ISOLATION OF ENTEROCOCCUS FAECALIS EXHIBITING CONJUGAL TETRACYCLINE FROM SWINE LOT OUTFLOW

Bradley J. Haack^a, Robert E. Andrews Jr.^a, and Thomas E. Loynachan^b

^aDepartment of Microbiology, Immunology, and Preventive Medicine and ^bDepartment of Agronomy, Iowa State University, Ames, Iowa 50011.

SUMMARY

Tn916 is a broad host range conjugal transposon, originally isolated from Enterococcus faecalis, that encodes tetracycline resistance (tet M). In addition to being self transmissible, Tn916 has been shown to mobilize non-self transmissible genetic elements, such as plasmids or chromosomal genes. With increased awareness and concern of genetic exchange in the environment, it was of interest to examine isolates of E. faecalis that contact environmental organisms for genetic elements similar to Tn916. Outflow samples obtained from an Iowa State University swine farrowing house were used to isolate E. faecalis for further study. Of the enterococci isolated, 71% were found to be resistant to tetracycline. To screen these isolates for conjugal activity, a microassay mating procedure was developed. The assay allows for conjugal activity to be determined for a large number of isolates with substantial savings of time and media. Using this microassay, 50 to 100 isolates may be tested with results available within 48 hr. Among the tetracycline-resistant enterococci isolated from the outflow samples, approximately 34% were able to pass the tetracycline resistance phenotype to Bacillus thuringiensis.

INTRODUCTION

Recently, there has been increasing interest in gene transfer and the acquisition of antibiotic resistance by bacteria in the environment. Particular emphasis has been placed on release of genetically engineered microorganisms (GEMs). One concern is that, once a GEM is introduced into the environment, the engineered DNA may be acquired by resident microbes resulting in disruption of the ecosystem homeostasis. This may be particularly significant if the newly acquired DNA provides a selectable advantage to the recipient (Stotzky and Babich, 1986).

In nature, the most important method by which bacteria exchange DNA seems to be conjugation (van Elsas, 1992). A wide range of bacterial genera have been shown to participate in conjugative genetic exchange. Conjugation requires a series of genes that encode conjugal functions. In Gram-positive bacteria, particularly within the genera Streptococcus and Enterococcus, conjugal genes frequently occur on plasmids as well as on transposons (Clewell, 1990). Besides encoding conjugal functions, these elements often carry antibiotic-resistance genes, as well as genes encoding resistance to heavy metals and bacteriocin production.

Conjugal elements may be, in part, responsible for the recent increase in pathogenic bacteria showing multiple resistance to many common antibiotics. The soil ecosystem is rich with antibiotic-resistance genes due to the prevalence of antibiotic-producing organisms. Some conjugal elements could transfer these genes from soil organisms to other bacteria. Multiple antibiotic resistance in infectious organisms may, at least in part, have developed through exposure to resistance genes from terrestrial organisms that have been picked up by or mobilized by conjugal elements.

The conjugal transposon Tn916 was originally isolated from Enterococcus faecalis and encodes resistance to tetracycline (Franke and Clewell, 1981). Tn916 is a well-studied example of a family of conjugal transposons including Tn918, Tn925, Tn1545 and several others. A recent study of Enterococcusisolates showed several with DNA homology to Tn916. The ubiquitous nature of E. faecalis in mammalian fecal material, and the abundance of conjugal elements isolated from these genera, make this microbe a possible vector for the delivery of conjugal elements into soil populations. Once in the soil ecosystem, conjugal elements may provide fluidity to the gene pool allowing mobilization of antibiotic resistance genes that might, in turn, be reintroduced into animal normal flora and/or pathogens. For this reason, it was of interest to examine isolates of this organism for conjugal elements similar to Tn916.

An Iowa State University swine facility was used as the source of isolates. Samples were acquired from the farrowing house where antibiotic feed additives were not in use. Because the outflow is eventually deposited onto agronomic soils, such wastes might be a method of introducing conjugal elements into the soil.

To screen the large number of isolates obtained, a microassay was developed to allow efficient and cost-effective screening of the initial isolates. Once the isolates were identified as possessing a conjugal tetracycline resistance gene, selected cultures were assayed through conventional mating procedures (Naglich and Andrews, 1988a; b) to determine frequencies of transfer and thus confirm the data generated by the microassay. The goal of this research was to examine the extent of conjugal tetracycline resistance present in swine outflow.

MATERIALS AND METHODS

Isolation of Enterococci. An outflow sample was obtained from an Iowa State University hog farrowing house. The sample was diluted and pour plates were prepared. To determine total culturable bacteria, brain heart infusion agar (BHI) (Difco, Detroit, MI) was used. For total culturable tetracycline resistant bacteria, the BHI was supplemented with 10 µg/ml tetracycline (Sigma Chemical Company, St. Louis, MO). For enumeration and isolation of enterococci, pour plates were prepared with KF Streptococcus agar (KF) (Difco). This medium was supplemented with 10 µg/ml tetracycline to isolate tetracycline resistant (Tetr) enterococci. From the KF agar plates, 100 typical Tetr enterococci were selected for conjugal studies and confirmed to be enterococci by the method of Knudtson and Hartman (1992).

Determination of Conjugal Activity. To determine if the Tetr within the isolates was conjugal, a microassay was devised to allow screening of a large number of isolates efficiently (Figure 1). The test isolates were grown overnight on BHI agar with selection. A colony from the overnight plate was mixed on a nitrocellulose membrane (0.45 µm) (Millipore) with a colony from an overnight LB agar culture of a selectable recipient. The recipient used in these experiments was Bacillus thuringiensis subsp. israelensisAN142 (Neor, 200 µg/ml) (Naglich and Andrews, 1988b). The mating mixture was incubated 24 hrs at 37C. Conjugants were detected by isolation streak of the mating mixture onto LB agar supplemented with 10 µg/ml tet and 200 µg/ml kanamycin (Sigma). Following identification, conjugal activity of 10 isolates was confirmed and frequencies of transfer determined by the conventional mating method of Naglich and Andrews (1988a).

As a positive control for conjugal transfer, E. faecalis CG110 (Chr::Tn916) (Gawron-Burke and Clewell, 1982) was mated with AN142 by the microassay technique. The frequency of Tn916 transfer was also determined by the method of Naglich and Andrews (1988a). To assure that the putative conjugants did not result from spontaneous mutation of the recipient to tetracycline resistance, AN142 was incubated separately on a membrane, followed by isolation streak onto LB (tet, kan). Spontaneous mutation was not detected during any mating performed.

RESULTS

Tetracycline Resistance. Concentrations of culturable organisms, tetracycline resistant organisms, enterococci and tetracycline resistant enterococci were determined by differential plate counts. The total culturable bacteria in the outflow sample were determined to be 5.5 x 10⁷ CFU/ml. Of the total bacteria cultured, 1.6 x 10⁷ CFU/ml or 29% were tetracycline resistant (Figure 2). The total concentration of enterococci was determined to be 2.9 x 10⁵ CFU/ml. Although this represented only a small portion of the total cultured organisms (Figure 3), 2.1 x 10⁵ CFU/ml or 71% of the total enterococci were resistant to tetracycline (Figure 4).

Conjugal Activity. One hundred tetracycline-resistant isolates were screened for conjugal transfer of Tetr into B. thuringiensis. When the microassay was employed, 34 of the 100 isolates were found to possess a conjugal tetracycline resistance gene (Figure 5). A group of 10 conjugation-positive isolates and 2 conjugation-negative isolates were mated with B. thuringiensis AN142 by the conventional mating protocol of Naglich and Andrews (1988) to determine frequencies of transfer (Table 1). Thus, the sensitivity of the microassay was confirmed by the conventional mating procedure. Moreover, the positive isolates showed that detectable transfer of Tetr occurred near the average value for the positive control CG110. The conjugation-negative isolates showed no detectable transfer of Tetr to the recipient.

DISCUSSION

The dissemination of antibiotic resistance determinants among bacteria is becoming an increasing problem in the treatment of disease. Because most antibiotics originated from soil organisms, the majority of the genes encoding resistance to these antibiotics may also have originated in soil. When a resistance gene such as that for tetracycline is acquired by a conjugal element, the gene becomes movable within the host range of that element. Some conjugal elements, such as Tn916, have a very broad host range.

The enterococci were chosen for this work because they have been shown to possess many conjugal resistance genes. Furthermore, because the deposition of animal fecal material containing high numbers of enterococci, as well as other enteric organisms, on agronomic soils is a common practice to increase soil fertility, this may be a method for the introduction of conjugal genes into the environment. Studies have shown the ability of conjugal elements to transfer between organisms in the soil (van Elsas, 1992). The work presented here confirms the presence of conjugal resistance genes in the swine facility outflow that is normally deposited onto soil.

Of the total cultured bacteria, 29% showed resistance to tetracycline. Because the sample was obtained from the farrowing house, tetracycline was not in use. Therefore, there was no selective advantage for Tetr organisms. However, studies have shown that some resistance determinants can persist in populations, even without selective pressure (Langlois et al., 1988). For this reason, numbers of tetracycline resistant organisms in the population may remain high. More interesting is the high level of resistance among the Enterococcus population. Clearly, there is a significant population of resistant enterococci present in the swine herds tested. These results are strengthened by data from Knudtson and Hartman (1993) who found 88% tetracycline resistance among enterococci isolates from swine carcasses at the time of slaughter. Furthermore, even though data presented herein show 71% of the Enterococcuspopulation to be resistant to tetracycline, these enterococci represent only 1% of the total tetracycline resistance within the culturable population. Therefore, the major portion of the resistant population has yet to be studied.

Although high numbers of resistant organisms among certain populations of bacteria may give some grounds for concern, the linking of the resistance to a conjugal element is a serious cause for concern. Because conjugation may allow the resistance genes to be transferred to many other organisms including pathogens, it was necessary to determine what portion of these genes were conjugal. Using the microassay developed herein, the level of conjugal Tetr was determined with substantial savings of time and media. Additionally, the assay was proven to be as accurate as the conventional mating method usually used. Of the 100 Tet^r isolates tested, 34% were found to conjugally transfer the resistance gene. Importantly, this conjugal transfer was cross genera. Furthermore, the frequency of resistance transfer by the enterococcal isolates is comparable to that of Tn916. This would seem to indicate a conjugal system similar to that of the Tn916-like conjugal elements. Because there is some evidence that Tn916 is conjugal in the soil environment (Natarajan and Oriel, 1992), finding elements that function like this element raises concern about what genes are being transferred into and among the soil microflora.

REFERENCES

Clewell, D.B. 1990. Movable genetic elements and antibiotic resistance in enterococci. Euro. J. of Clin. Micro. and Infect. Dis. 9:90-102.

Franke, A.E. and D.B. Clewell. 1981. Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid. J. of Bac. 145:494-502.

Gawron-Burke, C. and D.B. Clewell. 1982. A transposon in Streptococcus faecalis with fertility properties. Nature 300:281-4.

Knudtson, L.M. and P.A. Hartman. 1992. Routine procedures for isolation and identification of enterococci and fecal streptococci. Appl. and Env. Micro. 58:3027-3031.

Knudtson, L.M. and P.A. Hartman. 1993. Antibiotic resistance among enterococcal isolates from environmental and clinical sources. J. of Food Protect. 56:489-492.

Langlois, B.E., K. A. Dawson, I. Leak, and D.K. Aaron. 1988. Antimicrobial resistance of fecal coliforms from pigs in a herd not exposed to antimicrobial agents for 126 months. Vet. Micro. 18:147-153.

Naglich, J.G., and R.E. Andrews, Jr. 1988a. Introduction of the Streptococcus faecalis transposon Tn916into Bacillus thuringiensis subsp. israelensis. Plasmid 19:84-93.

Naglich, J.G. and R.E. Andrews. Jr. 1988b. Tn916-dependent conjugal transfer of pC194 and pUB110 from Bacillus subtilis into Bacillus thuringiensis subsp. israelensis. Plasmid 20:113-126.

Stotzky, G. and H. Babich. 1986. Survival of, and genetic transfer by, genetically engineered bacteria in natural environments. Adv. in Appl. Micro. 31:93-138.

van Elsas, J.D. 1992. In: Genetic Interactions Among Microorganisms in the Natural Environment (E.M. Wellington and J.D. van Elsas, eds.), pp. 17-39, Pergamon Press Limited, Headington Hill Hall, Oxford.

Figure 1. Microassay developed to detect conjugal transfer of tetracycline resistance. The assay allows quick and const effective detection of resistance transfer within 48 h of isolation.

Figure 2. Percentage of total culturale bacteria, from the Iowa State University swine lot, resistant to tetracycline. Determined by plate count on BHI agar supplemented with 10 µg/mi tetracycline vs. total culturable organisms on BHI without selection.

Figure 3. Percentage of tetracycline resistance attributed to entrococci present in an outflow sample from the Iowa State University swine lot. Determined by plate count on KF Streptococcus agar supplemented with 10 µg/ml tetracycline vs total tetracycline resistant organims on BHI supplemented with 10 µg/ml tetracycline.

Figure 4. Percentage of tetracycline resistance present in the Enterococcus population isolated from the outflow of the KF Streptococcus agar supplemented with 10 µg/ml tetracycline vs. total enterocci cultured on KF Streptococcus agar without selection.

Figure 5. Percentage of tetracycline resistant entrococci isolated from the outflow of the Iowa State University swine lot exhibiting a conjugal tetracycline resistance gene. Determined by applying the conjugal micrassay described herein on 100 tetracyline resistant Enterococcus isolates.