Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
Isolation of Klebsiella pneumoniae from lake water LUCYM. CAMPBELL, GLENDAMICHAELS,'RICHARDD. KLEIN,A N D IVANL. ROTH Depor/t>retrrof Mic~obiology.Utri\,er~iryofGro,gitr, AIlrer1.r. GA. U.S.A.30602 otitl Dc~por~r~~etrr c?f'Biology,Goitre.~i~ille Jlrtrio~.C o l l q e , Grtitle.s~~ille. GA, U.S.A.30501
CAMPBELL, L. M., G . MICHAELS. R . D. K L E I N and , I . L. ROTH. 1976. Isolation of KleOsic~lln pt~errt?lot~itre from lake water. Can. J. Microbial. 22: 1762-1767. The question of the importance of aquatic borne Kleb.siell(t prlerrt?iotiitrc~to p ~ ~ b lhealth ic has been argued as the 01-ganism has appeared often in both polluted effluents and oligotrophic systems. Using a selective medium. double violet agar. which also differentiates K . pt1c,1r1t1ot1i(1e~ from other organisms Found in aquatic systems, we sampled water from three ponds and a large can be isolated man-made lake, over a 3-month period. Results indicate that K . ptirr~t??ot~icre consistently and in high numbersfrom eutrophic waters even when environmental stress reduces total numbers, but that isolations from cleanerwaters al-e er~xtic.We conclude that mere isolation of the organism as an occasiontii contaminant PI-obablydoes not present a hazard to public health.
Introduction Lake and stream waters are surveyed routinely in the United States for presence of coliforms and fecal coliforms as one parameter in determining severity of domestic and industrial pollution and the effects on bacterial water quality. Fecal coliforms can be identified by their ability to ferment lactose with gas production at 44.5 "C, and between 93.0 and 98.7'z of coliforms isolated from feces of humans and warm-blooded animals meet this criterion (Geldreich 1972). It has been reported that about 30 to 40% of healthy warm-blooded animals carry KlebsieNa p n ~ ~ r r ~ ~ o tas l i anormal e intestinal flora, and about 20% of healthy humans also have carried the organism (Bordner and Carroll 1972). Nosocomial infections with the organism are fairly common and are tliought to be associated frequently with patients who are also carriers (Selden e t a l . 1971). It seems obvious that appearance of high numbers o f K . /1t1e~rrnot7iaein tlie aquatic environment, particularly in potable o r I-ecreational waters, should have significant public health impact. However, there is some controversy as to the public health importance of aquatically associated K . ptieut~lotriae, particularly concerning fecal versus nonfecal origins. For example, Kriittel (1975) recently reporin surted that the occurrence of K. pne~rt~lotlicre face samples of streams, rivers, and lakes was not ubiquitous and isolation was associated with that of Esclrerichia coli. These observations disagreed with those of Duncan and Razzell (1972), 'Department of Biology, Gainesville Junior College, Gainesville, GA, U.S.A. 30501.
who isolated K. pneumoniae from limited access watersheds, other watersheds of varying accessibility, and paper and pulpmill effluents. They concluded that the lack of documented cases of K. pneunloniae - caused water-borne infections indicated the organism was of little concern t o public health. It has been reported from laboratory experiments that potentially pathogenic members of the family Enterobacteriaceae are nonfastideous and can grow in highly dilute salts supplemented witli a single suitable carbon source (Hendricks and Morris011 1967). The dilute media and incubation temperatures utilized approximated conditions found in a cold mountain stream, a n d results showed that members of Enterobacteriaceae would multiply and not merely survive under these conditions. This investigation is concerned witli isolation of K . pt1~~1t77017iae from a large man-made lake and three smaller ponds using double violet agar (Campbell and Roth 1975). This medium has been developed to aid in differentiation between K. pne~rt7lo11iaeand E ~ r t e r o b a c t e raeroget7c.s a n d to facilitate the presumptive identification of tlie former after 24 h of incubation.
Materials and Methods Lake Sidney Lanier near Gainesville, Georgia, was sampled over a 3-month period a t 10 stations about the lake and on a tributary. The sampled stations are described in Table I , and located on a mapof Lake Lanier (Fig. I ) . This is a large lake formed by a hydroelectric dam and is used primarily for recreational purposes. T h e lake at average summer pool covers 38 000 acres with a shoreline of 540 mi and a maximum depth of about 200 ft. Duplicate surface samples were taken by dropping a
1763
CAMPBELL E T AL.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
TABLEI . Sample stations on Lake Sidney Lanier Stat~on No. 1
2 3 4 5 6
7
8
9 10
Descr~ption Lake Lanier Islands Beach, a heavily used resort area Just offshore 20-50 ft from Lake Lanier Islands sewage-treatment plant Sw~niniingarea a t Holly Park, a county recreatlon area Just offshore (about 100 ft) from Linwood sewage-treatment plant Chattahoochee River channel, deep water, about 7 ml above dam Two M ~ l eCreek (end of nav~gablearea, less than 3 ft deep), clean area, no industry or agr~culture About nil from mouth of Flat Creek Mouth of Flat Creek, a heavlly polluted area receiv~ng sewage-plant effluent (including both doniest~cwastes and poultry-processing plant effluent) End of navigable area (less than 3 ft deep) of Flat Creek Free flowing area of Flat Creek below poultryprocessing plant and above sewage-treatment plant
:
A pond at a local textile mill producing polyester and cotton yarn and fabric was sampled in the same manner a s Campus Lake and Meniorial Park Lake. This pond is 900 ft by 300 ft by 6 ft deep and is enclosed by the fence surrounding the mill. It is fed by springs in the lake bottom and receives waste water froni the mill's airconditioning system. There is little r u n o f froni the area and the pond does not receive process water from the mill. It is not used for recreational purposes and has no waterfowl. Samples (100 nil) were processed in the same nianner as the Menlorial Park Lake samples. Sa~iipleswere taken from four stations on the textile mill pond. Cotton samples for analysis for the presence of K. pnerrrr7onioe were removed from bales with a pair of sterile forceps and placed in sterile whirl-pack bags. Onehalf-gram samples of cotton were weighed aseptically and introduced into bottles containing 100 nil of sterile water. The bottles were shaken vigorously for 1 niin and the water was filtered through 0.45-pm Millipore membrane filters. The cotton was retained in the bottle by means of a sterile glass rod. The filters were placed on double violet agar plates and incubated at 37 "C for 24 h. The fecal coliforni and fecal streptococcus analyses were done using the membrane-filter t e c h n i q ~ ~ eass detailed in Siarlc/orcl rrretllocls for tlrc esotrritlntiot~of )voter on(/ btwste Ivo/er, 13/11 ecl.
Results Lake Lanier
. . . . . .. .. . .. . .. . . . . .. . .. . .. . ........... . .
. .
.. . ... . . . .... .
. .
... .. .. .. .. .. .. . . . . . . . . . ... .. . . .. .... . ,
Ten stations, with a surface and subsurface Nuclepore filter onto the water surface (Crow et (11. 1975), retrieving it with a pair of sterile forceps, and placing it (10 cm) sample taken at each, were monitored on on a plate of double violet agar containing per litre of Lake Lanier over a 3-month period. Klehsiellc~ deionized water: violet red bile agar (Difco), 41.5 g ; and ptie~itnonicre was isolated from five of 10 surface methyl violet 2 8 (Allied Chemical Corp.), 2.0 g (Canipsan~plingsover the test period (Table 2). Only bell and Roth 1975). Duplicate subsurface samples were once was K. pne~itiloniae isolated from a subtaken 10 cni below the surface, the saniple placed in a sterile Petri dish, and a Nuclepore filter dropped on the surface sample, at a station on Flat Creek water as with surface saniples. Colonies were counted (station lo), a shallow tributary which receives and picked to confirmatory biocheniicals as described effluent from a poultry-processing plant and a previously (Campbell and Roth 1975). The biochemical city sewage-treatment plant (intermittently chlormedia ~ ~ s were e d triple sugar agar (TSI) (Difco), niotilityindol-oriiithine decarboxylase (MIO) (Difco), and Chrisinated). The number 10 sanipling station is, tiansen's urea agar (Difco). however, below the poultry-processing plant and Memorial Park Lake is a sniall man-made pond of 6 above the sewage-treatment plant. Two of the acres with a niaxiniuni depth of 15 ft and a minimum five surface samples from which K. pne~rtiloniae depth of 2 ft used niostly for decorative purposes and was isolated were also from Flat Creek stations nonswimniing recreation. It receives r ~ ~ n o ffroni f the surrounding park area which is surrounded by an urban (9-S and 10-S) (Table 2). Only one station on housing area. The lake is inhabited by a flock of waterFlat Creek (10-S) yielded isolates of K. ptle~ifowl. The pond experienced a heavy algal bloonl in late t7iotlicie on all sampling dates. Three other stasummer of 1975. Surface samples (100 ml) were taken at tions on the lake yielded occasional samples that three stations as described in Stcrtrrlorcl tr~eilrocls(13th ed). and processed by filtration throc~gh0.45-prn Millipore were positive for K. ptie~itnotiioe, but the organnienibrane filters within 30 niin of collection. Plated filters ism was not consistently isolated from any of were handled as with Lake Lanier saniples. them. Campus Lake is a sniall man-made pond (500 ft in Colonies of K. pne~rnioniaewere distinguished length, 100 ft wide at the narrower end, 200 ft wide at the from other isolates by the distinct morphology wider end, and about 10 ft deep) located on the campus of Gainesville Junior College, Gainesville, Georgia. It is on double violet agar, a pale lavender 'gumdrop' stocked with fish and several ducks frequent the pond. colony ranging from 2 to 5 mm in diameter. The pond is primarily spring fed, but also receives r ~ ~ n o f f When such colonies were picked to confirnlatory from the campus. Surface saniples (100 nil) were taken at media, they produced acid slant, acid butt, gas, four collection sites at the water's edge and processed in the same mariner as the Meniorial Park Lake saniples. no hydrogen sulfide on TSI; they were non-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
1764
CAN. J. MICROBIOL. VOL. 22. 1976
FIG.1. Sample stations (see Table 1) on Lake Sidney Lanier.
motile, indol-variable, ornithine decarboxylasenegative on motility-indol-ornithine decarboxylase deeps (MIO), and urease-positive.
Memorial Park Lake Surface samples of water taken from this small
pond yielded K. p~leutnoniae on each occasion, including a period in late August and early September when a heavy algal bloom lowered total numbers (Table 3). Samples were taken from three locations at the pond's edge within the area affected by the bloom, and numbers of
1765
CAMPBELL E T AL.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
TABLE 2. Isolation of K. pt~c,lrtnot~iae,fecal coliforms, and fecal streptococci from Lake Sidney Lanier. Stations which yielded no organisms on any sampling date are not included Average* count per Nuclepore filter 17/6/76
8/7/76
22/7/76
17/8/76
Station
Kp
FC
FS
Kp
FC
FS
Kp
FC
FS
1-S 2-S 3-S 4-S 6 6-S 7-S 8 8-S 9 9-S 10 10-S
0 0 0 0 0 0 0 0 0 0 3 0 T
0 0 0 0 0 0 0 0 1 0 20 0 T
0 0 0 0 0 0 0 0 0 0 0 1 49
0 0 1 0 0 1 0 0 0 0 11.5 6.5 T
18 3 180 0 0 13 9 I 28 8 26 T
0 0 0 0 0 0 2 0 0 0 0
0 0 3.5 0 0 0 0 0 0 0 T 0 T
0 0 0 5 0 0 0 1
0 0 7 0 0 0 I 0 0 0 9 4 T
4 T
-
7 T 2 T
Kp
FC
0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 84 0 0 T T 1 2
9/9/76 FS
Kp
FC
FS
0 0 0 0 0 0 0 0 0 0 2 2 0
0 0 1 1 0 0 0 0 0 0 2 0 T
0 0 0 0 0 6 0 0 5 0 120 5 T
0 0 0 0 1 0 0 0 0 0 1 2 T
NOTE:Surface samples are designated with the terminal "S". Sample williout the "S" designation are subsurface samplcs. T h e sampling stations are described in Table I a n d shown on the m a p o f Lake Lanier (Fig. I). 'Average of t ~ v osamples per station. ADDREVIATIONS USED:T = too numerous to count. K p = Klebsiellapncv,i~oniaecount. F C = Fecal coliform count. FS = Fecal streptococci count. - = no data.
TABLE 3. Comparison of numbers of K. pneutnoniae with total numbers of bacteria isolated on double violet agar from Memorial Park Lake, Athens, Georgia Organisms per 100 ml (average of three stations) Date
Klebsiella pnelmionioe
18/8/76* 2018176 5/9/76 11/9/76 12/9/76 26/9/76t 3019176
100 200 100 267 200 1000 533
TABLE 4. Isolation of K. ptlelcniot~iaeand fecal coliforms from Campus Lake Organisms per 100 ml (average of four stations) Date
K. pt~elrtno~~ineFecal coliforms
Total
700
1 500 2 000 2 500 4 000 10 000 6 000
'Heavy algal bloom. ?Algal bloom cleared. NOTE:T = LOO numerous to count.
klebsiellae isolated dropped concomitantly with total numbers until the bloom cleared in midSeptember. Campus Lake Samples from the surface of Campus Lake yielded K. pneurnor~iae each time samples were taken (Table 4). While the numbers of K. pneuinoniae in Campus Lake were not as high as those in Memorial Park Lake, the organisms were consistently present.
TABLE 5. Isolation of K. pnelrmot~ioeand fecal coliforms from a textile mill pond Organisms per 100 ml (average of four stations) Date
K. pt~euniotlioe
Fecal coliforms
1/7/76 29110176
30 5
0 0
Textile Mill Pond The textile mill pond was sampled only twice, but K . pneurlzoniae was isolated on both of those occasions (Table 5). The count of the sample taken on October 29, 1974, was the lowest count obtained in the small bodies of water we sampled. We suspected that the textile mill pond might be contaminated with raw cotton and that cotton might be the source of the K. pneutnoniae in this pond. The results of our analysis of raw
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
1766
C A N . J . MlCROBl O L . V O L . 22. 1976
TABLE 6. Isolation of K. p~~elrtirotzioe and fecal colifornis from bales of unprocessed cotton on 25/5/76
Memorial Park Lake and Campus Lake, there is a constant waterfowl population, From our study it appears that neither pond is able t o clear itself of K. p/ieun~oniaedespite periodic rainfalls K. ptie~rt,~ottirre/g Fecal colifol.m/g Bale and dry periods. In the case of Memorial Park Lake, a heavy algal bloom in late summer, 1975, precipitated a drop in total bacterial numbers from 4200 CFU/100 ml to a low of 700 C F U / 100 ml. While numbers of K. pne~c1?7oniaealso dropped, a t no time was the organism completely absent. Thus K. pne~onoiziae could be considTABLE 7. Biochemical characteristics used in the ered t o be ubiquitous in the surface layers of identification of E. oer.ogetles, E. coli, and K. ptrerrthese small ponds. While the textile mill pond was t,~ot~ioe only sampled twice, it did yield K. pneumoniae on both occasions. The October 29, 1975, E. K. sample gave the lowest K. pnewnonirre count we creroE. ptrc.lcTest genes coli ~~zo~iioe recorded for any of the small ponds. This low count in the textile mill pond as compared t o the Lactose fermentation + + + Memorial Park Lake and Campus Lake could + rl * Ornithine decarboxylase probably be attributed to the absence of waterLysine decarboxylase + rl + Motility ++fowl at the textile mill pond which is fenced. + Urease There was some evidence that the textile mill Indol + + pond might be contaminated with cotton fibers. *d, Delayed. Bales of cotton are off-loaded from trucks on a cotton samples taken from bales at this textile loading dock adjacent to the textile mill pond. Since K. pneurnoniae has been isolated from a mill are shown in Table 6. The biocliemical cliaracteristics and reactions number of plant sources (Duncan and Razzell 1972) it seems likely that cotton may be conused in the confirmatory tests are listed in Table taminated with this organism. We analyzed 7. several samples of cotton and found that the Discussion cotton was indeed contaminated with K. pne~cDouble violet agar was developed to isolate 1?7onirre as shown in Table 5. Apparently K. and select for K. p17e~olio17icre from contaminated p17e~rtnoniueis widely distributed in nature. Lake Lanier is a much larger, deeper system waters or other sources where Klebsiellrr might occur. In this study, we investigated the distri- and apparently does not have a constant K. on a large lake and three piielm?onicie population, even in the surface bution of K. p17e~c1no17irie smaller ponds used for recreational or decora- layers. Of the five surface samples that yielded tive purposes t o determine if the organism is positive cultures of K. pne~olioniae,two stations, indeed ubiquitous and if the numbers fluctuate 9-S and 10-S, were in Flat Creek which is exor remain stable. Klebsielln p17e~trl7017iaecan be posed t o effluent from a chicken-processing plant thought of as an indicator organism while being and a sewage-treatment plant. At only one stasimultaneously considered as a potential patho- tion was the organism consistently isolated, most gen. Although questions have been raised re- of the isolations being occasional. It is possible garding its importance as a threat t o public that the greater dilution capacity of Lake Lanier health, investigations have clearly labeled kleb- contributes to the apparent clearing of the siellae isolated from aquatic systems and those organism from the system in most of the areas from clinical sources as identical (Seidler e t ril. sampled. Klebsiellap17e~cnioniaewas isolated only 1975 and Matsen er a/. 1974). It is likely that the once from a subsurface sample which would mere presence of the organism poses little threat indicate that its occurrence is generally limited t o the well-being of the public, but that the to the surface. We have found that there is essentially nc threat becomes more viable as numbers increase difference in enumeration of K. piieunio~iine on and other species are excluded. In two of the more stagnant systerlls examined, double violet agar and tryptic soy agar (unpub.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
CAMPBELL ET AL.
lished data). This fact allowed for rapid qualitative and quantitative analysis of water samples for presence of K. pneumoniae, without the necessity of picking large numbers of colonies to biochemical media for confirmation. Only those colonies suspected of being K. pneutnoniae were confirmed by use of auxiliary media. Colonies grown from Memorial Park Lake samples on double violet agar for 24 h were confirmed as K. pneutnoniae 100% of the time. Plates from Lake Lanier were held for up to 72 h before colonies were picked to confirmatory media and were confirmed with 88% accuracy. The precision in identifying K. pneurnoniae from the appearance of colonies on double violet agar has been further confirmed by our study on river water in which we achieved an average of 85.4x accuracy (Campbell et al. 1976). As colonial morphology tends to alter slightly when plates are held at room temperature, these results indicate that plates should be read promptly. Distribution of K. pneunloniae in lakes, therefore, can be determined quickly and accurately by use of double violet agar, without need for confirmation of colonies. The question of the importance of K. pneunloniae and its distribution to the ~ u b l i chealth has never been resolved. Some iorkers have been able to isolate K. pneutnoniae from exceptionally clean water systems, as well as polluted ones (Duncan and Razzell 1972). Others have found' the organism occurring in conjunction with fecal coliforms and feel that the presence of the organism represents deterioration of the environment (Knittel 1975). We have not found the organism to be ubiquitous, although it is widely distributed in nature as evidenced by the recovery of K. pne~mmoniae from raw cotton. What we have discovered is that the organism is a major constituent of the flora in eutrophic systems, with numbers and consistency in isolation over a period of time related directly to the degree of pollution encountered. While the Dresence of fecal coliforms amears to be correlated with the presence of K. pneulnoniae in between Lake, there is fecal coliforms, fecal and K. ptleulnonine in the much larger lake, Lake Lanier. The presence of K. pfieur)yonjae in the textile mil] pond and the absence of fecal coliforms there is likely due to the more Or less seeding of that pond with K. prleurlloniae from the cotton along with no appreciable input of coliforms. L.
1767
Thus in our analyses we have established that K. pneurnoniae does occur in natural waters in the absence of fecal coliforms; however, at some sites, the presence of K. pneur?loniae is correlated with a fecal coliform population. Acknowledgment This work was supported in part by research grant number R-803341-01-0 from the U.S. Environmental Protection Agency. We thank Dr. Warren Cook and the Biology Department of Georgia State University for the use of aquatic sampling equipment. The able technical assistance of Beatriz Chapman is gratefully acknowledged. A M E R I C APUBLIC N HEALTHASSOCIATION. 1971. Standard methods for the examination of water and waste water, 13th ed. American Public Health Association Inc.. N.Y. BORDNER, R. H., and B. J. CARROLL. 1972. Proceedingsof seminar on the significance of fecal coliform in industrial wastes. U.S. Environmental Protection Agency, Denver, Co. L. M., and I. L. ROTH.1975. Methyl violet: a CAMPBELL, selective agent for differentiation of Klebsiellti P I I ' I I tnotzitie from Et~te~~obrrcter rierogenes and other Gramnegative organisms. Appl Microbiol. 30(2): 258-261. CAMPBELL, L. M., I. L. ROTH, and R. D. K L E I N .1976. Evaluation of double violet agar in the isolation of Klebsiellrr ptlerrtnonicre from river water. Appl. Environ. Microbiol. 31(2): 213-215. CROW,S. A,, D. G. AHEARN.W. L. COOK,and A. W. B O U R Q U I N1975. . Densities of bacteria and fungi in coastal surface films a s determined by a membraneadsorption procedure. Limnol. Oceanogr. 20(4): 644646. D U N C A ND. , W., and W. E. RAZZELL.1972. Klebsiello biotypes among coliforms isolated from forest environments and farm produce. Appl. Microbiol. 24(6): 933-938. GELDREICH, E. E. 1972. Buffalo Lake recreational water quality: a study in bacteriological data interpretation. Water Res. 6: 913-924. HENDRICKS, C. W., and S . M . MORRISON. 1967. Muitiplication and growth of selected enteric bacteria in clear mountain stream water. Water Res. 1: 567-576. KNITTEL, M. D. 1975. Occurrence of Klebsielln pnerrttzorzirie in surface waters. Appl Microbiol. 29(5): 595-597. MATSEN,J. M.. J . A. S P I N D L E Rand , R.0 . BLOSSER.1974. Characterization of Klebsiellri isolates from natural receiving waters and comparison with human isolates. Appl. Microbiol. 28(4): 672-678. S E I D L E RR. , J., M. D. KNITTEL,and C. BROWN. 1975. potential pathogens in the environment: cultural reactions and nucleic acid studies on Klebsiella pneltmoniae from clinical and environmental sources'. Appl. MicrobiOl.29(6): 819-825. SELDEN,R . , S . LEE. W. L . L. WANG,and J. V . BENNETT. 1971. Nosocomial Klebsiello infections: intestinal colonizatlon as a reservoir. Ann. Intern. Med. 74(5): 657-664.
Isolation of Klebsiella pneumoniae from lake water LUCYM. CAMPBELL, GLENDAMICHAELS,'RICHARDD. KLEIN,A N D IVANL. ROTH Depor/t>retrrof Mic~obiology.Utri\,er~iryofGro,gitr, AIlrer1.r. GA. U.S.A.30602 otitl Dc~por~r~~etrr c?f'Biology,Goitre.~i~ille Jlrtrio~.C o l l q e , Grtitle.s~~ille. GA, U.S.A.30501
CAMPBELL, L. M., G . MICHAELS. R . D. K L E I N and , I . L. ROTH. 1976. Isolation of KleOsic~lln pt~errt?lot~itre from lake water. Can. J. Microbial. 22: 1762-1767. The question of the importance of aquatic borne Kleb.siell(t prlerrt?iotiitrc~to p ~ ~ b lhealth ic has been argued as the 01-ganism has appeared often in both polluted effluents and oligotrophic systems. Using a selective medium. double violet agar. which also differentiates K . pt1c,1r1t1ot1i(1e~ from other organisms Found in aquatic systems, we sampled water from three ponds and a large can be isolated man-made lake, over a 3-month period. Results indicate that K . ptirr~t??ot~icre consistently and in high numbersfrom eutrophic waters even when environmental stress reduces total numbers, but that isolations from cleanerwaters al-e er~xtic.We conclude that mere isolation of the organism as an occasiontii contaminant PI-obablydoes not present a hazard to public health.
Introduction Lake and stream waters are surveyed routinely in the United States for presence of coliforms and fecal coliforms as one parameter in determining severity of domestic and industrial pollution and the effects on bacterial water quality. Fecal coliforms can be identified by their ability to ferment lactose with gas production at 44.5 "C, and between 93.0 and 98.7'z of coliforms isolated from feces of humans and warm-blooded animals meet this criterion (Geldreich 1972). It has been reported that about 30 to 40% of healthy warm-blooded animals carry KlebsieNa p n ~ ~ r r ~ ~ o tas l i anormal e intestinal flora, and about 20% of healthy humans also have carried the organism (Bordner and Carroll 1972). Nosocomial infections with the organism are fairly common and are tliought to be associated frequently with patients who are also carriers (Selden e t a l . 1971). It seems obvious that appearance of high numbers o f K . /1t1e~rrnot7iaein tlie aquatic environment, particularly in potable o r I-ecreational waters, should have significant public health impact. However, there is some controversy as to the public health importance of aquatically associated K . ptieut~lotriae, particularly concerning fecal versus nonfecal origins. For example, Kriittel (1975) recently reporin surted that the occurrence of K. pne~rt~lotlicre face samples of streams, rivers, and lakes was not ubiquitous and isolation was associated with that of Esclrerichia coli. These observations disagreed with those of Duncan and Razzell (1972), 'Department of Biology, Gainesville Junior College, Gainesville, GA, U.S.A. 30501.
who isolated K. pneumoniae from limited access watersheds, other watersheds of varying accessibility, and paper and pulpmill effluents. They concluded that the lack of documented cases of K. pneunloniae - caused water-borne infections indicated the organism was of little concern t o public health. It has been reported from laboratory experiments that potentially pathogenic members of the family Enterobacteriaceae are nonfastideous and can grow in highly dilute salts supplemented witli a single suitable carbon source (Hendricks and Morris011 1967). The dilute media and incubation temperatures utilized approximated conditions found in a cold mountain stream, a n d results showed that members of Enterobacteriaceae would multiply and not merely survive under these conditions. This investigation is concerned witli isolation of K . pt1~~1t77017iae from a large man-made lake and three smaller ponds using double violet agar (Campbell and Roth 1975). This medium has been developed to aid in differentiation between K. pne~rt7lo11iaeand E ~ r t e r o b a c t e raeroget7c.s a n d to facilitate the presumptive identification of tlie former after 24 h of incubation.
Materials and Methods Lake Sidney Lanier near Gainesville, Georgia, was sampled over a 3-month period a t 10 stations about the lake and on a tributary. The sampled stations are described in Table I , and located on a mapof Lake Lanier (Fig. I ) . This is a large lake formed by a hydroelectric dam and is used primarily for recreational purposes. T h e lake at average summer pool covers 38 000 acres with a shoreline of 540 mi and a maximum depth of about 200 ft. Duplicate surface samples were taken by dropping a
1763
CAMPBELL E T AL.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
TABLEI . Sample stations on Lake Sidney Lanier Stat~on No. 1
2 3 4 5 6
7
8
9 10
Descr~ption Lake Lanier Islands Beach, a heavily used resort area Just offshore 20-50 ft from Lake Lanier Islands sewage-treatment plant Sw~niniingarea a t Holly Park, a county recreatlon area Just offshore (about 100 ft) from Linwood sewage-treatment plant Chattahoochee River channel, deep water, about 7 ml above dam Two M ~ l eCreek (end of nav~gablearea, less than 3 ft deep), clean area, no industry or agr~culture About nil from mouth of Flat Creek Mouth of Flat Creek, a heavlly polluted area receiv~ng sewage-plant effluent (including both doniest~cwastes and poultry-processing plant effluent) End of navigable area (less than 3 ft deep) of Flat Creek Free flowing area of Flat Creek below poultryprocessing plant and above sewage-treatment plant
:
A pond at a local textile mill producing polyester and cotton yarn and fabric was sampled in the same manner a s Campus Lake and Meniorial Park Lake. This pond is 900 ft by 300 ft by 6 ft deep and is enclosed by the fence surrounding the mill. It is fed by springs in the lake bottom and receives waste water froni the mill's airconditioning system. There is little r u n o f froni the area and the pond does not receive process water from the mill. It is not used for recreational purposes and has no waterfowl. Samples (100 nil) were processed in the same nianner as the Menlorial Park Lake samples. Sa~iipleswere taken from four stations on the textile mill pond. Cotton samples for analysis for the presence of K. pnerrrr7onioe were removed from bales with a pair of sterile forceps and placed in sterile whirl-pack bags. Onehalf-gram samples of cotton were weighed aseptically and introduced into bottles containing 100 nil of sterile water. The bottles were shaken vigorously for 1 niin and the water was filtered through 0.45-pm Millipore membrane filters. The cotton was retained in the bottle by means of a sterile glass rod. The filters were placed on double violet agar plates and incubated at 37 "C for 24 h. The fecal coliforni and fecal streptococcus analyses were done using the membrane-filter t e c h n i q ~ ~ eass detailed in Siarlc/orcl rrretllocls for tlrc esotrritlntiot~of )voter on(/ btwste Ivo/er, 13/11 ecl.
Results Lake Lanier
. . . . . .. .. . .. . .. . . . . .. . .. . .. . ........... . .
. .
.. . ... . . . .... .
. .
... .. .. .. .. .. .. . . . . . . . . . ... .. . . .. .... . ,
Ten stations, with a surface and subsurface Nuclepore filter onto the water surface (Crow et (11. 1975), retrieving it with a pair of sterile forceps, and placing it (10 cm) sample taken at each, were monitored on on a plate of double violet agar containing per litre of Lake Lanier over a 3-month period. Klehsiellc~ deionized water: violet red bile agar (Difco), 41.5 g ; and ptie~itnonicre was isolated from five of 10 surface methyl violet 2 8 (Allied Chemical Corp.), 2.0 g (Canipsan~plingsover the test period (Table 2). Only bell and Roth 1975). Duplicate subsurface samples were once was K. pne~itiloniae isolated from a subtaken 10 cni below the surface, the saniple placed in a sterile Petri dish, and a Nuclepore filter dropped on the surface sample, at a station on Flat Creek water as with surface saniples. Colonies were counted (station lo), a shallow tributary which receives and picked to confirmatory biocheniicals as described effluent from a poultry-processing plant and a previously (Campbell and Roth 1975). The biochemical city sewage-treatment plant (intermittently chlormedia ~ ~ s were e d triple sugar agar (TSI) (Difco), niotilityindol-oriiithine decarboxylase (MIO) (Difco), and Chrisinated). The number 10 sanipling station is, tiansen's urea agar (Difco). however, below the poultry-processing plant and Memorial Park Lake is a sniall man-made pond of 6 above the sewage-treatment plant. Two of the acres with a niaxiniuni depth of 15 ft and a minimum five surface samples from which K. pne~rtiloniae depth of 2 ft used niostly for decorative purposes and was isolated were also from Flat Creek stations nonswimniing recreation. It receives r ~ ~ n o ffroni f the surrounding park area which is surrounded by an urban (9-S and 10-S) (Table 2). Only one station on housing area. The lake is inhabited by a flock of waterFlat Creek (10-S) yielded isolates of K. ptle~ifowl. The pond experienced a heavy algal bloonl in late t7iotlicie on all sampling dates. Three other stasummer of 1975. Surface samples (100 ml) were taken at tions on the lake yielded occasional samples that three stations as described in Stcrtrrlorcl tr~eilrocls(13th ed). and processed by filtration throc~gh0.45-prn Millipore were positive for K. ptie~itnotiioe, but the organnienibrane filters within 30 niin of collection. Plated filters ism was not consistently isolated from any of were handled as with Lake Lanier saniples. them. Campus Lake is a sniall man-made pond (500 ft in Colonies of K. pne~rnioniaewere distinguished length, 100 ft wide at the narrower end, 200 ft wide at the from other isolates by the distinct morphology wider end, and about 10 ft deep) located on the campus of Gainesville Junior College, Gainesville, Georgia. It is on double violet agar, a pale lavender 'gumdrop' stocked with fish and several ducks frequent the pond. colony ranging from 2 to 5 mm in diameter. The pond is primarily spring fed, but also receives r ~ ~ n o f f When such colonies were picked to confirnlatory from the campus. Surface saniples (100 nil) were taken at media, they produced acid slant, acid butt, gas, four collection sites at the water's edge and processed in the same mariner as the Meniorial Park Lake saniples. no hydrogen sulfide on TSI; they were non-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
1764
CAN. J. MICROBIOL. VOL. 22. 1976
FIG.1. Sample stations (see Table 1) on Lake Sidney Lanier.
motile, indol-variable, ornithine decarboxylasenegative on motility-indol-ornithine decarboxylase deeps (MIO), and urease-positive.
Memorial Park Lake Surface samples of water taken from this small
pond yielded K. p~leutnoniae on each occasion, including a period in late August and early September when a heavy algal bloom lowered total numbers (Table 3). Samples were taken from three locations at the pond's edge within the area affected by the bloom, and numbers of
1765
CAMPBELL E T AL.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
TABLE 2. Isolation of K. pt~c,lrtnot~iae,fecal coliforms, and fecal streptococci from Lake Sidney Lanier. Stations which yielded no organisms on any sampling date are not included Average* count per Nuclepore filter 17/6/76
8/7/76
22/7/76
17/8/76
Station
Kp
FC
FS
Kp
FC
FS
Kp
FC
FS
1-S 2-S 3-S 4-S 6 6-S 7-S 8 8-S 9 9-S 10 10-S
0 0 0 0 0 0 0 0 0 0 3 0 T
0 0 0 0 0 0 0 0 1 0 20 0 T
0 0 0 0 0 0 0 0 0 0 0 1 49
0 0 1 0 0 1 0 0 0 0 11.5 6.5 T
18 3 180 0 0 13 9 I 28 8 26 T
0 0 0 0 0 0 2 0 0 0 0
0 0 3.5 0 0 0 0 0 0 0 T 0 T
0 0 0 5 0 0 0 1
0 0 7 0 0 0 I 0 0 0 9 4 T
4 T
-
7 T 2 T
Kp
FC
0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 84 0 0 T T 1 2
9/9/76 FS
Kp
FC
FS
0 0 0 0 0 0 0 0 0 0 2 2 0
0 0 1 1 0 0 0 0 0 0 2 0 T
0 0 0 0 0 6 0 0 5 0 120 5 T
0 0 0 0 1 0 0 0 0 0 1 2 T
NOTE:Surface samples are designated with the terminal "S". Sample williout the "S" designation are subsurface samplcs. T h e sampling stations are described in Table I a n d shown on the m a p o f Lake Lanier (Fig. I). 'Average of t ~ v osamples per station. ADDREVIATIONS USED:T = too numerous to count. K p = Klebsiellapncv,i~oniaecount. F C = Fecal coliform count. FS = Fecal streptococci count. - = no data.
TABLE 3. Comparison of numbers of K. pneutnoniae with total numbers of bacteria isolated on double violet agar from Memorial Park Lake, Athens, Georgia Organisms per 100 ml (average of three stations) Date
Klebsiella pnelmionioe
18/8/76* 2018176 5/9/76 11/9/76 12/9/76 26/9/76t 3019176
100 200 100 267 200 1000 533
TABLE 4. Isolation of K. ptlelcniot~iaeand fecal coliforms from Campus Lake Organisms per 100 ml (average of four stations) Date
K. pt~elrtno~~ineFecal coliforms
Total
700
1 500 2 000 2 500 4 000 10 000 6 000
'Heavy algal bloom. ?Algal bloom cleared. NOTE:T = LOO numerous to count.
klebsiellae isolated dropped concomitantly with total numbers until the bloom cleared in midSeptember. Campus Lake Samples from the surface of Campus Lake yielded K. pneurnor~iae each time samples were taken (Table 4). While the numbers of K. pneuinoniae in Campus Lake were not as high as those in Memorial Park Lake, the organisms were consistently present.
TABLE 5. Isolation of K. pnelrmot~ioeand fecal coliforms from a textile mill pond Organisms per 100 ml (average of four stations) Date
K. pt~euniotlioe
Fecal coliforms
1/7/76 29110176
30 5
0 0
Textile Mill Pond The textile mill pond was sampled only twice, but K . pneurlzoniae was isolated on both of those occasions (Table 5). The count of the sample taken on October 29, 1974, was the lowest count obtained in the small bodies of water we sampled. We suspected that the textile mill pond might be contaminated with raw cotton and that cotton might be the source of the K. pneutnoniae in this pond. The results of our analysis of raw
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
1766
C A N . J . MlCROBl O L . V O L . 22. 1976
TABLE 6. Isolation of K. p~~elrtirotzioe and fecal colifornis from bales of unprocessed cotton on 25/5/76
Memorial Park Lake and Campus Lake, there is a constant waterfowl population, From our study it appears that neither pond is able t o clear itself of K. p/ieun~oniaedespite periodic rainfalls K. ptie~rt,~ottirre/g Fecal colifol.m/g Bale and dry periods. In the case of Memorial Park Lake, a heavy algal bloom in late summer, 1975, precipitated a drop in total bacterial numbers from 4200 CFU/100 ml to a low of 700 C F U / 100 ml. While numbers of K. pne~c1?7oniaealso dropped, a t no time was the organism completely absent. Thus K. pne~onoiziae could be considTABLE 7. Biochemical characteristics used in the ered t o be ubiquitous in the surface layers of identification of E. oer.ogetles, E. coli, and K. ptrerrthese small ponds. While the textile mill pond was t,~ot~ioe only sampled twice, it did yield K. pneumoniae on both occasions. The October 29, 1975, E. K. sample gave the lowest K. pnewnonirre count we creroE. ptrc.lcTest genes coli ~~zo~iioe recorded for any of the small ponds. This low count in the textile mill pond as compared t o the Lactose fermentation + + + Memorial Park Lake and Campus Lake could + rl * Ornithine decarboxylase probably be attributed to the absence of waterLysine decarboxylase + rl + Motility ++fowl at the textile mill pond which is fenced. + Urease There was some evidence that the textile mill Indol + + pond might be contaminated with cotton fibers. *d, Delayed. Bales of cotton are off-loaded from trucks on a cotton samples taken from bales at this textile loading dock adjacent to the textile mill pond. Since K. pneurnoniae has been isolated from a mill are shown in Table 6. The biocliemical cliaracteristics and reactions number of plant sources (Duncan and Razzell 1972) it seems likely that cotton may be conused in the confirmatory tests are listed in Table taminated with this organism. We analyzed 7. several samples of cotton and found that the Discussion cotton was indeed contaminated with K. pne~cDouble violet agar was developed to isolate 1?7onirre as shown in Table 5. Apparently K. and select for K. p17e~olio17icre from contaminated p17e~rtnoniueis widely distributed in nature. Lake Lanier is a much larger, deeper system waters or other sources where Klebsiellrr might occur. In this study, we investigated the distri- and apparently does not have a constant K. on a large lake and three piielm?onicie population, even in the surface bution of K. p17e~c1no17irie smaller ponds used for recreational or decora- layers. Of the five surface samples that yielded tive purposes t o determine if the organism is positive cultures of K. pne~olioniae,two stations, indeed ubiquitous and if the numbers fluctuate 9-S and 10-S, were in Flat Creek which is exor remain stable. Klebsielln p17e~trl7017iaecan be posed t o effluent from a chicken-processing plant thought of as an indicator organism while being and a sewage-treatment plant. At only one stasimultaneously considered as a potential patho- tion was the organism consistently isolated, most gen. Although questions have been raised re- of the isolations being occasional. It is possible garding its importance as a threat t o public that the greater dilution capacity of Lake Lanier health, investigations have clearly labeled kleb- contributes to the apparent clearing of the siellae isolated from aquatic systems and those organism from the system in most of the areas from clinical sources as identical (Seidler e t ril. sampled. Klebsiellap17e~cnioniaewas isolated only 1975 and Matsen er a/. 1974). It is likely that the once from a subsurface sample which would mere presence of the organism poses little threat indicate that its occurrence is generally limited t o the well-being of the public, but that the to the surface. We have found that there is essentially nc threat becomes more viable as numbers increase difference in enumeration of K. piieunio~iine on and other species are excluded. In two of the more stagnant systerlls examined, double violet agar and tryptic soy agar (unpub.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 10/01/14 For personal use only.
CAMPBELL ET AL.
lished data). This fact allowed for rapid qualitative and quantitative analysis of water samples for presence of K. pneumoniae, without the necessity of picking large numbers of colonies to biochemical media for confirmation. Only those colonies suspected of being K. pneutnoniae were confirmed by use of auxiliary media. Colonies grown from Memorial Park Lake samples on double violet agar for 24 h were confirmed as K. pneutnoniae 100% of the time. Plates from Lake Lanier were held for up to 72 h before colonies were picked to confirmatory media and were confirmed with 88% accuracy. The precision in identifying K. pneurnoniae from the appearance of colonies on double violet agar has been further confirmed by our study on river water in which we achieved an average of 85.4x accuracy (Campbell et al. 1976). As colonial morphology tends to alter slightly when plates are held at room temperature, these results indicate that plates should be read promptly. Distribution of K. pneunloniae in lakes, therefore, can be determined quickly and accurately by use of double violet agar, without need for confirmation of colonies. The question of the importance of K. pneunloniae and its distribution to the ~ u b l i chealth has never been resolved. Some iorkers have been able to isolate K. pneutnoniae from exceptionally clean water systems, as well as polluted ones (Duncan and Razzell 1972). Others have found' the organism occurring in conjunction with fecal coliforms and feel that the presence of the organism represents deterioration of the environment (Knittel 1975). We have not found the organism to be ubiquitous, although it is widely distributed in nature as evidenced by the recovery of K. pne~mmoniae from raw cotton. What we have discovered is that the organism is a major constituent of the flora in eutrophic systems, with numbers and consistency in isolation over a period of time related directly to the degree of pollution encountered. While the Dresence of fecal coliforms amears to be correlated with the presence of K. pneulnoniae in between Lake, there is fecal coliforms, fecal and K. ptleulnonine in the much larger lake, Lake Lanier. The presence of K. pfieur)yonjae in the textile mil] pond and the absence of fecal coliforms there is likely due to the more Or less seeding of that pond with K. prleurlloniae from the cotton along with no appreciable input of coliforms. L.
1767
Thus in our analyses we have established that K. pneurnoniae does occur in natural waters in the absence of fecal coliforms; however, at some sites, the presence of K. pneur?loniae is correlated with a fecal coliform population. Acknowledgment This work was supported in part by research grant number R-803341-01-0 from the U.S. Environmental Protection Agency. We thank Dr. Warren Cook and the Biology Department of Georgia State University for the use of aquatic sampling equipment. The able technical assistance of Beatriz Chapman is gratefully acknowledged. A M E R I C APUBLIC N HEALTHASSOCIATION. 1971. Standard methods for the examination of water and waste water, 13th ed. American Public Health Association Inc.. N.Y. BORDNER, R. H., and B. J. CARROLL. 1972. Proceedingsof seminar on the significance of fecal coliform in industrial wastes. U.S. Environmental Protection Agency, Denver, Co. L. M., and I. L. ROTH.1975. Methyl violet: a CAMPBELL, selective agent for differentiation of Klebsiellti P I I ' I I tnotzitie from Et~te~~obrrcter rierogenes and other Gramnegative organisms. Appl Microbiol. 30(2): 258-261. CAMPBELL, L. M., I. L. ROTH, and R. D. K L E I N .1976. Evaluation of double violet agar in the isolation of Klebsiellrr ptlerrtnonicre from river water. Appl. Environ. Microbiol. 31(2): 213-215. CROW,S. A,, D. G. AHEARN.W. L. COOK,and A. W. B O U R Q U I N1975. . Densities of bacteria and fungi in coastal surface films a s determined by a membraneadsorption procedure. Limnol. Oceanogr. 20(4): 644646. D U N C A ND. , W., and W. E. RAZZELL.1972. Klebsiello biotypes among coliforms isolated from forest environments and farm produce. Appl. Microbiol. 24(6): 933-938. GELDREICH, E. E. 1972. Buffalo Lake recreational water quality: a study in bacteriological data interpretation. Water Res. 6: 913-924. HENDRICKS, C. W., and S . M . MORRISON. 1967. Muitiplication and growth of selected enteric bacteria in clear mountain stream water. Water Res. 1: 567-576. KNITTEL, M. D. 1975. Occurrence of Klebsielln pnerrttzorzirie in surface waters. Appl Microbiol. 29(5): 595-597. MATSEN,J. M.. J . A. S P I N D L E Rand , R.0 . BLOSSER.1974. Characterization of Klebsiellri isolates from natural receiving waters and comparison with human isolates. Appl. Microbiol. 28(4): 672-678. S E I D L E RR. , J., M. D. KNITTEL,and C. BROWN. 1975. potential pathogens in the environment: cultural reactions and nucleic acid studies on Klebsiella pneltmoniae from clinical and environmental sources'. Appl. MicrobiOl.29(6): 819-825. SELDEN,R . , S . LEE. W. L . L. WANG,and J. V . BENNETT. 1971. Nosocomial Klebsiello infections: intestinal colonizatlon as a reservoir. Ann. Intern. Med. 74(5): 657-664.
