Minimal Medium Recovery of Heated Salmonella typhimurium LT2

Transcription

1 Journal of General Microbiology (I973), 74, Printed in Great Britain 267 Minimal Medium Recovery of Heated Salmonella typhimurium LT2 By R. F. GOMEZ, A. J. SINSKEY, R. DAVIES" AND T. P. LABUZA Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massuchusetts , U.S.A. (Received 30 August 1972; revised 25 September 1972) SUMMARY Exponential phase Salmonella typhimurium LT 2, S. thompson and S. heidelberg grown in a glucose-salts medium (M-9), harvested at 37 "C and heat-treated at 50 "C exhibit a higher recovery on M-9 agar than on Trypticase Soy agar with yeast extract. This phenomenon is analogous to 'minimal medium recovery' of irradiated bacteria. Supplementation of M-9 agar with yeast extract or Casamino acids resulted in lower viable counts of heat-treated bacteria than on M-9 agar alone. Heat-treated bacteria incubated at 37 "C in M-9 broth or distilled water recovered their ability to grow on TSY agar. This recovery is inhibited by hydroxyurea. INTRODUCTION Recovery of heat-treated micro-organisms depends upon the isolation or enumeration media employed. For example, heated Staphylococcus aureus are less tolerant to salt (Iandolo & Ordal, I 966). The practice of preliminary resuscitation in non-selective enrichment media has been proposed (Mossell & Ratto, 1970). This procedure should also allow for the recovery of 'metabolically injured' cells (Straka & Stokes, 1959) which, after a variety of physical stresses, are unable to multiply on simple defined media and require nutrient supplementation. We show here, for heated Salmonella typhimurium LT 2, that the phenomenon of 'metabolic injury' depends on pretreatment growth conditions and very often the opposite result can be obtained. In accord with recent reports of thermally induced DNA breakage in Escherichia coli (Bridges, Ashwood-Smith & Munson, 1969 ; Sedgwick & Bridges, 1972) and its restitution (Woodcock & Grigg, I972), we present evidence of the repair of thermal damage and its possible dependence on DNA synthesis. METHODS Organisms. Salmonella typhimurium strain DB 2 I, an isolate of LT 2, was obtained from D. Botstein, Department of Biology, Massachusetts Institute of Technology. Salmonella heidelberg and S. thompson were obtained from the collection of the Department of Nutrition and Food Science, Massachusetts Institute of Technology. Growth media. TSY, BBL Trypticase Soy broth supplemented with 037L Difco Yeast Extract. M-9, a modification of the chemically defined medium of Adams (1959) containing (g/rooo ml distilled water): Na,HPO,, 7; KH,PO,, 3 ; NaCl, 0.5; NH,Cl, I ; MgSO,. 7H,O, 0.25; dextrose, 2. * Present address: National College of Food Technology, University of Reading, St George's Avenue, Weybridge, Surrey, U.K. 18 M I c 74

2 268 R. F. GOMEZ, A. J. SINSKEY, R. DAVIES AND T. P. LABUZA At times bacteria were also plated on M-9 supplemented with nucleosides, vitamins or amino acids. Nucleosides : adenosine, guanosine, thymidine, cytidine, and uridine (10 pg/ml of each) ; vitamins : thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxine, folic acid, inositol, choline, biotin, p-aminobenzoic acid, vitamin B,, (10 pgfml of each) ; amino acids : glutamine, alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, asparagine (10 pg/ml of each). Heat treatment. Bacteria from exponential phase cultures of Salmonella typhimurium LT2, grown at 37 "C in TSY or M-9 broth, were harvested by centrifugation, washed and resuspended in distilled water at 37 "C at density of 3-0 to 6.0 x IO* bacteria/ml. The suspensions were incubated at 50 "C for various times, serial dilutions were made in phosphate buffer (0.067 M, ph 7) at room temperature and 0.1 ml samples were spread on the surface of TSY and M-9 agar plates at room temperature. The plates were incubated at 37 "C. TSY plates were counted after 24 h and M-9 plates after 48 h. Post-heat treatment incubation. An exponentially growing culture of Salmonella typhimurium L T in ~ M-9 was harvested, washed and resuspended in distilled water. All of these operations were conducted at 37 "C to avoid cold shock. The suspension was divided into two portions, one being held for 18 min at 37 "C (control culture), the other for 18 min at 50 "C. After 18 min both suspensions were diluted (I: 20) into M-9, TSY or distilled water, and incubated at 37 "C. Enumeration was performed on M-9 and TSY agar plates immediately after heat treatment and during the incubation period. When the post-heat treatment incubation was done in water, the bacteria were filtered on to Millipore membranes (HA 0.45 pm; 25 mm), washed twice with distilled water and resuspended immediately after heating to free them of leakage materials. Control experiments showed 100% recovery of bacteria from the membranes. Eflect of hydroxyurea after heat treatment. Hydroxyurea (Sigma Chemical Co., St Louis, Missouri, U.S.A.) was recrystallized from absolute alcohol and introduced at a concentration of 0.1 M in the post-heat treatment M-9 incubation broth. To avoid any carry-over of hydroxyurea on to the plating media, 3 ml samples of the broth were filtered through Millipore membranes (HA 0.45 pm; 25 mm), washed twice with 5 ml of M-phosphate buffer (ph 7.0), resuspended in 3 ml of phosphate buffer and plated on M-9 and TSY agar plates. RESULTS Fig. I shows that, when the culture was grown in TSY broth, the classical 'metabolic iniury ' was observed after heat treatment, TSY agar yielding higher recovery of thermally stressed bacteria than M-9 agar during the early stages of heat treatment. On the other hand, when the culture was grown in M-9 before exposure to heat, reduction of viable counts on TSY agar relative to M-9 was observed, particularly during the first 60 min of heating. This phenomenon is similar to the 'minimal medium recovery' effect observed in radiation sensitive organisms (Ganesan & Smith, 1968; Ganesan & Smith 1970). The effect was independent of the composition of the suspending and heating menstrua (Table I) and brain heart infusion and other chemically defined and undefined enriched media behaved similarly to TSY (Table 2), with the exception of M-9 supplemented with nucleosides or vitamins. ' Minimal medium recovery ' after heat treatment has been demonstrated for several serotypes of salmonellas (Table 3). Neither Salmonella heidelberg nor S. thompson showed any significant 'metabolic injury' when grown in TSY broth. In general, 'minimal medium recovery' was the phenomenon most often observed.

3 Recovery of heated salmonellas oo lo-' '.y-m TSY grown culture \ \v g.- Y & g, > *E $ 10-4 M-9 plates,=&. -= TSY plates Minutes in water at 50 'C., Fig. I. Survival curve of Salmonella typhimuvium L T ~ in water at 50 "C. Dotted lines, culture grown in TSY broth prior to heat treatment; solid lines, culture grown in M-g broth prior to heat treatment; 0 and plated on TSY agar; A and +, plated on M-g agar. Table I. Efect of heating inen~truum on the plating eficiencies of M-9 and TSY agar for Salmonella typhimurium LT~, previously grown in M-9 after 25 min in water at 50 "C Surviving fraction 7-A- Heating liquid M-9 TSY Distilled water 2'0 x x IO-~ M-PO,~- buffer 6.2 x 1o-I 2-3 x IO-~ Skimmed milk 7.2 x x IO-~ When the cultures were grown in M-9 medium, counts on M-9 agar were always significantly higher than on TSY agar even before harvesting and heating. This suggests that the lower recovery of heated organisms on TSY compared with M-9 agar was not totally due to thermal treatment but may reflect an inability of the cultures to cope with a shift up from M-9 broth to TSY agar and that this situation is aggravated by heat treatments. When the organisms were suspended in M-9 after heat treatment (Fig. 2) the control culture showed an exponential increase in both TSY and M-g plate counts after 60 min, the M-9 plate counts being slightly higher than the TSY plate counts. The M-9 plate counts of the heat-treated culture remained constant for 4 h and then increased at the same rate and in the same fashion as the control. The TSY plate counts of the heat-treated culture, which initially showed the reduced viability effect, started increasing immediately and reached the same level as the M-9 plate counts after 4 h of incubation. We interpret this as resulting from the repair of heat-induced damage, as it is too rapid to be due to division in M-9 medium. The behaviour of heated and control organisms in TSY is shown in Fig. 3. The control culture responded to the shift up from M-9 to TSY, after holding for 18 min at 37 "C in distilled water, by a slight decrease in viability on both plating media. Exponential growth 18-2

4 270 R. F. GOMEZ, A. J. SINSKEY, R. DAVIES AND T. P. LABUZA Table 2. Effect of plating media on the recovery of Salmonella typhimurium LT2, previously grown in M-9, after 25 min in water at 50 "C Plating medium M-9 M-g + nucleosides (I o pglml) M-9 + vitamins (10,ug/ml) M-9+ Casamino acids (0.25 %) M amino acids (10,ug/ml) M % yeast extract Brain heart infusion TSY M % trypticase soy Surviving fraction I'OX x '4 x x I O-~ I'OX x IO-~ 3.2 x IO-~ 1.5 x 10-* 1-2 x I O-~ Table 3. Efect of growth medium prior to heat treatment OYE the plating eflciency on M-9 and TSY agar for Salmonella heidelberg and S. thompson after heat treatment in water at 50 "C Growth Minutes M-9 plate counts Organism medium at 50 "C TSY plate counts Salmonella heidelberg M ' TSY 0 1' I '4 Salmonella thompson M I5 4' '0 TSY I00 4'90 was resumed after I h. In contrast, holding in TSY after heat treatment resulted in a marked decrease in the counts on M-9 agar and a small increase in the counts on TSY agar. In other words, as far as it can be detected, holding in TSY broth resulted in the death of the vast majority of heat-treated bacteria. The results of incubation in distilled water after heating are shown in Fig. 4. Viable counts of the control cultures remained constant for the duration of the experiment as did M-9 plate counts of the heated culture, but TSY plate counts increased to within 70 yo of the M-9 counts, indicating that the majority of bacteria were able to repair the heat-induced damage without the aid of any exogenous materials. Incubation in M-9 broth containing 0.1 M-hydroxyurea (Fig. 5) caused a decrease in the recovery of heated bacteria from thermal injury; only 1% of the 'heat-injured' cells regained their ability to grow on TSY agar. In addition, 95% of the cells lost their ability to grow on M-9 agar plates. Hydroxyurea at 0.1 M was strictly bacteriostatic to control cultures within the periods used in this experiment. DISCUSSION Although reports dealing with 'metabolic injury' are abundant (Nelson, I 943 ; Allwood & Russell, 1966; Harries & Russell, 1966; Russell & Harries, 1968), our investigations have shown that the opposite phenomenon, ' minimal medium recovery ', is observed when

5 Reco very of heated salmonellas 27 1 M-9 plates A M-9 nlateq ~ - Heat-treated culture 0 ~~ ~ Minutes in M-9 broth after heat treatment Fig. 2. Post-heat treatment incubation in M-9 medium. Bacteria were held in water at 37 "C (open symbols) or at 50 "C (closed symbols) for 18 min. Both cultures were then diluted (I :20) into M-9 broth at 37 "C and plated at intervals on TSY agar (0 and 0) and M-9 agar (A and A). 1 O'O 10' 1 O8 * s 10' 02 aj 5 I ' 1 O4 I I I I I I / Minutes in TSY broth after heat treatment Fig. 3. Post-heat treatment incubation in TSY broth. Bacteria were either held in water at 37 'C (open symbols) or at 50 "C (closed symbols) for 18 min. Both cultures were then diluted (I :20) into TSY broth at 37 "C and plated at intervals on TSY agar (0 and 0) and M-g agar (A and A).

6 R. F. GOMEZ, A. J. SINSKEY, R. DAVIES AND T. P. LABUZA lo' I - TSY plates Control culture e A M-9 plates cr El lo6 3 0) I I I I 1 Fig. 4. Post-heat treatment incubation in distilled water. Bacteria were either held in water at 37 "C (open symbols) or at 50 "C (closed symbols) for 18 min. Both cultures were then filtered through Millipore membranes (HA 0.45 pm; 25 mm), resuspended in distilled water and plated at intervals during incubation at 37 "C on TSY agar (0 and 0) and M-g agar (A and A). 1 1 OR Y 3 : c- % s 107 lo I \ I- I /-/ - TSYplates With hydroxyurea I I i I I

7 Recovery of heated salmonellas 273 bacteria are grown in minimal media prior to heat treatment. Salmonella typhimurium L T~, grown in M-9 medium, is extremely sensitive to a shift up to either TSY broth or TSY agar after sublethal heat treatment (Fig. 3). Sensitivity to a shift-up from M-9 to TSY broth without heat treatment, as in Fig. 3, was not always observed. These findings substantiate those of Labots (1959), who has reported that the addition of yeast extract to milk containing sublethally heated coliform bacteria, previously grown in milk, causes a decrease in viable numbers, and that dilution with water retarded the death rate. Mukherjee & Bhattacharjee (1970) have observed an increase in counts on nutrient agar during incubation in phosphate buffer of heat-treated Escherichia coli. However, they did not report the high recovery on minimal media agar seen in our investigations (Fig. 2). In radiation-sensitive strains of Escherichia coli minimal medium recovery has been extensively studied by Ganesan & Smith (1968, 1971). They hypothesize that the phenomenon is due to a recovery process after ultraviolet irradiation, that is inhibited by complex media and appears to require conditions which permit the synthesis of DNA. It should be noted that minimal medium recovery of irradiated bacteria is only observed in radiationsensitive strains. Woodcock & Grigg (1972) and Sedgwick & Bridges (1972) demonstrated the presence of single- and double-stranded breaks in the DNA molecule of Escherichia coli after heat treatment and the former group showed the restitution of these during incubation in phosphate buffer. Our experiments indicate that hydroxyurea not only blocks repair of thermal injury but in addition it is bactericidal to heat-injured cells (Fig. 5). Hydroxyurea inactivates protein fraction B 2 of the ribonucleotide diphosphate reductase system without significantly affecting protein or RNA synthesis, thus depleting the cell of DNA synthesis precursors (Krakoff, Brown & Reichard, 1968). If the effect of hydroxyurea is only that reported by Krakoff et al. (1968), then deoxyribonucleotides are required for the repair of thermal injury and their absence results in the inactivation of heat-treated organisms. If DNA breaks are thermally induced in Salmonella typhimurium LT 2 and subsequently repaired, when provided with suitable conditions, then such a repair requires de novo accumulation of deoxyribonucleotide diphosphates and possibly reincorporation into the DNA molecule. The extreme sensitivity of M-9-grown bacteria to rich media after thermal treatment questions the validity of the practice of pre-enrichment procedures in rich media for the isolation of food-borne pathogens. The preprocessing history of the micro-organisms is likely to affect their recovery in rich media and, if their physiological state resembles that of M-9-grown bacteria, then a priori, the pre-enrichment step should lower the sensitivity of the isolation procedure. This work is Contribution no from the Department of Nutrition and Food Science, Massachusetts Institute of Technology and was supported by Public Health Service Grant FD We thank Professor Dr D. A. A. Mossel of the Central Institute of Nutrition and Food Research, Zeist, The Netherlands, and Dr Ir. 5. Stadhouders of the Netherlands Institute for Dairy Research for their interest in this work. We appreciate the helpful comments of Dr B. A. Bridges of the University of Sussex. REFERENCES ADAMS, M. H. (1959). Bacteriophages, 1st edn., p New York: Interscience Publishers. ALLWOOD, M. C. & RUSSELL, A. D. (1966). Some factors influencing the revival of heat-damaged Staphylococcus aureus. Canadian Journal of Microbiology 12, I

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