Time-Temperature Effects on Salmonellae and Staphylococci in Foods: II. Thermal Death Time Studies

Thermal death time studies were conducted at 5 F intervals from 130 to 150 F with strains of salmonellae and enterotoxigenic staphylococci. Heat-resistant Salmonella senftenberg strain 775W, Staphylococcus aureus strains 196E and Ms149, and non-heat-resistant Salmonella manhattan were studied in custard, chicken à la king, and ham salad.

The F₁₄₀ values (minutes of exposure at 140 F required to effect 100% destruction) were as follows: S. senftenberg 775W in custard 78, and chicken à la king 81.5; S. manhattan in custard 19, and chicken à la king 3.1; S. aureus 196E in custard 59, and chicken à la king 47; S. aureus Ms149 in custard 53, and chicken à la king 40.

The end points of survival-kill at all the test temperatures for both salmonellae and staphylococci in ham salad were considerably less than for the other foods studied.

D₁₄₀ values (minutes of exposure at 140 F required to effect a 90% reduction in numbers) were also calculated from the data and presented.

Values for z_F and z_D (slope of the thermal-death-time and decimal-reduction-time curves) are also presented and discussed in relation to type of food, organism, and temperature.

These data indicate that heating perishable foods of the type studied to 150 F and holding every particle of food at this temperature for at least 12 min reduces 10 million or less salmonellae or staphylococci per gram to nondetectable levels.

The same degree of destruction is achieved in similarly contaminated foods when held at 140 F for 78 to 83 min.

On the basis of the calculation procedures employed, it is estimated that 45-min exposure at 140 F would be necessary to reduce 1,000 organisms per gram to nondetectable levels.

     

Thermal Resistance of Salmonellae Isolated from Dry Milk

Salmonella anatum, S. binza, S. cubana, S. meleagridis, S. newbrunswick, and S. tennessee isolated from dry milk, and S. senftenberg 775W were studied for heat resistance to determine whether these organisms would survive pasteurization as recommended by the 1965 Pasteurized Milk Ordinance of the U.S. Public Health Service. Thermal inactivation determinations were made on washed cells of the test microorganisms suspended in sterile whole milk. Excluding S. senftenberg, D values ranged from 3.6 to 5.7 sec at 62.8 C, from 1.1 to 1.8 sec at 65.6 C, and from 0.28 to 0.52 sec at 68.3 C. Corresponding values for S. senftenberg were 34.0, 10.0, 1.2, and 0.55 sec for respective exposure temperatures of 65.5, 68.3, 71.7, and 73.9 C. The present milk pasteurization processes as recommended by the Public Health Service will inactivate all seven strains of salmonellae studied, provided that the initial concentration does not exceed a calculated 3 × 10¹² salmonellae per ml of milk.     

Drug Resistance of Staphylococci

Induction of chloramphenicol (CM) resistance in Staphylococcus aureus was investigated using 166 CM derivatives and analogues. It was found that 18 compounds of the samples used were able to induce resistance to CM in Staphylococcus aureus S1477 harboring an inducible CM-resistance determinant. Most of the samples which had a nitrophenyl moiety and a D- or DL-threo isomer in its steric configuration were found to have inducer activity for CM resistance. Competent inducers are D-isomer CM derivatives which have another substituent in place of the hydroxyl group at carbon atom 3 in the propanediol of the drug.     

Drug Resistance of Staphylococci

According to an epidemiological survey of drug resistance in Staphylococcus aureus, it was found that there are three types of cross-resistance to macrolide antibiotics (EM, erythromycin: OM, oleandomycin: LM, leucomycin: SP, spiramycin), i.e., resistance to EM, OM, LM, and SP, to EM and OM, and inducible resistance to “EM, OM”. In the inducible strains of S. aureus, EM and OM are active inducers, and the optimal concentrations of the inducers are 0.1 μg/ml and 1.0 μg/ml, respectively. The induction of high resistance (800 μg/ml or more) to both EM and OM occurred within 10 min exposure to 0.1 μg/ml of EM, and the resistance of induced cells was lost after overnight growth in the absence of inducer. After 1 to 3 hr exposure to 1.0 μg/ml of OM, the inducible strains acquired high resistance (100 μg/ml or more) to EM and to a lesser extent, resistance to OM, and the acquired resistance was lost when grown in antibiotic free media. When a known concentration of EM was mixed with the induced cells or with a crude extract from induced cells which had acquired high resistance to EM and OM, the antibiotic activity of EM was still retained in the mixture, indicating that the induced mixture or the extract from the induced cells was incapable of antibiotic (EM) inactivation under the test conditions.     

Drug Resistance of Staphylococci

It was reported that resistance to tetracycline (TC) in some staphylococcal strains was lost irreversibly without loss of resistance to other drugs when cultured at high temperature, and that the determinant for TC resistance exists on a plasmid. According to the transductional analysis of TC resistance in Staphylococcus aureus E169 it was found that the genes which govern resistance to tetracycline and streptomycin are located close together on a single genetic element. When TC resistance in MS146, one of our stock cultures, was transduced to E169S which had lost TC resistance, TC resistance in transductants was found to be unstable at elevated temperatures. By contrast, TC resistance in transductants MS353 TC^r, to which TC resistance was transduced from E169, was indicated to be stable even when cultured at high temperature. From these results, it is strongly suggested that instability of TC resistance in E169 is not accounted for by the genetic properties of the genes which govern TC resistance but those of host cell (E169) itself.     

Drug Resistance of Staphylococci

Examination of cross resistance to macrolide antibiotics in erythromycin resistant staphylococcal strains isolated from clinical sources in the United States showed that there were two types of cross resistance, group A (13.4%) and group C (86.6%). Group A possessed multiple resistance to the macrolide antibiotics, erythromycin, oleandomycin, leucomycin and spiramycin, and was also resistant to lincomycin. In group C, resistance to erythromycin alone or to both erythromycin and oleandomycin could be induced by exposure to erythromycin.     

Drug Resistance of Staphylococci

On the basis of resistance patterns to penicillins, strains of Staphylococcus aureus highly resistant to penicillin-G which were isolated from clinical sources, could be classified into 2 groups, A-1 and A-2. Group A-2 is a minor group which accounts for about 10% of these strains and exhibits lower degree of resistance to penicillin-G and phenethicillin than that exhibited by group A-1 strains. However, there was no marked difference between both groups in inducible production of penicillinase. The genetic determinants for penicillinase synthesis in strains of both groups were transduced into a recipient and the characteristics of the determinants were compared with each other under identical conditions. The results suggested that the genetic determinants for penicillinase synthesis in groups A-1 and A-2 strains differed from each other in both the structural gene for penicillinase production and the regulatory gene for the formation of the repressor.     

Drug Resistance of Staphylococci

Induction of chloramphenicol (CM) resistance in Staphylococcus aureus was investigated by using several CM derivatives. It was found that dl-threo-1-p-nitrophenyl-2-dichloroacetamino-1,3-dichloropropane has high antibacterial activity but low activity of induction for CM resistance. In spite of low antibacterial activity, induction of CM resistance occurred after prior treatment with dl-threo-1-p-nitrophenyl-2-dichloroacetamino-3-chloropropane-1-ol. It was found that dl-chloramphenicol di-acetate, dl-threo-1-p-nitrophenyl-2-dichloroacetamino-3-bromopropane-1-ol and dl-threo-1-phenyl-2-dichloroacetamino-1,3-propanediol have induction ability in spite of the absence of antibacterial activity. Other derivatives were classified into two groups; (1) low antibacterial activity and induction of CM resistance and (2) loss of both activities.     

One-Day Fluorescent-Antibody Procedure for Detecting Salmonellae in Frozen and Dried Foods

The indirect fluorescent-antibody technique was used to examine 422 food samples for the presence of salmonellae. A cultural phase involving a 16-hr preenrichment in buffered nutrient broth-milk medium followed by a 4- to 5-hr subculture into fresh medium of the same composition was evaluated. This procedure yielded a sufficient population of salmonellae so that no false-negative results were obtained. Of the 31 false-positives obtained, 12 samples yielded positive cultural results upon extensive subculture of the original enrichment broths. Yeast cells and both vegetative and spore forms of bacilli were observed to fluoresce when stained with anti-Salmonella serum. Efforts to ascertain the cause of these cross-reactions and several alternate explanations are discussed.     

Timed-Release Capsule Method for the Detection of Salmonellae in Foods and Feeds

A new method was developed for the detection of injured and uninjured salmonellae in foods and feeds. The steps of pre-enrichment in a nonselective broth and selective enrichment in a selective medium were combined into a single procedure. This was achieved by the gradual release of selective agents from wax-coated gelatin capsules added at the time of inoculation of nonselective basal broths. Pre-enrichment in lactose broth was combined with selective enrichment in tetrathionate or selenite-cystine broth by using timed-release capsules containing iodine or selenite. Five different categories of foods and feeds, naturally contaminated with salmonellae, were examined to compare the efficiencies of the capsule methods with conventional procedures. Combination of the separate steps of pre-enrichment and selective enrichment into a single procedure was feasible and resulted in substantial savings of labor and materials.     

Heat Resistance of Salmonellae in Concentrated Milk

The heat resistance of Escherichia coli, Salmonella typhimurium, and Salmonella alachua in milk solutions containing 10, 30, 42, and 51% (w/w) skim milk for total solids was determined. Increased milk-solids level effected a significant increase in the heat resistance of each organism. Although E. coli was more heat-resistant than both strains of Salmonella in 10% milk, the situation was reversed in 42 and 51% milk. Prior growth temperature was found to exert a profound effect on the heat resistance of S. typhimurium. Growth of S. typhimurium in 42% milk solids for 24 hr did not greatly enhance the thermal resistance of the organism when heated in a fresh 42% solids concentrate. Application of a partial vaccum during heating greatly diminished the decimal reduction times of S. typhimurium and E. coli and, in addition, virtually eliminated the protective effect of increased solids level.     

Resistance of Salmonellae Isolated in 1962 to Chlortetracycline

Among 652 salmonellae examined, Salmonella typhimurium displayed a much higher incidence of resistance to chlortetracycline than did other serotypes. Among S. typhimurium cultures from man, the incidence of resistance (38%) increased markedly over that of cultures isolated 3 years previously (13.9%), whereas a similar increase was not noted among cultures from fowls. Resistance among cultures from animals other than fowls (72.5%) was due largely to the high incidence of resistance among cultures from bovine sources (93.7%).     

Effects of Ingredients Used in Condensed and Frozen Dairy Products on Thermal Resistance of Potentially Pathogenic Staphylococci

A cell suspension of Staphylococcus aureus (196E) was injected into raw skim milk which contained different concentrations of sugar, serum solids, fat, stabilizers, and emulsifiers. The ingredient samples were exposed for the desired length of time in a constant-temperature water bath (60 C). Standard plate counts were made, and the number of surviving organisms was determined. Regression coefficients for each ingredient concentration were calculated and plotted against the per cent ingredient concentration to give an indication of protective action. Analyses of variance were conducted on bacterial counts to test the protective action of each ingredient. A comparison of the number of survivors in different sugar concentrations showed that with up to 14% sugar all the organisms were killed within 30 min. In sugar concentrations above 14%, the number of survivors increased regularly with each increase in sugar concentration up to 57%, which was the maximum used. In concentrations of serum solids above 9%, some organisms survived 35 min of heat treatment. Butter fat, stabilizer, and emulsifier did not offer any protective action in the concentrations observed.     

Comparison of Two Methods for the Isolation of Salmonellae From Imported Foods

Two methods for the detection of salmonellae in foods were compared in 179 imported meat and egg samples. The number of positive samples and replications, and the number of strains and kinds of serotypes were statistically comparable by both the direct enrichment method of the Food Hygiene Laboratory in England, and the pre-enrichment method devised for processed foods in the United States. Boneless frozen beef, veal, and horsemeat imported from five countries for consumption in England were found to have salmonellae present in 48 of 116 (41%) samples. Dried egg products imported from three countries were observed to have salmonellae in 10 of 63 (16%) samples. The high incidence of salmonellae isolated from imported foods illustrated the existence of an international health hazard resulting from the continuous introduction of exogenous strains of pathogenic microorganisms on a large scale.     

Resistance of Salmonellae Isolated in 1959 and 1960 to Tetracyclines and Chloramphenicol

The resistance of cultures of Salmonella typhimurium to tetracyclines and chloramphenicol has been examined periodically. Although none of 200 cultures isolated prior to 1948 was resistant to the antibiotics, 5% of 100 cultures from man and 9% of 100 cultures from fowls which were isolated in 1956 and 1957 were resistant to tetracyclines. Among 158 cultures isolated from man and 100 cultures isolated from fowls in 1959 and 1960, 13.9 and 29%, respectively, were resistant to tetracyclines. In the last series, cultures resistant to chloramphenicol were found for the first time. Among 150 cultures of other Salmonella serotypes from man and 137 similar cultures isolated from fowls in 1959 and 1960, 5.3 and 8%, respectively, were found resistant to tetracyclines. There is no obvious explanation for the higher percentage of resistant strains occurring in S. typhimurium than in other serotypes.     

Novobiocin Resistance and the Classification of Staphylococci and Micrococci

S ummary . Isolates of staphylococci and micrococci, 105 in all, classified according to Baird-Parker's classification (Baird-Parker, 1963,1965) were examined for their sensitivity to novobiocin. All of the staphylococci were sensitive to novobiocin as also were strains of Micrococcus luteus and M. roseus. Almost all strains belonging to Micrococcus subgroups 1–6 were resistant to novobiocin.     

Drug Resistance of Staphylococci VI. Genetic Determinant for Chloramphenicol Resistance

Naturally occurring strains of staphylococci which are resistant to chloramphenicol (CM) inactivate this antibiotic. One of the inactivation products of CM showed the chromatographic behavior of 3-acetoxychloramphenicol. Induction of resistance occurred after prior exposure to subinhibitory concentrations of the antibiotic. The resistance of induced populations, as well as CM-inactivation ability, was decreased when they were grown in CM-free medium. The CM-inactivation property was transduced together with CM resistance. Transductional analysis and CM-resistance elimination experiments indicated that CM resistance in naturally occurring strains of staphylococci is mainly accounted for by inactivation of the drug.