Natural Genetic Transformation of Clinical Isolates of Escherichia coli in Urine and Water

Transfer of plasmid-borne antibiotic resistance genes in Escherichiacoli wild-type strains is possible by transformation under naturallyoccurring conditions in oligotrophic, aquatic environments containingphysiologic concentrations of calcium. In contrast, transformationis suppressed in nitrogen-rich body fluids like urine, a commonhabitat of uropathogenic strains. Current knowledge indicatesthat transformation of these E. coli wild-type strains is ofno relevance for the acquisition of resistance in this clinicallyimportant environment.

INTRODUCTION

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Introduction

Horizontal gene transfer is a recognized process that allowsthe rapid spread of antibiotic resistance genes inside and outsideof hospitals, thereby impeding antimicrobial chemotherapy (17).Resistance determinants are readily acquired and disseminatedwithin and among bacterial populations by (i) conjugation, (ii)transduction, and (iii) transformation, virtually without barriersbetween species (6, 12). Natural genetic transformation is characterizedby the uptake of free DNA by a recipient bacterium, its chromosomalintegration or extrachromosomal stabilization, and its expression,which leads to a new phenotype (7, 11). Calcium appears to playa pivotal role for the development of bacterial competence,an inducible property of many bacterial species and a prerequisitefor transformation, in both gram-positive and gram-negativebacteria (18, 19, 25). Laboratory strains of Escherichia coliwere shown to be transformable by a nonphysiological Ca²⁺ concentrationof 100 mM (9, 22). Recombination- and DNase-deficient laboratorystrains are generally used, with a temperature shift from 0to 37°C after the addition of free DNA (9, 22, 27). Suchconditions are never encountered in the original biotope ofE. coli (i.e., the mammalian gastrointestinal and urogenitaltracts). Consequently, this species was not considered to betransformable in its natural habitats (11, 26). However, therehave been indications that the induction of competence in E.coli is controlled physiologically rather than physicochemically(4), and natural transformation of laboratory strains of E.coli has been demonstrated to occur in river, spring, and mineralwater (2) and in foodstuffs (1) but not yet in the body fluidsof mammals. Our intention was to evaluate whether wild-typeclinical isolates of E. coli were naturally transformable infreshwater, as has already been reported for laboratory strains,and to evaluate the impact of transformation on the disseminationof antibiotic resistance genes under conditions of clinicalsignificance.

Bacterial strains and plasmid DNA.

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Bacterial strains and plasmid...

Eight ampicillin-sensitive, nonpathogenic E. coli isolates fromsputum or swab samples of the oropharynxes of hospitalized patientswith sinusitis or otitis were chosen at random without additionalcriteria of selection. In addition, we tested four pathogenicstrains which were isolated from two patients with urogenitalinfections. From each patient, a single strain was isolatedfrom feces (S 988 and S 1018) and from urine (U 988 and U 1018),all of which showed identical restriction fragment length polymorphismbanding patterns. E. coli strain DH5 ${alpha}$ (number C2007-1; Clontech,Palo Alto, Calif.) was used as a control in each experiment.Supercoiled pUC18 (Roche, Vienna, Austria) and pGFP (Clontechnumber 6097-1) plasmid DNAs were purified by anion-exchangechromatography (Qiagen, Hilden, Germany).

Transformation assays.

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Transformation assays.

Transformation assays were performed as described by Sambrooket al. (22). Briefly, log-phase cells were harvested and thenumber of viable cells per milliliter (usually 10⁸ cells/ml)was determined by agar plating of serial dilutions. The bacteriawere washed with the selected water samples or CaCl₂ solutions(0 to 100 mM), resuspended in 200 µl of the same solution,and incubated for 1 h (test range, 1 min to 72 h) at the appropriatetemperature. Then, supercoiled pUC18 or pGFP plasmid DNA wasadded at various concentrations ranging from 1.25 ng/ml to 1.25µg/ml and for different time periods (1 to 60 min). Mostof the experiments were performed with DNA incubation for 10min. A standard shift from 0 to 37°C was applied, usuallyfor 10 min. After addition of 500 µl of Luria-Bertanibroth, all samples were incubated at 37°C for 45 min, and100 µl of the transformation mix was transferred ontoLuria-Bertani agar plates containing ampicillin (100 µg/ml).The transformation frequency (T_f) was established by calculatingthe ratio of the number of transformants per viable cell permilliliter (detection limit, 10^–7). The transformationefficiency (T_e) was calculated as the number of transformantsper microgram of plasmid DNA applied in a volume of 1 ml. Themean number of transformants obtained during three transformationassays was used for calculation.

Transformation assays in human urine were performed as describedabove, with the exception that all incubations were performedat 37°C in human urine in order to mimic natural conditions.Void urine was collected from healthy volunteers and sterilizedby filtration (Millex-GV; Millipore, Bedford, Mass.). For someexperiments, urine was treated at 80°C for 10 min to inactivatethe DNases. Ca²⁺ concentrations were measured with a calciumdetection kit, 587-A, from Sigma (St. Louis, Mo.); pH was determinedwith pH indicator strips (Merck, Darmstadt, Germany). The bacteriawere incubated in 1 ml of urine on a thermomixer for 24 h. Theywere then pelleted, washed with fresh pretreated urine, resuspendedin 500 µl of urine, and incubated for another 4 h undervigorous shaking. After addition of plasmid DNA to 200 µlof the preincubation solution (final concentration, 125 ng/ml),the bacteria were incubated for 24 h. A 500-µl volumeof fresh urine was then added, and bacterial growth was enabledfor an additional 4 h. Transformants were detected as describedabove.

Transformation in CaCl₂ solutions and in mineral water.

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Transformation in cacl2...

As is shown in Table 1, three (EC1, EC2, EC7) out of the eightnonpathogenic E. coli isolates persistently yielded transformantsupon application of the standard procedure. The other strainseither were not transformable (EC3, EC6) or showed fluctuatingtransformation (EC4, EC5). All four pathogenic strains yieldedtransformants in three consecutive experiments. EC1 always showedthe highest number of transformants. With this strain, competencecould be induced with a CaCl₂ concentration as low as 1 mM,but T_fs were always modest percentages of the values obtainedwith the laboratory strain DH5 ${alpha}$ (11% for 100 mM CaCl₂, 3% for50 mM, 1% for 10 mM). Transformants could be induced with EC1and DH5 ${alpha}$ by incubation in bottled, carbonized mineral water (Preblauer,Austria), which contained 2.9 mM Ca²⁺. No transformants wereobserved when the strains were incubated in other mineral waterswith lower Ca²⁺ contents, in Coca Cola, or in orange or multivitaminjuice.

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TABLE 1. Transformation of wild-type E. coli strains and DH5 ${alpha}$ ^a

Additional parameters influencing transformability.
As is shown in Table 2, 1 min of incubation in CaCl₂ was sufficientto generate transformants, indicating that induction of competenceis a rapid process. A steep rise in T_e was detectable after5 min of incubation with EC1 and after 10 min with DH5 ${alpha}$ . WithEC1, 1.25 ng of DNA/ml was sufficient to generate transformantswhen it was incubated in CaCl₂ at a concentration of 10 mM orhigher. With 100 mM CaCl₂ and 10⁹ cells/ml, the T_f increasedwith rising DNA concentrations whereas the efficiency of DNAtransfer remained constant, as one expects in a system not saturatedwith DNA. With DH5 ${alpha}$ (10⁸ cells/ml), the T_e decreased with risingDNA concentrations, but the T_f essentially remained stable.If one assumes that wild-type and laboratory strains behavesimilarily in this scenario, what we observed may be explainedby a surplus of plasmid molecules compared to the number ofcompetent cells available in a DNA-saturated environment.

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TABLE 2. Parameters influencing transformation of E. coli wild-type and laboratory strains in water^a

Transformants could also be detected after temperature shiftsfrom 25 to 37°C (but not with constant incubation at 25or 37°C) with a minimum of 10 mM CaCl₂. Unlike with DH5 ${alpha}$ ,the number of EC1 transformants did not increase along withthe concentration of calcium, indicating that the transformabilityof wild-type strains is more stringently controlled than thatof the laboratory strain by the kind of temperature shift.

Transformation in human urine.
No transformant was detected with any of the uropathogenic strains,nonpathogenic strains, or strain DH5 ${alpha}$ in urine (pH 6.8 to 7.5,heat inactivated and nontreated) with constant incubation at37°C (Table 1). Agarose gel analysis of the pGFP plasmidindicated complete DNA degradation within 5 to 10 min in nontreatedurine. Variations of the transformation protocol (i.e., incubationof E. coli strains on ice for 24 h prior to addition of plasmidDNA, heat shock at 0 to 37°C, and use of plasmid DNA concentrationsof up to 1.25 µg/ml) also did not lead to the formationof transformants. A single positive transformation in one outof three experiments could be detected with DH5 ${alpha}$ (T_f, 3.0 x 10^–8;T_e, 2.7 x 10¹) after DNase inactivation (by heat) and afterthe urine sample was spiked with calcium (final Ca²⁺ concentration,12 mM).

Relevance of different horizontal gene transfer mechanisms for the dissemination of antibiotic resistance.
The transfer of antibiotic resistance genes in E. coli by conjugationin soil (3), protozoa (23), body fluids (13), and the intestine(16) has been reported and most probably plays a decisive rolein the spreading of resistance genes in clinical settings (24).Concerning the phenomenon of transformation in body fluids,the data are scarce. Natural transformation of gram-positivepathogens like Streptococcus pneumoniae has been unequivocallydemonstrated for humans (21) and mice (5, 20). The developmentof genetic competence and natural transformation have been shownfor Bacillus subtilis in milk (29) and for Streptococcus gordoniiin human saliva (15). Inhibitory effects of rumen fluid andovine saliva on the development of the competence of Streptococcusbovis have also been described (14). Bauer et al. (1) have demonstratedE. coli T_fs below 10^–6 in milk. Recently, vesicle-mediatedtransfer of virulence genes has been reported, a process whichmay facilitate the dissemination of DNA fragments in DNase-richenvironments like urine (28).

We have detected the transformation of E. coli wild-type strainsin water at Ca²⁺ concentrations as low as 1 to 2.9 mM, whichare readily available in mineral waters and body fluids (proximalileum, 4.2 mM; urine, 5.9 mM; saliva, 2.3 mM) (10). However,transformants were not detectable in urine. DNA degradationseverely impairs the uptake of free DNA in DNase-rich body fluidslike urine, but an additional process functioning against transformationcan be inferred. (i) A strong correlation has been found betweenthe T_e and the formation of poly-ß-hydroxybutyrate(PHB)–calcium–polyphosphate complexes, which arepresumably channels for DNA transfer in the cytoplasmic membranesof E. coli cells (8). (ii) PHB synthesis is also a way to storecarbon in an osmotically inert form when nitrogen becomes thelimiting growth factor. (iii) Induction of competence preferentiallyoccurs during starvation caused by a nutrient imbalance, i.e.,when the C/N ratio is high (2, 11). Thus, the absence of transformationin urine may be due to the fact that E. coli grows initiallyin a nutrient-rich medium (>10⁹ CFU/ml) without the needfor PHB synthesis. Under starvation conditions during stationaryphase, a low C/N ratio in urine is expected. Neither conditionis favorable for the induction of competence. The situationmay be reversed in milk that contains an abundance of sugar,leading to a C/N ratio greater than 1 at starvation.

Conclusion.
The transformability of E. coli (clinical isolates and laboratorystrains) is restricted by the environmental conditions prevailingin its habitat. Although genetic transformation of E. coli mayoccur under natural conditions in aquatic, calcareous environments,urine suppresses the rate of transformation to below the detectionlimit. This observation indicates that natural transformationof E. coli wild types is most probably of no relevance for theacquisition of antibiotic resistance determinants in body fluidslike urine. Resistant phenotypes may be spread predominantlyby bacterial conjugation and vesicle-mediated transfer in clinicalsettings. However, we cannot rule out the occurrence of transformationin other natural environments (e.g., mammalian intestine) orfor other pathogroups of E. coli.

ACKNOWLEDGMENTS

This work was supported in part by grants of the Austrian FederalMinistry of Health and Consumer Protection, BKA, the OesterreichischeNationalbank, and the Austrian Society of Chemotherapy.

We are grateful to Konstantin Schwarz for providing the pathogenicE. coli strains and the related restriction fragment lengthpolymorphism data. We thank warmly Edith Mlynar and Tina Hauslfor excellent technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Department of Internal Medicine I, Division of Infectious Diseases, General Hospital of Vienna, Waehringerguertel 18-20, A-1090 Vienna, Austria. Phone: 0043/1/40400-4440. Fax: 0043/1/40400-5167. E-mail: markus.woegerbauer{at}akh-wien.ac.at.

REFERENCES

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