtiter plate was then incubated The pellet was then washed three times zolium (INT) (Sigma Chemical Co., St. at 37~ for 6 hr. The INT-stained cells in DCFS and resuspended in 20 ml of Louis, MO). For plate counts dilutions of were preserved using O. 1 ml of formalin DCFS water. The cell density of the sus- the samples were plated onto BCYE agar and stored at 4~ before enumeration. pension was determined using acridine (BDMS, Cincinnati, OH) and were then INT-stained cells were stained with a L. orange direct counts (AODC) and stored incubated for 10 days at 37~ Direct INT pneumophila-specific FAb (Genetic Sys- at 4~ before use. Water-grown cells were prepared by inoculating 4x10 s cells of the same L. pneumophila strain into two liters of - lOO DCFS tap water (cell density, 2 x 10 s cells/ ml). Plate counts were performed to track the growth of this strain. After 35 6o days, the culturable cell density was 4x 10 s cells/ml; there may have been additional viable, but nonculturable, cells. - 20 This cell suspension was used directly for ,---.. -5 ,,i--, chlorination experiments. t- 0 B o 1 100 O Chlorination of L. pneumophila 9i--,03 -1 DCFS tap water (800 ml at pH 8.6) was 60 -- 13_ placed in a chlorine-demand-free beaker f- Q.) -3 and placed on a magnetic stirrer. The JQ CO stirrer was set to create a 5-cm vortex in 03 20 the center of the solution. Agar-grown L. rr pneumophila cells were added to achieve c- 0 0 C , a final density of -107 cells/ml. Water- o I 10o 03 grown cells were used directly. The cells o-- Q~ ,,i--, were stirred at room temperature for 30 (i) c- min. A 75-ml aliquot was removed be- 03 -I m 60 fore the addition of chlorine. A mea- v O sured dose (10, 5, or 2.5 mg/1) of sodium --~ -3 hypochlorite solution (Fisher Scientific, > Cincinnati, OH) was added, and aliquots - 20 were removed for analyses at 1, 2, 4, 8, ~,- -5 I l I I I i.ll 0 16, and 32 min and at 24 hr. The dosages iv T ~ T used are typical of the range of concen- ! ~ 100 trations and exposure times used to dis- infect potable waters. As samples were o collected, the chlorine residual was neu- ~ 60 tralized using 0.1 ml of 10% (wt/vol) sodium thiosulfate and placed on ice before analyses. An additional sample was -~_ 20 collected at 5 min, and the chlorine re-

sidual determined using a Hach colori- - ~ . . . , ii ~ 0 metric test kit (Loveland, CO). Although 0 8 16 24 32 64 1440 the chlorine residual was not measured Contact Time (min) after 24 hr, all samples had pungent chlorinous odors. Each experiment with FIGURE 1 Results of chlorine disinfection and agarose gel analysis of PCR products. (O) L. pneumophila percent survival based on viable plate count determination; (Z~) percent of initial the agar-grown cells was performed in PCR signal from the 108-bp amplicon; ([2) percent of initial PCR signal from the 168-bp ampl- triplicate for each chlorine dose. The ex- icon; (O) percent of initial PCR signal from the 650-bp amplicon. (A,B,C) Agar-grown cells periment using the water-grown cells chlorinated with 10, 5, and 2.5 mg/1, respectively; (D) water-grown cells chlorinated at 5 mg/l. was performed in triplicate using a chlo- Percent survival of 10 -6 indicates no viable cells. Controls with no chlorine treatment still rine dose of 5 mg/1. Controls with no showed strong PCR signals (no loss of intensity) at 24 hr for the 108-, 168-, and the 650-bp chlorine added were also run. amplicons.

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terns, Seattle, WA), and the percentage of and 108-bp amplicon PCR products was lerton, CA). The signal intensity was actively respiring cells was determined mixed, 5 ILl of gel-loading dye was standardized against the intensity before using a Nikon Labophot microscope added, and the samples were electro- chlorine disinfection. with an epifluorescence attachment. phoresed on a 3% agarose gel in Tris-ac- etate EDTA buffer. The gel was then Hybridization Analysis of PCR PCR Analyses stained with ethidium bromide (EtBr) Products and photographed under a UV light tran- Ten milliliters of each sample was fil- silluminator. The signal intensity of rep- For the 108- and 168-bp amplicons, the tered through a Millipore Durapore licate samples was determined using a EnviroAmp Legionella kit detection sys- HVHP membrane filter (25-ram-diam., commercially available densitometer tem was used. The 650-bp amplification 0.45-1~M pore size). The filter was placed program (Biomed Instruments, Inc., Ful- products were detected using a 50-mer into a tube containing 2 ml of DNA ex- traction reagent (Perkin-Elmer Corp.,

Alameda, CA) with the trapped cells fac- j t A ing into the tube, and it was then vor- - 100 texed for 30 sec. The tube was boiled for 10 rain to extract the DNA, and 20 i~l was used as the template for PCR reactions. The 108- and 168-bp amplicons were 60 amplified using the EnviroAmp Le- gionella kit, whereas the 650-bp amplicon was amplified using a previously - 20 published protocol with slight modifica- -5 c- __ _ , , l ~- .-7--'-'-'--'--'---c tions. (7) The EnviroAmp Legionella kit :3 T 0 supplied the master mix for PCR, and 65 O 1 lOO (O p.1 of this mix was aliquoted into a Ge- neAmp Reaction Tube (Perkin-Elmer (1) 03 -1 Corp.) and overlayed with mineral oil. 6o 13_ 03 The extracted sample was added, and 15 E: p.l of 25 mM MgC12 was added for a total (1) -3 ET) ~ reaction volume of 100 i~l; PCR was then 03 00 o - 20 performed with the step-cycle recom- > -5 rr mended by the manufacturer with a ~. L J~ t I J J._l O c- /, o 13_ DNA Thermal Cycler (Perkin-Elmer O I i 100 model 480). After PCR the amplification 03 products were stored at -20~ (p o0 c-- For the 650-bp amplicon, the PCR re- 03 -I action mix contained 1• PCR reaction m -'( 60 buffer (10• buffer contains 500 mM KC1, o 03 -3 and 100 mM Tris-HCl at pH 8.3), 200 p.M > o~ each of the deoxynucleoside triphos- > - 20 phates, 0.35 p.M each of the primers, and ~'--:3 -5 2.5 units of AmpliTaq DNA polymerase CO 0 D (Perkin-Elmer Cetus) and was overlayed IO0 with mineral oil. Twenty microliters of extracted template was added, along o with 15 p.1 of 25 mM MgC12. PCR was per- -- _1 formed using the following step-cycle: 60 one cycle of 3 min at 96~ followed by 25 cycles of a two-temperature PCR cycle with denaturation at 95~ for 0.5 min, 20 followed by primer annealing and exten- 0 sion at 50~ for 1 min. Following PCR 16 24 2"64 1440 the products were stored at -20~ These conditions have been found pre- Contact Time (min) viously to be optimal and to permit even FIGURE 2 Results of chlorine disinfection and hybridization analysis of PCR products. ( 9L. single-cell detection. (7) pneumophila percent survival based on viable plate count determination; (A) percent of initial PCR signal from the 108-bp amplicon; ([]) percent of initial PCR signal from the 168-bp ampli- Agarose Gel Analysis of PCR con; (C)) percent of initial PCR signal from the 650-bp amplicon. (A,B,C) Agar-grown cells chlo- Products rinated with 10, 5, and 2.5 mg/l, respectively; (D) water-grown cells chlorinated at 5 mg/l. Percent survival of 10-6 indicates no viable cells. Controls with no chlorine treatment still showed strong Ten microliters each of the 650-, 168-, PCR signals (no loss of intensity) at 24 hr for the 108-, 168-, and the 650-bp amplicons.

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single-stranded oligonucleotide probe (5'- TABLE 1 The Predicted Number of Chromosomal Lesions per 106 bp Required to TTTGGGGAAGA ATITFAAAAATCAAG- Give Rise to the Corresponding Decrease in PCR Product Intensity Based on the GCATAGATGTTAATCCGAAGCAA-3') la- Poisson Distribution, Assuming One Lesion in an Amplicon Inhibits Amplification beled with digoxgenin and detected using a chemiluminescent detection sys- Decrease in signal Amplicon size (bp) tem according to the manufacturer's in- intensity (%) 168 650 1000 a 2000 a structions (Genius System, Boehringer 10 625 b 161 105 53 Mannheim, Indianapolis, IN). The signal 20 1,327 343 223 112 intensity was determined using the same 40 5,452 1,409 916 458 commercially available software pro- 80 9,577 2,475 1,609 805 gram used for the agarose gels, and all 90 13,707 3,542 2,303 1,152 signals were standardized against the ini- 99 27,409 7,082 4,605 2,303 tial signal obtained before chlorination. 99.9 41,116 10,695 6,908 3,454 99.99 54,818 14,165 9,210 4,605 99.999 68,448 17,687 11,500 5,750 RESULTS AND DISCUSSION aNot analyzed in this study; presented for comparison purposes only. After chlorine disinfection of L. pneumo- bCalculated using the following formula: phila, the PCR signals on agarose gels Number of lesions/lO6 bp = - [In(1 - x)](lO6/n) and blots using the 108- and 168-bp am- where x is the proportion of intact amplicons (i.e., 10% decrease in signal intensity equals 90% plicons were significantly more persis- intact amplicons), and n is bp/amplicon. tent than the signals seen when the 650- bp amplicon was used (Figs. 1 and 2). At higher chlorine dosages (10 and 5 mg/1) using agar-grown cells, no PCR signals gel analysis of PCR products we were ples in which large amplicons are found were detected after 24 hr of chlorine only able to detect changes in product could trigger remedial actions, such as contact time. At lower chlorine dosages intensity when there was a 30% differ- periodic flushing or chlorination. At the or using water-grown cells, PCR signals ence in starting template number, very least, larger amplicons could be were obtained after 24 hr of contact time whereas using hybridization analysis we used to determine when plate counts with the 168-bp amplicon only. Agar- could detect 10% changes in starting am- should be performed. grown cultures of L. pneumophila were plicon copy number (data not shown). In summary, we found that the 650- inactivated in <8, 2, and 1 rain, with In most PCR procedures for environ- bp amplicon of the L. pneumophila mip chlorine dosages of 2.5, 5, and 10 mg/1, mental monitoring small amplicons are gene failed to amplify more rapidly after respectively (Figs. 1 and 2). Water-grown used (<500 bp) to optimize sensitiv- chlorine disinfection than a 168-bp am- cells of the same strain were significantly ity. (1'z'1~ Data presented here indi- plicon within the 650-bp amplicon or a more resistant to chlorine disinfection, cate that larger amplicons (>500 bp) are distinct 108-bp amplicon. The loss of sig- and complete inactivation was obtained better correlated with viability after nal from the 650-bp amplicon appeared in under 8 min using a chlorine dose of chlorine disinfection and may provide to correlate with contact time and chlo- 5 mg/1. Increased chlorine resistance in more relevant information. Statistical rine dosage. This behavior is consistent water-grown cultures has been reported analysis supports this hypothesis; the with expectations for a viability assay. previously. (8~ Survival of viable noncul- number of chromosomal lesions neces- The loss of the signal from the 650-bp turable cells of L. pneumophila has been sary to produce a detectable reduction in amplicon was independent of the reported for water stored for 35 days signal intensity is inversely proportional method of cultivation for the test strain without chlorination. (9~ No actively re- to the amplicon length (Table 1); a sin- before disinfection. This would suggest spiring cells (INT positive) were detected gle lesion should block PCR amplifica- that an amplicon sequence in environ- using FAb--INT counts in any sample af- tion regardless of amplicon size. mental bacteria would be lost in a simi- ter the addition of chlorine. As seen in When monitoring microorganisms lar fashion after chlorine disinfection. Figures 1 and 2, the decline in the signal ubiquitous in aquatic environments, like obtained using the larger amplicon cor- Legionella species, the use of a two-tiered related better with chlorine inactivation PCR approach is warranted, with one tier ACKNOWLEDGMENTS than the corresponding decrease seen in of tests being used to detect the presence This work was supported by material the smaller amplicon signal. Loss of de- or absence of specific microorganisms support from Roche Molecular Systems tectable PCR amplification signal has and the second tier being used to deter- and The Perkin-Elmer Corporation (En- also been reported for an 800-bp ampli- mine whether these microorganisms are viroAmp Legionella kits). In addition, con of Legionella. (9) viable. A test using a smaller amplicon technical assistance was provided by The differences seen in the results ob- could be used for screening samples, and Martin J. Boyce and Stephanie Stickler. tained using agarose gel and hybridiza- positive samples could be evaluated us- tion analysis of PCR products is related ing a larger amplicon. Ideally, nested to the quantitative reliability of each amplicons should be used in these as- REFERENCES procedure; hybridization is several or- says, because studies with carcinogens ders of magnitude more sensitive than have shown that DNA damage occurs in 1. Atlas, R.M., S. Sayler, R.S. Burlage, and agarose gel visualization. Using agarose a nonrandom manner. (13) Those sam- A.K. Bej. 1992. Molecular approaches for

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PCR Methods and Applications 185 Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Effect of amplicon size on PCR detection of bacteria exposed to chlorine.

S C McCarty and R M Atlas

Genome Res. 1993 3: 181-185

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