Chilling invokes different morphologies in two ... - Wiley Online Library

Journal of Applied Microbiology 1998, 84, 820–826

Chilling invokes different morphologies in two Salmonella enteritidis PT4 strains L.E. Phillips1, T.J. Humphrey1 and H.M. Lappin-Scott2 1

PHLS Food Microbiology Research Unit and 2Department of Biological Sciences, Hatherly Laboratories, Exeter, UK 6230/05/97: received 9 May 1997, revised 27 August 1997 and accepted 29 September 1997

The reduction in chemical preservatives in food processing has resulted in more refrigerated (chilled) products. However, the effect of chilling on Salmonella enteritidis PT4 isolates has received relatively little attention. This study investigates the effect of chilling on two Salm. enteritidis PT4 isolates, denoted E and I. These isolates differ in their tolerance to heat, acidification, survival on surfaces, and behaviour in animal models. E routinely shows greater tolerance and pathogenicity than I. Chilling invokes profound cell elongation and heterogeneity in E which corresponded to a 90% sublethal injury; neither such substantial cell elongation nor significant injury was seen in I. The ability to recover resistance to desoxycholate coincided with a reduction to normal cell size. Incomplete cell division and failure of the septum to form is a likely hypothesis for cell elongation although outer membrane changes could be responsible. Possible links are suggested between cell elongation of the heat- and acid-tolerant strain and pathogenicity. L .E . P H IL LI P S, T. J . H UM P HR EY A ND H. M . L AP P IN -S C OT T. 1998.

INTRODUCTION

Control of the internationally important food-borne pathogen Salmonella which is transmitted by a wide variety of agricultural products and processed foods (Socket and Roberts 1991; Jay 1992) remains central to both the food industry and surveillance services (Wyatt et al. 1995). In order to grow, most bacteria require a moderate temperature and pH range and high water availability. Control treatments are successfully accomplished when one or more of these requirements are not fulfilled. Nevertheless, salmonellas routinely survive within extreme environments by inducing specific stimulons which may promote survival (Kolter et al. 1993; Foster and Spector 1995). It is perhaps not surprising that outbreaks continue to be reported in which a variety of treated or preserved foodstuffs are implicated (Socket and Roberts 1991; Advisory Committee on the Microbiological Safety of Food 1993; Wei et al. 1995). Demand for a reduction in additives and chemical preservatives in foodstuffs (Waites and Arbuthnott 1990) has made it necessary to refrigerate these additive-free products Correspondence to: Lisa E. Phillips, Oxoid Ltd, Wade Road, Basingstoke, Hampshire RG24 8PW, UK (e-mail: [email protected]).

to prevent spoilage. The minimum growth temperature for enteric organisms such as Escherichia coli and Salmonella ranges from 5 to 8 °C (Matches and Liston 1968; Shaw et al. 1971; Mossel et al. 1981; Jay 1992). Hence, their growth, together with that of mesophiles, is prevented by refrigeration, although lower minimum growth temperatures have been recorded for the growth of salmonellas in food (D’Aoust 1991). Detection of pathogens within foods frequently requires a recovery period necessary for the repair of damaged cells (Andrews 1986). This period is prolonged by the presence of low cell numbers and is further increased by cold storage of the bacteria (Mackey and Derrick 1982). The effects of chilling on acid and heat tolerance of pathogens have received relatively little attention, although preexposure to 4 °C has been shown to increase heat sensitivity in Salm. enteritidis PT4 (Humphrey 1990). Given recent work on differences between tolerance and virulence in closely related Salm. enteritidis PT4 isolates (Humphrey et al. 1995, 1996), it is likely that differences may also exist in response to low temperature. This work aimed to define further Salm. enteritidis PT4 strains E and I which differ in their surface survival, heat, acid and H2O2 sensitivities and pathogenicity © 1998 The Society for Applied Microbiology

C HI LL I NG TW O SA LM O NE LL A EN TE R IT ID I S P T4 S TR AI N S 821

(Humphrey et al. 1995, 1996). The effects of chilling (4 °C) on the morphology, injury, viability and recovery of these closely related PT4 strains are reported in this paper. MATERIALS AND METHODS Cultures

Salmonella enteritidis PT4 strains E and I were used throughout this study. Strains were maintained at 4 °C on nutrient agar slopes between experiments, and subcultured prior to use on Columbia Blood Agar with 5% lysed blood at 37 °C. Buffered Peptone Water (BPW) was used in culture preparation and chilling because of its wide use as a non-selective pre-enrichment broth in the recovery of Salmonella (Edel and Kampelmacher 1973). All media used were manufactured by Oxoid Ltd (Basingstoke, UK). Routine purity checks were made by streaking onto Xylose Lysine Desoxycholate (XLD) and MacConkey agar. Once chilled, purity was ensured by keeping the vials closed until sampling and each vial was discarded. No bacteria other than Salm. enteritidis PT4 were isolated during this study. Morphology and cell size of chilled cells of E and I

Cell morphologies of strains E and I were visualized using the nucleic acid stain acridine orange (Zimmerman et al. 1978). This method of viewing the cells was chosen because the quality and clarity of the cells was superior to that seen using the Gram stain technique. Stained cells were filtered onto pre-blackened 0·22 mm pore size polycarbonate membranes (Poretics Corporation, Livermore, USA) and air dried. Immersion oil was placed onto the membranes, a coverslip placed on top and further oil added for viewing. Cells were viewed using the ×100 objective of an Olympus CH-2 light microscope (Olympus Instruments, London, UK) and illuminated by a BH2-RFCA fluorescence light source as described by Walsh et al. (1995). Images were initially passed through a high performance CCD video camera (CUHU). Image capture and subsequent image editing were accomplished on a Macintosh 7200/90 computer using Scion Image LG5 operating software. Scion Image is an adaptation of the Public domain NIH-Image 1.59 program (developed at the National Institute of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov or a floppy disc from the National Technical information service, Springfield, VA, USA, part number PB95–500195GEI). Culture viability and injury of Salmonella enteritidis strains during chilling

Stationary phase cultures (18 h) of strains E and I grown statically in BPW at 37 °C were standardized to an OD of

0·2 at 600 nm (20·005) with fresh BPW using a CE 1010 spectrophotometer (Cecil Instruments, Cambridge, UK). Each strain was diluted in BPW to 5 × 105 ml−1. Aliquots (3 ml) of this suspension were dispensed in sterile glass bijoux and refrigerated immediately (Sikafrost Comfort, Siemens, Germany) to 4 °C (20·5 °C). This volume decreased in temperature from 22 °C to 4 °C in approximately 23 min. Triplicate samples were removed at regular intervals and appropriate serial dilutions were made in Maximum Recovery Diluent (MRD). These were plated in duplicate using a spiral plater WASP (Don Whitely Scientific, Shipley, UK) on Columbia Blood and XLD agars. Lag times of chilled cells

Lag periods of the chilled populations were determined using the Bioscreen C Analyser System (Labsystems Corporation, Helsinki, Finland), a fully automated temperature-controlled means of monitoring culture absorbance. Chilled cultures were serially diluted in MRD to ¾20 cells ml−1 and inoculated in 10 × 400 ml volumes into the multiwell plate. The plate was incubated at 37 °C in the Bioscreen where culture absorbance readings were recorded at 30 min intervals. Data were collated via the Bioscreen-Link computer software Version 4.09 and converted to MS EXCEL 5.0. After 70 d at 4 °C, remaining bijoux were shifted to 37 °C. Samples were removed at intervals and plated on Columbia Blood and XLD agar. Statistics

Experiments were repeated at least three times and statistics were calculated using the computer package MS EXCEL 5.0. RESULTS The effect of short-term chilling on the morphology and viability of strains E and I

Stationary phase cells of the two isolates were chilled in BPW at 4 °C for a period of 12 d during which time samples were removed for light microscopy, viable counts on blood and XLD agars and recovery at 37 °C. At intervals, 1 ml volumes of the chilled suspensions were sampled then fixed and stained for photography (Fig. 1a–h). The Salm. enteritidis strains displayed markedly different morphologies under the chilled conditions. At day 1 both populations comprised typical rod-shaped cells of between 2 and 3 mm in length (Fig. 1a,e). These lengths did not change until after 5 days at 4 °C; maximum lengths at this point were 15 mm and 10 mm for E and I, respectively (Fig. 1b,f). Cell lengths increased slightly over the following 3 days (data not shown) to reach 20 mm and 15 mm for E and I (Fig. 1c,g).

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 820–826

822 L .E . P H IL LI P S E T A L .

Fig. 1 Light micrographs of acridine orange-stained cells of Salmonella enteritidis PT4 strains E (a–d) and I (e–h) after 1, 6, 9 and 12

days of chilling in BPW. Distance between bars represents 10 mm

Significant elongation occurred between day 9 and 12 where typical cells of E often measured over 100 mm in length with individual cells recorded at 150 mm (Fig. 1d). Strain I did not elongate to the same extent during this period and reached a

corresponding maximum cell length of 35 mm after 12 days of chilling (Fig. 1h). In addition to microscopy, samples were serially diluted and plated onto blood and XLD agars. Both PT4 isolates



remained viable in BPW chilled for 12 d (Fig. 2). There was no significant decrease (P ¾ 0·05) in the viable cell count for isolate I during the experiment but E decreased significantly over the same period (P − 0·05). A significant increase in the viable count of strain I was observed after chilling for 3 d and a similar increase was observed in E after 24 h (P − 0·05). Results from the XLD counts suggested that neither population was significantly injured until day 12, at which time strain E sustained a 93% injury (S.E. 3·49) and I, only 29% (S.E. 8·05). Chilling at 4 °C for a period of 12 d increased the recovery times of both strains but there was no significant difference between the strains (data not shown). The effect of prolonged chilling on morphology and recovery of strains E and I

PT4 isolates were chilled for an extended period (a total of 70 d) under the conditions described previously. Cell lengths of both isolates were recorded at hourly intervals during incubation at the recovery temperature of 37 °C (Fig. 3a,b). Both isolates were affected by long-term chilling. After 70 d at 4 °C a variety of cell sizes, ranging from 30 mm to 130 mm, was recorded for E signifying a heterogeneous population of Salm. enteritidis. The majority of cells within I remained between 0 and 10 mm in length after long-term chilling despite some cells elongating to between 30 and 40 mm (Fig. 3b). The elongated cells of isolate E gradually reduced in size to their normal dimensions after 4 h at 37 °C (Fig. 3a). After 1 h at this temperature, the majority of cells in E measured between 50 and 60 mm in length; this decreased to 30–40 mm after 2 h, to 20–3 mm after 3 h and finally to between 0 and 10 mm after 4 h recovery. After incubation at 37 °C for 4 h both strains measured between 0 and 10 mm in length

Fig. 3 The effect of warming on Salmonella enteritidis PT4 strains E (a) and I (b) following prolonged chilling for 70 d in BPW. Samples incubating at 37 °C were removed at 1 h intervals for cell sizing

(Fig. 3a,b). Although the cells had resumed their normal morphology they remained in lag phase (Fig. 4). Counts on blood and XLD agars were taken at hourly intervals from the warmed (37 °C) samples (Fig. 4). Following prolonged chilling, both the blood and XLD agar counts of the strains showed no significant difference (P ¾ 0·05) as shown by the reading at 0 h (Fig. 4). After 3 h at 37 °C, XLD counts for E increased and by 5 h, blood and XLD counts were the same (P ¾ 0·05). Counts on XLD for I began to increase after 5 h and the blood counts after 7 h. However, these counts remained significantly different until after 9 h. Fig. 2 Viability of Salmonella enteritidis PT4 strains E and I

during 12 days of chilling in BPW at 4 °C. (Ž) E ; () I. Error bars represent S.E., n 5

DISCUSSION

This study has identified different responses to chilling at 4 °C between isolates of Salm. enteritidis PT4, with profound



Fig. 4 Effect of 70 days of chilling in BPW on the recovery at

37 °C of Salmonella enteritidis PT4 strains E (circles) and I (triangles). Open symbols, blood agar; closed symbols, XLD agar

elongation being shown by the more pathogenic isolate E. During prolonged (× 6 d) storage at 4 °C the culture broths containing this isolate became significantly more turbid than those containing the isolate I. Filament formation has been reported in organisms at elevated (Terry et al. 1966) and low (Shaw 1968) temperatures; on exposure of lon mutants to u.v. irradiation (Gottesman and Zipser 1978); following nutrient deprivation (Nelson et al. 1996); and with growth on caffeine (Jenson and Woolfolk 1985). Mutants defective in nucleoid segregation or partition (par) mutants have been found in E. coli and Salm. typhimurium (Ryter 1968; Schmid 1990). In some cases, the mutant populations contain cells that are extremely varied both in cell dimensions and in nucleoid morphology (Schmid 1990). Other mutations, such as those causing partial inhibition of ftsZ, have been reported to increase cell size (Tetart et al. 1992). Temperature-sensitive cell division mutants of Salm. typhimurium which showed an envelope alteration not only produced filaments at 42 °C, but also cells with increased diameters (Shannon et al. 1974). This was due to continued increase in biomass at the restrictive temperature coupled with a decrease in cell elongation resulting in wider cells (Shannon and Rowbury 1975). All of the above describe unbalanced growth where some part of the cell division process is compromised. The majority of low temperature-induced filamentation has been studied using E. coli (Shaw 1968). Nevertheless, similarities exist between work on E. coli at 6 °C and this study. These similarities include the time required for filaments to initiate (6 d), filament length (150 mm), and recovery of regular morphotypes once the cultures were returned to higher temperatures. However, the strains used by Shaw (1968) displayed branching which was not apparent from light micrographs of either PT4 strain (Fig. 1a–h). The

results from this study suggest that the DNA had replicated and segregated under the chilled conditions but that septum formation was inhibited causing unbalanced growth which may explain the heterogeneous distribution of cell size shown by E (Fig. 3a). Light micrographs also suggested an increase in the width of chilled cells (Fig. 1a–h), an observation previously reported in a cell division mutant (HD-20) of Salm. typhimurium (Shannon and Rowbury 1975). However, the apparent increased width in this instance may be partly due to a strong fluorescence being received by the camera from larger cells with more bound nucleic acid stain. This appears to be the first quantitative report of Salm. enteritidis PT4 elongating to such a degree in response to a chilled nutrientrich medium. Isolate E showed a loss of viability (Fig. 2) and surviving cells were largely unable to grow on selective media (Fig. 4). Smith et al. (1994) observed a similar result when Salm. typhimurium reduced in number by 1 log unit after 11 d at −1·8 °C. More recently, Nychas and Tassou (1996) also demonstrated the survival of Salm. enteritidis at 3 °C under the more stringent modified atmospheres which, again, did not permit growth. The increase in cell numbers of strain I (Fig. 2) may have been due to reductive division occurring during the first 24 h of chilling. Similarly, Smith et al. (1994) observed slight increases in total cell numbers and decreases in cell size within the first week of exposure to −1·8 °C, and suggested reductive division as a likely factor. It is possible that growth and division could explain the increase seen with I, even though growth has not been reported at 3 °C, and the majority of strains will only grow very slowly at 7 °C (Mossel et al. 1981). In addition, translation initiation, as well as binding and transport of amino acids, was inhibited in E. coli at 8 °C (Broeze et al. 1978). Strain E also showed a similar increase in viable counts after chilling for 3 d and, again, the same explanation may be applied. The relationship between lag time and time taken to recover resistance to selective media is an important criterion of repair (Mackey and Derrick 1982). Sublethally injured cells of Salm. typhimurium took up to 14 h to repair despite a 9 h lag time (Mackey and Derrick 1982). In this study, increases in blood and XLD counts were simultaneous when chilled cells of E and I were warmed to 37 °C (Fig. 4). The differences between the strains observed after 12 d at 4 °C were not continued after 70 days of storage as both strains exhibited over a 90% injury. The more rapid recovery of E at 37 °C suggests that elongation does not reduce the ability to recover and begin exponential growth and may possibly enable a more rapid recovery. These results confirm the work of Mackey (1984) which suggests that refrigeration in broth causes extensive sublethal injury to salmonellae and that differences may occur over a shorter time period in terms of injury and morphological manifestations. Reasons for variations in bacterial sensitivity



to cold remain unknown but are not related to any single properties such as the Gram reaction or morphology (Mackey 1984). This study has shown that very closely related strains of the same phage type have different responses to the important control measure of chilled storage just as they do to high temperature. As Salmonella can grow at temperatures very close to the desired refrigeration limits (Matches and Liston 1968), it is essential to understand how important pathogens such as Salm. enteritidis PT4 behave and respond to refrigerated conditions and further work needs to be undertaken to predict how prevalent pathogens will respond.

ACKNOWLEDGEMENTS

The authors are very grateful to Oxoid Ltd for funding this research. The authors would specifically like to thank Dr D. Swaine and Mr P. Stephens (Oxoid Ltd) and Mr R. Holbrook (Unilever Research) for their technical advice, and Mr R.A. Davey for practical assistance.

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