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NEW TECHNIQUES FOR ANALYSIS OF MICROBIAL GENE TRANSFER WITHIN A RHABDITIS SOIL NEMATODE.

ABSTRACT. Using small samples and a tedious, time consuming method, we previously demonstrated that soil nematodes of the genus Rhabditis ingested bacteria: digesting some; concealing and excreting others in viableform; allowing bacterial genetic interactions; and releasing transconjugants. New techniques were developed including a phenol red dextrose broth-metabolic inhibition test (PRDB/ MIT) that enable large-scale and rapid screening for transconjugants. Milliliter samples were obtained and processed using microfuge tubes. Screening for bacteria was accomplished by exposing populations of surface sterilized nematodes to multi-selective media. Transconjugant presence was demonstrated by culture turbidity and medium pH indicator color change under selective conditions. Internalized hybrids from individual nematodes were also detected by tracks of growth left on agar surfaces containing several selective components, a result of inoculation by nematode defecation. The new method enables rapid screening for t ransfer of genetic characteristics from an introduced bacterial population and may be an important screen for gene transfer from environmentally released genetically engineered bacteria into native bacterial populations.

KEY WORDS: Nematodes; environmental microbiology; microbial interactions; gene transfer

INTRODUCTION

Nematode species of the genus Rhabditis have been reported worldwide in many different kinds of soils. Rhabditis has been extracted from beach sand, an area of very low nutrients (Adamo, unpublished), as well as water supplies (Roman and Rivas, 1972), water from treatment plants (Abrams and Mitchell, 1978; Mott et al., 1981), compost, dung, on various insects, in earthworms (Thorne, 1961), in or on plants (Ferris et al., 1972; Wharton, 1986), birds (Schmidt, 1973), mammals (Monrad, 1985) and humans (Cheng, 1968; Beaver and Jung, 1985). In 1960 Chang and others reported that two Rhabditidae nematodes, Diplogaster nudicapitatus and Cheilobus quadrilabiatus were able to carry Salmonella and Shigella through a water treatment system, releasing these pathogens, as viable bacteria, in drinking water.

Rhabditis is an excellent laboratory model to study internal gene transfer because (a) these microbial worms are bacterial feeders which vector viable bacteria (Chang et al., 1960; Adamo et al., 1993), (b) this nematode is globally available and (c) this organism ingests a wide range of bacteria. In our laboratory alone we have successfully propagated Rhabditis on Escherichia coli (eight different genetic strains), Acetabacter aceti, Serratia marcessens, Enterobacter aerogenes, Sarcina luten, Bacillus cereus, B. megaterium, B. subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Several of these microbes are used in DNA recombinant studies. Our initial interest was to determine if genetically engineered microorganisms (GEMs) deliberately or accidentally released into an environment would transfer genes to native bacterial populations while within the Rhabditis. The bacterial genetic interactions would take place by transformation, conjugation or transduction within the digestive tracts of these nemato des. These sequestered sites could protect the GEM and, therefore, facilitate the transfer of recombinant DNA sequences from the GEMs to indigenous bacteria. If recombinant bacteria were protected from various treatment processes while inside the nematodes any gene transfer to native species that occurred within the nematode would lead to the general release of genetically engineered DNA sequences in native bacteria.

Early studies (Adamo et al., 1993; Adamo and Gealt, 1996b) showed that E. coli on the smooth surfaces of pins were killed with 1% bleach after a 1 minute exposure. A 2 minute exposure with 2% bleach was necessary on rough surfaces for complete killing of the bacteria. The 2% bleach-2 min exposure treatment had no effect on survival of either the nematodes or the bacteria contained within the nematodes. Dead nematodes acted essentially like a fomite, i.e., pin, allowing no protection from chlorine bleach treatment. Live nematodes were still active in 2% chlorine bleach after 4 h of exposure, exhibiting no abnormal behavior or change in reproductive capabilities. Living bleach-treated nematodes excreted protected, ingested bacteria as wastes that could be recovered on selective media. The bleach merely sterilized the surface of the nematode. In another study, we showed that a feeding time of less than one hour was required to internalize and protect viable bacteria (Stadnick et al., 1993).

Using a calibrated microscope and photomicrographs, the major physical characteristics of the microbes were assessed (Adamo and Gealt, 1995; 1996a). The measurements included the volume of the bacterium and that of the intestinal tract of the nematode so that an accurate estimate of the maximal carrying capacity of an adult male worm could be calculated (1.2 x [10.sup.6] bacteria per nematode). Surface sterilization with bleach was used on individual nematodes, which were then dissected in 10[micro]L of sterile water using a dental root canal file and an optical surgeons knife. This technique allowed bacteria to be extruded and serial dilutions of the bacteria to be prepared in microfuge tubes. The bacteria were then plated out on selective media. Resulting colony counts indicated that about 27% of the bacteria estimated to be carried by each nematode were viable, i.e., an average of 3.2 x [10.sup.5] bacteria per nematode.

These data clearly suggested the great potential for gene exchange within the nematode. Not only was the viable number of bacteria high, the population was also within a very confined space. Thus, the nematodes gathered bacteria, protected, concealed and concentrated them. These conditions suggested a high probability of interaction events and, indeed, conjugation was detected within the intestinal tract of Rhabditis and transconjugants were identified using various selective media (Adamo and Gealt, 1994; 1996a; Leighton et al., 1995). MacConkey and eosin methylene blue (EMB) were used to differentiate E. coli phenotypes based to their ability to utilize lactose ([Lac.sup.+]/[Lac.sup.-]). These and other media were also supplemented with various antibiotic combinations to detect genetically marked parents and transconjugants (Adamo and Gealt, 1996a).

The nematode manipulation techniques used in the earlier published work required handling individual nematodes using a dental root canal file. This was tedious and time consuming. The purpose of the current study was to develop a new technique that would allow relatively large nematode samples to be rapidly screened for internally formed bacterial transconjugants.

MATERIALS AND METHODS

Nematodes

Nematodes extracted from soil samples taken from Toms River and Island Heights, NJ as previously described using a combination of sieving and Bauermann funnel filtration were purged, surface sterilized and maintained on pure cultures of E. coli (Adamo and Gealt, 1996b). Nematodes were examined microscopically, measured, and individuals of the genus Rhabditis were selected for further study. The progeny of an individual line of the isolated nematode were maintained and used in all experiments.

Media

All bacterial strains were maintained on Nutrient Agar plates or in Nutrient Broth (Difco). Lactose utilization was verified on EMB agar or MacConkey agar (Difco) with lactose as the carbon source. Phenol red dextrose broth (PRDB, Difco), was used for all metabolic inhibition tests. Rhabditis were maintained on bacteria grown on nutrient agar. Antibiotics were used at the following concentrations: nalidixic acid (nal) at 20 [micro]g/ml, ampicillin (amp) at 50 [micro]g/ml and streptomycin (str) at 25 [micro]/ml.

Bacteria [1]

In all cases the nematodes were grown on a pure culture of E. coli B ([lack.sup.+], [nal.sup.S], [str.sup.S], [amp.sup.S]), except for sequential feeding experiments in which E. coli [chi]1997 ([lac.sup.-], [nal.sup.R], [str.sup.S], [amp.sup.S]) and E. coli MJ100 ([lack.sup.+], [nal.sup.S]; plasmid R100JA [[str.sup.R], [amp.sup.R]]) were used (Adamo and Gealt, 1996a). All cultures were incubated for 24 h at 37[degrees]C.

Microfuge tubes as culture tubes

Experiments were designed to determine the effectiveness of microfuge tubes (1.5 mL) as culture tubes. Thirty microfuge tubes were loaded with 1 mL of sterile nutrient broth. Five control tubes were left open, the lids of the tube left ajar. Another five control tubes were closed, the lids snapped shut. All control tubes were incubated at room temperature. Five more control tubes were left open and five were closed and these ten were incubated at 37[degrees] C. The final ten tubes were inoculated with 0.1 mL E. coli B, of which five tubes left open and five closed. These last ten tubes were incubated at 37[degrees] C. All tubes were observed after 24 h for bacterial growth as measured by turbidity and for formation of a pill, i.e., sediment, after being centrifuged for 5 mm at 14,000 RPM.

Metabolic inhibition test (MIT)

Phenol red dextrose broth (PRDB) was tested as a potential metabolic inhibition test (MIT) and selective medium, when supplemented with antibiotics to differentiate bacterial phenotypes. Twenty microfuge tubes were each loaded with 1 mL of PRDB. Five of these were supplemented with streptomycin at a dosage of 25 [micro]g/mL, and were inoculated with 0.1 mL E. coli MJ100. Another set of five tubes supplemented with nalidixic acid at a dosage of 20 [micro]g/mL was inoculated with 0.1 mL E. coli [chi]1997. The last 10 PRDB tubes were not supplemented with any antibiotic. Five of these were inoculated with 0.1 mL E. coli MJ100 and the other five with 0.1 mL E. coli [chi]1997. All tubes were incubated at 37[degrees] C and observed after 24 h for a color change in the medium, indicative of growth. Replicated plate counts were prepared to determine the colony forming units (CFU)/mL of each of the above treatments.

Bacterial conjugation study

PRDB was use in a MIT to detect transconjugants. Microfuge tubes were loaded with 1 mL of sterile PRDB supplemented with an antibiotic and inoculated with 0.1 mL of bacteria. Two tubes of PRDB supplemented with streptomycin were inoculated with bacteria, one with E. coli [chi]1997 and the other with E. coli MJ100. Two tubes of PRDB supplemented with nalidixic acid were inoculated, one with E. coli x1997 and the other with E. coli MJ100. Another two tubes of PRDB, this time supplemented with ampicillin were inoculated, one with the E. coli [chi]1997 strain and the other with E. coli MJ100. All cultures were incubated at 37[degrees] C for 24 h and observed for color change indicative of bacterial growth.

A microfuge tube was loaded with 1 mL of sterile nutrient broth and inoculated with 0.1 mL of E. coli 1997, 0.1 mL of E. coli MJ100 and incubated at 37[degrees] C. The mixed culture was used as an inoculum for three microfuge tubes of PRDB supplemented with nalidixic acid and streptomycin. One of the tubes was inoculated after a two-hour incubation of the mixed culture. The other two tubes were inoculated after 4 and 6 h of incubation. All three tubes were incubated at 37[degrees] C for 24 hours and observed for transconjugants.

Bacterial conjugation of internal nematode origin

The surface of an established Rhabditis culture (7 day old) growing on E. coli [chi]1997 in a culture dish was washed with 5 mL of an 18 h broth culture of E. coli [chi]1997 and allowed to stand for 30 min. Four 1-mL samples were taken from the washings, containing nematodes, and each was loaded into a 1.5 mL microfuge tube. The nematodes in each sample were then subjected to the previously established 2% chlorine bleach - 2 min treatment, which surface sterilized the nematodes while internalized bacteria were protected (Adamo et al., 1993).

The new, rapid, technique for this treatment is shown in Fig. 1. Each 1 mL microfuge sample was centrifuged for 2 min at 14,000 RPM. The supernatant was decanted, 1 mL of 2% chlorine beach added and mixed in each nematode sample. After a 2 min treatment the samples were centrifuged again for 2 min at 14,000 RPM. The bleach supernatant was removed and replaced with 1 mL of sterile water. The nematodes were washed and centrifuged as before.

The wash water was then decanted and the surface sterilized nematodes resuspended in 1mL of a nutrient broth culture of E. coli MJ100. The Rhabditis/E. coli cultures were then added to an 18 h spread-plate of E. coli MJ100 and allowed to feed for 1,2,4 or 6 hours. Extra broth culture of E. coli MJ100 was added to the culture plate to compensate for absorption. After feeding on the E. coli MJ100 for 1 h, 1 mL of nematode/bacteria suspension removed from the plate was loaded into a microfuge tube and centrifuged as before. The supernatant was removed, replaced with 2% chlorine bleach and mixed for 2 min. The sample was centrifuged as before and washed after removal of the bleach. The wash consisted of PRDB supplemented with nalidixic acid and ampicillin. After removing the wash by centrifuging, the surface-sterilized nematodes were resuspended in 1 mL of fresh sterile PRDB supplemented with nalidixic acid and ampicillin. The culture was then incubated at 37[degrees]C for 24 h, allowing any viable, internal tra nsconjugant bacteria to grow. The feeding nematodes were also removed and subjected to the same surface sterilization procedure after 2, 4 and 6 hours. Surface sterilized nematodes processed as described above were also transferred to sterile EMB and MacConkey plates supplemented with nalidixic acid and ampicillin to confirm that recovered bacteria were E. coli of the proper phenotype.

RESULTS AND DISCUSSION

Microfuge tubes as culture tubes

The results of experiments designed to determine the effectiveness of microfuge tubes as culture tubes, utilizing E. coli B as a model bacterium, are shown in Table I. No visual turbidity was observed in uninoculated sterile 1 mL nutrient broth culture tubes incubated at 37[degrees]C or room temperature nor were any sediments (pills) observed after the tubes were centrifuged at 14,000 RPM for 5 mm. These results were the same when tubes were closed or when left open. In those cases where the tubes were inoculated with E. coli B and incubated at 37[degrees]C visual turbidity and sediment (pill) were observed in all samples whether the tubes were closed or left open. The results indicated that microfuge tubes would make effective culture tubes and the technique did not required tube closure to be effective and maintain sufficient sterility. Indeed, an open tube worked equally as well as a closed one for these experiments. Thus the techniques utilized were appropriate for this study.

Metabolic inhibition test (MIT)

In earlier studies (Adamo, 1996) PRDB was used in a MIT to determine the titer of bacteriophage. In this study PRDB was supplemented with antibiotics to determine usefulness as a metabolic inhibition test in differentiating bacterial phenotypes. Table II shows the results of tests conducted with E. coli [chi]1997 and E. coli MJ100 grown in PRDB and PRDB supplemented with streptomycin or nalidixic acid. When PRDB was utilized without any antibiotic both genetic strains of E. coli showed growth and the media changed from a red to yellow color. When streptomycin was added to PRDB, the [chi]1997 ([str.sup.S]) did not grow and the medium remained red, while MJ100 ([str.sup.R]) showed growth and the medium turned yellow. When nalidixic acid was the supplement [chi]l997 ([nal.sup.R]) was not inhibited, so growth and color change were observed. The medium remained red for MJ100 ([nal.sup.S]) in nalidixic acid, indicating metabolic inhibition. In those cases that lacked metabolic inhibition color change, indicative of growth, was evident within 4 hours. Overall the results clearly suggested that PRDB supplemented with antibiotic was an excellent metabolic inhibition test system to rapidly differentiate certain genetic strains of E. coli.

Bacterial conjugation study

The results of experiments designed to detect transconjugants using PRDB and antibiotic supplements are shown in Table III. The data indicate that E. coli [chi]1997 was susceptible to streptomycin and ampicillin, while resistant to nalidixic acid. Further, E. coli MJ100 was susceptible to nalidixic acid and resistant to both streptomycin and ampicillin. When the two bacterial cultures were mixed and tested in PRDB supplemented with nalidixic acid and streptomycin transconjugants were detected 2, 4 and 6 hours after mixing. Thus, the E. coli MJ100 was capable of transferring its plasmid to the E. coli [chi]1997 under these "test tube" conditions.

Bacterial conjugation of internal nematode origin

The results of new techniques developed to detect transconjugants of internal nematode origin are shown in Table IV. Transconjugants were detected, from nematodes initially grown on E. coli x1997, utilizing PRDB supplemented with nalidixic acid and ampicillin 4 and 6 hours after feeding on the MJ100 strain of E. coli. The nematodes were surface sterilized between feedings and prior to being subjected to the metabolic inhibition test media containing a combination of both antibiotics, each of which was inhibitory to one of the parent bacterial strains. The results clearly showed transconjugants of internal nematode origin. The washes and surface sterilizations eliminated any external source of transconjugants. As a further indicator that the transconjugants were of internal origin, nematodes were transferred onto solid media. The nematodes left tracks, visible under the dissecting microscope, on both EMB and MacConkey agar media (data not shown). These tracks were the defecation trail of the nematode as it mov ed across the agar surface. The transconjugants' inability to use lactose was also evident. Overall these new techniques were much less time consuming and tedious allowing for massive rapid screening for transconjugants.

It is also important to note that these experiments yielded, for the first time, a definite indication of the time delay required for the movement of a specific bacterium through the gut of the nematode.

When the [chi]1997 and MJ100 strains of E. coli are mixed directly in the culture tube, transconjugants are observed in as little as 2 h (and might be apparent even earlier if more frequent time points had been taken). On the other hand, no transconjugants were observed until 4 h when conjugation takes place within the nematode vector. This indicates that there was at least a 2-h delay in the appearance of transconjugants. This delay represents the time from the ingestion of the plasmid donor (MJ100) by the nematode to the time defecation of the transconjugant occurs. This technique now represents a rapid screen for bacterial gene transfer within Rhabditis, a eukaryote of significant academic and industrial interest.

ACKNOWLEDGMENT

We would like to thank Steve Scribellito for his helpful graphic suggestions and assistance.

(1.)Abbreviations used: lac for lactose utilization, str for streptomycin resistance, nal for nalidixic acid resistance, amp for ampicillin resistance; S = sensitive; R = resistant; + = utilizer (as in [lac.sup.+]) and - = nonutilizer

LITERATURE CITED

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ADAMO, J. A. 1996. MIT titration of bacteriophage. ASM Focus, Am. Soc. Micro. 3(1): 7-8.

ADAMO, J. A., AND M. A. GEALT. 1996a. A demonstration of bacterial conjugation within the alimentary canal of Rhabditis nematodes. FEMS, Micro. Eco. 20:15-22.

_____. and _____. 1996b. Vectors and Fomites. The Am. Bio. Teach. 58(8):484-489.

_____. and _____. 1995. Bacterial viability in a soil nematode - A necessary precursor to the study of internalized bacterial gene transfer. Abst. Ann. Meet. Am. Soc. Micro. Washington, DC: 333.

_____. and _____. 1994. Bacterial gene transfer in nematodes. Abst. Ann. Meet. Am. Soc. Micro. Las Vegas, NV: 344.

ADAMO, J. A., STADNICK, C. M. AND M. A. GEALT. 1993. A nematode model system for the study of gene transfer in soil organisms. Abst. Ann. Meet. Am. Soc. Micro. Atlanta, GA: 385.

BEAVER, P. C. AND JUNG, R. C. 1985. Animal Agents and Vectors of Human Disease. Lea and Febiger, Philadelphia, PA. 5th Ed. 281 pp.

CHANG, S. L., BERG, G., CLARKE, N. A. AND KABLER, P.W. 1960. Survival and protection against chlorination of human enteric pathogen in free-living nematodes isolated from water supplies. Am.]. Trap. Med. Hyg. 9:136-142.

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 Growth studies in microfuge, nutrient
 broth, tubes.
INCUBATION TUBE GROWTH
TEMPERATURE INOCULUM (OPEN-CLOSED) TURBIDITY [1] "PILL" [2]
Room None O - -
Room None C - -
37[degrees]C E. coli B O + +
37[degrees]C E. coli B C + +
37[degrees]C None O - -
37[degrees]C None C - -


(1.)Turbidity: + = growth; - = no growth.

(2.)"Pill": += pill; - = no pill (14,000 RPM for 5 min)
 Metabolic inhibition test (MIT)
 utilizing phenol red dextrose
 broth (PRDB).
 INOCULUM GROWTHS [1] PLATE COUNTS [2]
MEDIA (E. COLI STRAIN) (COLOR CHANGE) (CFU x [10.sup.7]/ML)
PRDB [chi]1997 + 3.8 a
PRDB MJ-100 + 2.6 a
PRDB + Str [3] [chi]1997 - 0.0004 b
PRDB + Str MJ-100 + 3.3 a
PRDB + Nal [4] [chi]1997 + 4.3 a
PRDB + Nal MJ-100 - 0.0002 b


(1.)Growth: + = growth, color change from red to yellow; - = no growth media remains red.

(2.)Means of 3 replicates each. Those means followed by the same letter are statistically the same, those by a different letter are significantly different. P=0.05.

(3.)Str = Streptomycin supplement at a dose of 25 [micro]g/mL.

(4.)Nal = Nalidixic Acid supplement at a dose of 20 [micro]g/mL.
 Detection of transconjugants utilizing
 the phenol red dextrose broth (PRDB)
 metabolic inhibition test (MIT).
 INOCULUM HARVEST TIME [1] GROWTH [2]
MEDIA (E. COLI STRAIN) (HOURS) (COLOR CHANGE)
PRDB+Str [3] [chi]1997 0 -
PRDB+Str MJ-100 0 +
PRDB+Nal [4] [chi]1997 0 +
PRDB+Nal MJ-100 0 -
PRDB+Amp [5] [chi]1997 0 -
PRDB+Amp MJ-100 0 +
PRDB+Nal+Str [chi]1997/ MJ-100 2 +
PRDB+Nal+Str [chi]1997/ MJ-100 4 +
PRDB+Nal+Str [chi]1997/ MJ-100 6 +


(1.)Harvest Time: Hours sample taken after mixing two strains; 0 = no mixing.

(2.)Growth: + = growth, color change from red to yellow; - = no growth media remains red.

(3.)Str = Streptomycin supplement at a dose of 25 [micro]g/mL.

(4.)Nal = Nalidixic Acid supplement at a dose of 20 [micro]g/mL.

(5.)Amp = Ampicillin supplement at a dose of 50 [micro]g/mL.
 Detection of transconjugants, of internal nematode
 vector origin, utilizing the phenol red dextrose
 broth (PRDB) metabolic inhibition test (MIT).
 INOCULUM
 E. COLI STRAIN HARVEST TIME [1] GROWTH [2]
MEDIA (1ST/2ND FEEDING) (HOURS) (COLOR CHANGE)
PRDB+Nal+Amp3 [chi]1997/MJ-100 1 -
PRDB+Nal+Amp [chi]1997/MJ-100 2 -
PRDB+Nal+Amp [chi]1997/MJ-100 4 +
PRDB+Nal+Amp [chi]1997/MJ-100 6 +


(1.)Harvest Time: Hours sample taken after second of the two sequential feedings.

(2.)Growth: + = growth, color change from red to yellow; - = no growth, media remains red.

(3.)Nal = Nalidixic Acid supplement at a dose of 20 [micro]g/mL; Amp = Ampicillin supplement at a dose of 50 [micro]g/mL.
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Author:ADAMO, JOSEPH A.; GEALT, MICHAEL A.
Publication:Bulletin of the New Jersey Academy of Science
Article Type:Statistical Data Included
Geographic Code:1USA
Date:Sep 22, 1999
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