Heat Resistance in Liquids of Enterococcus spp., Listeria spp., Escherichia coli, Yersinia enterocolitica, Salmonella spp. and Campylobacterspp

Abstract

The aim of the work was to collect, evaluate, summarize and compare heat resistance data reported for Campylobacter, Enterococcus, Escherichia, Listeria, Salmonella and Yersinia spp. The work was limited to resistance in liquids with pH values 6–8. Results obtained under similar experimental conditions were sought. Thermal destruction lines for the various bacterial groups studied were constructed using log₁₀ D values and treatment temperatures. There was a good linear relationship between log₁₀ D and temperature with Escherichia coli, listerias and salmonellas. For campylobacters, enterococci and yersinias the relationships were weaker but, nevertheless, present. Using the slopes of the lines and their 95% confidence limits, z values and their 95% confidence limits were calculated. z values were compared with z values obtained from reports. The equations for the lines were also used for calculation of predicted means of D values at various treatment temperatures. 95% confidence limits on predicted means of D values and on predicted individual D values were also calculated. Lines and values are shown in figures and tables. Differences in heat resistance noted between and within the bacterial groups studied are discussed.

Sammanfattning

Värmeresistens i vätskor hos Enterococcus-, Listeria-, Escherichia-, Yersinia- , Salmonella- and Campylobacter-arter.

Introduction

Microbiologists now and then need heat resistance data for various microorganisms. In the literature, data of this kind are frequently based on reports from few investigations. To collect the data required, however, may be a laborious and time-consuming task for the individual user. The literature is generally extensive and many factors that may have influenced the results reported must be taken into consideration (for general information on influencing factors, see e.g. [71, 163, 137]). Furthermore, the presentations of results often differ essentially.

The aim of the present work was to collect, summarize, evaluate and compare heat resistance data reported for Campylobacter, Enterococcus, Escherichia, Listeria, Salmonella and Yersinia spp. As it was well known that considerably more heat resistance results were published from investigations with liquids than from those with other heating menstrua, it was considered appropriate to base the work on results obtained in liquids. Moreover, results of this kind could be expected to reflect the inherent heat resistance of the bacteria investigated better than those obtained in more complex heating menstrua.

Reports published until 2000 were studied. Data produced under experimental conditions as similar as possible were sought. This meant that results from some kinds of experiments were excluded. The various types of excluded data are given below under the different subheadings in Experimental conditions. It should be mentioned here that extensive reviews of heat resistance data reported for Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. have been published recently by [162, 43] and [42], respectively. However, the aims and the selections and analyses of data in these reviews differ from those in the present work.

Bacteria

The work deals with the following bacteria: Campylobacter jejuni/coli, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria seeligeri, Listeria welshimeri, Salmonella spp. and Yersinia enterocolitica. Some of these bacteria are well-known food-associated human pathogens, others are utilized – enterococci and E. coli – or proposed – L. innocua [56, 48] – as indicators. Some types of E. coli also appear as food-linked human pathogens [119, 121, 122, 27, 29, 86, 10, 174, 62, 93] and enterococci have recently emerged as one of the leading causes of nosocomial, non-food-associated, infections [94].

Experimental conditions

Growth of test bacteria

In most cases the bacteria were grown in conventional media. In some investigations the growth media were milk, liquid egg products or clarified cabbage juice. The pH values of the media were given in some cases. The values varied from 5.6 to 7.4. Enterococci, E. coli, listerias and salmonellas were incubated aerobically at 30–37°C and Y. enterocolitica aerobically at 25–37°C. Campylobacters were grown microaerobically at 35–43°C. In the great majority of cases the bacteria were incubated for 12–48 h, i.e. they could be considered to have reached the late logarithmic or stationary growth phase. At stationary growth phase, bacterial heat resistance is at a maximum [46, 173, 99, 102, 9, 125, 77, 86, 105, 93, 131, 132].

Heat resistance results obtained for bacteria grown under carbon, glucose or nitrogen starvation or other stress conditions (see e.g. [125, 87, 105]) were not used in the present work.

Conditions between growth and heat treatment

Results recorded for bacteria subjected to stress conditions prior to heat treatment were not used: sublethal heat shock (see e.g. [108, 110, 111, 19, 20, 121, 122, 12, 82, 54, 53, 153], alkaline stress [81, 84], acid stress [49, 103, 175], osmotic stress [89] or other types of stress (see e.g. [12, 54, 53]).

Heating menstrua

Heating menstrua used were milk and liquid milk products, broths, physiological saline and other salt solutions, liquid egg products, diluted soups, scalding waters used at chicken or pig slaughter, and some other liquids. Heat resistance results obtained in menstrua with pH values of approx. 6–8 were used in the present work, as the bacterial species investigated are known to have their maximum heat resistances in this pH range (see e.g. [5, 99, 174, 60, 80, 148, 128, 10, 131, 138]). Results from experiments where salts, fats, carbohydrates, proteins or other substances were added to the heating menstrua with the aim of influencing the heat resistance of the test bacteria were excluded (see e.g. [100, 22, 5, 66, 170, 31, 3, 133, 10, 96]).

Heat treatment

Various methods of heat treatment were applied, e.g. heating in water baths using glass capillary tubes, sealed glass tubes, glass ampoules or polythene pouches completely immersed in the water, test tubes placed with the water level to the bases of the test tube plugs, flasks or cups placed with the menstruum levels under the water level and in some cases shaken, and heating using pasteurizers, two-phase slug flow heat exchangers [15, 18, 21, 20, 97, 29], submerged-coil heating apparatuses [3, 89, 88, 10, 90], thermoresistometers [141, 131, 132] and an "attemperated dilution blank method" [113, 114].

Results from experiments using rising heating temperatures [169, 109, 140, 161] were excluded.

Recovery of heat-treated bacteria

In the great majority of cases the recovery of heat-treated bacteria was performed on agar plates. Enterococci and E. coli were incubated aerobically at 30–37°C for 24 h-7 days, listerias, salmonellas and Y. enterocolitica aerobically at 25–37°C for 24 h-7 days and campylobacters microaerobically at 37–43°C for 24–72 h. In some studies anaerobic recovery was used: L. monocytogenes [95, 63], E. coli [121, 122, 58, 10, 63, 62] and salmonellas [177, 10, 63]. Most Probable Number (MPN) techniques were applied in some investigations. Procedures for repair of heat-injured bacteria were studied by [1, 127, 116, 157, 158, 63].

Results from experiments where heat-treated bacteria were recovered on selective or other media known to inhibit growth of heat-injured cells were excluded.

Types of collected data and statistical analysis

D and z values were collected from the studied literature. The D value is the time of heat treatment required at a certain temperature to destroy 90% of the bacterial cells, and the z value is the number of degrees of temperature change needed to change the D value by a factor of 10 [163]. When not reported, D values were, where possible, calculated from bacterial counts and periods of time of heat treatment given in texts, tables or figures. Some z values were worked out from reported or calculated D values and reported treatment temperatures.

For each of the bacterial species/groups studied, the log₁₀ of D values recorded were plotted vs temperature and a thermal destruction line [163] was fitted using the method of least squares [30]. The equation for the line is log₁₀ D = a - bt, where D is the decimal reduction time in s, a the intercept, -b the slope and t the treatment temperature in °C. The degree of linear relationship between the temperatures used and the logarithms of D values recorded was expressed by the coefficient of correlation, r [30]. Using the absolute and inverse values of the slope and its 95% confidence limits, the z value and its 95% confidence limits were calculated [163, 30].

95% confidence limits on predicted means [30] of D values were calculated (the predicted mean is the same as D in the equation). 95% confidence limits on predicted individual D values [30] were also figured out (From a practical point of view it may be more interesting to know these limits than those on predicted means).

Summaries of data

Reported z values are summarized in Table 1. Reported and calculated z values taken together are given in Table 2, where z values figured out in the work by means of the equation mentioned, etc. are also shown. Thermal destruction lines for the bacteria studied, except those for L. innocua, L. ivanovii, L. seeligeri and L. welshimeri, are depicted in Figures 1, 2, 3, 4, 5, 6, 7, where 95% confidence limits on predicted individual log₁₀ D values are also illustrated graphically. In Table 3, some D values at these limits are shown for the seven bacterial groups and also for L. innocua. Equations for the thermal destruction line of L. innocua and that of L. ivanovii, L. seeligeri and L. welshimeri taken together, are given below under the headings Listeria innocua and Listeria ivanovii, L. seeligeri and L. welshimeri, respectively.

Table 1 z values reported from investigations where the experimental conditions laid down in this study were fulfilled.

Table 2 z values obtained using the slopes of thermal destruction lines constructed in the study and their 95% confidence limits and, for comparison, summaries of reported and calculated z values.

Table 3 Heat resistance values at 4 temperatures for bacteria studied in the work. The values are based on results reported from investigations where the experimental conditions laid down in the work were fulfilled.

Figure 1

Heat resistance data (Mean ± SD) recorded at the different treatment temperatures used and fitted thermal destruction line (-) for Enterococcus faecium. The equation for the line is log₁₀ D = 9.3080 - 0.10412t (r = -0.84748; total number of log₁₀ D values = 195). The 95% confidence limits on predicted individual log₁₀ D values are shown by (- -). The figure is based on data from: [69, 179, 85, 170, 148, 113, 114, 140, 68, 98, 136, 154, 94, 120, 144].

Figure 2

Heat resistance data (Mean ± SD) recorded at the different treatment temperatures used and fitted thermal destruction line (-) for Enterococcus faecalis. The equation for the line is log₁₀ D = 8.9359 - 0.10531t (r = -0.72968; total number of log₁₀ D values = 244). The 95% confidence limits on predicted individual log₁₀ D values are shown by (- -). The figure is based on data from: [145, 172, 99, 173, 179, 9, 26, 85, 152, 170, 33, 35, 59, 148, 113, 114, 140, 12, 94, 54, 53].

Figure 3

Heat resistance data (Mean ± SD) recorded at the different treatment temperatures used and fitted thermal destruction line (-) for Listeria monocytogenes. The equation for the line is log₁₀ D = 12.3787 - 0.17401t (r = -0.95631; total number of log₁₀ D values = 474). The 95% confidence limits on predicted individual log₁₀ D values are shown by (- -). The figure is based on data from: [15, 8, 18, 40, 16, 41, 52, 21, 50, 67, 127, 160, 45, 51, 101, 138, 164, 13, 19, 55, 95, 104, 112, 3, 17, 56, 97, 115, 139, 20, 49, 73, 116, 48, 157, 158, 161, 7, 89, 88, 133, 134, 105, 123, 135, 149, 23, 63, 90, 131, 147, 96].

Figure 4

Heat resistance data (Mean ± SD) recorded at the different treatment temperatures used and fitted thermal destruction line (-) for Escherichia coli. The equation for the line is log₁₀ D = 11.6471 - 0.16768t (r = -0.97349; total number of log₁₀ D values = 332). The 95% confidence limits on predicted individual log₁₀ D values are shown by (- -). Data used are from: [46, 92, 155, 24, 142, 102, 143, 22, 47, 66, 35, 39, 169, 1, 91, 178, 37, 87, 119, 121, 122, 58, 2, 27, 28, 29, 86, 165, 10, 174, 175, 63, 62, 93, 150]. Thermal destruction line for an unusually heat-resistant strain of E. coli reported by [72] is also shown (- • -).

Figure 5

Heat resistance data (Mean ± SD) recorded at the different treatment temperatures used and fitted thermal destruction line (-) for Yersinia enterocolitica. The equation for the line is log₁₀ D = 10.4176 - 0.14896t (r = -0.86082; total number of log₁₀ D values = 88). The 95% confidence limits on predicted individual log₁₀ D values are shown by (- -). The figure is based on data from: [70, 57, 126, 106, 37, 156, 159, 168, 153, 132].

Figure 6

Heat resistance data (Mean ± SD) recorded at the different treatment temperatures used and fitted thermal destruction line (-) for Salmonella spp. The equation for the line is log₁₀ D = 12.9511 - 0.19282t (r = -0.92147; total number of log₁₀ D values = 647). The 95% confidence limits on predicted individual log₁₀ D values are shown by (- -). Data used are from: [155, 4, 130, 100, 38, 124, 166, 32, 141, 60, 61, 125, 5, 47, 66, 146, 31, 34, 118, 39, 64, 31, 167, 75, 80, 108, 128, 14, 36, 109, 110, 6, 19, 77, 76, 111, 159, 81, 151, 82, 84, 103, 177, 176, 83, 133, 134, 123, 165, 10, 149, 63, 74, 117]. Thermal destruction line for the extremely heat-resistant Salm. senftenberg 775W is also shown (- • -) ; for references, see text.

Figure 7

Heat resistance data (Mean ± SD) recorded at the different treatment temperatures used and fitted thermal destruction line (-) for Campylobacter jejuni/coli. The equation for the line is log₁₀ D = 10.3432 - 0.15717t (r = -0.89853; total number of log₁₀ D values = 112). The 95% confidence limits on predicted individual log₁₀ D values are shown by (- -). The figure is based on data from: [44, 65, 11, 25, 171, 129, 78, 128, 79, 37, 156, 159].

Comments and further information

D and r values

The order of death of bacteria subjected to heat at a constant lethal temperature is often logarithmic [71, 163, 137], i.e. when the logarithm of survivors is plotted against the time of heating, the curve obtained, the survivor curve, is a straight line. The D value can then easily be calculated using the slope of the line. Deviations from the logarithmic order of death, however, are rather frequent and non-logarithmic survivor curves of some different types are obtained [71, 163, 137]. Deviations of this kind often make determinations of D values difficult.

The r values, varying from -0.92147 to -0.99405, obtained for Salmonella spp., E. coli and the 3 Listeria groups indicate very good linear relationships [30] between the log₁₀ D values recorded and the treatment temperatures used. The r values, varying from -0.72968 to -0.89853, recorded for Ent. faecalis, Ent. faecium, Y. enterocolitica and Camp. jejuni/coli indicate weaker but, nevertheless, good linear relationships [30]. The following should be noted here: The number of Y. enterocolitica strains investigated is low. The results reported, however, indicate that great variation in heat resistance exists between strains of this species. As to enterococci, non-logarithmic survivor curves were reported in several works [179, 33, 35, 148, 113, 68, 12].

Listeria monocytogenes

[107] published a similar review of the heat resistance of L. monocytogenes. Equations were given for heat treatments in: (a) various menstrua and (b) milk. The treatments in (b) had been performed by a sealed tube method (b1) or a slug flow heat exchanger (b2). The equations for (a), (b1) and (b2) were log₁₀ D = 10.888 - 0.14519t, log₁₀ D = 11.931 - 0.1635t and log₁₀ D = 10.126 - 0.1348t (D is in s in the equations). The means of D values obtained by the 3 equations for 55, 60, 65 and 72°C are shown in Table 4. The means in (a), (b1) and (b2) except that in (b2) for 55°C are higher than the corresponding ones (c) recorded for L. monocytogenes in the present work (Table 3). The differences between (a) and (c) may, at least to some extent, be explained by the fact that some of the heating menstrua in (a) were solids. The differences between (b1) or (b2) and (c) are therefore of greater interest, as all data for these 3 groups were obtained in liquids. A probable explanation of these differences is that heat resistance data for several "new" strains have been published later than the review by [107] and have thus been included in the present work. Furthermore, the methods of determining the heat resistance of bacteria have been widely discussed in recent years and some improvements or new procedures have been introduced. Factors of this kind may also have contributed to the differences.

Table 4 D values for Listeria monocytogenes according to the review by [107].

Listeria innocua

The non-pathogenic L. innocua is of special interest as it has, as mentioned, been proposed to be used as an indicator organism to evaluate thermal processes for lethality to L. monocytogenes. To function satisfactorily in this respect it is desirable that the indicator has heat resistance equal to or greater than the average heat resistance of L. monocytogenes or, more desirably, has heat resistance equal to that of the most resistant strains of this species. In the present work, heat resistance results for L. innocua were found in 5 reports [138, 112, 56, 48, 133]. The equation for the thermal destruction line constructed was log₁₀ D (D in s) = 14.2559 - 0.20077t (r = -0.95519). The average heat resistance values at 55, 60 and 65°C calculated for L. innocua were greater than those for L. monocytogenes (Table 3), but none of analysed differences between means of D values were statistically significant. As to L. innocua, however, only 36 D values were reported totally and the D values obtained at the individual treatment temperatures used were few, 1–4. The most heat-resistant strain of the L. innocua strains investigated was reported by [138]. D values determined at 58, 60, 63 and 65°C using a culture medium as heating menstruum were 2.7 to 5.4 times greater than the average D values found in the present work for L. monocytogenes at these temperatures. [56] tested L. innocua strain ATCC 33091 in buffer and in skim milk at 56, 60 and 66°C. In buffer, the D values were lower at 56 and 60 but higher at 66°C than the corresponding average values for L. monocytogenes. When L. innocua PFEI (strain ATCC 33091 containing a plasmid which did not alter its heat resistance) was tested in skim milk, all D values obtained at these temperatures were higher, 1.5 to 2.1 times, than the values mentioned for L. monocytogenes. [133] determined D values for a L. innocua strain isolated from raw egg. The tests were performed in egg yolk. D values obtained at 61.1, 63.3 and 64.4°C were 2.5 to 2.9 times longer than the corresponding average values for L. monocytogenes. The results reported indicate that L. innocua may have greater average heat resistance than L. monocytogenes. However, as mentioned, only few heat resistance results are reported for L. innocua and more research on this matter is required.

Listeria ivanovii, L. seeligeri and L. welshimeri [17] studied the heat resistance of L. ivanovii, L. seeligeri and L. welshimeri. One strain of each species was tested in milk at 52.2, 57.8, 63.3 and 68.9°C. The equation for the 3 species taken together is log₁₀ D (D in s) = 11.3419 - 0.15713t; r = -0.99405. All means of D values obtained for the 4 treatment temperatures were lower than the corresponding means noted in the present work for L. monocytogenes. The differences between the means were statistically significant for the values obtained at 52.2 and 57.8°C (p < 0.05 and < 0.001) but not for those obtained at 63.3 and 68.9°C. In view of the low number of D values, 24, reported for L. ivanovii, L. seeligeri and L. welshimeri and the fact that only one strain of each of these species was tested, no conclusion, however, should be drawn about differences in average heat resistance between these species and L. monocytogenes.

Salmonella

[124] studied the heat resistance of 300 Salmonella isolates and gave D_57°C values for 123 strains. The well-known extremely heat-resistant Salm. senftenberg 775W and 19 other strains of Salm. senftenberg were among the tested isolates. The resistance of the 19 strains was similar to that of the majority of isolates. Ng concluded that strains of salmonellae as resistant to heat as Salm. senftenberg 775W are rare. A similar conclusion was drawn by [146] who compared the heat resistance of Salm. senftenberg 775W with that of 20 strains of Salm. senftenberg isolated from herring meal.

The heat resistance of Salm. senftenberg 775W was also tested by [4, 130, 38, 166, 32, 141, 60, 125, 5, 66, 33, 34, 64, 31, 77] and [10]. The thermal destruction line fitted to the data (number of D values = 54) reported by the investigators mentioned is shown in Fig. 6. The equation for the line is log₁₀ D (D in s) = 12.8001 - 0.17111t (r = -0.94992).

In a screening of 221 Salmonella isolates, [5] found that 2 strains, one of Salm. senftenberg tested earlier by [38] and one of Salm. bedford, had D_60°C values similar to that of Salm. senftenberg 775W. Baird-Parker et al. considered it possible, although unlikely, that the Salm. senftenberg strain was identical to Salm. senftenberg 775W (the strain was isolated from home-killed meat in the United Kingdom and Salm. senftenberg 775W from dried eggs in the United States). The authors determined D values in heart infusion broth for the Salm. bedford strain and for Salm. senftenberg 775W. D values obtained at 50, 55 and 60°C were 350, 18.8 and 4.3 min for the Salm. bedford strain and 268, 36.2 and 6.3 min for Salm. senftenberg 775W. For comparison, it may be mentioned that the D values obtained for Salm. senftenberg 775W using the equation constructed in the present study are 293, 40.8 and 5.7 min at these temperatures.

Escherichia coli

[72] investigated an E. coli strain noted for its heat resistance. Tests were performed in milk. The thermal destruction line based on the data (number of D values = 22) reported by Holland and Dahlberg is shown in Fig. 4. The equation for the line is log₁₀ D (D in s) = 14.7478 - 0.19777t (r = -0.99403). The z value is 5.1°C. The author of the present work is unaware of whether this E. coli strain has been subjected to further heat resistance studies.

Concluding remarks

The design of the present study required that some differences in composition, etc. of heating menstrua used and in methods used for heat treatment and for recovery of heat-treated bacteria had to be accepted when heat resistance data were collected from the literature. This meant that experimental factors of varying character might have influenced the magnitude of heat resistance values used in the work. Statistical analyses of the results of these fairly numerous influences could not be achieved on the basis of available information. Scrutiny of heat resistance values chosen according to the stipulations laid down in the study, however, indicated that value differences probably caused by differences in experimental conditions were, in most cases, small or moderate.

The summary heat resistance values recorded -especially those for L. monocytogenes, E. coli and salmonellas which are based on large numbers of data – may give useful information on what is at present known about the heat resistance that the bacteria reviewed show in liquid heating menstrua with pH values of approx. 6–8. It should, however, be emphasized that they may, and often do, show heat resistance of different magnitude in other types of heating menstrua.