Vol. 57, No. 8INFECTION AND IMMUNITY, Aug. 1989, p. 2331-23380019-9567/89/082331-08$02.00/0Copyright © 1989, American Society for Microbiology

Interaction of Drug Resistance Plasmids and Bacteriophages withDiarrheagenic Strains of Escherichia coli

DAVID E. BRADLEYFaculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AIB 3V6

Received 19 October 1988/Accepted 20 April 1989

Seven transfer-derepressed plasmids from different incompatibility groups in Escherichia coli K-12 were

tested for their ability to enter 43 strains of diarrheagenic E. coli (mostly enteropathogenic E. coli clinicalisolates) representing 12 serogroups and including rough and semirough mutants (characterized by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis). Strains in some serogroups were more competent as

recipients of plasmids than were those in others. Five test plasmids in an E. coli K-12 (rough) donor transferredsignificantly less efficiently to two smooth strains than to their rough or semirough isogenic derivatives. Whenthe same smooth and rough strains were used as donors, the plasmids transferred to E. coli K-12 equally well.These results suggested that the 0-antigenic lipopolysaccharide side chains of diarrheagenic E. coli isolatesshielded the outer membrane receptors for conjugative pili, thus preventing plasmid entry. The differentreceptors for eight bacteriophages were also covered by 0 side chains. In addition, a limited survey of clinicalisolates for drug resistance markers and resident plasmids was carried out.

There are four principal classes of diarrheagenic Esche-richia coli (21): enteropathogenic E. coli: (EPEC), entero-toxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), andenterohaemorragic E. coli (EHEC). Each is associated withparticular serogroups of organisms defined by the 0 somaticantigens of the outer membrane lipopolysaccharide (LPS).In some countries, antibiotics may be used excessively in thetreatment of diarrheagenic E. coli. This provides selectivepressure enabling plasmids carrying antibiotic resistancegenes to infect pathogenic bacteria by conjugation. Plasmidtransfer can be likened to bacteriophage infection in thatboth involve a receptor-mediated interaction followed byDNA transfer into the recipient. In conjugation, cell contactis established by conjugative pili (protein filaments or rods[7]), which attach to protein or perhaps LPS receptors (1).The pili are then thought to pull the cells together byretracting, DNA transfer following through the point ofcontact. There are two basic forms of conjugative pili: rigidrods that function efficiently only on a surface, and flexiblefilaments that work equally well on a surface or in a liquid(13). Van der Ley et al. (32) have shown that the longpolysaccharide 0 side chains (see reference 28 for a review)are able to prevent protein-specific bacteriophages frominfecting bacteria; they shield receptors (22, 32) from the tailcomponents that recognize them. Similarly, the 0 sidechains might well prevent the entry of at least some types ofplasmid by blocking conjugative pilus receptors, as wassuggested for Salmonella typhimurium by Sanderson et al.(30).The principal objective of this work was to investigate the

effects of different serotypes of0 side chains on the ability ofself-transmissible plasmids to enter clinical isolates of diar-rheagenic (smooth [S]) E. coli and some rough (R) orsemirough (SR) derivatives. Whether a plasmid can infect astrain may depend on the nature of any plasmids resident inthat strain. If a resident plasmid is incompatible (16) with theincoming one, entry may be prevented by surface exclusion.If a new plasmid is acquired by a strain through conjugation,it could be passed on to other pathogenic recipients of adifferent serogroup, perhaps mobilizing nontransferable res-ident plasmids with undesirable traits such as drug resis-

tance. Resident plasmids are thus relevant to this study, anda limited survey is presented. Since bacteriophages, bothplasmid specific and plasmid nonspecific, could exert somecontrol on diarrheagenic E. coli strains, the degree of shield-ing provided by 0 side chains against infection by phages hasbeen assessed.

MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. Clinical

isolates of diarrheagenic E. coli used in this study are listedin Table 1. Those with the prefix F are from the collection ofM. Finlayson, Provincial Laboratory of Public Health, Uni-versity of Alberta, Edmonton, Alberta, Canada. Others weresupplied by S. Ratnam, Newfoundland and Labrador PublicHealth Laboratories, St. John's, Newfoundland, Canada,and H. Lior, Laboratory Centre for Disease Control, Ot-tawa, Ontario, Canada (prefixes R and L, respectively).Strain 1337 (prefix S, full designation EM87-1507MS/1337),supplied by M. Schoonderwoerd, Alberta Agriculture, Ed-monton, Alberta, Canada, was isolated from a calf. StrainsB75 and B76 (prefix W) were O+K- and O-K- derivatives,respectively, of the encapsulated strain E69 (09:K30:H12;Pro- His- Trp- Strr) of I. 0rskov and F. 0rskov, StatensSeruminstitut, Copenhagen, Denmark, constructed and sup-plied by C. Whitfield, Department of Microbiology, Univer-sity of Guelph, Guelph, Ontario, Canada. Strain B76 grewpoorly when made resistant to rifampin (Rif), and so it couldnot be used in some experiments. Some strains have beendescribed elsewhere (11). H and K antigens have not beenincluded in Table 1 since they were not available for allstrains, and in any case K antigens are not distinguishablefrom 0 antigens for the serogroups used here (28). Strainnumbers have been abbreviated in some cases. E. coli C2(prototroph), a derivative of strain C (ATCC 13706), wasused to propagate bacteriophage lambda, and E. coli K-12strains JE2571 (Leu- Thr- Thi- Lac- Pil- Fla-) and J53(Pro- Met- Pil+ Fla') were used as plasmid hosts, and thefirst was used as an example of an R strain (then designatedJE2571R). R and SR strains, together with one that had losta few 0-antigen repeating units (semismooth [r]; seeResults), were found when novobiocin-resistant (Nbc) or Rif'

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2332 BRADLEY INFECT. IMMUN.

TABLE 1. Transfer of plasmids from E. coli K-12 to diarrheagenic E. coli of different serogroups"

Transconjugant growth of following plasmiddSerogroup Strainb Class'

R905 R144drd3 pMG110 pIN25 N3 Sa RP4

o55e F 19736-2S EPEC + (+) + + + (+) +F 19950-2S EPEC (+) (+) (+) + + + +F 17023-2S EPEC + + (+) (+) (+) (+) +L EC2-2S EPEC - (+) (+) (+) - + +L EC225-2S EPEC - + + (+) (+) - +F 5788-2S EPEC - + - + - + +F 13721-2S EPEC - (+) - - (+) - -F 7176-2S EPEC - - - - - - -

0111 F 2470-2S EPEC - - - - - - -S 1337-2S VTEC - - - - - - -L 1002-2S EPEC - (+) - - - - -F 2568-2S EPEC - - - - - - -F 6378-2S EPEC - - - - - - -F 10732-2S EPEC - - - - - - -F EIU84-2S EPEC - + - + (+) - +

0125 F 16213-2S EPEC - (+) - + - + -L EC297-2S EPEC - - - - - + +L EC231-2S EPEC - (+) - - - - -F 5866-2S EPEC - - - - - + +B 5866-2R None - (+) (+) - (+) + +F 19017-2S EPEC - (+) (+) - - + +F 2218-2S EPEC - - - - - - -F 6881-2S EPEC - + - + - - +B 6881-2SR None - + - + + + +

0126 F 6354-2S EPEC - - - - - - -F 6027-2S EPEC - + - - - - -F 16185-2S EPEC - - - - - - -F 18137-2S EPEC - - - - - - (+)L EC232-2S EPEC + - - - - - +L 1709-2S EPEC - - - - - - +L 9401-2S EPEC - (+) - - - + (+)

0157 R 7777-2S EHEC - + - + - + +R 19386-2S EHEC - - - - - - (+)R 1140-2S EHEC + + + (+) +f + +R 2166-2S Asym - + + - + + +R 8868-2S Asym - + (+) - - + (+)R 9492-2S EHEC - + - (+) - + +R 1756-2S EHEC - - - - - (+) -

09 W B75-2S NT + - - + + + +W B76-2R None + - - + + + +

026 F 19461-2S EPEC - (+) - - - + (+)0119 F 19392-2S EPEC - - - - - - -0124 F 18031-2S EIEC (+) + - + - + (+)0127 R 1492-2S EPEC - + + (+) (+) - +0128 F 19547-2S EPEC + (+) - (+) (+) + (+)

B 19547-2r None (+) + - (+) + + +0142 F 22539-2S EPEC (+) + (+) + + + +

a Transfer by cross-streak mating, plasmid-selecting drugs being chloramphenicol for R905, kanamycin for R144drd3, tetracycline for pMG110, kanamycin forpIN25, tetracycline for N3, chloramphenicol for Sa, and kanamycin for RP4. Counterselection was with rifampin, but occasionally with streptomycin for B76Rsince B76-2R did not grow very well. When possible, negative results were checked by alternative drug selection.

b The prefix letter B refers to strains constructed for this work other than being made Rif, and the remaining letters refer to the persons donating the strains:F, M. Finlayson; L, H. Lior; R, S. Ratnam; S, M. Schoonderwoerd; W, C. Whitfield. The letters are not included in strain numbers elsewhere. The suffix -2indicates a rif' mutant, an additional S indicates smooth, R indicates rough, SR indicates semirough (lost all 0-antigen repeating units save one), and r indicatessemismooth (lost only a few high-molecular-weight 0-antigen repeating units, in this case five).

' Classes based on serogroups as listed by Levine (21). VTEC, verotoxin-producing E. coli; Asym, asymptomatic; None, presumably asymptomatic; NT, nottested.

d Symbols for transconjugant growth: +, confluent or nearly confluent; (+), a few colonies (1 to 50); -, no transfer. All plasmids transferred + between K-12strains.

' An additional 055 strain was tested because of some variability within this serogroup, notably by strain 7176-2S.f 1337 and 1140 were Tcr, so N3::Tn9 was used (chloramphenicol selection). EIU84 was Cmr, so Sa::Tn7 (trimethoprim selection) was used.

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PLASMID ENTRY INTO DIARRHEAGENIC E. COLI 2333

mutants of clinical strains were being isolated by spreadinglarge inocula of wild-type strains on plates containing ri-fampin or novobiocin. Colonies showing very rough mor-phology were subcultured to similar plates, and growth fromthese was suspended in 0.9% (wt/vol) NaCl in small tubes.When the tubes were placed in boiling water for 45 min andthen removed and allowed to cool, R strains agglutinated,often before cooling; others with changed 0 side chains wereidentified by increased sensitivity to a set of bacteriophages.Representatives were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silverstaining (see below).The plasmids used are classified by incompatibility (Inc)

and listed by Jacob et al. (19) unless otherwise indicated:R905 (IncV, temperature sensitive, replicating and transfer-ring at <30°C), pMG110 (IncHII [33]), pIN25 (IncT, temper-ature sensitive [14]), N3 (IncN), Sa (IncW), RP4 (IncP),pMR5 (temperature-sensitive mutant of RP1 which is thesame as RP4 [29]), and R144drd3 (IncI1 + B; laboratoryconstructed transfer-derepressed mutant [24]). R144drd3uses both rigid and flexible conjugative pili for conjugation ina liquid (9). For convenient selection of the IncFI plasmid F(no drug resistance), F: :Tn521 (transposon resistance topotassium tellurite [12]) or occasionally F: :Tn5 (resistance tokanamycin; constructed for this work) was used in its place.Drug resistances for the other plasmids are given in thereferences cited.

Bacteriophages used were kindly supplied as follows: K3(the same as K3hl), C. Manoil; TuIa and TuIb, J. Tommas-sen; P1 vir, D. E. Taylor; 6SR, K. Sanderson; 09-1, C.Whitfield. The remainder (see Tables 5 and 6) were from mycollection. Tests with phages P1, lambda, and T5 wereduplicated with isolates from the American Type CultureCollection (P1, ATCC 25404-Bl; lambda, ATCC 10798; T5,ATCC 11303-B5). Information on the bacteriophages usedand their specific protein receptors (see Table 6) was ob-tained from various sources (22, 23, 25, 32). Plasmid-specificphages in Table 5 have been reviewed (8) or describedindividually (6, 15, 20, 27, 31).Media, matings, transposon insertions, bacteriophage sen-

sitivity tests. Routine medium was brain heart infusion agar(BBL Microbiology Systems) with or without antibiotics orother additives at the following concentrations (microgramsper milliliter): ampicillin, 400; chloramphenicol, 25; genta-micin, 10; kanamycin, 100; nalidixic acid, 20; novobiocin,100; potassium tellurite, 5; rifampin, 100; streptomycin, 200;spectinomycin, 30; and tetracycline, 10. Sulfonamides andtrimethoprim were present at 1 mglml. Tryptone soya agar(Oxoid Ltd.) was used for trimethoprim plates. M9 minimalmedium with 2% glucose was used for selecting prototrophicrecipients in matings with auxotrophic donors.A rapid, qualitative, cross-streak mating method (2) was

modified to obtain maximum sensitivity. Heavy inocula ofdonor and recipient organisms were grown on nutrient agarfor 3 to 5 h. Recipient strains were spread thickly and evenlyin a drop of broth on one-half of a selective plate (rifampinwith an appropriate drug). When the plate was dry, a loopfulof donor organism was streaked from the uncovered to thecovered half of the plate. After incubation, the presence of 1to 10 transconjugant colonies on the covered area of thecross-streak indicated a transfer frequency of between 1 x

lo-4 and 1 x 10-3 transconjugants per donor per h, whileconfluent growth indicated >5 x 10-3 transconjugants perdonor per h. Qualitative and quantitative broth and platematings were performed as described previously (13). Trans-positions of Tnl (Apr) used plasmid pMR5 (see above),

1 2 3 4 5 6 7 8 9 10 11

Rc Ra JE2571R B75S B376R 56S6-2S 56656-2R "881-2S 68Bt-2SP 13547-2S 19547-2r

FIG. 1. Photographs of LPS 0-antigen repeating units fromdifferent SDS-PAGE silver-stained gels. To make the lanes exactlycomparable, gels were run under identical conditions with the samecalibration ladder included in each. Prints of the same size couldthen be obtained by matching the ladders in the enlarger. Lanes 1and 2 show LPS (obtained from Sigma) from strains J5 (Rc) andEH100 (Ra), respectively. JE2571R (lane 3) is an E. coli K-12 strain;B75S (lane 4) is 09, and B76R (lane 5) is its Ra derivative; lanes 6and 7 show a similar 0125 pair, but 6881-2S (lane 8; also 0125) gavean SR derivative (lane 9) with a single 0-antigen repeating unit(arrowed). In lane 11, strain 19547-2r had lost five high-molecular-weight 0-antigen repeating units relative to its S parent (lane 10).

which was transferred by overnight plate mating at 30°C tothe strain carrying the target plasmid, as a source. Trans-conjugants were cured of pMR5 in broth incubated at 44°C,loss of pMR5 being indicated by loss of its Kmr marker. Theinsertion of Tnl into the target plasmid was indicated by theretention of Apr. Confirmation that Tnl had inserted into atarget plasmid and not the bacterial chromosome was ob-tained by agarose gel electrophoresis (see below). The spottest for bacteriophage sensitivity was as described previ-ously (5).SDS-PAGE with silver staining of LPS. SDS-PAGE and

silver staining were done as described by Hitchcock andBrown (17); only LPS preparations treated with proteinaseK were used. Both stacking and separating gels containedSDS. LPS from E. coli Ra and Rc chemotypes (strainsEH100 and J5, respectively) was from Sigma Chemical Co.DNA preparation and estimation of plasmid molecular

weights. DNA was prepared by the method of Birnboim andDoly (4). Agarose gel electrophoresis was done with plasmidDNA supercoils, which were compared with standard plas-mids.

RESULTS

Characteristics of 0-antigen-deficient mutants of diarrhe-agenic E. coli strains. The following isogenic pairs werestudied by SDS-PAGE with silver staining to characterizetheir LPS content (Fig. 1): B75S, B76R, 5866-2S, 5866-2R,6881-2S, 6881-2SR, 19547-2S, 19547-2r (R, S, SR, and r referto the phenotypes). Rif' mutants (suffix 2) were not used forB75S and B76R, since the wild-type strains grew better. Thefastest-migrating band represented unsubstituted core LPS(lipid A plus R core), whereas the slower-migrating ladder ofbands represented LPS molecules substituted with progres-sively increasing numbers of 0-antigen tri- to pentasaccha-ride units (17, 18). The following numbers of bands werevisible on the actual gels: B75S (09), 10; 5866-2S (0125), 22;6881-2S (0125), 27; 19547-2S (0128), 21; 19547-2r (derivativeof 19547-2S), 16. Lanes 1 and 2 show LPS from Rc and Rastrains, respectively; E. coli K-12 strain JE2571R, clearly of

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2334 BRADLEY

TABLE 2. Effect of S, R, and SR phenotypes on the ability of 0125 diarrheagenic E. coli strains to receive plasmids

Mating Transfer frequency for recipients' Ratio of Transfer frequency for recipients' Ratio ofPlasmid" systeMb frequencies frequenciessystem5866-2S 5866-2R (R/S)' 6881-2S 6881-2SR (SR/S)'

F::Tn521 L 7.3 x 10-7 6.9 x 10-5 95 7.1 x 10-4 2.1 x 10-1 296R144drd3 L 8.3 X 10-6 3.3 x 10-3 398 3.0 x 10-' 8.5 x 10-' 3N3 S 4.3 x 10-6 3.3 x 10-4 77 2.4 x 10-6 6.9 x 10' 287,500RP4 S 1.3 x 10-2 1.6 x 10-2 1 6.0 x 10-3 3.6 x 10-2 6Sa S 3.0 x 10-2 5.8 x 10-2 2 3.3 x 10-5 1.9 X 10-3 58

a Donors were E. coli K-12 strain JE2571 or J53 derivatives.b Mating system used: L, liquid (broth); S, surface (plate).c Number of transconjugants per donor per hour. R/S or SR/S shows the ratio of transfer frequencies into R or SR recipients to S recipients, for the parent strain

indicated. Markers used for selection: F::Tn521, potassium tellurite; R144drd3, kanamycin; N3, tetracycline; RP4, kanamycin; Sa, chloramphenicol.

the Ra chemotype, is in lane 3. Other R strains were similar(see also reference 18). Strain 6881-2SR (lane 9) retainedsmall amounts of the LPS band substituted by a single0-antigen repeating unit (arrowed), thus corresponding tothe SR mutant found by Hitchcock and Brown (17). At theother extreme, strain 19547-2r (lane 11) appeared to have anaverage 0-antigen length that was reduced by about 5 of 21repeating units. The correlation between these results andsensitivities to plasmids and bacteriophages is discussedbelow.

Autoagglutination of the SR strain 6881-2SR with its single0-antigen repeating unit occurred after boiling in saline, aswas the case with R strains. This test does not thereforeexclude the possibility that one 0-side-chain sugar group ispresent. Strain 19547r, which contained LPS with up to 16repeating units, did not agglutinate at all.

Ability of plasmids to enter strains representing different E.coli serogroups. Seven drug resistance plasmids were se-lected for derepression of transfer systems, different incom-patibility groups, and representation of both surface andsurface plus liquid conjugation systems. They were tested bycross-streak mating for transfer into diarrheagenic E. colistrains of different serogroups (Table 1), together with the R,SR, and r derivatives described above. Confluent transfer(similar to a cross with an E. coli K-12 recipient), reducedtransfer, or no visible transfer was recorded (see Materialsand Methods). Being qualitative, individual transfer testswere less significant than the patterns of transfer for singlestrains and overall patterns within a given serogroup. Moststrains of serogroups 055 and 0157 received the majority ofthe plasmids, but plasmids did not transfer so well into 0125strains and transferred very poorly into representatives ofserogroups 0111 and 0126.With some exceptions, there was a degree of similarity in

plasmid sensitivity patterns among strains within a sero-group. If the single strains representing additional sero-groups were representative of their group, strains in sero-groups 09, 0124, 0127, 0128, and 0142 would probably besusceptible to plasmid entry, unlike those in serotypes 026and especially 0119. These generalizations are naturally oflimited value.There were plasmid sensitivity differences between 5866-

2S and 5866-2R and between 6881-2S and 6881-2SR; the Rand SR strains received more plasmids than did any S strainin the 0125 serogroup, including their parents. The 09 strainB75-2S and its derivative B76-2R had the same plasmidsensitivity patterns as did the pair 19547-2S and 19547-2r.Some plasmids had wider serogroup host ranges than others(see Discussion).

Effect of R and S phenotypes on the ability of plasmids toenter strains of diarrheagenic E. coli. To demonstrate un-

equivocally that plasmid entry could be blocked by 0 sidechains, quantitative matings with five plasmids were done todetermine whether the SR strain and a representative Rstrain (Ra chemotype) were better recipients than theirisogenic S parents. Table 2 shows the improvement obtained(ratio of frequencies for R and S) when most (6881-2SR) orall (5866-2R) 0 side chains were absent. This value wassubstantial (>10) in more than half the experiments. Thepresence of a single 0-antigen repeating unit in strain 6881-2SR did not appear to affect plasmid entry; 6881-2SR wasgenerally more competent as a recipient of plasmids thanstrain 5866-2R was. This was probably due to DNA restric-tion when F::Tn521 transferred poorly into 5866-2R. Cer-tainly the F pilus receptor was covered by 0125 side chains,as demonstrated by the 6881-2S and 6881-2SR matings(Table 2). A similar situation existed with the plasmid N3.The inhibition of plasmid transfer by 0 side chains on therecipient indicated that either the donor conjugative pili wereprevented from reaching receptor molecules or that thecell-to-cell surface contact required for pore formation andDNA transfer was being blocked, or both. The matter wasresolved by determining transfer frequencies in situations inwhich the donor rather than the recipient was covered by 0side chains. S donors were compared with R ones with E.coli K-12 as the recipient (Table 3), plasmids and hosts beingchosen for a significant R/S transfer frequency ratio in Table2 (strains 5866-2R and 5866-2S carrying R144drd3 grew toopoorly for use). When plasmid host strain 6881-2S, 6881-2SR, or 5866-2S was the donor, transfer frequencies wereclose to those obtained with E. coli K-12 isogenic matings

TABLE 3. Plasmid transfer frequencies for serogroup 0125 S, R,or SR isogenic donors in matings with the R recipient

E. coli K-12 strain JE2571-1

Donor strain Transferfrequency'

5866-2S(F::Tn521) ............... 4.4 x5866-2R(F::Tn521).............. 6.5 x6881-2S(F::Tn521) ............... 9.9 x6881-2SR(F::Tn521) .............. 5.6 xJE2571-2R(F::Tn521) ............... 1.1 x5866-2S(N3) ............... 1.1 x5866-2R(N3)............... 4.0 x6881-2S(N3) ............... 1.6 x6881-2SR(N3) ............... 2.0 xJE2571-2R(N3)............... 9.3 x

lo-,10-2lo-,lo-,100100lo-'100100l16

' Number of transconjugants per donor per hour. Matings for F::Tn521were done in broth with potassium tellurite transconjugant selection, and N3matings were done on plates with tetracycline selection. Counterselectionagainst all donors was done with nalidixic acid (see Materials and Methods forconcentrations).

6 Frequency from reference 13.

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TABLE 4. Drug resistances and plasmid content ofdiarrheagenic E. coli strainsa

Strain Sero- Drug resistance' Plasmid(s)Strain group (transfer frequency)' present (kb)d

19736 055 Apr (2.9 x 10-3), Smr, 63, 52, 7.0,Sur (both 8.3 x 10-5) 3.8

EC225 055 None (NT), Apr Tnl 55, 7.2, 4.7(<1.4 x 10-7)

2568 0111 None (NT) 83, 66, 11,9.0, 5.8

6378 0111 None (NT) 83, 66, 13,10

EC297 0125 None (NT) None6881 0125 None (NT), Apr Tnl 89, 31, 14,

(<1.1 X 10-7) 6.0, 5.45866 0125 None (NT) None18137 0126 None (NT) 70, 3.11709 0126 None (NT) 135, 3.87777 0157 None (NT), Apr Tnl 92

(<2.0 x 10-8)1140 0157 Tcr (<1.6 x 10-7), Sur 92, 4.1

(<2.4 x 10-6)B75S 09 Smr (NT, chromosomal) 135, 86, 4.119461 026 None (NT) None19392 0119 Apr (<7.7 x 10-8) 110, 9.2, 8.1,

6.3, 4.018031 0124 None (NT), Apr Tnl (4.6 116

X 10-6)1492 0127 None (NT) 84, 64, 4.119547 0128 None (NT) 110, 89, 7522539 0142 Apr, Smr, Sur, Tcr 110, 93, 8.3,

(all <2.0 x 10-7)e 6.4, 3.8

a Two representative strains from each group in Table 1 were selected,together with the single representatives of serogroups. The same order ispreserved. Approximate molecular weights of plasmids were estimated (seeMaterials and Methods).

b Drugs tested: ampicillin, chloramphenicol, gentamicin, kanamycin, strep-tomycin, spectinomycin, sulfonamides, tetracycline, and trimethoprim.

c Number of transconjugants per donor per hour by plate mating (suitablefor all conjugation systems). NT, Not tested. If all markers did not transfertogether, frequencies are given for each. If no markers transferred, the limit ofdetection for each is given. For strains indicated, resident plasmids werelabeled with transposon Tnl (Apr) to allow transconjugant selection withampicillin.

d Determined by agarose gel electrophoresis. While relevant, resourcesprevented the determination of plasmid incompatibility groups.

e None of the markers transferred. The limit of detection given is an averagefrequency obtained for the four markers (range, <4.1 x 10-8 to <6.7 x 10-7transconjugants per donor per h).

(Table 3, JE2571-2R donors). With 5866-2R donor strains,frequencies were somewhat lower than with S donors,probably because this host strain grew more slowly than theothers, perhaps synthesizing fewer conjugative pili. 0 sidechains therefore had no effect when present on donor cells,but only when on recipients, so that cell-to-cell contact wasnot prevented.

It appeared that the mutation causing the R phenotype,which was selected with rifampin or novobiocin (see Mate-rials and Methods), was most frequent in 0125 strains,because the only Ra mutants were from this serogroup.Unsuccessful attempts were made to isolate R mutants fromall serogroups by growing 25 strains with rifampin and 7 withnovobiocin.

Resident plasmids of diarrheagenic strains of E. coli. Of 18test strains (wild type), 14 were not resistant to any of aseries of test drugs (Table 4; the B75S Smr chromosomalmarker was laboratory selected). Strain 1337 from a calf wasnot included, since it had several drug resistance plasmidmarkers, undoubtedly owing to the selective pressure of

TABLE 5. Sensitivity of R, SR, and S strains of specific plasmid-bearing diarrheagenic E. coli to bacteriophages

adsorbing to conjugative pili

Sensitivity to bacteriophages of following plasmidb:Host Sero- FC R144drd3 N3 RP4

strain" group

Qp fd I(x PR64 IKe PR4 PRR1 PR4

B75-2S 09 + + - - - - - -B76-2R None NT NT + +5866-2S 0125 (+) (+) + + + + - +5866-2R None + + + + + + + +6881-2S 0125 + (+) + (+) (+) + - +6881-2SR None + (+) + (+) (+) + (+) +19547-2S 0128 NT NT - - (+) - - (+)19547-2r 0128 NT NT + (+) - - - (+)

a Tests were done on host strains carrying the plasmids indicated.b All plasmids were transfer-derepressed synthesizing conjugative pilus

receptors constitutively. Grading of spot test clearing: +, slightly hazy toclear; (+), well defined but hazy or isolated plaques; -, no visible clearing;NT, not tested because the plasmid could not be transferred to the test strain(owing to poor growth with B76-2R). When both phages for a plasmid failed togive any clearing, the plasmid was checked for transfer derepression bycross-streak mating. Phage types: Q1, Iax, PRR1, RNA containing (shaftadsorbing); fd, PR64 (equivalent to PR64FS), IKe, filamentous (tip-adsorb-ing); PR4, lipid containing (tip adsorbing). None of the host strains withoutplasmids was lysed by any of the phages.

c F::Tn5 (Kmr) or F::Tn521 (Ter) was used (see Materials and Methods).

antibiotics used in cattle feed. Two strains (0125 isolatesEC297 and 5866) had no detectable plasmids, but the remain-der, including those with no drug resistance markers, allharbored one or more plasmids large enough to contain bothtransfer operons and Inc genes (>ca. 30 kilobase pairs [kb]).Most isolates had a variety of small plasmids (<15 kb).Only one of the four strains carrying drug resistance

markers (19736, Apr Smr Sur) was able to transfer them to E.coli K-12 strain JE2571-1; furthermore, the transfer frequen-cies were low. When the 116-kb resident plasmid of strain18031 (0124) was tagged with transposon Tnl, it was able totransfer the added Apr marker at a low frequency. The otherthree transposon-labeled resident plasmids were nontrans-ferable.

Bacteriophage sensitivity patterns of diarrheagenic E. coli.Since none of the strains used was encapsulated, capsular Kantigens did not influence the following observations. Inmost cases, plasmid-specific phages, which adsorb to conju-gative pili, were not prevented from infecting S strains by theO side chains (Table 5). However, RNA phage Ia and thefilamentous phage PR64, which adsorb to the shafts and tipsof thin flexible I1 pili, respectively (see Materials and Meth-ods) (9, 15), infected B76-2R(R144drd3) and 19547-2r(R144drd3) but not their S parents carrying R144drd3.Thus, 09 and 0128 antigenic side chains inhibited Ia andPR64 infection, but 0125 side chains did not. Likewise, theP-pilus-specific RNA phage PRR1 (shaft adsorbing) infectedstrains 5866-2R and 6881-2SR but not their S parents.However, the tip-adsorbing lipid phage PR4 was able toinfect R and S strains equally well. This was not surprising,since the phages are structurally different and have differentmodes of infection (10). The S strain 19547-2S was lysedweakly by filamentous phage IKe, but its r derivative 19547-2r was not; the reason for this was not investigated. In casesin which phages did lyse their plasmid-carrying hosts, 0 sidechains did not prevent constitutive pilus expression andfunction (probably retraction), since both are required forviral infection.The effect of 0 side chains on sensitivity to 10 plasmid-

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TABLE 6. Sensitivity of diarrheagenic E. coli strains to bacteriophages not adsorbing to conjugative pili

No. of Sensitivity to following bacteriophageb:Strain Serogroup plasmids T2 T5 T6 K3 TuIb Tula Lambda' P1 6SR 09-1

entering" (OmpF, Ttr) (TonA) (Tsx) (OmpA) (OmpC) (OmpF) (LamB) (LPS) (LPS) (09)

JE2571R None 7 + + + + + + - - + -

B75-2S 09 5 + - - + (+) - - - +B76-2R None 5 + + + _ + + _ (+) +5866-2S 0125 3 - - - - - - - - - -5866-2R None 5 + + + - + + - (+) +6881-2S 0125 3 (+) - - - - (+) - - - -6881-2SR None 5 + + + - + + - +19547-2S 0128 6 + - - - - + - - - -19547-2r 0128 6 + - + - + +19461-2S 026 3 - - + - - - - - - -17023-2S 055 7 + - (+) + - (+) -2568-2S 0111 0 - - + + - - - - - -19392-2S 0119 0 - - (+) + - (+) - - - -18031-2S 0124 5 - - - - - - - - - -1709-2S 0126 1 (+) - (+) - - - - - - -1492-2S 0127 5 - - (+) + + - - - - -22539-2S 0142 7 - - + - - - - - - -2166-2S 0157 5 + - - + - (±) - - - -

a From Table 1. Seven plasmids were tested for entry by cross-streak mating; the number shown indicates those which transferred at a detectable level (seeMaterials and Methods).

b Grading of spot test clearing; +, slightly hazy to clear; (+), well defined but hazy or isolated plaques; -, no visible clearing. Receptor proteins or LPS arein parentheses (09 = 09 0 side chains).

Phage lambda lysed E. coli C2 on which it was propagated. Strain JE2571 did not plate the phage, since it had been lysogenized by it.

nonspecific bacteriophages with known cell envelope recep-tors was demonstrated by comparing the phage lysis patternsof strains whose 0-antigen repeating units had been reducedor removed, with those of their S parents. R and SR strainswere sensitive to more phages then their S parents were(Table 6). When the wild-type S strain was resistant to aphage and the R derivative was sensitive, it was concludedthat the protein or LPS receptor for the phage was shieldedby the 0-antigenic side chains. The sensitivity of strain19547-2r to two more phages than its S parent is discussedbelow. An anomaly which could not be explained was foundwith B75-2S, which plated the OmpA-specific phage K3, andits R derivative B76-2R, which did not. Several S strainsfrom different serogroups (Table 1) were also tested; therewas no correlation between the number of plasmids thatcould enter an S strain and the number of phages that couldinfect it. For example, strains 2568-2S and 19392-2S wereresistant to all the plasmids tested in Table 1 and yet weresensitive to three phages. Conversely, strain 18031-2S re-ceived five of seven plasmids yet was resistant to all thebacteriophages. Phage lambda did not lyse any of the strains,many of which were doubtless lysogenised by a similarphage, and 09-1 was specifc for the 0 side chains of its Shost B75-2S. Phages T5, P1, and 6SR lysed only R strains(P1 also lysed 6881-2SR), suggesting that the protein TonAand the LPS core were susceptible to shielding by 0 sidechains (see Discussion).

Rif' clones of S strains (suffix-2S in Tables 1 and 6)generally gave the same phage sensitivity patterns as didtheir wild-type parents, with the following exceptions.19547S (wild type) was resistant to all 10 phages, whereas19547-2S was sensitive to T2 and Tula (suggesting thatOmpF had been exposed), and 19547-2r was sensitive to T2,T6, TuIb, and Tula (suggesting that OmpC, OmpF, and Tsxwere exposed). 17023-2S differed from its wild-type parent inthat it acquired normal sensitivity to T2 and weak sensitivityto T6 and Tula; 19392-2S also acquired weak sensitivity toT6 and Tula.

DISCUSSION

This necessarily limited study leads to the conclusion thatsome serogroups of diarrheagenic E. coli are generallyreceptive to plasmids, whereas others are not; plasmid-receptive strains can easily acquire plasmid-borne resistanceto antibiotics. This is illustrated by 055 strain 19736; itsoverall sensitivity to plasmids (Table 1) probably accountsfor the presence of one or more transferable drug resistanceplasmids (Table 4). Some plasmids had a wider range ofrecipients than others. R144drd3 (Incml + B), RP4 (IncP),and Sa (IncW) transferred well into strains of all serogroupsexcept 0111 and 0126. RP4 (equivalent to R1822) has abroad interspecific host range (26), and this might be linkedwith its ability to enter strains of many serogroups. RP4 andSa transferred into almost the same diarrheagenic strains(Table 1), suggesting a common conjugative pilus receptor.5866-2S and 6881-2S differed in their competence to receiveplasmids, even though they were in the same serogroup(0125). The best interpretation of this is that the 0125 sidechains were arranged on outer membranes with differentmolecular configurations, so that different pilus receptorswere exposed. Alternatively, plasmid loss due to factorssuch as restriction could have occurred after entry. What-ever the reason, conclusions can be drawn only by compar-ing isogenic pairs of S, with R, SR, or r strains. With both5866 and 6881 pairs, R or SR mutants were sensitive to moreplasmids, showing that side chains covered pilus receptors.

Quantitative matings with R and S recipients (Table 2)demonstrated that the 0-antigenic layer inhibited plasmidtransfer when present on the recipient. However, the 0 sidechains had no effect when present on donor strains, conju-gative pili being able to grow through them and functionnormally (Table 3). These data showed that 0125 side chainsshielded conjugative pilus receptors for several plasmids.The isogenic pair B75-2S and B76-2R was an exception tothe observation that R strains were more competent toreceive plasmids than S ones; the probable reason was that

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PLASMID ENTRY INTO DIARRHEAGENIC E. COLI 2337

B75-2S was already able to receive five of the seven testplasmids. It must be remembered that there are othermechanisms by which a plasmid can be prevented fromentering a cell. Surface exclusion, which is determined by aresident plasmid and prevents entry by another plasmid ofthe same incompatibility group, is obviously relevant. An-other mechanism is DNA restriction. Neither would beaffected by the absence of 0 side chains, and they would beoperative in both members of isogenic R and S pairs.The absence of drug resistance markers in diarrheagenic

E. coli did not imply the absence of plasmids (Table 4).Indeed, strain 18031 (EIEC) carried a self-transmissibleplasmid with no drug resistance genes. Levine (21) describesplasmids which are characteristic of EPEC strains. Theirsizes are about 90 kb. They have been implicated in deter-mining pathogenicity traits, and some are undoubtedly rep-resented in Table 4. They could conceivably be mobilized bydrug resistance plasmids, transferring pathogenicity traits toother strains of E. coli. Like those in strain 18031, allplasmids are potential targets for drug resistance trans-posons which could originate from an incoming plasmid.The efficiency with which 0 side chains cover various

outer membrane proteins is demonstrated by resistance tomany bacteriophages (Table 6). The work of Van der Ley etal. (32), using R and S pairs of strains from serogroups 015(ETEC), 041, and 0156 (last two not classified), has beenextended here to 09, 0125, and 0128 serogroups (last twoEPEC). R strains were sensitive to more bacteriophagesthan were their S parents (also to more plasmids). Strains5866-2R and 6881-2SR differed in their sensitivity to phage6SR, which requires a complete LPS core for adsorption (seereference 3 for a review). Since both strains had 0125parents and differed only by the single 0-antigen repeatingunit present on 6881-2SR (Fig. 1), it might be inferred thatthis was sufficient to shield that part of the core acting as the6SR receptor. However, this is not necessarily so, sincemost of the LPS molecules of strain 6881-2SR look the sameas those of 5866-2R (Fig. 1); there is probably some otherreason for the different responses to phage 6SR. Both strainswere sensitive to phage P1, which also adsorbs to anincomplete LPS core (chemotype Rc [3]) terminating in aglucose residue linked to heptose. In addition, P1 can adsorbto a complete LPS core (Ra), such as was present on theabove strains. Strain 19547-2r was sensitive to phages T6 andTuIb, whereas its S parent was not. SDS-PAGE with silverstaining revealed a loss of five 0-antigen repeating units in19547-2r, and it can be inferred that they shielded the proteinreceptors of phages T6 and TuIb (Tsx and OmpC, respec-tively). If a strain received a drug resistance plasmid, itwould become susceptible to plasmid-specific phages. It wasfound here that 0 side chains did not usually cover receptorsfor these phages (Table 5), which may exert some control onthe spread of drug resistance plasmids by killing organismsharboring them.

ACKNOWLEDGMENTS

I thank Jeannette Whelan for excellent technical assistance,especially for preparing high-quality agarose and polyacrylamidegels. S. P. Howard, Memorial University, and C. Whitfield, Univer-sity of Guelph, were most helpful, particularly with respect toSDS-PAGE and silver staining. I also thank those who kindlysupplied bacterial strains and phages, particularly M. Finlayson,University of Alberta.The work was funded by grant MT5608 from the Medical Re-

search Council of Canada.

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