Characterization of Deoxyribonuclease-Positive Enterococci Isolated from Milk and Milk Products

348

V. K. BATISHl*, H. CHANDERl aodB. RANGANATHAN2

Dairy Bacteriology Division ofNational Dairy Research Institute, Karnal-132001 (Haryana), India and Southern Regional Station, National Dairy Research Institute, Adugodi, Bangalore-560030, India Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/4/348/1650913/0362-028x-45_4_348.pdf by guest on 28 September 2021 (Received for publication January 20, 1981)

ABSTRACT enterococci recovered from milk and milk products on the basis of biochemical, physiological and serological Five hundred and ten isolates of enterococci recovered from tests. The production of certain other extracellular milk and milk products were screened for deoxyribonuclease metabolites by these organisms was also studied. (DNase) production. Of the 166 (32.5(\7o) DNase-positive cultures, 29 (5.7%) produced thermonuclease that resisted boiling for 15 min. Although the incidence of thermolabile MATERIALS AND 1\IETHODS DNase-positive enterococci was 50.5% in Cheddar cheese, the majority of thermonuclease-producing enterococci was recover Analysis of milk and milk products for the presence ofenterococci ed from dried milks and infant foods. On the basis of Samples of milk and milk products which included raw and biochemical, physiological and serological tests, all DNase pasteurized milk, Cheddar cheese, butter, skim and whole milk powder, producing enterococci were characterized as S. faecalis var. baby foods and Kulfi, were collected aseptically from the local market jaecalis (21), S. foecalis var. zymogenes (9), S. faecalis var. as well as from the Institute's experimental dairy and examined for liquefaciens (90), S. faecium (23) and S. durans (16). The presence of enterococci using citrate azide agar (34). All the isolates predominant thermonuclease-positive type was S. faecalis var. were subjected to group D serological tests (J8) as well as Sherman's faecalis followed by S. faecalis var. zymogenes. Six strains of criteria (3 7) to determine if they were true enterococci. enterococci were found to be toxigenic when tested in ligated Deoxyribonuclease activity rabbit ileal loops, infant mice and rabbit skin. Enterococcal isolates were grown in Brain Heart Infusion broth for 18 h at 37 C and tested for the production of DNase using methyl green-DNA agar (Difco), pH 7.0. The medium was poured into disposable petri plates that were each divided into eight equal parts. The production of deoxyribonucleases (DNases) by Each part was spot-inoculated with the test culture using a loopful (4 streptococci groups A, B, C, F, G and L was first mm) of inoculum. After incubation for 24 h at 37 C, the plates were demonstrated by Tillett et al (42), Brown (10) and examined for formation of clear zones around the inoculated spots. The Gudding (20). Later DNase production was reported in thermostability of the enzyme was determined by boiling the culture filtrate for 15 min and transferring a 0.01 ml portion to one of the eight group D streptococci (enterococcus group) as well (8,40). wells (2 mm) in the agar plate. Formation of clear zones after 24 h Many of the enterococcal strains recovered from dairy indicated the presence of thermostable nuclease. products produced DNase which resisted boiling for 15 Microbiological tests min just like therrnonuclease (INase) of Staphylococcus DNase producing enterococci were tested for the production of aureus. catalase (41} and oxidase (27); motility (43); resistance to tellurite, Although the literature is continuing to provide us growth on 10 and 40"7o bile agar; hydrolysis of esculin; liquefaction of with detailed information on the production of gelatin; production of acid from glucose, maltose, mannitol and thermonuclease by S. aureus (29,31.33) as a major arabinose (12); production of tyrosine decarboxylase (J3); and hydrolysis of arginine (32) and hippurate (21). criterion for its identification (6,38), no systematic study has been made with respect to DNase or TNase Tests for production ofextracellularenzymes and toxigenicity producing enterococci. In the present investigation, an DNase-positive enterococci were also tested for the production of several extracellular enzymes using tryptone neopeptone dextrose attempt was made to characterize DNase-producing agar (fND) to which selected substrates were added. The TND medium was composed of L7"7o tryptone, 0.3o/o neopeptone (Difco), 0.25o/o

dextrose, 0.5%, sodium chloride 0.25o/o, K2HP04 and 1.5% agar. The 1Dairy Bacteriology Division of National Dairy Research Institute, pH of the medium was adjusted to 7.2. Esterase activity was detected on Kamal. the above medium containing indozylbutyrate (22). Amylase was 2Southern Regional Station, National Dairy Research Institute, assayed on agar containing starch. Production of protease was studied Bangalore. on TND agar containing 10% (v /v) sterile skim milk (11), while lipase

JOURNAL OF FOOD PROTECTION. VOL. 45, MARCH 1982 DNASE-POSITIVE ENTEROCOCCI 349

TABLE 1. The incidence enterococci in milk No. of No. of Deoxyribonuclease-producing Sample samples enterococci enterococci fhermolabile Thermostable Raw milk 12 90 23(25.6)a 3(3.3) Pasteurized milk 10 20 4(20.0) 0(0.0) Cheddar cheese 17 105 53(50.5) 3(2.9) Butter 10 15 0(0.0) 0(0.0) Skim milk powder 19 105 32(30.5) 11(10.5) Whole milk powder 8 20 9(45.0) 1(5.0) Infant foods 16 100 19(19.0) 9(9.0) Kulfi 15 55 26(47.3) 2(3.6) Total: 107 510 166(32.5) 29(5.7)

a Numbers in parentheses indicate percentage. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/4/348/1650913/0362-028x-45_4_348.pdf by guest on 28 September 2021 activity was determined using tributyrin agar \24). Lecithinase activity DNase-producing enterococci occurred in the form of was demonstrated using lecithin agar (47), while hemolysin production ovoid cells, singly or in small chains or pairs. The was detected on plates containing blood agar base (Difco) and diameter of the cells ranged from 0.9 to 1.2 ,.an and cells erythrocytes from rabbit's blood (13). Bergey's Manual of Determina tive Bacteriology (15) was used for identification of individual strains. were gram-positive. None of the cultures produced catalase or oxidase. Four cultures exhibited motility. One Toxigenicity tests hundred and eight cultures belonging to the above Thermostable DNase-producing enterococci were tested for their category exhibited growth on potassium tellurite agar. toxigenicity by suckling mice response test, ligated rabbit ileal loop All of the strains showed growth in 10 and 40% bile, reaction and skin permeability reaction (30). hydrolyzed esculin and produced tyrosine decarboxylase. Ninety-two strains hydrolysed hippurate, whereas gelatin RESULTS was liquefied by 90 strains. Acid was produced from glucose and maltose by all isolates. Mannitol fermenta tion with production of acid was exhibited by 139 strains. The incidence of DNase-producing enterococci re Only 23 strains produced acid from arabinose, while 100 covered from 107 samples of milk and milk products is strains produced acid from sorbitol. The results of the shown in Table 1. The incidence of thermolabile DNase microbiological tests are summarized in Table 3. positive enterococci was greatest in Cheddar cheese Table 4 shows data on the production of extracellular (50.5%) followed by Kulfi (47.3%) and whole milk powder enzymes and hemolysin by DNase-producing enterococ (45.0o/o). However, the incidence of thermonuclease cal isolates. Out of 149 enterococcal isolates, 110 producing enterococci was greatest in skim milk powder produced esterase, 19 protease and 21 lipase. Gelatinase (10.5%), followed by infant foods (9.0%). In contrast, not production was exhibited by 80 strains. However, none of a single thermostable DNase-producing enterococcus was recovered from any of the pasteurized milk and TABLE 3. Microbiological characteristics of deoxyribonu butter samples examined in this study. clease-producing enterococci isolated from milk and milk Table 2 shows some of the physiological and products. serological reactions ofDNase-producing enterococci. Of Test No. of cultures 166 DNase-positive isolates, 149 cultures reacted with postive/Total No. of group D antiserum, as well as exhibited growth at pH 9.6 cultures tested and also at 10 and 45 C. These isolates grew well in 6.5% Catalase 0/149 NaCl and resisted heating at 60 C for 30 min. On the Oxidase 0/149 basis of the above tests, a11149 isolates were categorized Motility 4/149 as "true enterococci" according to Sherman's criteria. Resistance to tellurite 110/149 Growth in 10% bile 149/149 TABLE 2. Physiological and serological reactions of deoxyri Growth in 40o/o bile 149/149 bonculease-producing enterococci isolated .from milk and milk Esculin hydrolysis 149/149 Arginine decarboxylase 149/149 Tyrosine decarboxylase 149/149 No. of isolates Test Hippurate hydrolysis 92/149 positive/Total No. of isolates tested Gelatin liquefaction 80/149 Acid from: Growth at pH 9.6 149/166 Glucose 149/149 Growth in 6.5%sodium chloride 149/166 Maltose 149/149 Growth at 10 and 45 C 149/166 Mannitol 139/149 Resistance to 60 C for 30 min 149/166 Arabinose 23/149 Agglutinate with group D antiserum 149/166 Sorbitol 110/149

JOURNAL OF FOOD PROTECTION, VOL. 45, MARCH 1982 350 BATISH ET AL. the strains produced either lecithinase or amylase. three S. faecalis var. zymogenes, two S. faecalis var. Twenty-nine enterococcal isolates exhibited thermostable faecalis and one S. faecium, have been categorized as DNase activity. When tested on rabbit blood agar, only toxigenic. one enterococcal isolate displayed hemolytic activity, while 9 isolates were {1-hemolytic. Although all the TABLE 6. Toxigenic characteristics of thermonuclease-pro hemolytic strains produced thermonuclease, all the ducing enterococci recovered from milk and milk products. thermonuclease producers were not hemolytic. Type of enterococci Toxigenic response (No. of positive strains/ On the basis of the above physiological and serological No. of cultures tested) tests, as well as production of extracellular enzymes and Infant micea Ligated rabbit Rabbit skin hemolysins, all the enterococcal isolates in this study ileal loop permeabilityb were identified as follows: 80 strains were characterized S. faecal is var. as S. faecalis var. liqu(!faciens, 9 as S. jaecalis var. faecalis 3/19 2/19 2/19 zymogenes, 21 as S. faecalis var. faecalis, 23 of S. S.faecalis var.

jaecium and 16 as S. durans. Thermonuclease pro zymogenes 3/9 3/9 3/9 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/4/348/1650913/0362-028x-45_4_348.pdf by guest on 28 September 2021 duction was exhibited mainly by S. faecalis var. faecalis S.jaecium 111 111 1/1 and S. faecalis var. zymogenes as shown in Table 5. DNases of all 80 strains of S. faecalis var. liquefaciens were inactivated after boiling for 15 min. Among the Total 7/29 6/29 6/29 isolates of S. faecium, only one was capable of producing aThe ratio of intestinal weight/total body weight was 0.09 or thermonuclease. Although all four motile strains of more. enterococci belonged to S. faecium, none of them bAn inflammatory zone of 25 sq. mm or more was considered a produced thermonuclease. positive reaction. The toxigenic characteristics of thermonuclease producing enterococci are shown in Table 6. Of 29 thermonuclease-positive strains, 7 produced accumula DISCUSSION tion of fluid in abdomens of infant mice. When injected into rabbit ileal loops, 6 strains showed fluid accumula Enterococci include S. faecalis, and its varieties such tion. The 6 cultures also evoked a positive skin vascular as S. faecalis var. liquejaciens and S. faecalis var. permeability reaction in rabbits. On the basis of the zymogenes, S. jaecium and S. durans. These organisms above tests, these 6 strains of enterococci, comprised of are known to be causative agents of a wide variety of infections in human beings and animals. Although the TABLE 4. Production of extracellular products by deoxyri· presence of enterococci in food materials is indicative of bonuclease·producing enterococci isolated from milk and milk contamination from fecal sources (5,9), occasionally these organisms are also implicated in food spoilage (S) Extracellular product No. of positive cultures/ and food poisoning outbreaks (16,19,36). In view of their No. of cultures tested invariable presence in foods including milk and milk Esterase 110/149 products, enterococci may pose a serious threat to public Protease 191149 health by causing cross-infections among human beings. Lipase 21/149 Enterococci belong to group D streptococci since they Gelatinase 80/149 possess the group D antigen (7). The different Lecithinase 0/149 physiological and serological reactions of the enterococ Amylase 0/149 cal isolates are recorded in Table 2. Since the Thermonucleasea 29/149 characteristics of enterococci are markedly distinguish Coagulase 3/149 able from other group D streptococci, Kalina (25) Hemolysin activity on rabbit blood: proposed that they be included in a new genus, a-hemolysis 21!149 Enterococcus. {J-hemolysis 9/149 In the present investigation, all the samples of milk Non-hemolytic 119/149 and milk products examined showed the presence of a Resisted boiling for 15 min. enterococci. Several investigators (1-3,26,28,39,44,45) have reported the occurrence of enterococci in large

TABLE 5. Incidence ofdeoxyribonuclease-and thermonuclease·producing enterococci in milk and milk products. TypeofDNase No. of strains of enterococci S.faecalis S.faecalis S.faecalis S.faecium S. durans Total var. faecalis var. zymogenes var. liquefaciens Thermolabile 21 9 80 23 16 149

Thermostable 19 9 0 0 29

JOURNAL OF FOOD PROTECTION. VOL. 45. MARCH 1982 DNASE-POSITIVE ENTEROCOCCI 351

numbers in raw and pasteurized milk, butter, frozen correlation could be made between motility of the strain milk products and dried milk. In the present studies S. and its enterotoxigenicity. From the foregoing discussion faecalis var. liquefaciens was the predominant entero it may be concluded that no single test is as yet available cocci isolated from milk and milk products. Facklam (17) for the rapid and reliable differentiation of enterococci. reported that S. faecalis var. zymogenes and var. Although tests for both DNase production and hemolytic liquefaciens were the major subgroups of group D activity could be used readily for differentiation of streptococci isolated from human infections. various enterococcal strains, several other physiological DN ase production has been reported in several and serological tests are also needed to confirm the microorganisms including S. aureus (14), Bacillus species and varieties within this group. However, subtilis (8), Serratia marcescens (35) and Streptococcus thermostable DNase production may be used as a pyogenes and group A streptococci (46). The production criterion for determining the possible toxigenicity of of this enzyme in enterococci has not been studied in enterococci. detail, hence the occurrence of large numbers of

DNase-producing enterococci obtained from milk and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/4/348/1650913/0362-028x-45_4_348.pdf by guest on 28 September 2021 milk products in this study necessitated detailed REFERENCES characterization of these enterococcal isolates. Many of these enterococcal isolates were capable of producing thermostable DNase, the majority of such cultures being 1. Aleksieva, V. 1974. Enterococci in dried milk. Cited from Dairy Sci. Abstr. 37:346, 1975. recovered from dried milks. S.jaecalis var .faecalis and S. 2. Aleksieva, V. 1976. Enterococci in cream and butter. Cited from faecalis var. zymogenes were the predominant types Dairy Sci. Abstr. 38: 5752, 1976. among thermonuclease producers. Our findings are in 3. Aleksieva, V. 1977. Presence and freezing resistance of agreement with those of Thomas and Nambudripad (40) enterococci in ice cream. Cited from Food Sci. Techno!. 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