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

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.


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 re-sults reported must be taken into consideration (for general information on influencing factors, see e.g. Hansen & Riemann 1963, Stumbo 1973, Pflug & Holcomb 1983. Furthermore, the presentations of results often differ essentially. The aim of the present work was to collect, summarize, evaluate and compare heat resis-tance 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 Stringer et al. (2000), Doyle et al. (2001) and Doyle & Mazzotta (2000), respectively. However, the aims and the selections and analyses of data in these reviews differ from those in the present work.

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 (Elliker & Frazier 1938, White 1953, Krishna Iyengar et al. 1957, Lemcke & White 1959, Beuchat & Lechowich 1968, Ng et al. 1969, Humphrey et al. 1990, Jackson et al. 1996, Lou & Yousef 1996, Kaur et al. 1998, Pagán et al. 1998. Heat resistance results obtained for bacteria grown under carbon, glucose or nitrogen starvation or other stress conditions (see e.g. Ng et al. 1969, Jenkins et al. 1988, Lou & Yousef 1996 were not used in the present work.

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. Anellis et al. 1954, Krishna Iyengar et al. 1957, White 1963, Garibaldi et al. 1969a, Humphrey et al. 1981, Sanz Pérez et al. 1982, Okrend et al. 1986, Blackburn et al. 1997, Pagán et al. 1998. 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. Lategan & Vaughn 1964, Calhoun & Frazier 1966, Baird-Parker et al. 1970, Goepfert et al. 1970, Vrchlabski & Leistner 1970, Corry 1974, Anderson et al. 1991, Palumbo et al. 1995, Blackburn et al. 1997, Knight et al. 1999.

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 (Bradshaw et al. 1985, Bunning et al. 1986, 1988, Konvincic et al. 1991, Clementi et al. 1995, submerged-coil heating apparatuses (Anderson et al. 1991, Jørgensen et al. 1995, Blackburn et al. 1997, Juneja et al. 1998), thermoresistometers (Read et al. 1968, Pagán et al. 1998 and an "attemperated dilution blank method" (Magnus et al. 1986(Magnus et al. , 1988. Results from experiments using rising heating temperatures (Tsuchido et al. 1974, Mackey & Derrick 1987a, Quintavalla et al. 1988, Stephens et al. 1994 were excluded.

Heat resistance of bacteria
Baird -Parker et al. (1970), Gibson (1973) Campylobacter 2.8-5.81 4.8 ± 0.7 14 Blankenship & Craven (1982), Waterman (1982), jejuni/coli Sörqvist (1989), Sörqvist & Danielsson-Tham (1990) 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 (Stumbo 1973). 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 log 10 of D values recorded were plotted vs temperature and a thermal destruction line (Stumbo 1973) was fitted using the method of least squares (Colton 1974). The equation for the line is log 10 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 (Colton 1974). Using the absolute and inverse values of the slope and its 95% confidence limits, the z value and its 95% confidence limits were calculated (Stumbo 1973, Colton 1974). 95% confidence limits on predicted means (Colton 1974) of D values were calculated (the predicted mean is the same as D in the equation). 95% confidence limits on predicted individual D values (Colton 1974) 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 1-7, where 95% confidence limits on predicted individual log 10 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.

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 (Hansen & Riemann 1963, Stumbo 1973, Pflug & Holcomb 1983, 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 (Hansen & Riemann 1963, Stumbo 1973, Pflug & Holcomb 1983 (Colton 1974). 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, nonlogarithmic survivor curves were reported in several works (Zivanovic et al. 1965, Dabbah et al. 1971a,c, Sanz Pérez et al. 1982, Magnus et al. 1986, Gordon & Ahmad 1991, Boutibonnes et al. 1993). Mackey & Bratchell (1989) 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 log 10 D = 10.888 -0.14519t, log 10 D = 11.931 -0.1635t and log 10 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 Mackey & Bratchell (1989) 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.

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 (Quintavalla & Barbuti 1989, Foegeding & Stanley 1991, Fairchild & Foegeding 1993, Palumbo et al. 1995. The equation for the thermal destruction line constructed was log 10 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 re-ported 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 Quintavalla & Barbuti (1989). 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. Foegeding & Stanley (1991) 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. Palumbo et al. (1995) 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.
In a screening of 221 Salmonella isolates, Baird-Parker et al. (1970) found that 2 strains, one of Salm. senftenberg tested earlier by Davidson et al. (1966) Fig. 4. The equation for the line is log 10 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.  Food Prot. 1990, 53, 9-14. Foegeding PM, Stanley NW: Listeria innocua transformed with an antibiotic resistance plasmid as a thermal-resistance indicator for Listeria monocy-