- Email: [email protected]
Influence of freezing-thawing and refrigeration on R-plasmid (pRPJ24) stability in Enterobacter cloacae 94R
International Journal of Food Microbiology, 11 (1990) 143-150 Elsevier
143
FOOD 00320
Influence of freezing-thawing and refrigeration on R-plasmid (pRPJ24) stability in Enterobacter cloacae 94R A.H.G.P. Jayaratne, D.L. Collins-Thompson and J.T. Trevors Department of Enoironmental Biology, University of Guelph, Guelph, Ontario, Canada
(Received 25 October 1989; accepted 16 January 1990)
The effect of twenty freezing (-20°C) and thawing cycles of Enterobacter cloacae 94R cells containing the R-plasmid pRPJ24 inoculated into broth and ground beef meat samples'revealed no loss of resistance due to plasmid instability. In addition, low temperature storage at 4°C did not produce any significant loss of the tetracycline and kanamycin resistances encoded on the pRPJ24 plasmid. The results of this study indicated that indigenous R-plasmids like pRPJ24 are stable in resident recipients like E. cloacae 94R in ground beef. However, the proportion of viable cells containing the pRPJ24 plasmid decreased significantly after 20 freezing-thawingcycles over 14 days incubation at 4°C. Key words: Enterobacter cloacae; Plasmid; Stability; Freezing-thawing; Refrigeration; Meat
Introduction W i d e s p r e a d useage of subtherapeutic levels of antibiotics in a n i m a l feed has a n indirect effect o n the acquisition of antibiotic resistance(s) by bacterial strains that affect h u m a n s (Pohl, 1977). As a result there is n o w an e n o r m o u s reservoir of resistant organisms f o u n d in foods of a n i m a l origin ( D u p o n t a n d Steele, 1987). M e a t used for h u m a n c o n s u m p t i o n m a y c o n t a i n large n u m b e r s of these bacteria of a n i m a l origin (Babcock et al., 1973; K i m a n d Stephens, 1972; M o o r h o u s e et al., 1969; W a l t o n , 1970; W a l t o n a n d Lewis, 1971). G e n e s coding a n t i b i o t i c resistances i n these organisms are c o m m o n l y carried o n R - p l a s m i d s (Hardy, 1981). Plasmids controlling antibiotic resistances have been s h o w n to be transferred b e t w e e n Escherichia coli strains (Platt et al., 1986; Smith, 1969; Williams, 1977) a n d from E. coli to Shigella sonnei ( F a r r a r et al., 1972) u n d e r in vivo c o n d i t i o n s in h u m a n s . F u r t h e r m o r e , epidemiological evidence indicates meat a n d meat p r o d u c t s c o n t a m i n a t e d with a n t i b i o t i c - r e s i s t a n t S a l m o n e l l a strains are
Correspondence address." A.H.G.P. Jayaratne, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2Wl.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144
responsible for some salmonellosis outbreaks in humans (Holmberg et al., 1984; Spika et al., 1987) and thus may be of considerable concern to public health. The dissemination and distribution of antibiotic resistance determinants in environments such as meat is a function of the genetic potential o f the bacterial population (O'Brien et al., 1987). The transferability and stability of R-plasmids under natural conditions are also responsible for dissemination of drug-resistant genes. In situ transfer of R-plasmids among resident enterobacterial populations in meat and meat products has been recently reported (Jayaratne et al., 1987; Jayaratne et al., 1989). However, despite the intensive study of plasmids, relatively little is known about the stability of indigenous, naturally occurring R-plasmids in bacterial cells under various conditions in the environment. The objective of the present study was to determine the effect of low temperature; freezing-thawing and refrigeration conditions on the stability of a naturally occurring R-plasmid and its resistance determinants.
Materials and Methods
Effect of freezing and thawing on R-plasmid stability 9 ml of LB (Luria-Bertani) broth (Acumedia, Baltimore, MD, U.S.A.) supplemented with 10% ( v / v ) sterile glycerol and approximately 11 g of raw ground beef patties were used as the freezing media. A broth culture of Enterobacter cloacae 94R harbouring pRPJ24 (Jayaratne et al., 1989) was grown in LB broth for 18 h at 37°C with shaking at 100 rpm (approximately 10 9 cfu/ml). 1 ml of this culture was used to inoculate 9 ml of LB broth supplemented with 10% ( v / v ) sterile glycerol. The samples were prepared in sets of three tubes and contained approximately 2 x l0 s c f u / m l . All tubes were then frozen at - 2 0 ° C for 24 h. A series of 11 g of raw ground beef patties on sterile petri plates were prepared. All ground beef samples were inoculated with 0.1 ml of inoculum (2 x 1 0 9 c f u / m l ) from a LB broth culture and frozen at - 2 0 ° C for 24 h. The broth cultures were thawed at 20°C for 1 h and appropriate dilutions were plated on nonselective and selective MacConkey agar (BBL, Cockeysville, MD, U.S.A.) medium supplemented with 2 5 / ~ g / m l tetracycline or 2 5 / z g / m l kanamycin. After thawing, broth samples were returned to - 2 0 ° C for refreezing. The plating was carried out for 15 thawing and freezing cycles. All frozen raw ground beef samples were subjected to repeated thawing and freezing in a similar manner. Each time, three samples were blended separately with 99 ml of 0.1% ( w / v ) sterile peptone water and appropriate dilutions of those were plated on selective and nonselective MacConkey agar medium supplemented with appropriate antibiotic, as described earlier. All other samples were refrozen after each thawing cycle. Stability of the R-plasmid in both cases was monitored by estimating the bacterial numbers on both selective and nonselective growth media, with respect to freezing and thawing cycles. Initial numbers of bacteria per sample ( c f u / g or ml) were determined by plating appropriate dilutions of the original culture on both selective and nonselective MacConkey agar medium.
145
Effect of refrigeration
on R-plasmid stability A series of approximately 11 g of raw and sterilized ground beef patties were prepared. Sterilized ground beef patty samples were prepared by autoclaving at 121°C for 15 min. Both sterile and raw ground beef samples were inoculated with 0.1 ml of 0.1% ( w / v ) sterile peptone water dilution (approximately 107 c f u / m l ) of an E. cloacae 94R R-plasmid host culture, grown at 37°C for 18 h. This resulted in approximately 105 c f u / g inoculum per meat sample. All samples were incubated at 4°C. A set of uninoculated raw ground beef samples (approximately 11 g per sample) were used as the control to monitor the native enterobacterial population. Three of each inoculated raw and sterile ground beef samples and uninoculated control samples were analyzed daily for 2 weeks. Each sample was blended separately with 99 ml of 0.1% ( w / v ) sterile peptone water and the appropriate dilutions were plated on nonselective and selective MacConkey agar medium. Stability of R-plasmid in raw and sterilized meat samples at 4°C was monitored by estimating bacterial numbers on both selective and nonselective MacConkey agar media. Statistical analysis The data was analyzed using an Edi-Sci statistical program (Modesto, CA, U.S.A.). operated on an Apple II plus microcomputer. Significant differences between control and treatment means were tested using Student's t-test at P = 0.05.
Results and Discussion Repeated cycles of freezing and thawing of E. cloacae 94R, harbouring pRPJ24, in broth (Fig. 1) and raw ground beef samples (Fig. 2) did not lead to any loss of 160 Q X
140 120
LL I-Z
O o ILl .--I m < >
100 80 6O 40 20 0 0
6
8
10
12
14
16
18
20
FREEZING/I'HAWING CYCLES
Fig. 1. Influence of repeated freezing and thawing on stability of R-plasmid pRPJ24 in broth; represented as viable counts of surviving E. cloacae 94R host on (1~) MacConkey agar (control). (D) MacConkey agar supplemented with 25 # g / m l tetracycline hydrochloride, and (@) MacConkey agar supplemented with 25/Lg/ml kanamycin sulfate. The means of the selective platings in all freezing/thawing cycles were not significantly different from the control.
146 100
X ~
80
"1
0 Z ::3 0 (..? U.I _J en
60 40
20
< > 0 0
4
6
8
10
12
14
16
18
20
FREEZING / THAWING CYCLES
Fig. 2. Influence of repeated freezing and thawing on stability of R-plasmid pRPJ24 in ground beef; represented as viable counts of surviving E. cloacae 94R host on ([]) MacConkeyagar, ([]) MacConkey agar supplemented with 25 t~g/ml tetracycline hydrochloride, and (@) MacConkey agar supplemented with kanamycin sulfate. The means of the 2 selective platings in all freezing/thawing cycles were not significantly different from the control.
plasmid-encoded drug resistance as estimated by the comparison of viable counts of bacterial population carrying antibiotic resistance markers to that of the total population. Plating samples on nonselective and selective media containing antibiotics revealed no significant difference in the population estimates, indicating no significant loss of R-plasmid pRPJ24 from its host E. cloacae 94R (Figs. 1 and 2). However, in both in vitro and in situ conditions there was a considerable decrease in total population levels over 20 repeated freezing and thawing cycles examined. This may be the result of cell injury caused during freezing and thawing. Meat and meat products are frequently subjected to similar freezing and thawing conditions, and the present investigation provided information on the stability of similar naturally-occurring R-plasmids under such conditions. However, since there is a high variability among R-plasmids in their behaviour under different conditions, it is not possible to generalize using these observations. The effect of freezing and thawing on indigenous, naturally-occurring R-plasmids has not been previously studied. However, Saida et al. (1986), investigated the effect of freezing and thawing on a R-plasmid under in vivo and in vitro conditions using E. coli C600 harbouring plasmid pBR322. They observed decreased viability of the cells and 100% maintainance of drug resistance with increased freezing and thawing times. These results are in agreement with the results obtained in the present study. They also observed a decrease in genetic transformation efficiency according to the increased freezing-thawing times. The effect of refrigeration storage on plasmid stability was examined using raw and sterile ground beef samples inoculated with E. cloacae 94R harbouring pRPJ24. The results indicated no significant change in the populations resistant to antibiotics over 14 days, when compared with the population estimates obtained from nonselective medium (Figs. 3 and 4). However, there was a significant decrease in the total population after a 14 day period. Monitoring indigenous enterobacterial
147
~o LL O9 Z O O rn
_< >
2
3
7
6
8
10
14
DAYS
Fig. 3. Survival of E. cloacae 94R host stability of R-plasmid pRPJ24 in raw ground beef samples at 4°C: as determined by the viable counts on ([]) MacConkey agar supplimented with 25 /~g/ml tetracycline hydrochloride and (D) MacConkey agar supplimented with 25 ~tg/ml kanamycin sulfate. The means of the selective platings were not significantly different from the control over the 14 day period.
populations in raw ground beef samples on storage at 4°C, over two weeks, displayed a rapid increase in their numbers after 3 days. They reached apprioximately 108 c f u / g after a 2 week period. Even at high population levels of indigenous bacteria (approximately 108 cfu/g), the E. cloacae 94R harbouring pRPJ24 was maintained at approximately the same population densities (103 cfu/g), as in the experiment carried out with sterile ground beef samples (Figs. 3 and 4). Therefore, indigenous bacteria had no effect on survival of E. cloacae 94R harbouring pRPJ24. The decreasing populations of E. cloacae 94R observed over a 14 day period were not due to other inhibiting bacteria, but was the result of loss of viability at 4°C during prolonged storage. Devanas et al. (1986) observed a decline in population of 8
0
1
z
3
4
5 6 DAYS
7
8
10
14
Fig. 4. Survival of E. cloacae 94R host and stability of R-plasmid pRPJ24 in sterile ground beef samples at 4°C; as determined by the viable counts on, ([]) MacConkey agar, (n) MacConkey agar medium supplemented with 25 ~ g / m l tetracycline hydrochloride, and ([]) MacConkey agar medium supplemented with 25 p.g/ml kanamycin sulfate. The means of the selective platings were not significantly different from the control over the 14 day period.
148
E. coli strains PRC487 (pACYC175) and X1666 (pES019), when inoculated into
nonsterile soil. Similar observations have also been reported by Schilf and Klingmuller (1983), in populations of E. coli strains J5 (RP4) and JC5466 (pRD1), in Enterobacteriaceae strains (pRD1 and RP4), and in Pseudomonasfluorescens (pRD1) when inoculated into pond water or nonsterile soil. These results were similar to those obtained in the current study in meat. However, the decrease in populations in the present study is not as great as those reported in the soil environment. The rapid decrease of introduced bacterial populations in soil experiments have been attributed to the depletion of nutrients unlike in the present study, where the population decrease was presumably the result of loss of viability at low temperature during storage. Furthermore, unlike the present study, Devanas et al. (1986) did not observe any significant change in indigenous bacterial populations. Melling et al. (1977) examined the survival of R + and R - strains of E. coli in mixed culture in a chemostat, and demonstrated the importance of the bacterial environment (i.e., nutrients) on the survival of an R + strain in competition with an R strain. Furthermore, the relative numbers of the two strains were shown to have a significant effect on the outcome of such studies. In general, the R-plasmid pRPJ24 in host E. cloacae 94R was relatively stable in the present investigation. This plasmid is relatively large ( > 80 kbp) and thus is expected to be maintained at only a few copies in the host cell. The presence of low copy numbers of plasmids usually does not carry an extra burden on the energy metabolism of the host cell, as when a high number of copies of a smaller plasmid is present. Thus, one could expect it to be fairly stable in the host cell when exposed to various nonselective conditions. In contrast, Saida et al. (1986) showed the stability of high copy number pBR322 when exposed to freezing-thawing conditions. It is possible that other factors are involved in plasmid stability when exposed to nonchallenging conditions. The effect of plasmid size on survival of plasmid-containing strains of E. coli in soil was examined by Devanas et al. (1986). They concluded that the stability of plasmids in genetically similar host strains and of the host-plasmid system was not related to the size of the plasmid. For example, survival of strains containing smaller plasmids ( < 10 Mdal) did not differ significantly from that of strains containing larger plasmids ( > 25 Mdal). Similar observations were made by Zund and Lebek (1980), who noted that among natural plasmids with a wide range of sizes, there was no consistent relationship between size of the plasmid and growth rate of the host. Other researchers have investigated the stability and maintenance of plasmids under various growth limiting and nonlimiting conditions. Gowland and Slater (1984) examined the stability of drug resistance plasmids TP120 and R1 in E. co# K12 host under continous cultivation with carbon limiting condition. They demonstrated these plasmids were stable over 5 months without any change either in size or composition. Similar results were reported by Filonov et al. (1985), who examined RP4 (conjugative) and pBS94 (nonconjugative) R-plasmids in E. coli C600 in a chemostat culture, and found both plasmids to be stably preserved in bacterial cells. In another study, Devanas et al. (1986) examined the maintenance of plasmids in plasmid-containing strains of E. coli PRC487 (pACYC175) and X1666 (pES019).
149 T h e y d e m o n s t r a t e d n o l o s s o f p l a s m i d i n t h e soil e n v i r o n m e n t . I n c o n t r a s t , H e l l i n g et al. ( 1 9 8 1 ) o b s e r v e d a n i n i t i a l d e c l i n e i n t h e p r o p o r t i o n o f p l a s m i d - c o n t a i n i n g E. coli i n t h e t o t a l p o p u l a t i o n g r o w n i n c a r b o n - l i m i t e d c o n t i n o u s c u l t u r e . H o w e v e r , periodic changes in the population resulted in fluctuations in the proportion of p l a s m i d c o n t a i n i n g o r g a n i s m s a n d d e l a y e d t h e loss o f t h e p l a s m i d f r o m t h e population.
Acknowledgements This research was supported by a Commonwealth Scholarship to A.H.G.P.J. and N S E R C ( C a n a d a ) o p e r a t i n g g r a n t s t o D . L . C - T a n d J . T . T . S i n c e r e a p p r e c i a t i o n is e x p r e s s e d t o S. S p r o w l a n d J e a n n e H o g e t e r p f o r t y p i n g t h e m a n u s c r i p t .
References Babcock, G.F., Berryhill, D.L. and Marsh, D.H. (1973) R-factor of Escherichia coli from dressed beef and humans. Appl. Microbiol. 25, 21-23. Devanas, M.A., Rafaeli-Eshkol, D. and Stotzky, G. (1986) Survival of plasmid-containing strains of Escherichia coli in soil: effect of plasmid size and nutrients on survival of hosts and maintenance of plasmids. Curr. Microbiol. 13, 269-277. Dupont, H.L. and Steele, J.H. (1987) Use of antimicrobial agents in animal feeds: Implications for human health. Rev. Infect. Dis. 9, 447-460. Farrar, W.D. Jr., Margene, E., Guerry, P., Falkow, S., Drusin, L.M. and Roberts, R.B. (1972) Interbacterial transfer of R-factor in the human intestine. In vivo acquisition of R-factor mediated kanamycin resistance by a multiresistant strain of Shigella sonnei. J. Infect. Dis. 126, 27-33. Filonov, A.E., Rublevskaya, I.N. and Boronin, A.M. (1985) Stability and dynamics of R-plasmids in Escherichia coli populations under conditions of continous culturing. Mikrobiologiya 54, 227-232. Gowland, P.C. and Slater, J.H. (1984) Transfer and stability of drug resistance plasmids in Escherichia coli K12. Microb. Ecol. 10, 1-13. Hardy, K. (1981) Bacterial Plasmids. American Society for Microbiology, Washington, D.C. Helling, R.B., Kinney, T. and Adams, J. (1981) The maintenance of plasmid-containing organisms in populations of Escherichia coli. J. Gen. Microbiol. 123, 129-141. Holmberg, S.D., Osterholm, K.A., Senger and Cohen, M.L. (1984) Drug resistant Salmonella from animals fed antimicrobials. N. Engl. J. Med. 311,617-622. Jayaratne, A.H.G.P., Collins-Thompson, D.L. and Trevors, J.T. (1987) In situ conjugal transfer of plasmid-encoded multiple antibiotic resistance in Escherichia coli isolated from meat. Int. J. Food Microbiol. 5, 53-71. Jayaratne, A.H.G.P., Collins-Thompson, D.L. and Trevors, J.T. (1989) In vitro and in situ conjugal transfer of an R-plasmid among Enterobacteriacease species isolated from meat samples. FEMS Microbiol. Ecol. 62, 79-86. Kim, T.K. and Stephens, J.F. (1972) Drug resistance and transferable drug resistance of Escherichia coli isolated from ready to cook broilers. Poult. Sci. 51, 1165-1170. Melting, J., Elwood, D.C. and Robinson, A. (1977) Survival of R-factor carrying Escherichia coli in mixed cultures in the chemostat. FEMS Microbiol. Lett. 2, 87-89. Moorhouse, E.C., O'Grady, M.F. and O'Connor, H. (1969) Isolation from sausage of antibiotic-resistant Escherichia eoli with R-factors. Lancet 2, 50-52. O'Brien, T.F., Hqu, M.I., Acar, J.F., Burchall, J.J., Kleeberg, H.H., Kronvall, G., Ohashi, M., Pinto,
150 M.E., Rowe, B., Sng, E.H., Trabulsi, L.R., Fu, W. and Wiedmann, B. (1987) Resistance of bacteria to antimicrobial agents: Report of task force 2. Rev. Infect. Dis. 9 (Supplement 3), $245-$260. Platt D.J., Chesham, J.S. and Kristinsson, K.G. (1986) R-plasmid transfer in vivo: a prospective study. J. Med. Microbiol. 21, 325 330. Pohl, P., (1977) Relationship between antibiotic feeding in animals and emergence of bacterial resistance in man. J. Antimicrob. Chemother. 3 (Supplement C), 67-70. Saida, K., Kimura, D., Mikami, E. and Nakamura, Y. (1986) Effect of freezing-thawing on a drug-resistant plasmid. Fermet. Res. Inst. 66, 1-8. Schilf, W. and Klingmuller, W. (1983) Experiments with Escherichia coli on the dispersal of plasmids in environmental samples. Recomb. D N A Tech. Bull. 6, 101-102. Smith, H.W., (1969) Transfer of antibiotic resistance from animal and h u m a n strains of Escherichia coli to resident E. coli in the alimentary tract of man. Lancet 1, 1174-1176. Spika, J.S., Waterman, S.H., SooHoo, G.W., St. Louis, M.E., Pacer, R.E., James, S.M., Bissett, M.L., Mayer, L.W., Chiu, J.Y., Hall, B., Greene, K., Potter, M.E., Cohen, M.L. and Blake, P.A. (1987) Chloramphenicol-resistant Salmonella newport traced through hamburger to dairy farms. N. Engl. J. Med. 316, 565-570. Walton, J.R. (1970) Contamination of meat carcasses by antibiotic-resistant coliform bacteria. Lancet 2, 561-563. Walton, J,R. and Lewis, L.E. (1971) Contamination of fresh and cooked meats by antibiotic-resistant colifrom bacteria. Lancet 2, 255-257. Williams, P.H. (1977) Plasmid transfer in the h u m a n alimentary tract. FEMS Microbiol. Lett. 2, 91 95. Zund, P. and Lebek, G. (1980) Generation time-prolonging R-plasmids: Correlation between increases in the generation time of Escherichia coli caused by R-plasmids and their molecular sizes. Plasmid 3, 65-69.
143
FOOD 00320
Influence of freezing-thawing and refrigeration on R-plasmid (pRPJ24) stability in Enterobacter cloacae 94R A.H.G.P. Jayaratne, D.L. Collins-Thompson and J.T. Trevors Department of Enoironmental Biology, University of Guelph, Guelph, Ontario, Canada
(Received 25 October 1989; accepted 16 January 1990)
The effect of twenty freezing (-20°C) and thawing cycles of Enterobacter cloacae 94R cells containing the R-plasmid pRPJ24 inoculated into broth and ground beef meat samples'revealed no loss of resistance due to plasmid instability. In addition, low temperature storage at 4°C did not produce any significant loss of the tetracycline and kanamycin resistances encoded on the pRPJ24 plasmid. The results of this study indicated that indigenous R-plasmids like pRPJ24 are stable in resident recipients like E. cloacae 94R in ground beef. However, the proportion of viable cells containing the pRPJ24 plasmid decreased significantly after 20 freezing-thawingcycles over 14 days incubation at 4°C. Key words: Enterobacter cloacae; Plasmid; Stability; Freezing-thawing; Refrigeration; Meat
Introduction W i d e s p r e a d useage of subtherapeutic levels of antibiotics in a n i m a l feed has a n indirect effect o n the acquisition of antibiotic resistance(s) by bacterial strains that affect h u m a n s (Pohl, 1977). As a result there is n o w an e n o r m o u s reservoir of resistant organisms f o u n d in foods of a n i m a l origin ( D u p o n t a n d Steele, 1987). M e a t used for h u m a n c o n s u m p t i o n m a y c o n t a i n large n u m b e r s of these bacteria of a n i m a l origin (Babcock et al., 1973; K i m a n d Stephens, 1972; M o o r h o u s e et al., 1969; W a l t o n , 1970; W a l t o n a n d Lewis, 1971). G e n e s coding a n t i b i o t i c resistances i n these organisms are c o m m o n l y carried o n R - p l a s m i d s (Hardy, 1981). Plasmids controlling antibiotic resistances have been s h o w n to be transferred b e t w e e n Escherichia coli strains (Platt et al., 1986; Smith, 1969; Williams, 1977) a n d from E. coli to Shigella sonnei ( F a r r a r et al., 1972) u n d e r in vivo c o n d i t i o n s in h u m a n s . F u r t h e r m o r e , epidemiological evidence indicates meat a n d meat p r o d u c t s c o n t a m i n a t e d with a n t i b i o t i c - r e s i s t a n t S a l m o n e l l a strains are
Correspondence address." A.H.G.P. Jayaratne, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2Wl.
0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144
responsible for some salmonellosis outbreaks in humans (Holmberg et al., 1984; Spika et al., 1987) and thus may be of considerable concern to public health. The dissemination and distribution of antibiotic resistance determinants in environments such as meat is a function of the genetic potential o f the bacterial population (O'Brien et al., 1987). The transferability and stability of R-plasmids under natural conditions are also responsible for dissemination of drug-resistant genes. In situ transfer of R-plasmids among resident enterobacterial populations in meat and meat products has been recently reported (Jayaratne et al., 1987; Jayaratne et al., 1989). However, despite the intensive study of plasmids, relatively little is known about the stability of indigenous, naturally occurring R-plasmids in bacterial cells under various conditions in the environment. The objective of the present study was to determine the effect of low temperature; freezing-thawing and refrigeration conditions on the stability of a naturally occurring R-plasmid and its resistance determinants.
Materials and Methods
Effect of freezing and thawing on R-plasmid stability 9 ml of LB (Luria-Bertani) broth (Acumedia, Baltimore, MD, U.S.A.) supplemented with 10% ( v / v ) sterile glycerol and approximately 11 g of raw ground beef patties were used as the freezing media. A broth culture of Enterobacter cloacae 94R harbouring pRPJ24 (Jayaratne et al., 1989) was grown in LB broth for 18 h at 37°C with shaking at 100 rpm (approximately 10 9 cfu/ml). 1 ml of this culture was used to inoculate 9 ml of LB broth supplemented with 10% ( v / v ) sterile glycerol. The samples were prepared in sets of three tubes and contained approximately 2 x l0 s c f u / m l . All tubes were then frozen at - 2 0 ° C for 24 h. A series of 11 g of raw ground beef patties on sterile petri plates were prepared. All ground beef samples were inoculated with 0.1 ml of inoculum (2 x 1 0 9 c f u / m l ) from a LB broth culture and frozen at - 2 0 ° C for 24 h. The broth cultures were thawed at 20°C for 1 h and appropriate dilutions were plated on nonselective and selective MacConkey agar (BBL, Cockeysville, MD, U.S.A.) medium supplemented with 2 5 / ~ g / m l tetracycline or 2 5 / z g / m l kanamycin. After thawing, broth samples were returned to - 2 0 ° C for refreezing. The plating was carried out for 15 thawing and freezing cycles. All frozen raw ground beef samples were subjected to repeated thawing and freezing in a similar manner. Each time, three samples were blended separately with 99 ml of 0.1% ( w / v ) sterile peptone water and appropriate dilutions of those were plated on selective and nonselective MacConkey agar medium supplemented with appropriate antibiotic, as described earlier. All other samples were refrozen after each thawing cycle. Stability of the R-plasmid in both cases was monitored by estimating the bacterial numbers on both selective and nonselective growth media, with respect to freezing and thawing cycles. Initial numbers of bacteria per sample ( c f u / g or ml) were determined by plating appropriate dilutions of the original culture on both selective and nonselective MacConkey agar medium.
145
Effect of refrigeration
on R-plasmid stability A series of approximately 11 g of raw and sterilized ground beef patties were prepared. Sterilized ground beef patty samples were prepared by autoclaving at 121°C for 15 min. Both sterile and raw ground beef samples were inoculated with 0.1 ml of 0.1% ( w / v ) sterile peptone water dilution (approximately 107 c f u / m l ) of an E. cloacae 94R R-plasmid host culture, grown at 37°C for 18 h. This resulted in approximately 105 c f u / g inoculum per meat sample. All samples were incubated at 4°C. A set of uninoculated raw ground beef samples (approximately 11 g per sample) were used as the control to monitor the native enterobacterial population. Three of each inoculated raw and sterile ground beef samples and uninoculated control samples were analyzed daily for 2 weeks. Each sample was blended separately with 99 ml of 0.1% ( w / v ) sterile peptone water and the appropriate dilutions were plated on nonselective and selective MacConkey agar medium. Stability of R-plasmid in raw and sterilized meat samples at 4°C was monitored by estimating bacterial numbers on both selective and nonselective MacConkey agar media. Statistical analysis The data was analyzed using an Edi-Sci statistical program (Modesto, CA, U.S.A.). operated on an Apple II plus microcomputer. Significant differences between control and treatment means were tested using Student's t-test at P = 0.05.
Results and Discussion Repeated cycles of freezing and thawing of E. cloacae 94R, harbouring pRPJ24, in broth (Fig. 1) and raw ground beef samples (Fig. 2) did not lead to any loss of 160 Q X
140 120
LL I-Z
O o ILl .--I m < >
100 80 6O 40 20 0 0
6
8
10
12
14
16
18
20
FREEZING/I'HAWING CYCLES
Fig. 1. Influence of repeated freezing and thawing on stability of R-plasmid pRPJ24 in broth; represented as viable counts of surviving E. cloacae 94R host on (1~) MacConkey agar (control). (D) MacConkey agar supplemented with 25 # g / m l tetracycline hydrochloride, and (@) MacConkey agar supplemented with 25/Lg/ml kanamycin sulfate. The means of the selective platings in all freezing/thawing cycles were not significantly different from the control.
146 100
X ~
80
"1
0 Z ::3 0 (..? U.I _J en
60 40
20
< > 0 0
4
6
8
10
12
14
16
18
20
FREEZING / THAWING CYCLES
Fig. 2. Influence of repeated freezing and thawing on stability of R-plasmid pRPJ24 in ground beef; represented as viable counts of surviving E. cloacae 94R host on ([]) MacConkeyagar, ([]) MacConkey agar supplemented with 25 t~g/ml tetracycline hydrochloride, and (@) MacConkey agar supplemented with kanamycin sulfate. The means of the 2 selective platings in all freezing/thawing cycles were not significantly different from the control.
plasmid-encoded drug resistance as estimated by the comparison of viable counts of bacterial population carrying antibiotic resistance markers to that of the total population. Plating samples on nonselective and selective media containing antibiotics revealed no significant difference in the population estimates, indicating no significant loss of R-plasmid pRPJ24 from its host E. cloacae 94R (Figs. 1 and 2). However, in both in vitro and in situ conditions there was a considerable decrease in total population levels over 20 repeated freezing and thawing cycles examined. This may be the result of cell injury caused during freezing and thawing. Meat and meat products are frequently subjected to similar freezing and thawing conditions, and the present investigation provided information on the stability of similar naturally-occurring R-plasmids under such conditions. However, since there is a high variability among R-plasmids in their behaviour under different conditions, it is not possible to generalize using these observations. The effect of freezing and thawing on indigenous, naturally-occurring R-plasmids has not been previously studied. However, Saida et al. (1986), investigated the effect of freezing and thawing on a R-plasmid under in vivo and in vitro conditions using E. coli C600 harbouring plasmid pBR322. They observed decreased viability of the cells and 100% maintainance of drug resistance with increased freezing and thawing times. These results are in agreement with the results obtained in the present study. They also observed a decrease in genetic transformation efficiency according to the increased freezing-thawing times. The effect of refrigeration storage on plasmid stability was examined using raw and sterile ground beef samples inoculated with E. cloacae 94R harbouring pRPJ24. The results indicated no significant change in the populations resistant to antibiotics over 14 days, when compared with the population estimates obtained from nonselective medium (Figs. 3 and 4). However, there was a significant decrease in the total population after a 14 day period. Monitoring indigenous enterobacterial
147
~o LL O9 Z O O rn
_< >
2
3
7
6
8
10
14
DAYS
Fig. 3. Survival of E. cloacae 94R host stability of R-plasmid pRPJ24 in raw ground beef samples at 4°C: as determined by the viable counts on ([]) MacConkey agar supplimented with 25 /~g/ml tetracycline hydrochloride and (D) MacConkey agar supplimented with 25 ~tg/ml kanamycin sulfate. The means of the selective platings were not significantly different from the control over the 14 day period.
populations in raw ground beef samples on storage at 4°C, over two weeks, displayed a rapid increase in their numbers after 3 days. They reached apprioximately 108 c f u / g after a 2 week period. Even at high population levels of indigenous bacteria (approximately 108 cfu/g), the E. cloacae 94R harbouring pRPJ24 was maintained at approximately the same population densities (103 cfu/g), as in the experiment carried out with sterile ground beef samples (Figs. 3 and 4). Therefore, indigenous bacteria had no effect on survival of E. cloacae 94R harbouring pRPJ24. The decreasing populations of E. cloacae 94R observed over a 14 day period were not due to other inhibiting bacteria, but was the result of loss of viability at 4°C during prolonged storage. Devanas et al. (1986) observed a decline in population of 8
0
1
z
3
4
5 6 DAYS
7
8
10
14
Fig. 4. Survival of E. cloacae 94R host and stability of R-plasmid pRPJ24 in sterile ground beef samples at 4°C; as determined by the viable counts on, ([]) MacConkey agar, (n) MacConkey agar medium supplemented with 25 ~ g / m l tetracycline hydrochloride, and ([]) MacConkey agar medium supplemented with 25 p.g/ml kanamycin sulfate. The means of the selective platings were not significantly different from the control over the 14 day period.
148
E. coli strains PRC487 (pACYC175) and X1666 (pES019), when inoculated into
nonsterile soil. Similar observations have also been reported by Schilf and Klingmuller (1983), in populations of E. coli strains J5 (RP4) and JC5466 (pRD1), in Enterobacteriaceae strains (pRD1 and RP4), and in Pseudomonasfluorescens (pRD1) when inoculated into pond water or nonsterile soil. These results were similar to those obtained in the current study in meat. However, the decrease in populations in the present study is not as great as those reported in the soil environment. The rapid decrease of introduced bacterial populations in soil experiments have been attributed to the depletion of nutrients unlike in the present study, where the population decrease was presumably the result of loss of viability at low temperature during storage. Furthermore, unlike the present study, Devanas et al. (1986) did not observe any significant change in indigenous bacterial populations. Melling et al. (1977) examined the survival of R + and R - strains of E. coli in mixed culture in a chemostat, and demonstrated the importance of the bacterial environment (i.e., nutrients) on the survival of an R + strain in competition with an R strain. Furthermore, the relative numbers of the two strains were shown to have a significant effect on the outcome of such studies. In general, the R-plasmid pRPJ24 in host E. cloacae 94R was relatively stable in the present investigation. This plasmid is relatively large ( > 80 kbp) and thus is expected to be maintained at only a few copies in the host cell. The presence of low copy numbers of plasmids usually does not carry an extra burden on the energy metabolism of the host cell, as when a high number of copies of a smaller plasmid is present. Thus, one could expect it to be fairly stable in the host cell when exposed to various nonselective conditions. In contrast, Saida et al. (1986) showed the stability of high copy number pBR322 when exposed to freezing-thawing conditions. It is possible that other factors are involved in plasmid stability when exposed to nonchallenging conditions. The effect of plasmid size on survival of plasmid-containing strains of E. coli in soil was examined by Devanas et al. (1986). They concluded that the stability of plasmids in genetically similar host strains and of the host-plasmid system was not related to the size of the plasmid. For example, survival of strains containing smaller plasmids ( < 10 Mdal) did not differ significantly from that of strains containing larger plasmids ( > 25 Mdal). Similar observations were made by Zund and Lebek (1980), who noted that among natural plasmids with a wide range of sizes, there was no consistent relationship between size of the plasmid and growth rate of the host. Other researchers have investigated the stability and maintenance of plasmids under various growth limiting and nonlimiting conditions. Gowland and Slater (1984) examined the stability of drug resistance plasmids TP120 and R1 in E. co# K12 host under continous cultivation with carbon limiting condition. They demonstrated these plasmids were stable over 5 months without any change either in size or composition. Similar results were reported by Filonov et al. (1985), who examined RP4 (conjugative) and pBS94 (nonconjugative) R-plasmids in E. coli C600 in a chemostat culture, and found both plasmids to be stably preserved in bacterial cells. In another study, Devanas et al. (1986) examined the maintenance of plasmids in plasmid-containing strains of E. coli PRC487 (pACYC175) and X1666 (pES019).
149 T h e y d e m o n s t r a t e d n o l o s s o f p l a s m i d i n t h e soil e n v i r o n m e n t . I n c o n t r a s t , H e l l i n g et al. ( 1 9 8 1 ) o b s e r v e d a n i n i t i a l d e c l i n e i n t h e p r o p o r t i o n o f p l a s m i d - c o n t a i n i n g E. coli i n t h e t o t a l p o p u l a t i o n g r o w n i n c a r b o n - l i m i t e d c o n t i n o u s c u l t u r e . H o w e v e r , periodic changes in the population resulted in fluctuations in the proportion of p l a s m i d c o n t a i n i n g o r g a n i s m s a n d d e l a y e d t h e loss o f t h e p l a s m i d f r o m t h e population.
Acknowledgements This research was supported by a Commonwealth Scholarship to A.H.G.P.J. and N S E R C ( C a n a d a ) o p e r a t i n g g r a n t s t o D . L . C - T a n d J . T . T . S i n c e r e a p p r e c i a t i o n is e x p r e s s e d t o S. S p r o w l a n d J e a n n e H o g e t e r p f o r t y p i n g t h e m a n u s c r i p t .
References Babcock, G.F., Berryhill, D.L. and Marsh, D.H. (1973) R-factor of Escherichia coli from dressed beef and humans. Appl. Microbiol. 25, 21-23. Devanas, M.A., Rafaeli-Eshkol, D. and Stotzky, G. (1986) Survival of plasmid-containing strains of Escherichia coli in soil: effect of plasmid size and nutrients on survival of hosts and maintenance of plasmids. Curr. Microbiol. 13, 269-277. Dupont, H.L. and Steele, J.H. (1987) Use of antimicrobial agents in animal feeds: Implications for human health. Rev. Infect. Dis. 9, 447-460. Farrar, W.D. Jr., Margene, E., Guerry, P., Falkow, S., Drusin, L.M. and Roberts, R.B. (1972) Interbacterial transfer of R-factor in the human intestine. In vivo acquisition of R-factor mediated kanamycin resistance by a multiresistant strain of Shigella sonnei. J. Infect. Dis. 126, 27-33. Filonov, A.E., Rublevskaya, I.N. and Boronin, A.M. (1985) Stability and dynamics of R-plasmids in Escherichia coli populations under conditions of continous culturing. Mikrobiologiya 54, 227-232. Gowland, P.C. and Slater, J.H. (1984) Transfer and stability of drug resistance plasmids in Escherichia coli K12. Microb. Ecol. 10, 1-13. Hardy, K. (1981) Bacterial Plasmids. American Society for Microbiology, Washington, D.C. Helling, R.B., Kinney, T. and Adams, J. (1981) The maintenance of plasmid-containing organisms in populations of Escherichia coli. J. Gen. Microbiol. 123, 129-141. Holmberg, S.D., Osterholm, K.A., Senger and Cohen, M.L. (1984) Drug resistant Salmonella from animals fed antimicrobials. N. Engl. J. Med. 311,617-622. Jayaratne, A.H.G.P., Collins-Thompson, D.L. and Trevors, J.T. (1987) In situ conjugal transfer of plasmid-encoded multiple antibiotic resistance in Escherichia coli isolated from meat. Int. J. Food Microbiol. 5, 53-71. Jayaratne, A.H.G.P., Collins-Thompson, D.L. and Trevors, J.T. (1989) In vitro and in situ conjugal transfer of an R-plasmid among Enterobacteriacease species isolated from meat samples. FEMS Microbiol. Ecol. 62, 79-86. Kim, T.K. and Stephens, J.F. (1972) Drug resistance and transferable drug resistance of Escherichia coli isolated from ready to cook broilers. Poult. Sci. 51, 1165-1170. Melting, J., Elwood, D.C. and Robinson, A. (1977) Survival of R-factor carrying Escherichia coli in mixed cultures in the chemostat. FEMS Microbiol. Lett. 2, 87-89. Moorhouse, E.C., O'Grady, M.F. and O'Connor, H. (1969) Isolation from sausage of antibiotic-resistant Escherichia eoli with R-factors. Lancet 2, 50-52. O'Brien, T.F., Hqu, M.I., Acar, J.F., Burchall, J.J., Kleeberg, H.H., Kronvall, G., Ohashi, M., Pinto,
150 M.E., Rowe, B., Sng, E.H., Trabulsi, L.R., Fu, W. and Wiedmann, B. (1987) Resistance of bacteria to antimicrobial agents: Report of task force 2. Rev. Infect. Dis. 9 (Supplement 3), $245-$260. Platt D.J., Chesham, J.S. and Kristinsson, K.G. (1986) R-plasmid transfer in vivo: a prospective study. J. Med. Microbiol. 21, 325 330. Pohl, P., (1977) Relationship between antibiotic feeding in animals and emergence of bacterial resistance in man. J. Antimicrob. Chemother. 3 (Supplement C), 67-70. Saida, K., Kimura, D., Mikami, E. and Nakamura, Y. (1986) Effect of freezing-thawing on a drug-resistant plasmid. Fermet. Res. Inst. 66, 1-8. Schilf, W. and Klingmuller, W. (1983) Experiments with Escherichia coli on the dispersal of plasmids in environmental samples. Recomb. D N A Tech. Bull. 6, 101-102. Smith, H.W., (1969) Transfer of antibiotic resistance from animal and h u m a n strains of Escherichia coli to resident E. coli in the alimentary tract of man. Lancet 1, 1174-1176. Spika, J.S., Waterman, S.H., SooHoo, G.W., St. Louis, M.E., Pacer, R.E., James, S.M., Bissett, M.L., Mayer, L.W., Chiu, J.Y., Hall, B., Greene, K., Potter, M.E., Cohen, M.L. and Blake, P.A. (1987) Chloramphenicol-resistant Salmonella newport traced through hamburger to dairy farms. N. Engl. J. Med. 316, 565-570. Walton, J.R. (1970) Contamination of meat carcasses by antibiotic-resistant coliform bacteria. Lancet 2, 561-563. Walton, J,R. and Lewis, L.E. (1971) Contamination of fresh and cooked meats by antibiotic-resistant colifrom bacteria. Lancet 2, 255-257. Williams, P.H. (1977) Plasmid transfer in the h u m a n alimentary tract. FEMS Microbiol. Lett. 2, 91 95. Zund, P. and Lebek, G. (1980) Generation time-prolonging R-plasmids: Correlation between increases in the generation time of Escherichia coli caused by R-plasmids and their molecular sizes. Plasmid 3, 65-69.