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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1987, p. 1768-1774 Vol. 53, No. 80099-2240/87/081768-07$02.00/0Copyright ©3 1987, American Society for Microbiology

Survival and Virulence of Copper- and Chlorine-StressedYersinia enterocolitica in Experimentally Infected Mice

AJAIB SINGH AND GORDON A. McFETERS*Department of Microbiology, Montana State University, Bozeman, Montana 59717

Received 6 February 1987/Accepted 11 May 1987

The effect of gastric pH on the viability and virulence of Yersinia enterocolitica 0:8 after exposure to sublethalconcentrations of copper and chlorine was determined in mice. Viability and injury were assessed with anonselective TLY agar (tryptic soy broth containing lactose, yeast extract, and agar) and two selective media,TLYD agar (TLY agar plus sodium deoxycholate) and CIN agar (cefsulodin-Irgasan-novobiocin agar). Bothcopper and chlorine caused injury which was manifested by the inability of the cells to grow on selective media.CIN agar was more restrictive to the growth of injured cells than TLYD agar. Injury of the exposed cells wasfurther enhanced in the gastric environment of mice. Besides injury, the low gastric pH caused extensive lossof viability in copper-exposed cells. Lethality in the chlorine-exposed cells was less extensive, and a portion ofthe inoculum (5.2 x 105 of 1 x 107 inoculated cells) reached the small intestine 5 min postinoculation. Noadverse effect on the injured cells was apparent in the small intestine, and a substantial revival (approximately70%) of the injury occurred in 3 to 4 h after intraluminal inoculation. The virulence of chlorine-stressed Y.enterocolitica in orally inoculated mice was similar to that of the control culture, but copper-stressed cellsshowed reduced virulence. Virulence was partly restored by oral administration of sodium bicarbonate beforethe inoculation of copper-exposed cells. Neutralization of gastric acidity had no effect on the virulence of thecontrol or chlorine-stressed cells. The results of this study indicate that the extensive injury caused by the lowgastric pH does not affect the virulence potential of chlorine-exposed cells. However, extensive cell death in themouse stomach is responsible for the reduced virulence of the copper-stressed bacteria.

Enumeration of coliforms is the most commonly employedmethod for evaluating the bacteriological quality of water.However, in recent years it has become increasingly evidentthat a variety of physical and chemical agents can causeinjury in indicator bacteria (6, 15, 18). This injury is mani-fested by a loss in the ability of microorganisms to growunder selective conditions that are not restrictive for undam-aged cells (1, 9). Thus, injured bacteria are not detectedwhen commonly accepted selective media are employed (14,15). In addition, injured cells may constitute the majority ofcoliforms present in treated drinking water (18). Thesefindings support the conclusion that the artifactually lowdetermination of the actual numbers of indicator organismspresent, or their apparent absence, resulting from injury canlead to a significant underestimation of the potential publichealth hazard (17). This can have serious consequences ifwaterborne pathogens are also present.

Pathogens, like indicator organisms, become injured afterexposure to stressors in water and become sensitive tovarious selective agents. Potential health hazards associatedwith stressed organisms are evident from the study offoodborne pathogens. The virulence of Staphylococcus aii-reus (8) and Salmonella gallinarum (26) remained unalteredafter injury caused by freeze-drying. Collins-Thomson et al.(4) reported undiminished enterotoxin production by S.aureus after recovery from heat injury. The virulence ofinjured waterborne pathogens has received attention onlyrecently. A reduced attachment of chlorine-stressed entero-toxigenic Escherichia coli (ETEC) for human leukocytes inan in vitro system was described by Walsh and Bissonnette(28). We reported injury induced by copper and chlorine insome of the waterborne bacterial pathogens and studied its

* Corresponding author.

effect on their virulence determinants (16, 23-25). The injurydescribed in these studies was induced under controlledlaboratory conditions that were similar to those described asinjuring coliforms in treated distribution water systems (6,14, 15). The chlorine-induced injury caused a loss in theability of ETEC and Salmonella typhimurium to attach toHenle cells, whereas Yersinia enterocolitica lost its ability toinvade HeLa cells (16). Copper-induced injury in Y. entero-colitica caused reduced virulence in mice after intraperito-neal inoculation (23). The in vitro recovery of copper-injuredETEC cells occurred in a nutrient-rich as well as in a definedmedium. This process was demonstrated by growth and theproduction of heat-stable toxin (24). These observationswere further extended to in vivo systems, thereby demon-strating recovery, growth, and enterotoxin production bycopper- and chlorine-injured ETEC cells in the small intes-tines of mice and rabbits (25).

It is evident that sublethally injured pathogens show atemporary reduction or inability to express their pathogenictraits. Hence, when placed in a nonselective in vitro envi-ronment or in a suitable in vivo system, recovery from injurytakes place and is followed by growth and expression ofpathogenic properties. However, the question whethersublethally damaged enteropathogens can cause diseaseafter passing through the healthy stomach without beingkilled or undergoing additional stress of exposure to lowgastric pH has not been addressed. Thus, the present inves-tigation was undertaken to determine the survival of copper-and chlorine-treated Y. enterocolitica in the upper gastroin-testinal tract of experimentally infected mice. The results ofthese studies show that copper- and chlorine-injured cellsresponded differently when introduced into the upper diges-tive system but that their pathogenic potential was retainedafter injury.

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MATERIALS AND METHODS

Test organism. Y. enterocolitica 0:8 E661 was obtainedfrom D. A. Schiemann, Department of Microbiology, Mon-tana State University, Bozeman. The culture was initiallygrown in tryptic soy broth supplemented with 0.3% yeastextract for 24 h and harvested in 1% peptone with 40%glycerol. Portions (1 ml) of this suspension were frozen andstored at -70°C for use as stock cultures.

Preparation of injured cells. The culture was grown intryptic soy broth containing 0.3% yeast extract at 22°C for 24h. The harvested cells were washed twice with sterilizedreagent-grade water (Milli Q water system; Millipore Corp.,Bedford, Mass.). Injury by copper was induced by suspend-ing approximately 5 x 108 cells per ml in 1.68 x 10-' Minorganic carbon buffer (6) containing 0.75 mg of copper perliter at 4°C as described earlier (24).To induce chlorine injury, we exposed the washed cells of

Y. enterocolitica (5 x 108 cells per ml) to 0.8 mg of chlorineper liter for 10 min at 4°C as described previously (25).

Assessment of injury. Injury in copper- and chlorine-exposed cells was assessed by the difference between thenumber of CFU per milliliter on nonselective (tryptic soybroth supplemented with 1% lactose, 0.3% yeast extract,and 1.5% agar; TLY agar) and a selective TLYD medium(TLY agar containing 0.1% sodium deoxycholate), and re-sults were expressed as percent injury within the populationstested. In addition, a highly selective medium, cefsulodin-Irgasan-novobiocin (CIN) agar (22) (Yersinia selective agar;Difco Laboratories, Detroit, Mich.), was used. This mediumgives high recovery of Y. enterocolitica from feces withoutany enrichment procedure and significantly reduces the levelof background organisms (22). Viable cell count on each ofthese media was done by surface plating the samples onduplicate plates after making appropriate dilutions.

Animals. Female CD-1 mice (8 to 10 weeks of age) wereused. The mice were obtained from Charles River BreedingLaboratories, Inc. (Wilmington, Mass.) and maintained atthe Animal Resources Center, Montana State University.Development of animal model. Experiments were done to

determine gastrointestinal motility and the appropriate timeintervals for assessing the viability and injury of the Y.enterocolitica cells after inoculation of mice. Evans blue dye(0.1 ml of 4% aqueous solution) was administered orally totwo groups (12 mice in each group) of normally fed andfasted (24 h) mice by using a 20-gauge, 1.5-in. (3.8-cm)curved animal-feeding needle (Popper and Sons, Inc., NewHyde Park, N.Y.). Mice in the third group (12 to 15 mice,fasted for 24 h) were injected directly into the stomachduring laparotomy under anesthesia. The movement of thedye from the stomach into the small intestine was assessedby measuring the colored portion of small intestine afterkilling three mice at each timed interval. The results (Table1) indicate that the dye moved rapidly in unanesthetizedanimals irrespective of their feeding status. It approachedthe ileocecal junction 15 min after inoculation, and clearingof the dye from the anterior portion of the small intestine wasobserved in 30 min. In anesthetized mice the movement ofthe dye was considerably slower, apparently owing to re-

duced gastrointestinal motility.To determine the effectiveness of sodium bicarbonate in

neutralizing the gastric acidity, we administered 0.1 ml of0.1, 0.5, or 1.0 M NaHCO3 orally to a group of 15 mice.Three animals were killed at 0-, 5-, 15-, 30-, and 45-minintervals, and their stomachs were externalized and incisedlongitudinally. The pH of stomach contents was determined

by using narrow-range pH indicator strips (EM Science,Cherry Hill, N.J.). The gastric acidity of the mice whichwere neutralized with 0.1 or 0.5 M NaHCO3 declined con-siderably within 5 to 15 min. However, the gastric pH of the1.0 M NaHCO3-inoculated mice remained at 7.4 up to 45 minafter administration. Thus, the latter treatment was em-ployed for neutralizing gastric acid in the later experiments.

Distribution of Y. enterocolitica after orogastric inoculationin mice. Food was withheld for 24 h before inoculation, butanimals used in these experiments had free access to water.For oral inoculation the method of Carter and Collins (2) wasused. Approximately 107 cells of untreated Y. enterocoliticain 0.1 ml of 0.9% NaCl (saline) solution were administeredorogastrically with a 21-gauge animal-feeding needle (Popperand Sons, Inc.) attached to a tuberculin syringe. Theinoculum also contained Evans blue dye (2 drops of 2%aqueous solution per ml) as a marker. At timed intervals themice were killed and the stomach and intestine from eachanimal were removed. The stomachs were incised and rinsedthoroughly with 10 ml of phosphate-buffered saline (pH 7.4)containing 0.1% gelatin. A portion of the intestine (markedby the dye) was removed, rinsed with sterile water, andplaced in 50 ml of cold sterile phosphate-buffered saline. Thetissue was homogenized for 1 min in a laboratory homoge-nizer (Hi-Speed model 45; The VirTis Co., Inc., Gardiner,N.Y.). The eluted sample from the stomach and the intesti-nal homogenate were surface plated on TLY, TLYD, andCIN agar after appropriate dilutions in sterilized Milli Qwater. The results were expressed as the total number ofviable cells recovered per stomach or small intestinal portionof the inoculated mice.

Survival of Y. enterocolitica after intragastric inoculation ofanesthetized mice. The mice were fasted for 24 h and anes-thetized by intraperitoneal administration of sodium pento-barbital (0.06 mg/g of body weight). The abdomen of eachmouse was opened by a midline incision, and the stomachwas exposed. A ligature was applied 1 to 2 cm below thepyloric end of the stomach. Y. enterocolitica cells (approx-imately 107 cells in 0.1 ml of saline solution) were inoculateddirectly into the stomach with a 30-gauge needle. Two micewere killed at timed intervals, and the stomach of eachmouse was removed and processed separately, as describedabove, to assess the survival and injury in the inoculatedbacterial preparations.

In some experiments the above procedure was modifiedby applying the ligature on the small intestine approximately10 to 12 cm below the pyloric end of the stomach. Thisallowed the movement of inoculum from the stomach into aspecified portion of the small intestine. After appropriateincubation two mice were killed, and both the stomach andthe ligated portion of the intestine from each mouse were

TABLE 1. Movement of Evans blue dye from the stomachinto the small intestine of mice

Intestinal length containing dye (cm)a

Time (min) Unanesthetized miceb Anesthetized

Normally fed Fasted mice" (fasted)

0 0.5 0.4 1.5 0.4 05 16.3 ± 2.7 13.6 ± 2.0 2.3 ± 1.0

15 38.0 ± 4.3 36.1 ± 3.8 7.8 ± 1.630 41.0 ± 2.1 41.8 ± 5.7 20.2 ± 2.2

a Mean±

standard deviation of at least three determinations.b Dye was delivered into the stomach with a feeding needle.C Dye was injected into the stomach after laparotomy.

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1770 SINGH AND McFETERS

processed separately as described above to assess the viablecounts on all three media. The tissues from animals whichdied before the end of incubation period were not processed.

Effect of sodium bicarbonate on gastric pH and survivabilityof Y. enterocolitica. Sodium bicarbonate (0.1 ml of 1.0 MNaHCO3) was administered orally to mice 10 min beforeinoculation. Copper-exposed Y. enterocolitica cells (107cells in 0.1 ml of saline solution) were injected directly intothe ligated stomach (ligated near the pyloric end) of anesthe-tized mice. After appropriate incubation the stomach wasprocessed as described earlier after the p-I was determined.

Intraluminal inoculation. Both copper- and chlorine-injured Y. enterocolitica cells were inoculated into theligated intestinal loops of anesthetized mice by the methodpreviously described (25), and the percentage of injured cellswas determined with TLY and TLYD media as describedabove.

Determination of lethality in mice after oral administrationof Y. enterocolitica. A total of 70 mice were fasted overnight,transferred into filter-topped cages (five mice in each cage),and divided into seven groups (10 mice in each group). Micein groups 1, 2, and 3 were inoculated orally with 2 x 107 cellsof uninjured, chlorine-injured, and copper-injured Y. entero-colitica, respectively. Groups 4, 5, and 6 were also inocu-lated with the corresponding culture preparations but re-ceived 0.1 ml of 1.0 M NaHCO3 15 min before inoculation,

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FIG. 1. Distribution of Y. enterocolitica in the stomach and smallintestine of orally inoculated mice. CFU on TLY (e---e) and TLYD(O---O) agar in the inoculum. CFU on TLY (0 *) and TLYD(0 O) agar in the stomach and CFU on TLY (O U) andTLYD (O OL) agar in the small intestine after inoculation. Thepercent injury was calculated as described in the text, and values are

shown in the parentheses. Each value represents the mean of fourdeterminations.

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FIG. 2. Viability and injury of untreated and copper-treated Y.enterocolitica before and after intragastric inoculation in anesthe-tized mice. CFU on TLY (S---@), TLYD (O----), and CIN (A---A)agar in untreated culture before inoculation and CFU on TLY(@ 0), TLYD (- *), and CIN (A A) agar in stomach ofafter inoculation. CFU on TLY (O---O), TLYD (O----O), and CIN(A---A) agar in a copper-treated culture before inoculation and CFUon TLY (O---O), TLYD (O O), and CIN (A A) agar instomach after inoculation. The percent injury was calculated asdescribed in the text, and values are shown in the parentheses. Eachvalue represents the mean of five to six determinations.

instead of the saline received by the first three groups. Group7 received 0.1 ml each of sodium bicarbonate and normalsaline.Feed was provided 1 h after the inoculation, and animals

were observed twice daily over a period of 3 weeks todetermine lethality by the method described by Laired andCavanaugh (12). Y. enterocolitica was isolated from the liverand spleen and the small intestine of the dead animals byusing CIN agar.

RESULTS

Distribution of Y. enterocolitica in upper gastrointestinaltract of orally inoculated mice. The mean values of fourdeterminations obtained from two separate trials (a total ofeight mice per trial were used; two mice were sacrificed ateach postinoculation time) are shown in Fig. 1. We foundthat a 10-fold reduction occurred immediately afterorogastric inoculation of untreated Y. enterocolitica cellsuspension and that the number of viable cells recoveredfrom the stomach continued to decrease with time. Theaverage number of CFU detected on the selective (TLYD)medium was lower than that on TLY agar, and the injuredpopulation varied from 45.5 to 82.1%.The average bacterial count obtained from the anterior 10

to 15 cm length of the small intestine (marked by the dye) 5

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TABLE 2. Injury in Y. enterocolitica in the upper gastrointestinal tract of mice

Stomach Small intestine"

Inoculum Time %Ijr"%Ijr(mimn) Viable countb % Injury Viable count % Injury(TLY) TLYD CIN (TLY) TLYD CIN

Unexposed 0 (42 + 15) x 105 54.8 59.5 ND" ND ND5 (11 ± 3) x 105 78.1 80.6 (19 ± 7) x 104 54.5 72.8

15 (25 ± 9) x 104 89.3 93.5 (25 ± 9) x 104 53.0 59.330 (19 ± 11) X 104 86.1 89.2 (29 ± 13) x 104 63.9 77.4

Chlorine exposed 0 (35 ± 11) x 105 81.1 86.8 ND ND ND5 (9 ± 2) x 105 97.0 98.9 (14 ± 6) x 104 92.5 92.9

15 (14 ± 12) x 104 98.4 99.1 (20 ± 13) x 104 96.5 98.030 (6 + 5) x 104 96.6 98.1 (23 ± 16) x 104 97.6 99.1

a Ligated anterior portion (10 to 12 cm) was used.Mean values + standard deviation of three trials (total of six to eight mice).Calculated as [(CFU on TLY agar - CFU on TLYD or CIN agar)/CFU on TLY agari x 100.

d ND, Not determined.

min postinoculation was 5.2 x 105 cells. The movement ofthe dye from the stomach into the entire small intestinallength was observed 15 to 30 min after inoculation, suggest-ing further evacuation of stomach contents into the smallintestine. However, the viable count obtained from the smallintestine did not increase with time (Fig. 1).

Viability of Y. enterocolitica after intragastric inoculation of

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enterocolitica before and after intragastric inoculation in anesthe-tized mice. CFU on TLY (@---@), TLYD (E---E), and CIN (A---A)agar in an untreated culture before inoculation and CFU on TLY(0- ), TLYD (-*), and CIN (A A) agar in stomachafter inoculation. CFU on TLY (O---0), TLYD (0I---L0), and CIN(A---/A) agar in a chlorine-treated culture before inoculation andCFU on TLY (0 0), TLYD (O 0), and CIN (A A) agarin the stomach after inoculation. The percent injury was calculatedas described in the text, and values are shown in the parentheses.Each value represents the mean of five to six determinations.

anesthetized mice. Intragastric inoculation was done afterapplying a ligature at the pyloric end of the stomach ofanesthetized mice to prevent movement of the inoculum.The values in Fig. 2 and 3 show the mean CFIJ obtainedfrom five to six mice. The enumeration of Y. enterocoliticacultures was done on TLY, TLYD, and CIN agar before andafter inoculation. The percentages of injured cells withinthese populations determined on TLYD and CIN agar areshown in parentheses.Recovery of the cells from the stomach immediately after

inoculation varied from 50 to 70%. A reduction in theviability determined by CFU on TLY agar and an enhancedsensitivity of the cells to the selective media used wasobserved after intragastric inoculation. The reduced viabilityin the control cells was not significantly different (Fig. 1)from that of chlorine-treated cells in the stomach (P = 0.05)at 0, 5, and 15 min (tested by Student's t test), althoughsignificantly reduced viability was observed at 30 minpostinoculation (P < 0.001). The viability of the copper-treated culture was more adversely affected in the stomachthan were buffer-suspended control cells (Fig. 2). The differ-ence between control and injured cell suspensions wassignificant after 5 min (P < 0.01) and 15 and 30 min (P <0.001) of gastric incubation.

Intragastric incubation (from 0 to 30 min) of copper- andchlorine-treated bacteria caused an extensive increase intheir sensitivity to TLYD and CIN agar, indicating thatsubstantial portions of the population became increasinglyinjured (Fig. 2 and 3). CFU obtained on CIN agar wereinvariably lower than those on TLYD agar.The mean pH (from five to six mice) of the uninoculated

stomachs of mice was 1.8 + 0.2, and values at 0, 5, 15, and30 min postinoculation were 2.10 + 0.3, 2.5 + 0.2, 2.4 + 0.4,and 3.0 + 0.4, respectively.

Injury in Y. enterocolitica in the stomach and small intestineof intragastrically inoculated anesthetized mice. A ligaturewas applied 10 to 12 cm below the pyloric end of mousestomachs to assess injury in the inoculated cells (control andchlorine stressed) in the gastric environment and after move-ment into the small intestine. This procedure allowed themovement of inoculum from the stomach, but cells remainedconfined within the ligated portion of the small intestine. Theresults obtained from three trials (8 to 12 mice per trial wereused; 2 to 3 mice were sacrificed at each timed interval) areshown in Table 2. An immediate decrease in the viable cellsof the untreated control inoculum (1 x 107 to 42 x 105 cellsat 0 min) was observed. Incubation up to 30 min showed a

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FIG. 4. Viability and injury of copper-treated Y. enterocolitica inthe stomach of mice after neutralizing the gastric acidity. CFU onTLY (O---O), TLYD (O---O), and CIN (A---A) agar in theinoculum. CFU on TLY (0 *), TLYD (0 O), and CIN(A A) agar in the stomach after inoculation. The percent injurywas calculated as described in the text, and the values are shown inthe parentheses. Each value represents the mean of threedeterminations.

further decrease (42 x 105 to 19 x 105 cells per stomachcontent). This could be explained by movement of theinoculum into the small intestine as well as cell death fromgastric acidity. The injury after different periods of incuba-tion varied between 54.8 and 93.5%, and lower cell countswere obtained on CIN agar compared with those on TLYDagar. Y. enterocolitica cells (1.9 x 105) were recovered fromthe small intestine 5 min after inoculation. Further increasesin bacterial numbers at 30 min were not observed.The chlorine-treated cells, like the untreated controls,

showed decreased viability (35 x 105 at 0 min to 6 x 104 at30 min) after intragastric inoculation. However, a largerproportion of the chlorine-treated inoculum was injured inthe stomach as determined with TLYD and CIN agar (81.1 to96.6% and 86.8 to 98.1%, respectively). Also, the chlorine-treated cells that moved into the small intestine showedmore injury (92.5 to 99%) compared with that of the un-treated inoculum (53.0 to 77.4%).

Intragastric incubation of copper-exposed Y. enterocoliticaafter neutralizing the gastric acidity. Administration of so-dium bicarbonate raised the stomach pH to 7.6. Copper-injured cells were inoculated 10 min later when pH 7.4 wasreached and remained unchanged during the entire period ofincubation. The number of CFU on TLY, TLYD, and CINagar in the inoculum before and after intragastric adminis-tration is shown in Fig. 4. The percent injury (determinedwith TLYD and CIN agar) is shown in the parentheses.There was no significant change in bacterial counts on TLY,TLYD, or CIN agar at various incubation times, indicatingthat the neutralized gastric contents did not alter the viabilityor injury of copper-treated Y. enterocolitica cells.

Effect of intraluminal incubation on injured Y. enterocoliticacells. The objective of this experiment was to determinewhether intraluminally inoculated Y. enterocolitica cells thatwere injured with copper and chlorine recovered their abilityto grow in the presence of sodium deoxycholate. In anearlier study (27) it was observed that copper-injured E. coli

TABLE 3. Effect of intraluminal inoculationa on copper- andchlorine-treated Y. enterocolitica in mice

Incubation bRecovery

Treatment Incuaion % Cells injuredb of injurytime(h)(%

Copper 0 76.5 ± 6.5 (n = 6)4 23.2 + 7.8 (n = 6) 69.6

Chlorine 0 87.8 ± 8.5 (n = 3)3 25.4 ± 4.9 (n = 3) 71.0

a Approximately 107 cells per intestinal loop were inoculated.b Injury was determined by using TLY and TLYD agar as described in the

text. Mean ± standard deviation of the mean. n, Number of loops examined.

recovered from injury and initiated active multiplication 4 hafter intraluminal incubation, whereas chlorine-treated cellsrequired 3 h. Hence, in this study determinations of viabilityand injury were made after 4 and 3 h, respectively. Theresults shown in Table 3 indicate that recovery of bothcopper- and chlorine-injured Y. enterocolitica (69.6 and71.0%, respectively) took place in the small intestine. Re-covery was manifested by the restoration of colony-formingability on a selective medium.

Virulence of injured Y. enterocolitica after orogastric chal-lenge in mice. Groups of mice (10 in each group) werechallenged with control as well as copper- or chlorine-treated Y. enterocolitica cells. Comparison of mouse lethal-ity in the groups with and without administration of sodiumbicarbonate before inoculation was made, and the results areshown in Fig. 5. Although extensive injury was expected inthe cells after intragastric inoculation of chlorine-treated Y.

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FIG. 5. Lethality in mice after orogastric inoculation ofuntreatad, chlorine-treated (A), and copper-treated (B) Y. enteroco-litica cells. Normal saline was administered before inoculation withuntreated (O---O), chlorine-treated (O *), and copper-treated(A) cells. NaHCO3 was administered before inoculation with un-treated (O---0), chlorine-treated (0 O), and copper-treated (A)cells.

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VIRULENCE OF INJURED Y. ENTEROCOLITICA 1773

enterocolitica, the injury did not appear to alter the virulenceof the cells in mice (Fig. 5A). Furthermore, sodium bicar-bonate administration did not enhance the virulence ofcontrol or chlorine-injured cells in mice. The lethality in-duced by cells stressed with copper (Fig. SB) was low whencompared with that of the control (50 and 90%, respec-

tively). Administration of sodium bicarbonate before oralinoculation enhanced the lethality from 50 to 80%.

DISCUSSION

Y. enterocolitica has been isolated from various animalsources (11, 27), although swine are recognized as theprimary reservoir of biotypes implicated in humanyersiniosis (27). Isolates from inanimate reservoirs such as

water are rarely of the same biotypes that cause humanillness (21). However, accidental contamination of drinkingwater has been documented in several outbreaks (7, 13, 20).

Several factors in drinking water are known to stressbacteria, but copper and chlorine appear to be most impor-tant in this process (6, 15, 17). Injury in Y. enterocoliticacultures after exposure to sublethal concentrations of copper(23) and chlorine (16) has been reported; this resulted in thereduced growth on TLYD agar. In the present investigationwe found that larger proportions of copper- and chlorine-exposed Y. enterocolitica manifested injury on CIN agar

than on TLYD agar. CIN agar is a highly selective mediumand is recommended for the recovery and isolation of Y.enterocolitica (22). However, the results of our study sug-

gest that stressed bacteria are not recovered on this mediumand that prior resuscitation is necessary. This finding is ofmore importance when examining stressful environmentssuch as disinfected water and wastewater for this organism.The low gastric pH of mice (ranging from 1.8 to 3.0) was

responsible for additional injury in Y. enterocolitica, and thiseffect was more dramatic in copper-stressed cells than inchlorine-stressed cells. Injury of E. coli in an in vitro acidenvironment was described by Przybylski and Witter (19).They reported injury in more than 99% of the viable cellsafter 60 min of exposure to 0.3 M sodium acetate buffer, pH4.2. We found that in addition to causing injury, a higherbacterial lethality was induced by gastric acidity in copper-

injured cells than in chlorine-injured cells. The reason forthis difference are not apparent, but it is possible thatvariations in severity and sites of cellular damage are respon-

sible. Intragastric incubation of copper-treated cells afterneutralizing the gastric pH did not produce any of theadverse effects.At 5 min postinoculation, 5.2% of the inoculated Y.

enterocolitica cells were recovered from the anterior portionof the small intestine. On extended incubation, the inoculumwas spread throughout the entire length and could result inlow recovery. This may be the reason for the reducednumbers of organisms detected at later time intervals fromthe small intestine.By using the technique of ligating the anterior portion of

the small intestine we demonstrated that various proportionsof both the unexposed and chlorine-exposed cultures be-came nonculturable on the selective media used and thatinjury increased in the population surviving in the stomach.Further cell death or injury was not observed in the cellsreaching the small intestine. These findings are consistentwith those reported by Dixon (5), who failed to observe theantibacterial action of the small intestines of rats on inocu-lated bacteria. An acid-induced injury in E. coli has beendescribed by Przbzylski and Witter (19) in an in vitro system.

These authors also observed the recovery of injury after thestressed bacteria were placed in a suitable environment. Wedetermined that stressed Y. enterocolitica cells were able torepair the injury in 3 to 4 h in the intestinal lumen of mice.Similar findings on the in vivo recovery of injured ETEC andenteroinvasive E. coli in the mouse gut have been reported(25).

Virulence as determined by lethality caused by controland chlorine-injured Y. enterocolitica in mice with or with-out prior neutralization of gastric acidity was similar. Theseresults demonstrate that gastric acidity causes extensive butreversible injury in chlorine-stressed Y. enterocolitica cells.It is apparent that the induced injury does not alter virulencein orally infected mice. Thus, neutralization of gastric acidityin our experiments did not show any effect in mouse viru-lence tests when chlorine-injured cells were used.Sodium bicarbonate has been used by several workers to

neutralize the gastric acidity (3, 10). It was shown thatconsiderably lower inocula of Vibrio cholerae were neededto induce diarrheal responses in human volunteers pretreatedwith sodium bicarbonate than the control group (3). It ispossible that rapid gastric emptying in mice and the ability ofboth uninjured and chlorine-injured Y. enterocolitica cells totolerate low gastric pH allowed sufficient numbers of viablecells to escape into the small intestine and initiate aninfection. However, in copper-exposed Y. enterocolitica,low pH caused extensive cell death. This may be due to theexistence of more severely injured cells after exposure tothis stressor. Thus, a lower number of viable cells mighthave reached the small intestine than is required to initiateinfection. Partial restoration of virulence for mice aftergastric neutralization provides support for this view.

In conclusion, we demonstrated that a large number of Y.enterocolitica cells lose their ability to grow on CIN agarafter exposure to sublethal concentrations of copper, chlo-rine, and an acidic gastric environment. This could signifi-cantly alter the interpretation of Y. enterocolitica enumera-tions from stressful environments; especially when CIN agaris used. Because of the possible difference in the sublethalcellular lesion(s) induced, copper-treated cells show greaterdeath on exposure to low gastric pH than the chlorine-exposed cells. However, the extent of injury within a popu-lation after exposure to the low pH of the stomach is furtherenhanced in the stressed cultures.

It is apparent that reduced viability and not increasedinjury of Y. enterocolitica in the mouse stomach is respon-sible for the diminished virulence observed in orally infectedmice. Also, the results of this study indicate that chlorine-and copper-stressed Y. enterocolitica cells retain virtuallyfull potential for causing pathogenesis after oral infections.Additional studies examining survival, multiplication, andpathogenicity of other enteric pathogens after exposure tovarious stress factors are required to provide a better under-standing of the role of injured cells in the process of oralinfection.

ACKNOWLEDGMENTSWe thank Anne Camper and Helga Pac for technical assistance,

Barry Pyle for critically reading the manuscript, and Nancy Burnsfor typing.

This study was supported by Public Health Service grant Al 19089from the NationalInstitute of Allergy and Infectious Disease.

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