INFLUENCE OF SOME FOOD INDUSTRY TECHNOLOGIES ON THE CHARACTERISTICS OF PSEUDOMONAS AERUGINOSA STRAINS

H r Ii z 0., Jindra L u k Ii S 0 v Ii: Influence of Some Food Industry Technologies on the Characteristics of Pseudomonas aeruginosa Strains. Acta vet. Brno, 57, 1988:169-181. An investigation was made into the effects of 2 and 3% concentration of NaCl,2.5% nitrite salting mixture, freezing and milk fermentation on the characteriStics. of 5 typical ~­ monas aeruginosa strains isolated from water (1), ·bull semen (2) and bovine mastitis (2). the results were as follows: 1. On examination of 75 re-isolated P. aeruginosa strains qualitative changes were found 12 times, being recorded in a total of 10 (13.3%) strains. they concerned only dubious to negative attempts at gelatin hydrolysis (9 times), pyoverdine loss (twice) and absence of visible gas from KN02 (once). Another frequent finding was the loss of blue-green pigmentati<?n on King' s agar A, but the production of pyocyanin in Gessard s medium was unaffected. 2. A total of 55 minor qUllJ'ltitative changes were found in 4 strains of the foregoing group and in 41 (54.7% other strains. they concerned delayed growth at 41"C (35 times), production of visible gas from KNO only after adaptation passage in nitrate broth (16 times); delayed oxidase reaction (twice), decreased motility (once) and delayed nitrate reduction (once). they occurred mainly in cases where 2 or 3% NaCl or nitrite salting mixture was added. 3. In 16 (21.3%) strains, decreased mortality of mice inoculated i.p. with 0.2 ml of 18to 24 h culture was recorded. Almost half of these strains came from meat-peptone broth containing 2% NaCl. Involved in this phenomenon were 12 (23.5%) out of 51 aberrant strains and 4 (16.6%) out of 24 typical strains. 4. Out of 5 virtually unchanged re-isolates, 1 strain from meat-peptone broth with 3% NaCl (1 week) and 2 strains from yoghurt (48 h) showed a decrease in LD50, on average, by 29%, nitrite salting mixture (2 weeks) the shifts of LD50 were only -13.1% and +1%, respectively.

the results were as follows: 1. On examination of 75 re-isolated P. aeruginosa strains qualitative changes were found 12 times, being recorded in a total of 10 (13.3%) strains.they concerned only dubious to negative attempts at gelatin hydrolysis (9 times), pyoverdine loss (twice) and absence of visible gas from KN0 2 (once).Another frequent finding was the loss of blue-green pigmentati<?n on King' s agar A, but the production of pyocyanin in Gessard s medium was unaffected.
2. A total of 55 minor qUllJ'ltitative changes were found in 4 strains of the foregoing group and in 41 (54.7% other strains.they concerned delayed growth at 41"C (35 times), production of visible gas from KNO only after adaptation passage in nitrate broth (16 times); delayed oxidase reaction (twice), decreased motility (once) and delayed nitrate reduction (once).they occurred mainly in cases where 2 or 3% NaCl or nitrite salting mixture was added.
3. In 16 (21.3%)strains, decreased mortality of mice inoculated i.p. with 0.2 ml of 18-to 24 h culture was recorded.Almost half of these strains came from meat-peptone broth containing 2% NaCl.Involved in this phenomenon were 12 (23.5%)out of 51 aberrant strains and 4 (16.6%)out of 24 typical strains.
Food industry tecnno10gies are directed towards prolonged keeping possibility, acquisition of a new quality or flavour and, in meat industry, also towards fresh appearance of the product.Undesired bacteria are looked upon as a whole and should be destroyed or at least stopped in their replication during the processing.
Numerous reports on isolation of Pseudomonas aeruginosa from raw milk (K i e 1 wei n 1968; 0 t t e e t a1.1978; Kat 0 n a and Lan y i 1982) or meat (B u r z y n s k a and Mac i e j s k a 1974; 0 r may .. et a1.1980 are, no doubt, of interest but offer no solution to this special problem.Similarly, little help in this respect can be derived from positive findings of P. aeruginosa in baby food (B u r z y n s k a and M ac i e j s k a 1974) or pasterurized milk (H a 1 ado v a and Lac 0v a 1979) which rather indicate additional contamination.
In other words, few data are available on the influence of technological factors on the characteristics of microbial species in general.These aspects also remained unnoticed in a monograph (J e d 1 i C k 0 v a 1981) and recent food industry manuals (A r p a i and Bar t 1 1977; S i 1 han k 0 v a 1983) published in our country.
The re-isolated strains were examined for 20 diagnostic features as specified in Table 1.In addition to this, antigenic structure (O-serovar) and virulence for white mice were retested.The techniques used in our study are in keeping with those described in standard manuals (C a r t e r 1973; Cowan and S t e e 1 1974; S t a r r et a1.1981; Krieg and HoI t 1984) and recent relevant reports (A r a i et al. 1970).They were cited in full in a previous study by M r Ii z (1987) who also suggested some improvements: 1) Relation to molecular oxygen is tested by inoculation into 0.3% meat--peptone agar containing 0.5 g sodium thioglycolate and 0.15 ml of 1% aqueous solution of resazurin per 1 litre medium.
' 2) Motility is assessed on the basis of diffuse growth after inoculation into semi-solid agar ad 1) supplemented with 0.1% KN0 3 • In negative and dubious cases the hanging drop technique is used in addition.
3) Denitrification ability is examined in anaerobic meat-peptone broth with 0.1% KNO and with an inverted Durham gas tube.Incubation at 37 °c is carried o~ for no longer than 10 days.Possible neg!1tiv.e(defective or anaerogenic) strains can be completed by 24-h passage 1n n1trate broth (P a l l e ron i and D 0 u d 0 r 0 f f 1972).
4) pyocyanin production is tested using a modification of G e s s a r d ( 1981) medium (1% glycerol and 2% Bacto-Protone or Neoptone Difco in distilled water) incubated at 30• C for 5 days.
The virulence of the strains was tested in groups of 2 mice each inoculated Lp. with 0.2 ml of 18-to 24-h broth culture and in separate experiments per os using contaminated feed tablets.Moreover, the LDso (R e e d and M u e n c h 1938) was determined in all 5 starting strains and in 5 virulent re-isolates.

Results
The effects of technological factors were found in 51 (68%) out of 75 P. aeruginosa re-isolates (Table 2).A total of 67 changes shown by the strains can be divided into two groups: a) Qualitative changes in the diagnostic sense were recorded 12 times, being found in 10 (13.3%) strains.They concerned only dubious to negative attempts at gelatin hydrolysis (9 times), loss of pyoverdine (twice) and absence of visible gas from KNO (once).Another frequent phenomenon was the loss ot blue-green pigmentation on King• s agar A, but the production of pyocyanin in the modified Gessard medium remained unaffected.b) A total of 55 minor quantitative chal!ges were found in 4 strains of the foregoing group and in 41 (54.7%) other strains.They concerned delayed growth at 41 • C (35 times), production of visible gas from KN0 2 only after adaptation passage in nitrate broth (16 times), delayed oxidase reaction (twice), decreased motility (once) and delayed nitrate reduction (once).They occured mainly in meat-peptone broth containing 2 or 3% NaCI or nitrite salting mixture.
In certain agreement with the aforementioned data was the finding of 16 (21.3%)cases of decreased mortality in i.p. inoculated mice.Involved in this phenomenon w~re 12 (23.5%)out of 51 aberrant strains and 4 (16.6%)out of typical strains.The highest frequency (7 times) was recorded for meat-peptone broth containing 2% NaCI from which also the majority (3) of entirely innocuous strains came.
All 75 mouse pairs fed P. aeruginosa cultures survived.
The antigenic structure (O-serovar) showed no changes during the experiments.
Table 3 shows the results obtained in 15 re-isolates each of the 5 starting strains.It can be seen that the number of changes ranged fro~ 10 to 15, averaging 13.4 (0.9 per re-isolate).Decreased mortality of mice was observed mainly in re-isolates of strain No. 39 from water and strain No. 85 from bull semen.
Bioassays for determination of the LD50 in the 5• starting strains and 5 virulent re-isolates were carried out almost concurrently and in immediate continuation of the density determination of their 18-to 24-h broth cultures (Table 4).Comparison of the results shows that the LD50 of re-isolates of strains Nos.73,85 and 105 was 17.9 to 43.1% lower, whereas in the re-isolates of strains Nos.39 and 118 the shifts of LD50 were only -13.1 and +1%, respectively.

Discussion
It is well-known that changes in the environment may produce more or less pronounced changes in the characteristics of bacteria.The extent and intensity of this variability are of many-sided importance, being of value, among other things, to bacteriological diagnostics.This question was therefore considered by us concurrently with our investigation into the effects of some technological processes on the survival of P. aeruginosa in foods (L u k a S 0 v a and M r a z 1986).-------------------------------------------------------------------------- The changes in the biochemical characteristics were mainly quantitative.In gelatin, however, where the proportion of dubious to negative cases was 12%, a qualitative aberration is indicated.In practice it means that this characteristic should be assigned the sign of d or v.' A rather surprising finding was the 29.3% absence of blue-green pigmentation on King -sagar A• which is generally regarded as specific and very sensitive.Even though it is probable that the proportion of positive results might be increased by cutting the cultures and shaking out possible pyocyanin into chloroform, it seems more useful to choose Gessard -s modified medium.The evidence from the bioassays on mice suggests that involved in the 16 dubious to negative results was not only the nutrient medium (mainly meat-peptone broth containing 2% NaCl) but also the biochemical lability of some re-isolates.In this connexion it should be noted that decreased mortality to complete innocuity was found mainly in aberrant strains (cca by 7%).
The determinations of LD50 suggest that the virulence of pseudomonads CJlh more or less increase in certain environments (particularly in yoghurt).From Table 4 it also appears that in routine bioassays on mice an i.p. dose of 0.2 ml of 18-to 24-h broth culture is preferable to the 0.1 ml dose used hitherto.
Of certain value is also the fact that all the re-isolates of P. aeruginosa were innocuous for mice when administered per os.In our view, this observation might be used for more favourable evaluation of microbial findings in feeds and some foods.
l I-IR.co !!j "t:ICO co ~~ i of the test for acetamide hydrolysis (A r a i et al. 1970) and the usefulness of the employed denitrification technique in pseudomonads.