International Journal of Food Microbwlogy, 12 (1991) 263-270 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50
263
FOOD 00388
Incidence of Aeromonas and Listeria spp. in red meat and milk samples in Brisbane, Australia A. I b r a h i m a n d I.C. M a c R a e Department of Microbiolog),, Umverslty of Queensland, Queensland, Austraha (Received 19 June 1990; accepted 4 December 1990)
A total of 150 samples, 50 each of beef, lamb and pork from l0 local retail stores in Brisbane metropolitan area as well as 150 raw bovine bulk milk tank samples obtained from Queensland United Foods (QUF), were examined for the presence of Aeromonas and l.zsterta spp. over a period of 1 year. Different sets of enrichment and plating media were used to recover the organisms with subsequent identification using conventional biochemical and serotyping techniques. A total of 509 isolates consisting of Aeromonas spp. (350) and l.,isteria spp. (159) were obtamed from 6070, 58~, 74~, 26.6~, 34~, 40c$, 30~, 2.6~ of samples of beef, lamb, pork and milk respectively. Motile aeromonads ( A. hydrophzla, A. sobria, A. caviae) and i..isteria innocua were isolated from all kinds of samples whereas /_ monocytogenes was only isolated from flesh food. A. hydrophila contributed the largest percentage of the motile aeromonads (607o). The majority o f / - monocytogenes cultures were serotype 4. Key words: Meat; Milk; Aeromonns; l.,isteria
Introduction Motile aeromonads and Listeria monocytogenes are the most recently added species to the growing llst of foodborne pathogens. Aeromonas hydrophila and A. sobria have been frequently isolated from clinical cases of human diarrhoea (Nishikawa and K.ishi, 1988; Burke and Gracey, 1986; Agger et al., 1985). Motile aeromonads have also been detected in different varieties of foods especially those of animal origin like meat and milk (Greenway, 1988; Okrend et al., 1987; Ternstrom and Molin, 1987; Palumbo et al., 1985). The coincidence of the previous findings has led Buchanan and Palumbo (1985) to suggest that aeromonads, in particular, A. hydrophila and ,4. sobria are significant foodborne pathogens. Outbreaks of listeriosis due to contamination of foods have been reported in different countries (Fleming et al., 1985; Schlech et al., 1983). In addition, 2500 cases of listeriosis were reported to occur in the U.S.A. every year (Todd, 1989). The organism has been detected in different foods including milk (Hayes et al., 1986), Correspondence address: I.C. Mac Rae, Department of Microbiology, Umversity of Queensland, Queensland, 4072 Australia.
264 vegetables (Heisick et al., 1989), meat (Johnson et al.. 1990, 1988) and chicken (Pini and Gilbert. 1988), Moreover. The World Health Orgamzation (WHO) now recognises that contamination of foodstuffs with L. monocrtogenes is the principal means of acquiring hsteriosis (WHO, 1988). Little is known about the incidence of these organisms in different foods m Australia, therefore, ~t was the aim of this study to check their prevalence in both red meat and milk.
Materials and Methods
Collectton of samples Red meat A total of 150 samples, 50 each of beef, lamb and pork (different cuts) were purchased at 10 local retail supermarkets, over a 1 year period, in regular consumer packages each of which was considered as one separate sample and transferred to the laboratory with a minimal delay and examined on the day of purchase. Milk A total of 150 raw bovine bulk milk tank samples were obtained from Queensland United Foods (QUF), the major milk processing plant for the city of Brisbane, and were examined on the same day. Preparation of samples Approximately, 20 g of meat was sampled using sterile scissors and scalpels. The sample was homogenized in a Stomacher 400 (Seward Medical, London) in 180 ml of the appropriate enrichment broth in a sterile Stomacher 400 bag for 2 rain. Milk samples (20 ml each) were directly added to the enrichment. Isolation and identification procedures A eromonas Samples were first enriched in alkaline peptone water (APW) 0.1% (pH 8.4-8.6) as recommended by Shread et al. (1981) and incubated at 28°C for 24 h. Loopfuls from the enrichment were streaked on starch ampicillin agar (SAA) prepared according to Palumbo et al. (1985) as well as Aeromonas medium base (Oxoid CM 833) supplemented with ampicillin (Sigma-A-9518) at 5 mg/l. Inoculated plates of SAA were incubated at 28°C for 24 h and examined for large, honey-yellow, amylase-positive colonies. Plates of Aeromonas medium base were incubated at 28°C for 48 h and examined for dark green opaque colonies with a dark centre. These were recorded as presumptive Aeromonas spp. Ltsteria Meat. Cold enrichment (Gray et al., 1948) in tryptose broth (TB) as well as primary and secondary enrichment media according to USDA recommendations
265 (McClain and Lee, 1988) known as University of Vermont enrichment broth No. 1 and 2 (UVM1 and UVM2) were used as follows: A sample portion was added to 180 ml of UVM1 as primary enrichment and incubated at 30°C for 24 h, after which 0.1 ml of this culture was transferred to 10 ml of UVM2 as secondary enrichment and incubated at 30°C for a further 24 h. Another portion was enriched in 180 ml of TB (Difco) and incubated at 4°C for up to 8 weeks with subsequent weekly plating. Loopfuls from UVM2 and TB were streaked onto Lithium chloride-phenyl ethanol-moxalactam agar (LPMA) (Lee and McCLain, 1986) and Listeria selective agar (LSA) plates (Curtis et al., 1989). Milk. A sample portion was added to 180 ml of FDA enrichment broth (EB) prepared according to Lovett et al. (1987) and incubated at 30°C for up to 7 days with subsequent plating after 1 and 7 days. Another portion was added to 180 ml of TB and treated as mentioned above. The enrichment was streaked on Modified McBride Agar (MMA) plates (Lovett et al., 1987) as well as LSA. LPMA and LSA plates were incubated at 30°C for 48 h and MMA plates at 35°C for 48 h. Plates of LPMA and MMA were examined using 45 ° incident transmitted light (Henry, 1933) and presumptive listeria colonies appeared blue to bluish grey whereas on LSA colonies appeared black with black halos and sunken centre. Identification h eromonas
Colonies were purified and examined for oxidase reaction and identified to the genus level by checking their biochemical profile on Aeromonas hydrophila medium (AHM) prepared according to Kaper et al. (1979). Cultures giving an alkaline reaction (purple colour) at the top of the medium and acid reaction (yellow colour) in the butt were considered Aeromonas spp. Subsequent speciation was carried out using the criteria of Popoff (1984). Listeria
Three to five colonies were picked, purified and confirmed by the following phenotypic tests: Gram-stain, catalase and oxidase reactions, umbrella-shaped motility pattern using motility test medium (Difco-0105-01-3), haemolysis on horse blood agar, aesculin hydrolysis, fermentation of mannitol, rhamnose and xylose (Bortolussi et al., 1985) and C A M P test (McClain and Lee, 1988). Isolates which proved to be L. monocytogenes were serotyped using Difco type 1 & 4 antisera.
Results and Discussion Aeromonas species were found to be prevalent in all kinds of samples (Table I) with the highest percentage in pork (74%) and lowest in raw milk (27%). The high incidence of aeromonads in red meat and milk has been confirmed by others in Australia and elsewhere (Majeed et al., 1989; Palumbo et aL, 1985; Kielwein et al.,
266 TABLE I Incidence of Aeromonas and L:sterla spp. in red meat and milk Type of sample
Number of samples exanuned
Aeromonas spp.
Ltsterta spp
Beef Lamb Pork Milk
50 50 50 150
30 (60) a 29 (58) 37 (74) 40 (27)
17 (34) 20 (40) 15 (30) 4 (3)
Figures m parentheses indicate the percentage of the posture samples
1969). T h e use of A P W as e n r i c h m e n t p r i o r to p l a t i n g was f o u n d to be an i m p o r t a n t f a c t o r in i n c r e a s i n g the i s o l a t i o n rate as r e c o m m e n d e d by m a n y i n v e s t i g a t o r s ( R o b i n s o n et al., 1986; M i l l e r s h i p a n d C h a t t o p a d h y a y , 1984). T h e r e was no a p p r e c i a b l e difference in the recovery rate b e t w e e n A e r o m o n a s m e d i u m a n d S A A . A l t h o u g h the f o r m e r was f o u n d to be m o r e efficient in r e d u c i n g the s w a r m i n g g r o w t h e n c o u n t e r e d in p o r k samples, S A A a l l o w e d easier d i f f e r e n t i a t i o n of a e r o m o n a d s from o t h e r m i c r o f l o r a . I n total, 350 isolates of A e r o m o n a s spp. were o b t a i n e d ( T a b l e II) a n d all were f o u n d to be motile. These isolates c o m p r i s e d 75 (21%) from beef, 69 (20%) f r o m l a m b , 87 (25%) from p o r k a n d 119 (34%) f r o m raw milk. Strains i d e n t i f i e d as A , h y d r o p h i l a were 212 ( n e a r l y 60%), A . s o b r m 34 (10%), A. c a v t a e 74 (21%) a n d 30 strains (9%) as a t y p i c a l a e r o m o n a d s . M o r e t h a n one A e r o m o n a s spp. were isolated from s o m e m e a t samples, however, each p o s i t i v e s a m p l e from raw milk c o n t a i n e d o n l y o n e species (results n o t shown). T h e i s o l a t i o n of a significant n u m b e r of a t y p i c a l A e r o m o n a s spp. seems to c o n f i r m the o p i n i o n of P o p o f f et al. (1981) that each o f the three species of m o t i l e a e r o m o n a d s r e p r e s e n t s at least two species o r subspecies in need of m o r e d i f f e r e n t i a t i n g p h e n o t y p i c profiles. T h e i n c i d e n c e o f L i s t e r t a spp. in the s a m p l e s is s u m m a r i z e d in T a b l e I. T h e highest p e r c e n t a g e was in l a m b (40%) whilst the lowest was in milk (3%). T h e isolated species o f h s t e r i a are listed in T a b l e III. L. m o n o c y t o g e n e s was r e c o v e r e d f r o m 24% o f beef, 16% o f l a m b a n d 10% of p o r k w h e r e a s the o r g a n i s m was not d e t e c t e d in milk at all. L. i n n o c u a was p r e s e n t in all s a m p l e s with d i f f e r e n t
TABLE II Dtstribuuon of Aeromonas spp. in flesh and dairy foods Type of sample
Number of stratus
Aeromonas spp.
Beef Lamb Pork Milk
75 69 87 119
54 51 65 42
4
11
6
7 3 20
6 15 42
5 4 15
Total
350
212
34
74
30
A. h.vdrophda
A. sobrla
A. cawae
Atypical aeromonads
267 TABLE III Dismbuuon of L*stena spp. m flesh and dairy foods Type of sample
1_ monoc.vtogenes
L. mnocua
Beef Lamb Pork Milk
12 (24) ~ 8 (16) 5 (10)
5 (10) 12 (24) 10 (20) 4 (3)
a Ftgures in parentheses indicate the percentage of the posture samples. percentages with the highest in lamb (24%) and the lowest in milk (3%). Serotyping of the L. m o n o c y t o g e n e s isolates is given in Table IV. The incidence of LJsteria spp. in raw meat was fairly low compared with the isolation rate found in other countries. In the U.S.A., the organism was estimated to be present in 70% of ground beef (Brackett, 1988) and in New Zealand 48% and 50% of retail lamb and pork, respectively, were found to be contaminated with the organism (Lowry and Tiong. 1988). However, in a large survey conducted in Sweden the organism was not detected in any of the three kinds of meat examined (Ternstrom and Molin, 1987). The isolation of L. i n n o c u a from raw milk in the present study is in keeping with the results of overseas investigators (Farber et al., 1988: Liewen and Plautz, 1988). A survey of 600 raw milk samples by New South Wales Dairy Corporation for L z s t e r i a spp. yielded 0.4% positive samples for L. i n n o c u a and no isolation of /- m o n o c v t o g e n e s (Sutherland, 1989). Although there have been a tremendous number of plating media formulated to recover iisteria from foods, there has been no consensus as to the best medium for direct plating. Our aim was to maximize the recovery of the organism from the samples examined, hence we used an integrated set of enrichment and isolation media. For meat samples, we used the enrichment broth developed by Donnelly and Baigent (1986) and that of Fraser and Sperber (1988) modified by McClain and Lee (1988) as primary and secondary enrichment, respectively. We found them to be superior to cold enrichment in detecting listeria with no major difference between L P M A and LSA as plating media. However, differentiation of listeria colonies was easier on LSA due to the presence of aesculin-ferric ammonium citrate as an
TABLE IV D~smbutlon of serotypes of /- monocytogenes m flesh and dairy foods Type of sample
Number of strains
Serotype
Beef Lamb Pork
31 20 15
6 3 2
4 22 10 12
Untyped 3 7 1
66
11
44
11
l
Total
268 indicator system. In addition, the Henry technique was found to be a subjective s y s t e m as m a n y c o l o n i e s p i c k e d u s i n g t h i s t e c h n i q u e t u r n e d o u t n o t to b e Ltster~a spp. upon confirmation. In conclusion, the opinion of Buchanan and Palumbo (1985) that motile a e r o m o n a d s , p a r t i c u l a r l y A . hydrophila a n d ,4. sobr~a, r e p r e s e n t n e w f o o d b o r n e pathogens cannot be ignored with the high isolation rate of such organisms in this s u r v e y . I n a d d i t i o n , t h e i s o l a t i o n o f L. m o n o c v w g e n e s f r o m m e a t w i d e n s t h e s c o p e o f its p u b l i c h e a l t h h a z a r d , e s p e c i a l l y s i n c e t h e o r g a m s m w a s d e t e c t e d in p r o c e s s e d m e a t in Q u e e n s l a n d b y t h e S t a t e H e a l t h D e p a r t m e n t ( p e r s o n a l c o m m u m c a t i o n ) .
Acknowledgement Mr. lbrahim was financially supported University of Queensland.
by Ernest Singer Scholarship
from the
References Agger, W.A., McCormick. J.D. and Gurwxth, M.J. (1985) Chnical and mzcrobioiog~cal features of Aeromonas h.vdrophila-associated diarrhoea. J. Chn. Microbiol. 21. 909-913. Bortolussi. R.. Schlech. W.F., III and Albriton. W.L. (1985) Listena. In: E.H. Lennette. A. Balows. W.J. Hausler Jr. and H.J. Shadomy (Eds.). Manual of Clinical M~crob~ology. 4th edn.. American Soc,ety for Microbiology, Washington, DC. pp. 205-208. Brackett, R.E. (1988) Presence and perststence of lasterJa monocvtogene In food and water Food Technol. 4, 162-164 Buchanan. R.L. and Palumbo, S.A. (1985) Aeromonas t!vdrophda and Aeromonas sohrla as potential food po,sontng species: a rev,ew. J. Food Safety 7, 15-29. Burke, V. and Gracey, M. (1986) Aeromonas species m human dlarrhoeal d~sease. J. Gastroenterol Hepatol. 1,237-249. Curtis, G.D.W., Mitchell. R.G.. King. A.F. and Griffin, E.J. (1989) A selective differenual medmm for the isolation of Ltsterta monoq.vtogenes. Lett Appl. Microblol. 8.95-98. Donnelly. C W. and Balgent, G.J. (1986) Method for flow cytometnc detecuon of Llsterta monocvtogene3 m milk. Appl. Environ. Microb,ol. 52, 689-695. Farber. J.M., Sanders, G.W. and Malcolm. S.A. (1988) The presence of LIster, a spp. m raw mdk m Ontario. Can..I. Microblol 34, 95-100. Fleming. DW.. Cochl. S.L.. MacDonald. K.L.. Brondum, J.. Hayes, P.S.. Phkyus. B.D.. Holmes, M.B, Auduner. A.. Broome. C V. and Re, ngold. A.L. (1985) Pasteurized mdk as a vehicle of refection m an outbreak of hstenosls N Eng. J Med. 312. 404-407. Fraser, J.A and Sperber. W.H (1988) Rapid detection of Ltsterta spp m food and environmental samples by escuhn hydrolys,s. J. Food Protect. 51. 762-765. Gray, M.L., Stafseth. H.J.. Thorp Jr. F.. Sholl. L.B. and Riley Jr.. W. (1948) A new techmque for ,solatmg l*sterellae from the bovine brain. J Bactenol. 55. 471-476. Greenway. G. (1988) Aeromonas--A future concern for the dairy and food industry. In: The Dairy Technologist. Food Research Institute. Victoria, Austraha. pp. 73-75 Hayes. P.S., Feeler. J C.. Graves. L M.. Ajellow. G.W. and Flemmg. D W. (1986) isolauon of L:sterta mon6wvtogenes from ray, mdk. Appl. Environ. Microblol 51, 438-4.40. Helslck. J.E.. Wagner. D.E., Nterman. M.L and Peeler. J T (1989) Ltsterta spp. found on fresh market produce. Appl Environ. Microblol. 55, 1925-1927.
269 Henry. B.S. (1933) Dtssociauon m the genus Brucella. J. Infect. Dis. 52. 374-402. Johnson, J.L.. Doyle. M.P. and Cassens, R.G. (1988) Survival of bsterla monocytogenes m ground beef Int. J. Food Mlcrobiol. 6, 243-247. Johnson, J.L.. Doyle. M.P. and Cassens. R.C. (1990) LIsteria monocvtogenes and other LIsteria spp. In meat and meat products. J. Food Protect. 53, 81-91. Kaper, J.. Seidler, R.J., Lockman, H. and Colwell. R.R. (1979) Medium for the presumpuve tdent~ftcauon of Aeromonos hydrophda and Enterobactenaceae. Appl Environ. Microblol. 38, 1023-1026. Kielwem, G., Gerlach, R. and Jhone, H. (1969) Untersuchungen tiber das Vorkomen yon Aeromonas hydroghda m Rohmilch. Arch. Lebensmittelhyg. 20. 34-38. Lee, W.H. and McClam, D. (1986) Improved Listeria monocvtogenes selecuve agar. Appl. Envaron. Mlcroblol. 52, 1215-1217. Ltewen, M.B. and Plautz, M.W. (1988) Occurrence of Llsterla monocytogenes in raw milk m Nebraska. J. Food Protect. 51,840-841. Lovett, J., Franc~s, D.W. and Hunt. J.M. (1987) Llsterm monoqytogenes m raw milk' Detectmn. mcidence, and pathogemctty. J. Food Protect. 50. 188-192. Lowry. P.D. and Tmng. I. (1988) The incidence of l, zsterla monocytogenes m meat and meat p r o d u c t s - factors affectmg distribuuon. Proc. 34th Int. Congress Meat Sci. Technol. pp. 528-530 Ma.leed, K., Eagan, A. and Mac Rae. I.C. (1989) Enterotoxlgenic aeromonads on retail lamb meat and offal. J. Appl. Bacteriol. 67, 165-170. McClam, D. and Lee, H.L. (1988) Development of USDA-FSIS method for ~solatmn of Ltsteria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal.Chem. 71. 660-664 MillershJp. S.E. and Chattopadhyay, B. (1984) Method for the isolatton of Aeromonas h~'drophda from faeces. J. Hyg. 92, 145-152. Ntshikawa, Y. and Kishi, T. (1988) Isolation and charactenzatton of mottle Aeromonas from human. food and envtronmental specimens. Epidem. Inf. 101, 213-223. Okrend, A.G.J.. Rose, B.E. and Bennett. B. (1987) Incidence and toxlgemclty of Aeromonas species m retail poultry, beef and pork. J. Food Protect. 50, 509-513. Palumbo, S.A., Maxano, F.. Williams, A.C., Buchanan, R.L. and Thayer, D.W. (1985) Starch ampicflhn agar for the quantltauve detection of Aeromonas hydrophda. Appl. Environ. Mtcrbml. 50, 1027-1030 Pmi, P.N. and Gilbert, R.J. (1988) The occurrence m the U.K. of Llsterm spp. m raw chicken and soft cheeses. Int. J. Food Mlcrobiol. 6, 317-326. Popoff. M. (1984) Aeromonas. In: N.R. K n e g (Ed.), Bergey's Manual of Systemauc Bactenolog), Vol. 1., Wilhams & Willons, Balumore, MD, pp. 545-548. Popoff. M., Coynauh, C., IOredlian, M. and Lemelin, M. (1981) Polynucleoude sequence relatedness a m o n g mottle Aeromonas species. Curr. Microbtol. 5. 109-114. Robmson, J., Beaman, J., Wagener. L. and Burke, V. (1986) Comparison of direct platmg wtth the use of enrichment culture for isolation of Aeromonas spp. from faeces. J. Meal. Mtcrobtol. 22, 315-317. Schlech. W.F.. Lav~gne, P.M.. Bortolussi, R.A., Allen, A.C., Haldane, E.V.. Wort, A.J.. Htghtower, A.W., Johnson, S.E., Kang, S.H., Ntcholls. E.S. and Broome, C.V. (1983) Epidemic hstertosts--evldence for transrmss.on by food. New Eng. J. Med. 308, 203-206. Shread, P., Donovan. T.3. and Lee, J.V. (1981) A survey of the mcidence of Aeromonas m h u m a n faeces. Soc. Gen. Mtcrobtol. Q. 8, 184. Sutherland. P.S (1989) Lasterm monocytogenes, in: K.A. Buckle, J.A. Dave),. M.J. Eyles. A.D Hockmg. K.G. V~ewton and E.J. Stuttard, (Eds.), Foodborne Microorgamsms of Pubhc Health Slgmflcance, 4th edn., A u s t r a h a n Institute of Food Science and Technology (AIFST). Nev. South Wales (NSW) Branch. pp. 28"7-311. Ternstrom. A. and Mohn, G. (1987) Incidence of potential pathogens on raw pork. beef and chicken m Sweden. with spec:al reference to Er~'stpelothrtx rhusmpathtae. J. Food Protect. 50, 141-146 Todd, E.C.D. (1989) Prehminary estimates of costs of foodborne dtseases m the Un.ted States. J. Food Protect 52. 595-601. World Health O r g a m z a t m n Working G r o u p (1988) Foodborne Listertosls. Bulletm WHO, 66, 421-428.
263
FOOD 00388
Incidence of Aeromonas and Listeria spp. in red meat and milk samples in Brisbane, Australia A. I b r a h i m a n d I.C. M a c R a e Department of Microbiolog),, Umverslty of Queensland, Queensland, Austraha (Received 19 June 1990; accepted 4 December 1990)
A total of 150 samples, 50 each of beef, lamb and pork from l0 local retail stores in Brisbane metropolitan area as well as 150 raw bovine bulk milk tank samples obtained from Queensland United Foods (QUF), were examined for the presence of Aeromonas and l.zsterta spp. over a period of 1 year. Different sets of enrichment and plating media were used to recover the organisms with subsequent identification using conventional biochemical and serotyping techniques. A total of 509 isolates consisting of Aeromonas spp. (350) and l.,isteria spp. (159) were obtamed from 6070, 58~, 74~, 26.6~, 34~, 40c$, 30~, 2.6~ of samples of beef, lamb, pork and milk respectively. Motile aeromonads ( A. hydrophzla, A. sobria, A. caviae) and i..isteria innocua were isolated from all kinds of samples whereas /_ monocytogenes was only isolated from flesh food. A. hydrophila contributed the largest percentage of the motile aeromonads (607o). The majority o f / - monocytogenes cultures were serotype 4. Key words: Meat; Milk; Aeromonns; l.,isteria
Introduction Motile aeromonads and Listeria monocytogenes are the most recently added species to the growing llst of foodborne pathogens. Aeromonas hydrophila and A. sobria have been frequently isolated from clinical cases of human diarrhoea (Nishikawa and K.ishi, 1988; Burke and Gracey, 1986; Agger et al., 1985). Motile aeromonads have also been detected in different varieties of foods especially those of animal origin like meat and milk (Greenway, 1988; Okrend et al., 1987; Ternstrom and Molin, 1987; Palumbo et al., 1985). The coincidence of the previous findings has led Buchanan and Palumbo (1985) to suggest that aeromonads, in particular, A. hydrophila and ,4. sobria are significant foodborne pathogens. Outbreaks of listeriosis due to contamination of foods have been reported in different countries (Fleming et al., 1985; Schlech et al., 1983). In addition, 2500 cases of listeriosis were reported to occur in the U.S.A. every year (Todd, 1989). The organism has been detected in different foods including milk (Hayes et al., 1986), Correspondence address: I.C. Mac Rae, Department of Microbiology, Umversity of Queensland, Queensland, 4072 Australia.
264 vegetables (Heisick et al., 1989), meat (Johnson et al.. 1990, 1988) and chicken (Pini and Gilbert. 1988), Moreover. The World Health Orgamzation (WHO) now recognises that contamination of foodstuffs with L. monocrtogenes is the principal means of acquiring hsteriosis (WHO, 1988). Little is known about the incidence of these organisms in different foods m Australia, therefore, ~t was the aim of this study to check their prevalence in both red meat and milk.
Materials and Methods
Collectton of samples Red meat A total of 150 samples, 50 each of beef, lamb and pork (different cuts) were purchased at 10 local retail supermarkets, over a 1 year period, in regular consumer packages each of which was considered as one separate sample and transferred to the laboratory with a minimal delay and examined on the day of purchase. Milk A total of 150 raw bovine bulk milk tank samples were obtained from Queensland United Foods (QUF), the major milk processing plant for the city of Brisbane, and were examined on the same day. Preparation of samples Approximately, 20 g of meat was sampled using sterile scissors and scalpels. The sample was homogenized in a Stomacher 400 (Seward Medical, London) in 180 ml of the appropriate enrichment broth in a sterile Stomacher 400 bag for 2 rain. Milk samples (20 ml each) were directly added to the enrichment. Isolation and identification procedures A eromonas Samples were first enriched in alkaline peptone water (APW) 0.1% (pH 8.4-8.6) as recommended by Shread et al. (1981) and incubated at 28°C for 24 h. Loopfuls from the enrichment were streaked on starch ampicillin agar (SAA) prepared according to Palumbo et al. (1985) as well as Aeromonas medium base (Oxoid CM 833) supplemented with ampicillin (Sigma-A-9518) at 5 mg/l. Inoculated plates of SAA were incubated at 28°C for 24 h and examined for large, honey-yellow, amylase-positive colonies. Plates of Aeromonas medium base were incubated at 28°C for 48 h and examined for dark green opaque colonies with a dark centre. These were recorded as presumptive Aeromonas spp. Ltsteria Meat. Cold enrichment (Gray et al., 1948) in tryptose broth (TB) as well as primary and secondary enrichment media according to USDA recommendations
265 (McClain and Lee, 1988) known as University of Vermont enrichment broth No. 1 and 2 (UVM1 and UVM2) were used as follows: A sample portion was added to 180 ml of UVM1 as primary enrichment and incubated at 30°C for 24 h, after which 0.1 ml of this culture was transferred to 10 ml of UVM2 as secondary enrichment and incubated at 30°C for a further 24 h. Another portion was enriched in 180 ml of TB (Difco) and incubated at 4°C for up to 8 weeks with subsequent weekly plating. Loopfuls from UVM2 and TB were streaked onto Lithium chloride-phenyl ethanol-moxalactam agar (LPMA) (Lee and McCLain, 1986) and Listeria selective agar (LSA) plates (Curtis et al., 1989). Milk. A sample portion was added to 180 ml of FDA enrichment broth (EB) prepared according to Lovett et al. (1987) and incubated at 30°C for up to 7 days with subsequent plating after 1 and 7 days. Another portion was added to 180 ml of TB and treated as mentioned above. The enrichment was streaked on Modified McBride Agar (MMA) plates (Lovett et al., 1987) as well as LSA. LPMA and LSA plates were incubated at 30°C for 48 h and MMA plates at 35°C for 48 h. Plates of LPMA and MMA were examined using 45 ° incident transmitted light (Henry, 1933) and presumptive listeria colonies appeared blue to bluish grey whereas on LSA colonies appeared black with black halos and sunken centre. Identification h eromonas
Colonies were purified and examined for oxidase reaction and identified to the genus level by checking their biochemical profile on Aeromonas hydrophila medium (AHM) prepared according to Kaper et al. (1979). Cultures giving an alkaline reaction (purple colour) at the top of the medium and acid reaction (yellow colour) in the butt were considered Aeromonas spp. Subsequent speciation was carried out using the criteria of Popoff (1984). Listeria
Three to five colonies were picked, purified and confirmed by the following phenotypic tests: Gram-stain, catalase and oxidase reactions, umbrella-shaped motility pattern using motility test medium (Difco-0105-01-3), haemolysis on horse blood agar, aesculin hydrolysis, fermentation of mannitol, rhamnose and xylose (Bortolussi et al., 1985) and C A M P test (McClain and Lee, 1988). Isolates which proved to be L. monocytogenes were serotyped using Difco type 1 & 4 antisera.
Results and Discussion Aeromonas species were found to be prevalent in all kinds of samples (Table I) with the highest percentage in pork (74%) and lowest in raw milk (27%). The high incidence of aeromonads in red meat and milk has been confirmed by others in Australia and elsewhere (Majeed et al., 1989; Palumbo et aL, 1985; Kielwein et al.,
266 TABLE I Incidence of Aeromonas and L:sterla spp. in red meat and milk Type of sample
Number of samples exanuned
Aeromonas spp.
Ltsterta spp
Beef Lamb Pork Milk
50 50 50 150
30 (60) a 29 (58) 37 (74) 40 (27)
17 (34) 20 (40) 15 (30) 4 (3)
Figures m parentheses indicate the percentage of the posture samples
1969). T h e use of A P W as e n r i c h m e n t p r i o r to p l a t i n g was f o u n d to be an i m p o r t a n t f a c t o r in i n c r e a s i n g the i s o l a t i o n rate as r e c o m m e n d e d by m a n y i n v e s t i g a t o r s ( R o b i n s o n et al., 1986; M i l l e r s h i p a n d C h a t t o p a d h y a y , 1984). T h e r e was no a p p r e c i a b l e difference in the recovery rate b e t w e e n A e r o m o n a s m e d i u m a n d S A A . A l t h o u g h the f o r m e r was f o u n d to be m o r e efficient in r e d u c i n g the s w a r m i n g g r o w t h e n c o u n t e r e d in p o r k samples, S A A a l l o w e d easier d i f f e r e n t i a t i o n of a e r o m o n a d s from o t h e r m i c r o f l o r a . I n total, 350 isolates of A e r o m o n a s spp. were o b t a i n e d ( T a b l e II) a n d all were f o u n d to be motile. These isolates c o m p r i s e d 75 (21%) from beef, 69 (20%) f r o m l a m b , 87 (25%) from p o r k a n d 119 (34%) f r o m raw milk. Strains i d e n t i f i e d as A , h y d r o p h i l a were 212 ( n e a r l y 60%), A . s o b r m 34 (10%), A. c a v t a e 74 (21%) a n d 30 strains (9%) as a t y p i c a l a e r o m o n a d s . M o r e t h a n one A e r o m o n a s spp. were isolated from s o m e m e a t samples, however, each p o s i t i v e s a m p l e from raw milk c o n t a i n e d o n l y o n e species (results n o t shown). T h e i s o l a t i o n of a significant n u m b e r of a t y p i c a l A e r o m o n a s spp. seems to c o n f i r m the o p i n i o n of P o p o f f et al. (1981) that each o f the three species of m o t i l e a e r o m o n a d s r e p r e s e n t s at least two species o r subspecies in need of m o r e d i f f e r e n t i a t i n g p h e n o t y p i c profiles. T h e i n c i d e n c e o f L i s t e r t a spp. in the s a m p l e s is s u m m a r i z e d in T a b l e I. T h e highest p e r c e n t a g e was in l a m b (40%) whilst the lowest was in milk (3%). T h e isolated species o f h s t e r i a are listed in T a b l e III. L. m o n o c y t o g e n e s was r e c o v e r e d f r o m 24% o f beef, 16% o f l a m b a n d 10% of p o r k w h e r e a s the o r g a n i s m was not d e t e c t e d in milk at all. L. i n n o c u a was p r e s e n t in all s a m p l e s with d i f f e r e n t
TABLE II Dtstribuuon of Aeromonas spp. in flesh and dairy foods Type of sample
Number of stratus
Aeromonas spp.
Beef Lamb Pork Milk
75 69 87 119
54 51 65 42
4
11
6
7 3 20
6 15 42
5 4 15
Total
350
212
34
74
30
A. h.vdrophda
A. sobrla
A. cawae
Atypical aeromonads
267 TABLE III Dismbuuon of L*stena spp. m flesh and dairy foods Type of sample
1_ monoc.vtogenes
L. mnocua
Beef Lamb Pork Milk
12 (24) ~ 8 (16) 5 (10)
5 (10) 12 (24) 10 (20) 4 (3)
a Ftgures in parentheses indicate the percentage of the posture samples. percentages with the highest in lamb (24%) and the lowest in milk (3%). Serotyping of the L. m o n o c y t o g e n e s isolates is given in Table IV. The incidence of LJsteria spp. in raw meat was fairly low compared with the isolation rate found in other countries. In the U.S.A., the organism was estimated to be present in 70% of ground beef (Brackett, 1988) and in New Zealand 48% and 50% of retail lamb and pork, respectively, were found to be contaminated with the organism (Lowry and Tiong. 1988). However, in a large survey conducted in Sweden the organism was not detected in any of the three kinds of meat examined (Ternstrom and Molin, 1987). The isolation of L. i n n o c u a from raw milk in the present study is in keeping with the results of overseas investigators (Farber et al., 1988: Liewen and Plautz, 1988). A survey of 600 raw milk samples by New South Wales Dairy Corporation for L z s t e r i a spp. yielded 0.4% positive samples for L. i n n o c u a and no isolation of /- m o n o c v t o g e n e s (Sutherland, 1989). Although there have been a tremendous number of plating media formulated to recover iisteria from foods, there has been no consensus as to the best medium for direct plating. Our aim was to maximize the recovery of the organism from the samples examined, hence we used an integrated set of enrichment and isolation media. For meat samples, we used the enrichment broth developed by Donnelly and Baigent (1986) and that of Fraser and Sperber (1988) modified by McClain and Lee (1988) as primary and secondary enrichment, respectively. We found them to be superior to cold enrichment in detecting listeria with no major difference between L P M A and LSA as plating media. However, differentiation of listeria colonies was easier on LSA due to the presence of aesculin-ferric ammonium citrate as an
TABLE IV D~smbutlon of serotypes of /- monocytogenes m flesh and dairy foods Type of sample
Number of strains
Serotype
Beef Lamb Pork
31 20 15
6 3 2
4 22 10 12
Untyped 3 7 1
66
11
44
11
l
Total
268 indicator system. In addition, the Henry technique was found to be a subjective s y s t e m as m a n y c o l o n i e s p i c k e d u s i n g t h i s t e c h n i q u e t u r n e d o u t n o t to b e Ltster~a spp. upon confirmation. In conclusion, the opinion of Buchanan and Palumbo (1985) that motile a e r o m o n a d s , p a r t i c u l a r l y A . hydrophila a n d ,4. sobr~a, r e p r e s e n t n e w f o o d b o r n e pathogens cannot be ignored with the high isolation rate of such organisms in this s u r v e y . I n a d d i t i o n , t h e i s o l a t i o n o f L. m o n o c v w g e n e s f r o m m e a t w i d e n s t h e s c o p e o f its p u b l i c h e a l t h h a z a r d , e s p e c i a l l y s i n c e t h e o r g a m s m w a s d e t e c t e d in p r o c e s s e d m e a t in Q u e e n s l a n d b y t h e S t a t e H e a l t h D e p a r t m e n t ( p e r s o n a l c o m m u m c a t i o n ) .
Acknowledgement Mr. lbrahim was financially supported University of Queensland.
by Ernest Singer Scholarship
from the
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