Vol. 57, No. 3
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 751-758
0099-2240/91/030751-08$02.00/0 Copyright C) 1991, American Society for Microbiology
Characterization of Caulobacters Isolated from Wastewater Treatment Systems JEAN D. MACRAE AND JOHN SMIT*
Department of Microbiology, University of British Columbia, Vancouver, British Columbia V6T I W5, Canada Received 31 August 1990/Accepted 3 January 1991
Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.
The first goal of wastewater treatment systems is the removal of nutrients, particulates, pathogens, and toxic materials from the effluent over as short a time interval as practicable (15). In primary treatment, particulates are removed by gravity settlement, screening, flotation, skimming, and the like, but to accomplish significant reductions in the soluble organic carbon, nitrogen, and phosphate in wastewater, the engineering solutions usually involve the use of microbial populations. By various means, the development of microbial communities or consortia result in the conversion of soluble nutrients into insoluble aggregates that are removed from wastewater, producing sludge and cleaner effluent (15). The microbial populations that develop in these treatment systems are a complex expansion of indigenous soil and freshwater bacteria, with selection for those bacteria that can grow quickly and compete for nutrients in an environment that has much higher nutrient loadings than is typical of soil and water (18). In most cases, the consortium that develops produces a variety of adhesive exopolysaccharides and other substances that result in the production of thick biofilms or coarse, particulate aggregates of bacteria (3, 7, 8). Beyond such generalizations, however, it has been difficult to characterize the bacterial composition of wastewater treatment systems (24, 27). Direct counting and standard plate counting techniques are hampered by aggregates, and the former is also hampered by difficulties in distinguishing living and dead or moribund cells. Many of the bacteria present cannot be cultured, because of either undetermined nutritional requirements or the inability to grow as colonies
on solid media (19). Even so, a number of studies have been done and numerous genera have been identified within sludge-forming bacterial populations (4, 11, 18). Caulobacters are stalked bacteria that are members of biofouling populations by virtue of an adhesive holdfast anchored at the tip of their stalk, as well as at the base of the single polar flagellum during the motile, swarmer-cell phase of these dimorphic bacteria (14, 20). As far as we can determine, there has not been a single reported instance of caulobacters being isolated from a municipal wastewater treatment facility. Since they have long been known to be found in pristine freshwater supplies and are often contaminants in distilled water plumbing, it is sometimes presumed (though not necessarily by other caulobacter researchers) that caulobacters would not be widespread in mesotrophic or organically rich environments (21, 23). Work in our laboratory, however, had provided some indication that caulobacters might not be restricted to nutrient-poor environments. Marine caulobacters were readily isolated from regions receiving significant input of organic material (1). Growth of laboratory strains occurred readily and to high cell density in media containing relatively high concentrations of carbon, nitrogen, and phosphate. We also knew that in phosphate-sufficient environments, some strains of caulobacters do not produce the long stalks that are characteristic of the genus in phosphate-limited situations and so can be difficult to identify by light microscopy.
In an effort to learn more about the actual range of habitats for caulobacters in the environment, we examined the environment of wastewater treatment systems, where organic carbon, nitrogen, and phosphate, as well as toxic metals, are available in relative abundance.
* Corresponding author. 751
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MAcRAE AND SMIT
APPL. ENVIRON. MICROBIOL.
TABLE 1. Sources of caulobacter isolates Wastewater treatment sources FWC8,-14,-19,-20
FWC9,-12 FWC11 FWC21 FWC15,-16,-18
FWC17 FWC22 FWC23,-24,-27-, -28,-31 FWC25 FWC26 FWC30 FWC29 FWC32,-33 FWC34 FWC35 FWC38 FWC37 FWC39,-40
FWC41,-42,-43 Non-wastewater treatment sources FWC1 FWC4,-5 FWC6,-7 FWC13 FWC45 a
Location (U.S. or Canada)
Type of facility
Strain
Secondary treatment, activated sludge process, Bio-P" Bench-scale secondary treatment system, Bio-P fed with landfill leachate Holding tank for raw influent sewage Bench-scale rotating biological contactor Primary treatment facility for greater Vancouver, British Columbia, area Secondary treatment facility, rotating biological contactor Secondary treatment facility, aerobic digestor Contour trench wastewater treatment system
University of British Columbia Department of Civil Engineering, test plant University of British Columbia Department of Civil Engineering University of British Columbia University of British Columbia lona Island, British Columbia
Langley, British Columbia Langley, British Columbia Takla, British Columbia
Secondary treatment facility, primary treatment region Secondary treatment facility, activated sludge Untreated influent sewage Secondary treatment facility, activated sludge system Trickle filter secondary treatment facility Secondary treatment facility, activated sludge system Trickle filter secondary treatment facility Secondary treatment facility, activated sludge system Secondary treatment facility, activated sludge system Secondary treatment facility, activated sludge system, Bio-P Secondary treatment facility, activated sludge system
Edmonton, Alberta, Gold Bar facility Edmonton, Alberta, Gold Bar facility Edmonton, Alberta, Gold Bar facility Bozeman, Mont. Coeur d'Alene, Idaho Pullman, Wash. Las Vegas, Nev.
Lake Washington Surface water in waterlogged area Tap water Surface water from a peat bog Stream water
Seattle, Wash. Bothell, Wash. Oakland, Calif. Richmond, British Columbia Burnaby, British Columbia
Las Vegas, Nev. Post Falls, Idaho
Kelowna, British Columbia Calgary, Alberta, Bonnybrook facility
Bio-P, Biological phosphate removal.
MATERIALS AND METHODS Sample sources and isolation techniques for caulobacters. Samples were taken from the various wastewater treatment facilities (Table 1) by simple liquid collection or, in the case of sampling trickling filters or contactors, by scraping bacterial growth from a surface and suspending in water. The samples were transported or mailed to the laboratory, diluted 1:1,000 in 0.01% peptone, and incubated aerobically at room temperature without shaking. After 3 to 7 days, a loopful of the liquid surface film (usually not a visible film) was diluted appropriately, plated onto a peptone-yeast extract (PYE) agar medium (20), and incubated at room temperature for several days. Colonies were individually examined by phase-contrast light microscopy. After standard purification procedures, isolates were grown in PYE liquid medium (supplemented with riboflavin at 2 ,ug/ml, if needed) and stored in 10% dimethyl sulfoxide at -70°C. A collection of 150 bacterial strains, judged not to be caulobacters, was prepared from wastewater treatment samples by dilution and plating, either on PYE or CGY (19) medium. The isolation of caulobacters from non-wastewater treatment sources was similar to that used for wastewater treatment sources, except for a longer enrichment period, and has been described (1, 20). Characterization of wastewater treatment caulobacters. In addition to gross characteristics, such as colony color, cell shape, and growth stimulation by riboflavin, a number of approaches were taken to define and distinguish individual strains of caulobacters.
(i) Total protein profiles. Late-logarithmic-phase cultures (5 ml) were washed by centrifugation and suspension with water, frozen, thawed and suspended in 250 ,u1 of 10 mM Tris-1 mM EDTA. Then, 1 ,ul of DNase (0.5 mg/ml), 20 ,ul of lysozyme (10 mg/ml), and 3 ,u1 of 1 M MgCl2 were added, and the mixture was incubated at room temperature for 10 to 15 min before being stored at -20°C. Protein concentrations were determined by a modified Lowry assay (13), and samples containing 40 ,ug of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10 to 17% gradient) using standard methods (10, 33). (ii) Western blot analysis. A portion of the isolates were examined by the Western blot (immunoblot) technique as previously described (29), using antibody prepared against the S layer of C. crescentus CB15 (28). (iii) Holdfast analysis. Variability in holdfast composition was judged by a fluorescein-coupled lectin binding assay as previously described (14). (iv) Antibiotic sensitivity. Difco Dispenso-discs were placed on PYE plates overlaid with soft agar containing an inoculum of log-phase cells. The plates were incubated at 30°C for 24 to 48 h, and the zones of inhibition were measured in millimeters. Scoring was based on the manufacturer's recommendations, except that the recommended medium was not used and "intermediate" values were scored as sensitive. For ampicillin, streptomycin, tetracycline, and chloramphenicol, the strains were also patched onto PYE plates prepared with various concentrations of the
CAULOBACTERS IN WASTEWATER TREATMENT
VOL. 57, 1991
-
~
I- :
^
753
I
FIG. 1. Phase-contrast microscopy of the air-water interface from a caulobacter isolation experiment (see Materials and Methods). The original inoculum was activated sludge from a secondary treatment system, modified to accomplish biological phosphate reduction from the effluent. The micrograph shows a region containing unusually high numbers of caulobacters. The long stalks are likely the result of limited levels of phosphate in the isolation scheme.
drugs and observed for growth following several days of incubation at room temperature. (v) Detection of plasmids. Strains were evaluated for the presence of plasmids by the method of Kado and Liu (9). (vi) Gene hybridization experiments. Colony hybridization was done by standard methods, using Gelman BioTrace RP filters (12). For restriction fragment length polymorphism (RFLP) analysis, chromosomal DNA was isolated as previously described (17); portions were digested with EcoRI, BamHI, and HindIII; and Southern blot hybridization was performed by standard methods (12). 32P-labeled probes were prepared by nick translation (12) of appropriate DNA fragments. One microgram of digested DNA was run per lane, and 3 x 10 cpm of probe was used in each hybridization. After hybridization, blots or lifts were washed at 55°C with 150 mM NaCI-10 mM EDTA-10 mM sodium phosphate, pH 7.4 (I x SSPE). This wash regimen corresponds to a moderate stringency, permitting up to approximately 30% base pair mismatch, assuming the caulobacters to have about 65% GC composition, as do characterized caulobacter strains (23). RFLP hybridization blots were exposed to autoradiographic film for 2 days and scored. The surface (S)-layer protein gene probe was prepared from a 4.4-kb HindIII-BamHI fragment containing the 130K gene of C. crescentus CB15 (29). Probe for the Caulobacter flagellin genes was prepared from the 0.9-kb SalI-EcoRI insert of
pCA161 (16), which hybridizes with all six of the flagellin genes of strain CB15. (vii) Electron microscopy. Examination for the presence of an organized paracrystalline S layer was done by suspending bacterial colonies in a small amount of water and preparing negative-stain grids with ammonium molybdate as previously described (30). Grids were examined in a Siemens 101A transmission electron microscope, operated at 60 kV. RESULTS Isolation of caulobacters. In general, caulobacters were readily isolated from virtually every type of municipal wastewater treatment system examined, ranging from passive contour trench systems to activated sludge or trickling filter systems to rotating biological contactors. The geographical and climatological locales ranged from the Nevada desert to northern British Columbia, Canada, inlands. Caulobacters could be detected at all points of treatment systems, except for the strongly anaerobic regions of anaerobic digestors used by many facilities to reduce sludge volume and generate methane gas. The procedure used to isolate caulobacters was similar to that used for isolating caulobacters from freshwater sources, which is based on the principle that caulobacters will become enriched, relative to other bacteria in the sample,
754
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MAcRAE AND SMIT
TABLE 2. Characteristics of caulobacters isolated from wastewater treatment facilities Strain
FWC9 FWC11 FWC12 FWC14 FWC15 FWC16 FWC17 FWC18 FWC19
FWC20 FWC21 FWC22 FWC23 FWC24 FWC25 FWC26 FWC27 FWC28 FWC29 FWC30 FWC31 FWC32 FWC33 FWC34 FWC35 FWC37 FWC38 FWC39 FWC40 FWC41 FWC42 FWC43
Cell
plasmids
S
layer
Colony color
shape
2 2 2 0 1 1 1 1 2 2 1 0 3 2 1 1 2 1 0 1 1 1 0 1 1 0 0 2 0 1 1 1
+ +
Tan Tan Tan White Tan Tan Tan Tan Tan Light yellow Tan Tan Yellow Tan Tan Tan Tan Tan Tan Red Tan Tan Tan Tan Tan Tan Bright yellow Tan Tan Tan Tan Bright yellow
Crescent Crescent Crescent Rod Crescent Crescent Crescent Crescent Crescent Crescent Rod Crescent Crescent Crescent Crescent Crescent Crescent Crescent Crescent Ovoid Crescent Crescent Crescent Crescent Crescent Crescent Rod Crescent Rod Crescent Crescent Rod
No. of
+
+ + + + + +
+
+ + + + +
+ + + + + +
+
+ +
-
when incubated for extended periods (1 to 2 months) in a medium with very low organic content. In the present situation, we routinely noted that caulobacters could be easily detected in surface films by phase-contrast microscopy in 1 week or less, obviating the need for any significant enrichment beyond the tendency of caulobacters to accumulate in the surface film (21, 23). The results suggested that caulobacters were abundant in most wastewater treatment samples as compared with other aquatic sources. Some samples had surprising levels of caulobacters. In a few instances, as many as 40% of the colonies seen on the initial PYE plates were caulobacters. Given the ambiguities regarding plating efficiency of wastewater treatment bacteria, it is not possible to equate such numbers to the actual proportion in the treatment systems, yet it seems fair to estimate that in these instances caulobacters were a significant fraction of the total. The use of the surface film as a source for isolating pure cultures, as compared with direct isolation from particulates, worked well, since the bacteria were much more dispersed and after brief mechanical disruption of a diluted sampling (with a vortex mixer), colonies containing a single species of bacteria were the most common occurrence. Also, the probable limitation of phosphate in the incubation medium encouraged long-stalk development and ready recognition of the caulobacters (Fig. 1). In contrast, stalked bacteria were seldom discernible by direct examination of the bacterial
Degree of rosette formation
Degree of hybridization with probes: S layer Flagellin
+++ + +++ + +++ + + +++ +++ + +++ + +++ + +++ ++ +++ + ++ +++ + +++ ++ + + ++ -+ + + +++ + +++ + +++ + +++ + +++ + + + +++ + -+++ +++ ++ +++ ++ + +++ +++ + ++ +++ + +++ ++ +++ ++ ++ +++
+++
++
+++ +++ +++ + +++ +++ +++ +++ +++
+++ ++ + +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+ +++ +++
+
aggregates that were typically present in most types of wastewater treatment samples. Characterization of caulobacter isolates. The results of most of the characterization studies are given in Tables 2 to 4. By far, most of the caulobacters seen and, subsequently, most of the preserved isolates had numerous characteristics that identified them as the typical caulobacter detected in wastewater treatment systems and much like the wellcharacterized C. crescentus strains. The cell bodies were crescent-shaped and produced relatively few rosettes in monoculture (a consequence of fused holdfasts of multiple cells), and, via negative-stain electron microscopy, nearly all showed the presence of a hexagonally packed, paracrystalline S layer. The S layers were visually indistinguishable from those produced by C. crescentus CB2 and CB15 (29, 30). Cells in colonies more than 5 days old showed virtually no rosettes, minimal adhesiveness to glass surfaces, minimal motility, and few visible stalks. In short, they were difficult to identify by textbook morphological characteristics as caulobacters yet were readily detected when one became familiar with what to expect. Although the typical caulobacters were visually similar, there were gross characteristics that distinguished them. There was considerable variety in holdfast composition, as judged by lectin-binding patterns, similar to that noted before for freshwater caulobacter strains (14). For many in
VOL. 57,
FWC14 FWC15 FWC16 FWC17 FWC18 FWC19 FWC20 FWC21 FWC22 FWC23 FWC24 FWC25 FWC26 FWC27 FWC28 FWC29 FWC30 FWC31 FWC32 FWC33 FWC34 FWC35 FWC37 FWC38 FWC39
FWC40 FWC41 FWC42 FWC43
DBA
Soybean
ConA
UEA1
WGA
Ricinus
-
-
-
-
+
-
-
-
-
-
-
-
+ +
-
+
+ + + + + + + + + + + +
-
-
+ + -
-
-
-
-
-
-
+
+
+ + -
+
+
+ +
-
+
+ +
-
+ +
-
Resistance to antibiotica
Strain
Result with lectina:
Strain
+ -
+
+
-
+
+
-
-
-
+
+ +
+ +
-
-
-
+
+
+
+ +
755
TABLE 4. Antibiotic resistance in caulobacter isolates
TABLE 3. Assessing caulobacter holdfast composition by lectin binding
FWC8 FWC9 FWC11 FWC12
_s
CAULOBACTERS IN WASTEWATER TREATMENT
1991
+ + -
+
+
a The assay was performed as previously described (14) with fluorescein isothiocyanate lectins. DBA, Dolichos biflorus agglutinin; Soybean, soybean agglutinin; ConA, concanavalin A; UEA1, Ulex europaeus agglutinin 1; WGA, wheat germ agglutinin; Ricinus, Ricinus communis agglutinin. Peanut agglutinin was also examined but gave negative results in every case.
the typical group, growth on the initial PYE solid medium occurred readily, but they subsequently grew slowly or unpredictably in PYE liquid medium. Normal growth rates were restored in most of these cases upon the addition of riboflavin. On the basis of current classification schemes for caulobacters, this would categorize them as C. vibrioides, the type strain for the Caulobacter group (23). Occasionally, we detected a caulobacter that to varying degrees did not follow the typical pattern. FWC14, -21, -30, -38, -40, and -43 are in that class. They had longer, more visible stalks and formed long, thin rod or ovoid cell bodies. They produced rosettes in monoculture more readily and lacked a visible S layer. To minimize duplication of strains, we did not preserve more than one typical caulobacter from a particular source, even though numerous typical caulobacter colonies might be noted, yet we always kept an atypical one. Thus, the atypical isolates are significantly overrepresented in the collection of 33 strains, as compared with their abundance in the samples we examined. Protein band profiles obtained by SDS-PAGE were a major criterion for distinguishing strains of caulobacters (Fig. 2). Unless significant differences were seen in other tests, strains with similar protein band profiles were consid-
Wastewater sources FWC8 FWC9
Cm
Tet
Em
Tm
r
r r r
r r r
r
s
s
r
r r
r r
s s
s
FWC11
r
FWC12 FWC14 FWC15 FWC16 FWC17 FWC18
s s
r
FWC19
FWC20 FWC21 FWC22 FWC23 FWC24 FWC25 FWC26 FWC27 FWC28 FWC29 FWC30 FWC31 FWC32 FWC33 FWC34 FWC35 FWC37 FWC38 FWC39 FWC40 FWC41 FWC42 FWC43 Non-wastewater sources C. crescentus CB15A C. crescentus CB2A C. vibrioides C. bacteroides FWC1 FWC4
FWC5
FWC6 FWC7 FWC13 FWC45
,' I
eIA
s s
s
s
s
r
r r r r r r
r r r
r r r r r r r r
s s
r
r r r r
r
S S
S S
S S
S S
s s
r r
r s
r
r r r
r
s s s
s s s s s s s
s
s r r s
r
s
s
r r s s
s
Aps Nal'
s s s s s s s s s s s s
s
Other(s)b
r r r
Smr
s s
s
r r
r
s
s
S S S
S S S
S S S
S S S
Aps Pens
s s s s s s s s s r s
s s s s s s s s s
s s r s s s s s
s
Pbs
r
r s
r r s
r
s s s s s s s s s
Smr Nals
Aps Pens Pbs Ap5 Pen' Pb5
a Abbreviations: r, resistant; s, sensitive; Cm, chloramphenicol; Tet, tetracycline; Em, erythromycin; Tm, tobramycin; Ap, ampicillin; Pen, penicillin; Sm, streptomycin; Pb, polymyxin B; Nal, nalidixic acid. b Except as noted, most isolates were resistant to ampicillin, penicillin, polymyxin B, and nalidixic acid and were sensitive to streptomycin.
ered to be the same. This resulted in dropping 2 strains from the original collection of 35. Despite the similarity in appearance of the S layers, antibody prepared against the C. crescentus CB15 S layer protein reacted with proteins of size and abundance similar to those of the C. crescentus CB15A S-layer protein in Western blot analysis in only a few of the wastewater treatment isolates (not shown). Colony lift hybridization (Fig. 3) and RFLP analysis with the S-layer gene probe under moderate stringency conditions gave a positive signal for nearly all caulobacter strains and one and occasionally two distinct bands on the Southern
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mw8 9 11 12 15 16 17 18 19 20 22 23 24 25
FIG. 2. SDS-PAGE of total proteins from caulobacter isolates. The numbers represent FWC strains (i.e., 8 = FWC8). 103 Molecular weight standards (mw) rabbit muscle phosphorylase, 97.4; bovine serum albumin, 66.2; hen egg white albumin, 42.7; bovine carbonic anhydrase, 31; soybean trypsin inhibitor, 21.5; hen egg white lysozyme, 14.4.
blots with all three enzymes (not shown). C. crescentus CB15, the source of the S-layer gene probe, gave a single band for the three enzymes. However, the strength of the
signal was faint or absent with all of the atypical strains (FWC14, -21, -30, -38, -40, and -43) and FWC23. These strains also had no other indication of an S layer. Highstringency washes (650C, O.1x SSPE) eliminated the signal for most of the caulobacters, indicating that the S-layer gene sequence was not highly conserved among the caulobacters, as compared with the C. crescentus CB15 gene used as a probe. The flagellin gene probe gave similar results, except that typically 2 to 3 bands were produced. Virtually no patterns of close relatedness among the strains, based on RFLP analysis, were apparent. That is, there were few instances of similarly migrating bands that
APPL. ENVIRON. MICROBIOL.
could be used to define subset groups of strains, as is often possible with closely related bacterial strains (5). Colony hybridization was also done with 150 randomly selected "non-caulobacter" wastewater treatment bacterial strains. None hybridized with the S-layer gene probe, and a single positive score was obtained with the flagellin gene probe. Most strains, both from wastewater and other sources, were resistant to ,-lactam antibiotics (ampicillin and penicillin), nalidixic acid, and (like many gram-negative bacteria) polymyxin B and were sensitive to streptomycin. Resistance to chloramphenicol, tetracycline, erythromycin, and (to a lesser extent) tobramycin was, however, significantly elevated among the wastewater treatment caulobacters, compared to those isolated from non-wastewater sources (Table 4). The one exception was FWC13, which was isolated from a municipal nature park, a source which may well be compromised by human and animal pollution. Plasmids were common among the isolates, in contrast to the general absence of plasmids noted by others for caulobacters from non-wastewater treatment sources (26). Most plasmids were large, estimated to be 50 kb or larger. Negative assignments must be regarded as tentative. We have found the plasmid detection method we used to be more reliable for large plasmids than other procedures, but it still occasionally fails to detect a known large plasmid. DISCUSSION Despite the general expectation that caulobacters would not be a significant component of high-organic-load wastewater treatment systems and the absence of reports to the contrary, we have found them to be readily detected in every system we examined. Indeed, we found them to be more readily isolated from wastewater than other water sources we have examined, to such an extent that a lengthy enrichment period in the absence of organics was unnecessary. This suggests that caulobacters may be a larger fraction of the total bacterial population in wastewater treatment systems than in more typical water and soil sources. It is perhaps not surprising, however, that caulobacters have not been reported previously in analyses of wastewater treatment systems. Direct examination of samples by microscopy rarely reveals a stalked bacterium among the clumps of bacterial cells usually seen in wastewater treatment samples. The typical caulobacter isolate has a relatively short stalk in phosphate-sufficient media (i.e., greater than 20 ,uM Pi [25, 32]), forms rosettes infrequently, and loses much of its motility and adhesiveness in colonies that are more than a few days old. When the typical isolates are evaluated by commercial schemes for bacterial identification, they either key to no species or give "poor"-to"acceptable" identifications for a variety of genera (not shown); caulobacters are not represented in the current schemes. Often, wastewater treatment samplings involve the use of saline solutions, presumably related to a desire to test for human pathogenic bacteria. Freshwater caulobacters generally do not grow in salinities greater than 50 to 100 mM. A relatively recent development in the wastewater treatment industry is the "biological" removal of phosphate in effluent (2, 15, 34). Phosphate is a key nutrient in the stimulation of eutrophication of water sources as a result of sewage discharge (15). The process involves control of the degree of aeration at certain stages of secondary treatment, resulting in the accumulation of phosphate into the bacterial population as polyphosphate (34). Since most of the bacterial
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Flagellin
757
Surface Array
FIG. 3. Colony hybridization of caulobacter isolates. Autoradiograms of colony lifts probed with the flagellin and surface array gene probes. For each autoradiogram: row 1, C. crescentus CB15A; row 2, FWC8, -9, -10, -11, -12, -13; row 3, FWC14, -15, -16, -17, -18, -19; row 4, FWC20, -21, -22, -23, -24, -25; row 5, FWC26, -27, -28, -29, -30, -31; row 6, FWC32, -33, -34, -35, -36, -37; row 7, FWC38, -39, -40, -41, -42, -43.
population becomes part of the sludge, effluent is rendered more free of phosphate and the sludge becomes more valuable as a fertilizer or soil amendment. The mechanism by which the polyphosphate accumulation occurs and the bacterial species in secondary treatment which accomplish this process are not well characterized, but essential characteristics seem to include the ability to store excess carbon as poly-p-hydroxybutyrate and similar alkanoate polymers, to store phosphate as polyphosphate, and to tolerate periods of reduced oxygen availability (2, 34). Acinetobacters have such characteristics and are often assumed to be a key genus involved with the biological phosphate removal process (11, 31); however, caulobacters have also been reported to be able to accumulate poly-phydroxybutyrate and polyphosphate (20, 21) and to grow in anaerobic conditions (1). We are presently evaluating whether the caulobacter strains isolated from wastewater treatment, and especially those isolated from treatment plants operating with biological phosphate removal processes, are active participants in the phosphate accumulation process, now that they are known to be present in the wastewater treatment environment. The apparent increase in resistance to antibiotics among the caulobacters, particularly those in common clinical use (e.g., tetracycline) or which are so-called second-generation antibiotics (e.g., erythromycin), is of concern. We presume that this pattern is the result of Caulobacters in wastewater treatment systems being in close proximity to antibioticresistant intestinal or otherwise human-associated bacteria for extended periods. The transfer of drug resistance determinants among bacteria is often assumed to be the result of interspecies conjugal plasmid transfer. Caulobacter strains have been shown to be quite capable of participating in such transfers (1, 6), and the high incidence of plasmids in the wastewater treatment strains may reflect that capability. In general, secondary wastewater treatment is considered to be a wise practice, resulting in the conversion of soluble organics and other nutrients, as well as toxic components, to a concentrated sludge (32). The sludge can be dealt with separately, and the effluent need not be treated as exten-
sively with disinfectants, such as chlorine compounds, leading to a more environmentally compatible effluent from treatment facilities. Yet secondary treatment systems may well increase the time and extent of contact between those discharged bacteria which carry drug resistance determinants, but persist only a short time in the environment, and resident bacteria, such as caulobacters. These bacteria are well adapted to persist in water supplies, and secondary treatment facilities may serve to facilitate the development of a stable reservoir of antibiotic resistance determinants within these bacteria, which persist in the environment and can in various ways be transferred back to human-associated bacteria. One effect, then, of secondary wastewater treatment may be a reduced lifetime for antibiotics used in clinical medicine. Most of the caulobacters demonstrated at least some homology with both the flagellin and S-layer gene probes. Most in the collection and, based on primary examination of the samples, a great majority of the caulobacters in wastewater treatment systems possess an S layer or, at least, hybridize significantly with the C. crescentus CB15 S-layer gene probe. The observation that some that do not exhibit a visible S layer but nevertheless contain DNA homologous to an S-layer gene may suggest that the S-layer genes are in some way important to most members of the caulobacter group. The absence of hybridization with both the flagellin and S-layer genes to a collection of random wastewater treatment isolates suggests that these probes could be used to develop an assay for direct assessment of the relative proportion of caulobacters among wastewater treatment bacteria. This approach avoids the problems associated with quantitation by standard microbiological techniques and is currently being evaluated in an attempt to more carefully define the role and extent of involvement of caulobacters in wastewater treatment practices. ACKNOWLEDGMENTS We thank the following individuals for assistance in wastewater treatment sample collection: Peggy Albertson, Penny Amy, John Barrett, Greg Bull, Larry Chow, Don Johnstone, Grace Nowak,
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Barry Rabinowitz, and Mark Watt. We acknowledge the technical assistance of Patti Edwards in several aspects, including the electron microscope examination of strains, and Mabel Wong, who assisted with the lectin labeling experiments and antibiotic sensitivity testing. We thank Angus Chu for providing some of the random isolates of sewage bacteria and for testing several caulobacter isolates in commercial bacterial identification schemes. We also thank William Oldham, Jeanne Poindexter, and William Ramey for discussions and helpful suggestions. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (OGP0036574), the U.S. Office of Naval Research (N00014-89-J-1749), and the U.S. National Institutes of Health (GM39055) to J.S. and by a Graduate Fellowship from NSERC to J.D.M. REFERENCES 1. Anast, N., and J. Smit. 1988. Isolation and characterization of marine caulobacters and assessment of their potential for genetic experimentation. Appl. Environ. Microbiol. 54:809-817. 2. Comeau, Y., K. J. Hall, R. E. W. Hancock, and W. K. Oldhain. 1986. Biochemical model for enhanced biological phosphorus removal. Water Res. 20:1511-1521. 3. Costerton, J. W., K.-J. Cheng, G. G. Geesey, T. I. Ladd, J. C. Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial films in nature and disease. Annu. Rev. Microbiol. 41:435-464. 4. Dias, F. F., and J. V. Bhat. 1964. Microbial ecology of activated sludge. Appl. Microbiol. 12:412-417. 5. Douglas, S. E., and N. Carr. 1988. Examination of genetie relatedness of marine Synechococcus spp. by using restriction fragment length polymorphisms. Appl. Environ. Microbiol. 54:3071-3078. 6. Ely, B. 1979. Transfer of drug resistance factors to the dimorphic bacterium Caulobacter crescentus. Genetics 91:371-380. 7. Fletcher, M. 1985. Effect of solid surfaces on the activity of attached bacteria, p. 339-362. In D. C. Savage and M. Fletcher (ed.), Bacterial adhesion. Plenum Publishing Corp., New York. 8. Fletcher, M. 1987. How do bacteria attach to surfaces? Miciobiol. Sci. 4:133-135. 9. Kado, C. I., and S. Liu. 1981. Rapid procedure for detectioni and isolation of large and small plasinids. J. Bacteriol. 145:13651373. 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
227:680-685. 11. Lotter, L. H., and M. Murphy. 1985. The identification of heterotrophic bacteria in an activated sludge plant with particular reference to polyphosphate accumulation. Water SA 11: 179-184. 12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 13. Markwell, M. A. K., S. M. Haas, L. L. Bieber, and N. E. Tolbert. 1978. A modification of the Lowry procedure to simplify protein determinations in membrane and lipoprotein samples. Anal. Biochem. 87:206-210. 14. Merker, R. I., and J. Smit. 1988. Characterization of the adhesive holdfast of marine and freshwater caulobacters. Appl. Environ. Microbiol. 54:2078-2085.
APPL. ENVIRON. MICROIBIOL. 15. Metcalf and Eddy, Inc. 1978. Wastewater enginieerinig: collection, treatment, disposal, and reuse, 2nd ed., p. 119-140 and 393-467. McGraw-Hill, New York. 16. Milhausen, M., and N. Agabian. 1983. Caulobacter flagelnll mRNA segregates asymmetrically at cell division. Nature (London) 302:630-632. 17. Mitchell, D., and J. Smit. 1990. Identification of the genes affecting production of the adhesion organelle of Caulobacter crescentus CB2. J. Bacteriol. 172:5425-5431. 18. Pike, E. B. 1976. Aerobic bacteria, p. 1-63. In C. R. Curds and H. A. Hawkes (ed.), Ecological aspects of used-water treatment, vol. 1. Academic Press, Inc. (London), Ltd., London. 19. Pike, E. B., E. G. Carrington, and P. A. Ashburner. 1972. An evaluation of procedures for enumeration of bacteria in acti vated sludge. Appi. Bacteriol. 35:309-321. 20. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. 21. Poindexter, J. S. 1981. Oligotrophy. Feast and famine existence. Adv. Microb. Ecol. 5:63-83. 22. Poindexter, J. S. 1981. The caulobacters: ubiquitous unusual bacteria. Microbiol. Rev. 45:123-179. 23. Poindexter, J. S. 1989. Geiius Caulobacter Henrici and Johnson 1935, 83AL, p. 192-1939. In J. T. Staley (ed.), Bergey's manual of systematic bacteriology, vol. 3. The Williams & Wilkins Co., Baltimore. 24. Prakasam, T. B. S., and N. C. Dondero. 1967. Aerobic heterotrophic bacterial populations of sewage and activated sludge. Appl. Microbiol. 15:1122-1127. 25. Schmidt, J. M., and R. Y. Stailer. 1966. I'he development of cellular stalks in bacteria. J. Cell Biol. 28:423-436. 26. Schoenlein, P. V., and B. Ely. 1983. Plasmids and bacteriocins in Caulobacter species. J. Bacteriol. 153:1092-1094. 27. Skinner, F. A., P. C. T. Jones, and J. E. Mollison. 1952. A comiparison of a direct- and plate-counting technique for the quantitative estimation of soil microbiology-organisms. J. Gen. Miciobiol. 6:261-271. 28. Siiiit, J., and N. Agabian. 1982. Cell surface patterniing and niorphogenesis: biogenesis of a periodic surface ari-ay during Caulobacter development. J. Cell Biol. 95:41-49. 29. Smit, J., and N. Agabian. 1984. Cloning of the major protein of the Caulobacter crescentus periodic surface layer: detection anid characterization of the cloned peptide by protein expression assays. J. Bacteriol. 160:1137-1145. 30. Smit, J., D. A. Grano, R. M. Glaeser, and N. Agabian. 1981. Periodic surface array in Caulobacter crescentus: fine structure and chemiiical analysis. J. Bacteriol. 146:1135-1150. 31. Stephenson, T. 1987. Acinetobacter: its role in biological phosphate removal, p. 313-316. In R. Ramadori (ed.), Biological phosphate removal from wastewaters. Pergamon Press, New York. 32. Walker, S. G., R. E. W. Hancock, and J. Smit. 1990. FEMS Microbiol. Lett., In press. 33. West, D. C., C. Lagenamr, and N. Agabian. 1976. Isolation and characterization of Caulobacter crescentus bacteriophage Cdi. J. Virol. 17:568-575. 34. Yeoman, S., T. Stephenson, J. N. Lester, and R. Perry. 1986. Biotechnology for phosphorus removal during wastewater treatment. Biotechnol. Adv. 4:13-26.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 751-758
0099-2240/91/030751-08$02.00/0 Copyright C) 1991, American Society for Microbiology
Characterization of Caulobacters Isolated from Wastewater Treatment Systems JEAN D. MACRAE AND JOHN SMIT*
Department of Microbiology, University of British Columbia, Vancouver, British Columbia V6T I W5, Canada Received 31 August 1990/Accepted 3 January 1991
Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.
The first goal of wastewater treatment systems is the removal of nutrients, particulates, pathogens, and toxic materials from the effluent over as short a time interval as practicable (15). In primary treatment, particulates are removed by gravity settlement, screening, flotation, skimming, and the like, but to accomplish significant reductions in the soluble organic carbon, nitrogen, and phosphate in wastewater, the engineering solutions usually involve the use of microbial populations. By various means, the development of microbial communities or consortia result in the conversion of soluble nutrients into insoluble aggregates that are removed from wastewater, producing sludge and cleaner effluent (15). The microbial populations that develop in these treatment systems are a complex expansion of indigenous soil and freshwater bacteria, with selection for those bacteria that can grow quickly and compete for nutrients in an environment that has much higher nutrient loadings than is typical of soil and water (18). In most cases, the consortium that develops produces a variety of adhesive exopolysaccharides and other substances that result in the production of thick biofilms or coarse, particulate aggregates of bacteria (3, 7, 8). Beyond such generalizations, however, it has been difficult to characterize the bacterial composition of wastewater treatment systems (24, 27). Direct counting and standard plate counting techniques are hampered by aggregates, and the former is also hampered by difficulties in distinguishing living and dead or moribund cells. Many of the bacteria present cannot be cultured, because of either undetermined nutritional requirements or the inability to grow as colonies
on solid media (19). Even so, a number of studies have been done and numerous genera have been identified within sludge-forming bacterial populations (4, 11, 18). Caulobacters are stalked bacteria that are members of biofouling populations by virtue of an adhesive holdfast anchored at the tip of their stalk, as well as at the base of the single polar flagellum during the motile, swarmer-cell phase of these dimorphic bacteria (14, 20). As far as we can determine, there has not been a single reported instance of caulobacters being isolated from a municipal wastewater treatment facility. Since they have long been known to be found in pristine freshwater supplies and are often contaminants in distilled water plumbing, it is sometimes presumed (though not necessarily by other caulobacter researchers) that caulobacters would not be widespread in mesotrophic or organically rich environments (21, 23). Work in our laboratory, however, had provided some indication that caulobacters might not be restricted to nutrient-poor environments. Marine caulobacters were readily isolated from regions receiving significant input of organic material (1). Growth of laboratory strains occurred readily and to high cell density in media containing relatively high concentrations of carbon, nitrogen, and phosphate. We also knew that in phosphate-sufficient environments, some strains of caulobacters do not produce the long stalks that are characteristic of the genus in phosphate-limited situations and so can be difficult to identify by light microscopy.
In an effort to learn more about the actual range of habitats for caulobacters in the environment, we examined the environment of wastewater treatment systems, where organic carbon, nitrogen, and phosphate, as well as toxic metals, are available in relative abundance.
* Corresponding author. 751
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TABLE 1. Sources of caulobacter isolates Wastewater treatment sources FWC8,-14,-19,-20
FWC9,-12 FWC11 FWC21 FWC15,-16,-18
FWC17 FWC22 FWC23,-24,-27-, -28,-31 FWC25 FWC26 FWC30 FWC29 FWC32,-33 FWC34 FWC35 FWC38 FWC37 FWC39,-40
FWC41,-42,-43 Non-wastewater treatment sources FWC1 FWC4,-5 FWC6,-7 FWC13 FWC45 a
Location (U.S. or Canada)
Type of facility
Strain
Secondary treatment, activated sludge process, Bio-P" Bench-scale secondary treatment system, Bio-P fed with landfill leachate Holding tank for raw influent sewage Bench-scale rotating biological contactor Primary treatment facility for greater Vancouver, British Columbia, area Secondary treatment facility, rotating biological contactor Secondary treatment facility, aerobic digestor Contour trench wastewater treatment system
University of British Columbia Department of Civil Engineering, test plant University of British Columbia Department of Civil Engineering University of British Columbia University of British Columbia lona Island, British Columbia
Langley, British Columbia Langley, British Columbia Takla, British Columbia
Secondary treatment facility, primary treatment region Secondary treatment facility, activated sludge Untreated influent sewage Secondary treatment facility, activated sludge system Trickle filter secondary treatment facility Secondary treatment facility, activated sludge system Trickle filter secondary treatment facility Secondary treatment facility, activated sludge system Secondary treatment facility, activated sludge system Secondary treatment facility, activated sludge system, Bio-P Secondary treatment facility, activated sludge system
Edmonton, Alberta, Gold Bar facility Edmonton, Alberta, Gold Bar facility Edmonton, Alberta, Gold Bar facility Bozeman, Mont. Coeur d'Alene, Idaho Pullman, Wash. Las Vegas, Nev.
Lake Washington Surface water in waterlogged area Tap water Surface water from a peat bog Stream water
Seattle, Wash. Bothell, Wash. Oakland, Calif. Richmond, British Columbia Burnaby, British Columbia
Las Vegas, Nev. Post Falls, Idaho
Kelowna, British Columbia Calgary, Alberta, Bonnybrook facility
Bio-P, Biological phosphate removal.
MATERIALS AND METHODS Sample sources and isolation techniques for caulobacters. Samples were taken from the various wastewater treatment facilities (Table 1) by simple liquid collection or, in the case of sampling trickling filters or contactors, by scraping bacterial growth from a surface and suspending in water. The samples were transported or mailed to the laboratory, diluted 1:1,000 in 0.01% peptone, and incubated aerobically at room temperature without shaking. After 3 to 7 days, a loopful of the liquid surface film (usually not a visible film) was diluted appropriately, plated onto a peptone-yeast extract (PYE) agar medium (20), and incubated at room temperature for several days. Colonies were individually examined by phase-contrast light microscopy. After standard purification procedures, isolates were grown in PYE liquid medium (supplemented with riboflavin at 2 ,ug/ml, if needed) and stored in 10% dimethyl sulfoxide at -70°C. A collection of 150 bacterial strains, judged not to be caulobacters, was prepared from wastewater treatment samples by dilution and plating, either on PYE or CGY (19) medium. The isolation of caulobacters from non-wastewater treatment sources was similar to that used for wastewater treatment sources, except for a longer enrichment period, and has been described (1, 20). Characterization of wastewater treatment caulobacters. In addition to gross characteristics, such as colony color, cell shape, and growth stimulation by riboflavin, a number of approaches were taken to define and distinguish individual strains of caulobacters.
(i) Total protein profiles. Late-logarithmic-phase cultures (5 ml) were washed by centrifugation and suspension with water, frozen, thawed and suspended in 250 ,u1 of 10 mM Tris-1 mM EDTA. Then, 1 ,ul of DNase (0.5 mg/ml), 20 ,ul of lysozyme (10 mg/ml), and 3 ,u1 of 1 M MgCl2 were added, and the mixture was incubated at room temperature for 10 to 15 min before being stored at -20°C. Protein concentrations were determined by a modified Lowry assay (13), and samples containing 40 ,ug of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10 to 17% gradient) using standard methods (10, 33). (ii) Western blot analysis. A portion of the isolates were examined by the Western blot (immunoblot) technique as previously described (29), using antibody prepared against the S layer of C. crescentus CB15 (28). (iii) Holdfast analysis. Variability in holdfast composition was judged by a fluorescein-coupled lectin binding assay as previously described (14). (iv) Antibiotic sensitivity. Difco Dispenso-discs were placed on PYE plates overlaid with soft agar containing an inoculum of log-phase cells. The plates were incubated at 30°C for 24 to 48 h, and the zones of inhibition were measured in millimeters. Scoring was based on the manufacturer's recommendations, except that the recommended medium was not used and "intermediate" values were scored as sensitive. For ampicillin, streptomycin, tetracycline, and chloramphenicol, the strains were also patched onto PYE plates prepared with various concentrations of the
CAULOBACTERS IN WASTEWATER TREATMENT
VOL. 57, 1991
-
~
I- :
^
753
I
FIG. 1. Phase-contrast microscopy of the air-water interface from a caulobacter isolation experiment (see Materials and Methods). The original inoculum was activated sludge from a secondary treatment system, modified to accomplish biological phosphate reduction from the effluent. The micrograph shows a region containing unusually high numbers of caulobacters. The long stalks are likely the result of limited levels of phosphate in the isolation scheme.
drugs and observed for growth following several days of incubation at room temperature. (v) Detection of plasmids. Strains were evaluated for the presence of plasmids by the method of Kado and Liu (9). (vi) Gene hybridization experiments. Colony hybridization was done by standard methods, using Gelman BioTrace RP filters (12). For restriction fragment length polymorphism (RFLP) analysis, chromosomal DNA was isolated as previously described (17); portions were digested with EcoRI, BamHI, and HindIII; and Southern blot hybridization was performed by standard methods (12). 32P-labeled probes were prepared by nick translation (12) of appropriate DNA fragments. One microgram of digested DNA was run per lane, and 3 x 10 cpm of probe was used in each hybridization. After hybridization, blots or lifts were washed at 55°C with 150 mM NaCI-10 mM EDTA-10 mM sodium phosphate, pH 7.4 (I x SSPE). This wash regimen corresponds to a moderate stringency, permitting up to approximately 30% base pair mismatch, assuming the caulobacters to have about 65% GC composition, as do characterized caulobacter strains (23). RFLP hybridization blots were exposed to autoradiographic film for 2 days and scored. The surface (S)-layer protein gene probe was prepared from a 4.4-kb HindIII-BamHI fragment containing the 130K gene of C. crescentus CB15 (29). Probe for the Caulobacter flagellin genes was prepared from the 0.9-kb SalI-EcoRI insert of
pCA161 (16), which hybridizes with all six of the flagellin genes of strain CB15. (vii) Electron microscopy. Examination for the presence of an organized paracrystalline S layer was done by suspending bacterial colonies in a small amount of water and preparing negative-stain grids with ammonium molybdate as previously described (30). Grids were examined in a Siemens 101A transmission electron microscope, operated at 60 kV. RESULTS Isolation of caulobacters. In general, caulobacters were readily isolated from virtually every type of municipal wastewater treatment system examined, ranging from passive contour trench systems to activated sludge or trickling filter systems to rotating biological contactors. The geographical and climatological locales ranged from the Nevada desert to northern British Columbia, Canada, inlands. Caulobacters could be detected at all points of treatment systems, except for the strongly anaerobic regions of anaerobic digestors used by many facilities to reduce sludge volume and generate methane gas. The procedure used to isolate caulobacters was similar to that used for isolating caulobacters from freshwater sources, which is based on the principle that caulobacters will become enriched, relative to other bacteria in the sample,
754
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MAcRAE AND SMIT
TABLE 2. Characteristics of caulobacters isolated from wastewater treatment facilities Strain
FWC9 FWC11 FWC12 FWC14 FWC15 FWC16 FWC17 FWC18 FWC19
FWC20 FWC21 FWC22 FWC23 FWC24 FWC25 FWC26 FWC27 FWC28 FWC29 FWC30 FWC31 FWC32 FWC33 FWC34 FWC35 FWC37 FWC38 FWC39 FWC40 FWC41 FWC42 FWC43
Cell
plasmids
S
layer
Colony color
shape
2 2 2 0 1 1 1 1 2 2 1 0 3 2 1 1 2 1 0 1 1 1 0 1 1 0 0 2 0 1 1 1
+ +
Tan Tan Tan White Tan Tan Tan Tan Tan Light yellow Tan Tan Yellow Tan Tan Tan Tan Tan Tan Red Tan Tan Tan Tan Tan Tan Bright yellow Tan Tan Tan Tan Bright yellow
Crescent Crescent Crescent Rod Crescent Crescent Crescent Crescent Crescent Crescent Rod Crescent Crescent Crescent Crescent Crescent Crescent Crescent Crescent Ovoid Crescent Crescent Crescent Crescent Crescent Crescent Rod Crescent Rod Crescent Crescent Rod
No. of
+
+ + + + + +
+
+ + + + +
+ + + + + +
+
+ +
-
when incubated for extended periods (1 to 2 months) in a medium with very low organic content. In the present situation, we routinely noted that caulobacters could be easily detected in surface films by phase-contrast microscopy in 1 week or less, obviating the need for any significant enrichment beyond the tendency of caulobacters to accumulate in the surface film (21, 23). The results suggested that caulobacters were abundant in most wastewater treatment samples as compared with other aquatic sources. Some samples had surprising levels of caulobacters. In a few instances, as many as 40% of the colonies seen on the initial PYE plates were caulobacters. Given the ambiguities regarding plating efficiency of wastewater treatment bacteria, it is not possible to equate such numbers to the actual proportion in the treatment systems, yet it seems fair to estimate that in these instances caulobacters were a significant fraction of the total. The use of the surface film as a source for isolating pure cultures, as compared with direct isolation from particulates, worked well, since the bacteria were much more dispersed and after brief mechanical disruption of a diluted sampling (with a vortex mixer), colonies containing a single species of bacteria were the most common occurrence. Also, the probable limitation of phosphate in the incubation medium encouraged long-stalk development and ready recognition of the caulobacters (Fig. 1). In contrast, stalked bacteria were seldom discernible by direct examination of the bacterial
Degree of rosette formation
Degree of hybridization with probes: S layer Flagellin
+++ + +++ + +++ + + +++ +++ + +++ + +++ + +++ ++ +++ + ++ +++ + +++ ++ + + ++ -+ + + +++ + +++ + +++ + +++ + +++ + + + +++ + -+++ +++ ++ +++ ++ + +++ +++ + ++ +++ + +++ ++ +++ ++ ++ +++
+++
++
+++ +++ +++ + +++ +++ +++ +++ +++
+++ ++ + +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+ +++ +++
+
aggregates that were typically present in most types of wastewater treatment samples. Characterization of caulobacter isolates. The results of most of the characterization studies are given in Tables 2 to 4. By far, most of the caulobacters seen and, subsequently, most of the preserved isolates had numerous characteristics that identified them as the typical caulobacter detected in wastewater treatment systems and much like the wellcharacterized C. crescentus strains. The cell bodies were crescent-shaped and produced relatively few rosettes in monoculture (a consequence of fused holdfasts of multiple cells), and, via negative-stain electron microscopy, nearly all showed the presence of a hexagonally packed, paracrystalline S layer. The S layers were visually indistinguishable from those produced by C. crescentus CB2 and CB15 (29, 30). Cells in colonies more than 5 days old showed virtually no rosettes, minimal adhesiveness to glass surfaces, minimal motility, and few visible stalks. In short, they were difficult to identify by textbook morphological characteristics as caulobacters yet were readily detected when one became familiar with what to expect. Although the typical caulobacters were visually similar, there were gross characteristics that distinguished them. There was considerable variety in holdfast composition, as judged by lectin-binding patterns, similar to that noted before for freshwater caulobacter strains (14). For many in
VOL. 57,
FWC14 FWC15 FWC16 FWC17 FWC18 FWC19 FWC20 FWC21 FWC22 FWC23 FWC24 FWC25 FWC26 FWC27 FWC28 FWC29 FWC30 FWC31 FWC32 FWC33 FWC34 FWC35 FWC37 FWC38 FWC39
FWC40 FWC41 FWC42 FWC43
DBA
Soybean
ConA
UEA1
WGA
Ricinus
-
-
-
-
+
-
-
-
-
-
-
-
+ +
-
+
+ + + + + + + + + + + +
-
-
+ + -
-
-
-
-
-
-
+
+
+ + -
+
+
+ +
-
+
+ +
-
+ +
-
Resistance to antibiotica
Strain
Result with lectina:
Strain
+ -
+
+
-
+
+
-
-
-
+
+ +
+ +
-
-
-
+
+
+
+ +
755
TABLE 4. Antibiotic resistance in caulobacter isolates
TABLE 3. Assessing caulobacter holdfast composition by lectin binding
FWC8 FWC9 FWC11 FWC12
_s
CAULOBACTERS IN WASTEWATER TREATMENT
1991
+ + -
+
+
a The assay was performed as previously described (14) with fluorescein isothiocyanate lectins. DBA, Dolichos biflorus agglutinin; Soybean, soybean agglutinin; ConA, concanavalin A; UEA1, Ulex europaeus agglutinin 1; WGA, wheat germ agglutinin; Ricinus, Ricinus communis agglutinin. Peanut agglutinin was also examined but gave negative results in every case.
the typical group, growth on the initial PYE solid medium occurred readily, but they subsequently grew slowly or unpredictably in PYE liquid medium. Normal growth rates were restored in most of these cases upon the addition of riboflavin. On the basis of current classification schemes for caulobacters, this would categorize them as C. vibrioides, the type strain for the Caulobacter group (23). Occasionally, we detected a caulobacter that to varying degrees did not follow the typical pattern. FWC14, -21, -30, -38, -40, and -43 are in that class. They had longer, more visible stalks and formed long, thin rod or ovoid cell bodies. They produced rosettes in monoculture more readily and lacked a visible S layer. To minimize duplication of strains, we did not preserve more than one typical caulobacter from a particular source, even though numerous typical caulobacter colonies might be noted, yet we always kept an atypical one. Thus, the atypical isolates are significantly overrepresented in the collection of 33 strains, as compared with their abundance in the samples we examined. Protein band profiles obtained by SDS-PAGE were a major criterion for distinguishing strains of caulobacters (Fig. 2). Unless significant differences were seen in other tests, strains with similar protein band profiles were consid-
Wastewater sources FWC8 FWC9
Cm
Tet
Em
Tm
r
r r r
r r r
r
s
s
r
r r
r r
s s
s
FWC11
r
FWC12 FWC14 FWC15 FWC16 FWC17 FWC18
s s
r
FWC19
FWC20 FWC21 FWC22 FWC23 FWC24 FWC25 FWC26 FWC27 FWC28 FWC29 FWC30 FWC31 FWC32 FWC33 FWC34 FWC35 FWC37 FWC38 FWC39 FWC40 FWC41 FWC42 FWC43 Non-wastewater sources C. crescentus CB15A C. crescentus CB2A C. vibrioides C. bacteroides FWC1 FWC4
FWC5
FWC6 FWC7 FWC13 FWC45
,' I
eIA
s s
s
s
s
r
r r r r r r
r r r
r r r r r r r r
s s
r
r r r r
r
S S
S S
S S
S S
s s
r r
r s
r
r r r
r
s s s
s s s s s s s
s
s r r s
r
s
s
r r s s
s
Aps Nal'
s s s s s s s s s s s s
s
Other(s)b
r r r
Smr
s s
s
r r
r
s
s
S S S
S S S
S S S
S S S
Aps Pens
s s s s s s s s s r s
s s s s s s s s s
s s r s s s s s
s
Pbs
r
r s
r r s
r
s s s s s s s s s
Smr Nals
Aps Pens Pbs Ap5 Pen' Pb5
a Abbreviations: r, resistant; s, sensitive; Cm, chloramphenicol; Tet, tetracycline; Em, erythromycin; Tm, tobramycin; Ap, ampicillin; Pen, penicillin; Sm, streptomycin; Pb, polymyxin B; Nal, nalidixic acid. b Except as noted, most isolates were resistant to ampicillin, penicillin, polymyxin B, and nalidixic acid and were sensitive to streptomycin.
ered to be the same. This resulted in dropping 2 strains from the original collection of 35. Despite the similarity in appearance of the S layers, antibody prepared against the C. crescentus CB15 S layer protein reacted with proteins of size and abundance similar to those of the C. crescentus CB15A S-layer protein in Western blot analysis in only a few of the wastewater treatment isolates (not shown). Colony lift hybridization (Fig. 3) and RFLP analysis with the S-layer gene probe under moderate stringency conditions gave a positive signal for nearly all caulobacter strains and one and occasionally two distinct bands on the Southern
756
MAcRAE AND SMIT
mw8 9 11 12 15 16 17 18 19 20 22 23 24 25
FIG. 2. SDS-PAGE of total proteins from caulobacter isolates. The numbers represent FWC strains (i.e., 8 = FWC8). 103 Molecular weight standards (mw) rabbit muscle phosphorylase, 97.4; bovine serum albumin, 66.2; hen egg white albumin, 42.7; bovine carbonic anhydrase, 31; soybean trypsin inhibitor, 21.5; hen egg white lysozyme, 14.4.
blots with all three enzymes (not shown). C. crescentus CB15, the source of the S-layer gene probe, gave a single band for the three enzymes. However, the strength of the
signal was faint or absent with all of the atypical strains (FWC14, -21, -30, -38, -40, and -43) and FWC23. These strains also had no other indication of an S layer. Highstringency washes (650C, O.1x SSPE) eliminated the signal for most of the caulobacters, indicating that the S-layer gene sequence was not highly conserved among the caulobacters, as compared with the C. crescentus CB15 gene used as a probe. The flagellin gene probe gave similar results, except that typically 2 to 3 bands were produced. Virtually no patterns of close relatedness among the strains, based on RFLP analysis, were apparent. That is, there were few instances of similarly migrating bands that
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could be used to define subset groups of strains, as is often possible with closely related bacterial strains (5). Colony hybridization was also done with 150 randomly selected "non-caulobacter" wastewater treatment bacterial strains. None hybridized with the S-layer gene probe, and a single positive score was obtained with the flagellin gene probe. Most strains, both from wastewater and other sources, were resistant to ,-lactam antibiotics (ampicillin and penicillin), nalidixic acid, and (like many gram-negative bacteria) polymyxin B and were sensitive to streptomycin. Resistance to chloramphenicol, tetracycline, erythromycin, and (to a lesser extent) tobramycin was, however, significantly elevated among the wastewater treatment caulobacters, compared to those isolated from non-wastewater sources (Table 4). The one exception was FWC13, which was isolated from a municipal nature park, a source which may well be compromised by human and animal pollution. Plasmids were common among the isolates, in contrast to the general absence of plasmids noted by others for caulobacters from non-wastewater treatment sources (26). Most plasmids were large, estimated to be 50 kb or larger. Negative assignments must be regarded as tentative. We have found the plasmid detection method we used to be more reliable for large plasmids than other procedures, but it still occasionally fails to detect a known large plasmid. DISCUSSION Despite the general expectation that caulobacters would not be a significant component of high-organic-load wastewater treatment systems and the absence of reports to the contrary, we have found them to be readily detected in every system we examined. Indeed, we found them to be more readily isolated from wastewater than other water sources we have examined, to such an extent that a lengthy enrichment period in the absence of organics was unnecessary. This suggests that caulobacters may be a larger fraction of the total bacterial population in wastewater treatment systems than in more typical water and soil sources. It is perhaps not surprising, however, that caulobacters have not been reported previously in analyses of wastewater treatment systems. Direct examination of samples by microscopy rarely reveals a stalked bacterium among the clumps of bacterial cells usually seen in wastewater treatment samples. The typical caulobacter isolate has a relatively short stalk in phosphate-sufficient media (i.e., greater than 20 ,uM Pi [25, 32]), forms rosettes infrequently, and loses much of its motility and adhesiveness in colonies that are more than a few days old. When the typical isolates are evaluated by commercial schemes for bacterial identification, they either key to no species or give "poor"-to"acceptable" identifications for a variety of genera (not shown); caulobacters are not represented in the current schemes. Often, wastewater treatment samplings involve the use of saline solutions, presumably related to a desire to test for human pathogenic bacteria. Freshwater caulobacters generally do not grow in salinities greater than 50 to 100 mM. A relatively recent development in the wastewater treatment industry is the "biological" removal of phosphate in effluent (2, 15, 34). Phosphate is a key nutrient in the stimulation of eutrophication of water sources as a result of sewage discharge (15). The process involves control of the degree of aeration at certain stages of secondary treatment, resulting in the accumulation of phosphate into the bacterial population as polyphosphate (34). Since most of the bacterial
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FIG. 3. Colony hybridization of caulobacter isolates. Autoradiograms of colony lifts probed with the flagellin and surface array gene probes. For each autoradiogram: row 1, C. crescentus CB15A; row 2, FWC8, -9, -10, -11, -12, -13; row 3, FWC14, -15, -16, -17, -18, -19; row 4, FWC20, -21, -22, -23, -24, -25; row 5, FWC26, -27, -28, -29, -30, -31; row 6, FWC32, -33, -34, -35, -36, -37; row 7, FWC38, -39, -40, -41, -42, -43.
population becomes part of the sludge, effluent is rendered more free of phosphate and the sludge becomes more valuable as a fertilizer or soil amendment. The mechanism by which the polyphosphate accumulation occurs and the bacterial species in secondary treatment which accomplish this process are not well characterized, but essential characteristics seem to include the ability to store excess carbon as poly-p-hydroxybutyrate and similar alkanoate polymers, to store phosphate as polyphosphate, and to tolerate periods of reduced oxygen availability (2, 34). Acinetobacters have such characteristics and are often assumed to be a key genus involved with the biological phosphate removal process (11, 31); however, caulobacters have also been reported to be able to accumulate poly-phydroxybutyrate and polyphosphate (20, 21) and to grow in anaerobic conditions (1). We are presently evaluating whether the caulobacter strains isolated from wastewater treatment, and especially those isolated from treatment plants operating with biological phosphate removal processes, are active participants in the phosphate accumulation process, now that they are known to be present in the wastewater treatment environment. The apparent increase in resistance to antibiotics among the caulobacters, particularly those in common clinical use (e.g., tetracycline) or which are so-called second-generation antibiotics (e.g., erythromycin), is of concern. We presume that this pattern is the result of Caulobacters in wastewater treatment systems being in close proximity to antibioticresistant intestinal or otherwise human-associated bacteria for extended periods. The transfer of drug resistance determinants among bacteria is often assumed to be the result of interspecies conjugal plasmid transfer. Caulobacter strains have been shown to be quite capable of participating in such transfers (1, 6), and the high incidence of plasmids in the wastewater treatment strains may reflect that capability. In general, secondary wastewater treatment is considered to be a wise practice, resulting in the conversion of soluble organics and other nutrients, as well as toxic components, to a concentrated sludge (32). The sludge can be dealt with separately, and the effluent need not be treated as exten-
sively with disinfectants, such as chlorine compounds, leading to a more environmentally compatible effluent from treatment facilities. Yet secondary treatment systems may well increase the time and extent of contact between those discharged bacteria which carry drug resistance determinants, but persist only a short time in the environment, and resident bacteria, such as caulobacters. These bacteria are well adapted to persist in water supplies, and secondary treatment facilities may serve to facilitate the development of a stable reservoir of antibiotic resistance determinants within these bacteria, which persist in the environment and can in various ways be transferred back to human-associated bacteria. One effect, then, of secondary wastewater treatment may be a reduced lifetime for antibiotics used in clinical medicine. Most of the caulobacters demonstrated at least some homology with both the flagellin and S-layer gene probes. Most in the collection and, based on primary examination of the samples, a great majority of the caulobacters in wastewater treatment systems possess an S layer or, at least, hybridize significantly with the C. crescentus CB15 S-layer gene probe. The observation that some that do not exhibit a visible S layer but nevertheless contain DNA homologous to an S-layer gene may suggest that the S-layer genes are in some way important to most members of the caulobacter group. The absence of hybridization with both the flagellin and S-layer genes to a collection of random wastewater treatment isolates suggests that these probes could be used to develop an assay for direct assessment of the relative proportion of caulobacters among wastewater treatment bacteria. This approach avoids the problems associated with quantitation by standard microbiological techniques and is currently being evaluated in an attempt to more carefully define the role and extent of involvement of caulobacters in wastewater treatment practices. ACKNOWLEDGMENTS We thank the following individuals for assistance in wastewater treatment sample collection: Peggy Albertson, Penny Amy, John Barrett, Greg Bull, Larry Chow, Don Johnstone, Grace Nowak,
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Barry Rabinowitz, and Mark Watt. We acknowledge the technical assistance of Patti Edwards in several aspects, including the electron microscope examination of strains, and Mabel Wong, who assisted with the lectin labeling experiments and antibiotic sensitivity testing. We thank Angus Chu for providing some of the random isolates of sewage bacteria and for testing several caulobacter isolates in commercial bacterial identification schemes. We also thank William Oldham, Jeanne Poindexter, and William Ramey for discussions and helpful suggestions. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (OGP0036574), the U.S. Office of Naval Research (N00014-89-J-1749), and the U.S. National Institutes of Health (GM39055) to J.S. and by a Graduate Fellowship from NSERC to J.D.M. REFERENCES 1. Anast, N., and J. Smit. 1988. Isolation and characterization of marine caulobacters and assessment of their potential for genetic experimentation. Appl. Environ. Microbiol. 54:809-817. 2. Comeau, Y., K. J. Hall, R. E. W. Hancock, and W. K. Oldhain. 1986. Biochemical model for enhanced biological phosphorus removal. Water Res. 20:1511-1521. 3. Costerton, J. W., K.-J. Cheng, G. G. Geesey, T. I. Ladd, J. C. Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial films in nature and disease. Annu. Rev. Microbiol. 41:435-464. 4. Dias, F. F., and J. V. Bhat. 1964. Microbial ecology of activated sludge. Appl. Microbiol. 12:412-417. 5. Douglas, S. E., and N. Carr. 1988. Examination of genetie relatedness of marine Synechococcus spp. by using restriction fragment length polymorphisms. Appl. Environ. Microbiol. 54:3071-3078. 6. Ely, B. 1979. Transfer of drug resistance factors to the dimorphic bacterium Caulobacter crescentus. Genetics 91:371-380. 7. Fletcher, M. 1985. Effect of solid surfaces on the activity of attached bacteria, p. 339-362. In D. C. Savage and M. Fletcher (ed.), Bacterial adhesion. Plenum Publishing Corp., New York. 8. Fletcher, M. 1987. How do bacteria attach to surfaces? Miciobiol. Sci. 4:133-135. 9. Kado, C. I., and S. Liu. 1981. Rapid procedure for detectioni and isolation of large and small plasinids. J. Bacteriol. 145:13651373. 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
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