Journul of Applied Bacteriology 1983, 54, 135-139 1 153/05/82

A note on the antibiotic susceptibilities of Clostridium perfringens serotypes isolated from meat

J . L . SMART*, M.L. TRUMAN* & M . F . S T R I N G E R ? *Agricultural Research Council Meat Research Institute, Langford, Bristol, BS18 7DY ?Food Hygiene Laboratory, Central Public Hrafth Laboratory, Colindale Avenue, London, N W 9 S H T , U K

Received 17 M a y I982 and accepted 22 July 1982

S M A R T . J .L . , TRUMAN, M . L . & S T R I N G E R , M . F . 1983. A note on the antibiotic susceptibilities of Clostridium perfringens serotypes isolated from meat. Journal of Applied Bacteriology 54, 135-1 39.

The susceptibilities to six antibiotics have been determined of Clostridium per- fiingens serotypes isolated from beef, pork and lamb carcases and from minced beef. Generally similar results were obtained to those of previous studies of mainly clinical isolates. Fourteen of the 229 strains tested were resistant to tetracycline; three of these, from the same beef carcase, were also resistant to erythromycin.

The constant widespread use of antibiotics in animal husbandry and veterinary and human medicine, coupled with current knowledge of transfer of antibiotic resistance, makes it essen- tial to monitor the susceptibilities of known pathogenic bacteria to antibiotics (Linton 1977). Clostridium perfringens is ubiquitous and associ- ated with disease in animals and foodborne ill- ness in man. Several antibiotic susceptibility studies, mainly on clinical isolates of Cl. perfringens, have been reported (Staneck & Washington 1974; Dornbusch et al. 1975; Sch- wartzman et al. 1977) but little is known of the antibiotic susceptibilities of isolates from other sources. Meat and meat products were involved in 134 (74%) of the 181 outbreaks of Cl. per- fringens food poisoning in which food was im- plicated in 1976-1978 (Hepner 1980). The antibiotic susceptibilities of Cl. perfringens serotypes isolated from beef, pork and lamb car- cases (Smart et al. 1979) and from minced beef (this report) were determined for comparison with previous studies and also to demonstrate whether any serotype, or group of serotypes, displayed marked resistance to particular anti- biotics. The results of serotyping Cl. perfringens isolates from retail minced beef are also presen- ted. 0021-8847/83/02W135 $02.00 0 1983 The Society for Applied Bacteriology

10

Materials and Methods

M E D I A

Trypticase broth (TPYGC). This comprised (% w/v): Trypticase (BBL) 5; Bactopeptone (Difco) 0.5; yeast extract (Oxoid) 0.2; glucose 0.4 and L-cysteine hydrochloride 0.05. The pH was adjusted to 6.8.

Blood agar (BA). Prepared as Smart et al. (1979).

Rohertson’s cooked meat medium ( C M M ) . Supplied by Southern Group Laboratory, Hither Green Hospital, London SE13.

Brain Heart Infusion agar (Difco) ( B H I A ) . Prepared according to the manufacturer’s in- structions and used as the base medium for anti- biotic susceptibility tests.

A N T I B I O T I C S

The antibiotics used were tetracycline (Lederle Laboratories Division, Cyanamid of Great Bri- tain Ltd., Gosport, Hampshire); erythromycin (Dista Products Ltd., Liverpool, England); benzyl penicillin (Glaxo Laboratories Ltd., Greenford, England); chloramphenicol (Parke Davis & Co., Hounslow, London, England); lin- comycin and clindamycin (Upjohn Co., Kala- mazoo, Michigan, USA).

136 J. L. Smart et al. O R G A N I S M S

The isolates tested were from retail minced beef (Roberts et al. 1980) and beef, pork and lamb carcases (Smart et al. 1979). They were identified as C1. perfringens and serotyped by methods previously described (Smart et al. 1979). Seroty- ping was carried out at the Food Hygiene Lab- oratory, Colindale, London. Only strains which could be serotyped were tested for their anti- biotic susceptibility, several strains of the same serotype being included when available. Of the 229 strains tested, 117 were from minced beef and 112 from carcases. Stock cultures were maintained in CMM at ambient temperatures.

S U S C E P T I B I L I T Y TESTING

For each antibiotic a two-fold dilution series was made in sterile water. The solutions were stored refrigerated (4°C) for up to 72 h but gen- erally were used immediately after preparation. Antibiotic (1 ml) was added to 99 ml of molten BHIA which had been cooled to 50°C to give the required final concentration, mixed thor- oughly and poured in to 9 cm diam. plastic Petri dishes in 16 ml volumes. After solidifica- tion the plates were placed in a hot air oven at

50°C for ca 30 min until the agar surface was dry. Subcultures of the CI. perfringens strains were made by inoculating 5 ml quantities of TPYGC in 7 ml McCartney bottles, with 0.2 ml from the stock CMM cultures. After incubation for 1 6 1 8 h at 37"C, the cultures were placed in groups of 12 in specially formed metal holders of the same diameter (9 cm) as a standard Petri dish, the bottles being arranged such that the centres of their mouths were equidistant. A 12- pronged inoculator made from 2 mm diam. nichrome wire, and formed to fit the culture bottles when arranged as described above, was used to inoculate the BHIA plates. The inocula- tor, stored in alcohol, was sterilized by flaming, cooled, then inserted into the bottoms of the opened culture bottles. Care was taken not to touch the sides of the bottles on removal; the inoculator was then lightly touched onto the surface of a plate. The inocula were allowed to dry prior to incubation under hydrogen in anaerobe jars for 24 h at 37°C. For each group of 12 cultures a control plate, containing no antibiotic, was inoculated and used to compare growth with that on antibiotic containing plates. Each strain was tested at least twice and the

Table 1. Serotypes of CI. perfringens isolated from minced beef

Isolates Number of

samples Total Number typed Serotypes (No. of samples if > 1)

A 35 114 53

1

5 3/4(5)

9/38

3/4(3) 5 7

11/27 14 16 17/52(3) 18

314 4 7

I 1 11/24 14

B 36 147 59

C 26 90 40

56 57 65/69 68 71 72 74

W4) 45 52(2) 65/69(3) 65/69/72(2) 66

65/69 65/69/72(3) 66/68 71

Totals 97 35 1 152 59 serotyps

A, Small family butchers; B, intermediate sized chain butchers; C, supermarkets.

Antibiotic susceptibility of C1. perfringens 137 minimum inhibitory concentrations (MICs) were recorded. Purity checks were performed on BA plates incubated aerobically and anaero- bically a t 37°C.

Results The serotyping results of the C1. perjringens iso- lates from minced beef are shown in Table 1. Of the 351 isolates, 142 were non-typable with the available sera and a further 57 could not be typed due to autoagglutination in saline. The remaining 152 typed isolates comprised 59 serotypes, a much smaller ratio of serotypes to typable isolates than was found previously on beef, pork and lamb carcases when 310 isolates which could be typed comprised 46 serotypes (Smart et al. 1979). Thirteen of the 46 carcase serotypes were isolated from beef carcases (Smart et al. 1979) and 10 of them were detected in the minced beef samples examined in this study.

Serotype 314, the most frequently implicated serotype in food poisoning outbreaks associated with beef, including minced beef (Stringer et al. 1979), was found in more minced beef samples (9) than any other serotype (Table 1). Serotypes 314, 23, 30, 34, 38, and 65/69 were found in minced beef samples from all three types of retail outlets but the great majority of the serotypes (41) occurred in one or more samples from one retail source only. The proportion of typeable isolates was similar for all retail sources.

The antibiotic susceptibility results of C1. per- fringens serotypes isolated from carcases and minced beef are shown in Table 2 and are essen- tially similar. By comparing in vitro determined MICs with the concentrations of antibiotic achieved in the bloodstream with different dosage schedules, Sapico et d. (1972) grouped 43 CI. perfringens strains isolated from clinical material into one of three groups-susceptible, intermediate or resistant. Organisms having MICs < the blood concentrations achieved with ordinary dosage schedules were classified as sus- ceptible, those with MlCs within the range of blood concentrations achieved with high dosage schedules and without major risk of toxicity as intermediate, and above these levels as resistant. To group our test organisms we have adopted this system using the concentrations of anti- biotic closest to those cited by Sapico et al. (1 972). Those serotypes considered on this basis to be resistant to particular antibiotics are listed in Table 3.

Clindamycin. All the isolates were considered to be susceptible (< 3.1 pg/ml). The distribution of MICs was bimodal in that 110 isolates were inhibited between 0.004 pg/ml and 0.064 pg/ml whereas the remaining 1 19 isolates required between 0.125 pg/ml and 1.0 pg/ml for inhi- bition. A similar bimodal distribution of MICs was reported by Mathias et al. (1974) for 30 clinical C1. perfringens isolates though the con- centration ranges required for inhibition were higher.

Table 2. Antibiotic susceptibility of CI. perfringens serotypes isolated from beef, pork and lamb carcases and from minced beef

MIC Clindamycin Lincomycin Erythromycin Tetracycline Penicillin Chloramphenicol &nl C M C M C M C M C M C M ~-

0,004 0.008 0.016 0.032 0,064 0,125 0.25 0.5 1 2 4 8

16 32 128

- - - - - - - - 3 - - - 6 - - - - - - 1 8 -

4 18 - 19 52 - - 14 26 - 16 5 - 2 -

1 - 58 28 - 6 3 1 6 6 - -

5 2 22 17 - 1 47 1 28 18 - 23 16 12 26 - 1 4 3 2 1 6 9 - 33 10 2 21 2 - 5 5 3 1 9 - 10 20 29 16 , 4 28 - 9 - 1 1 - - 31 23 69 42 1 2 - - 43 - _ - 5 33 43 5 1 9 - - 62

- - - 1 - 1 7 - - 6 - - - 1 3 2 8 3 - -

- - - -

- _ -

- ~

-

1 3 2 3

42 35 30

1

Number of strains isolated from carcases (C) or minced beef (M) susceptible to given levels of antibiotic

138 J. L. Smart et al. Table 3. Antibiotic-resistant serotypes of C1. perfingens

Antibiotic Serotype Source MIC ( f@-4

Lincomycin 7/36 minced beef 16 Erythromycin 314 16

314 1 same beef carcase* 16 16

minced beef 16 314

20162 29 minced beef 16

Tetracycline 3/4 16 314 1 same beef carcase* 16

32 16

314 pig carcase

16 314t 3/4t pig carcase

pig carcasc 16 16

314 minced beef

16 7 minced beef 11/24 minced beef 128

lamb carcase 16 pig carcase 16 30 lamb carcase 16 minced beef 16 45

65/69/72 minced beef 32

314

251

321:

* The same isolates from the same beef carcase. 5 From carcases sampled consecutively. 1 From the same lamb carcase.

Lincomycin. Serotype 7/36, isolated from minced beef, was considered resistant (MIC 2 16 pgiml); the other serotypes were sus- ceptible having MICs < 4 pgiml. The distribu- tion of MICs of all of the isolates tested was bimodal, this was particularly obvious with the carcase isolates.

Erythromycin. Susceptible isolates (< 2 pgiml) totalled 147, intermediates 77 (> 4 pg/ml-< 8 pg/ml) and resistant 5 (> 16 pgiml). The resist- ant serotypes and sources are shown in Table 3.

Tetracycline. The isolates considered suscep- tible totalled 183 (< 2 pgiml), intermediate 32 (2 4 pgim1-G 8 pgiml) and resistant 14 (2 16 pg/rni). The resistant serotypes (Table 3), each isolated from different minced beef samples, were 314, 7, 45, 65/69/72 (MICs 16 pg/ml) and 11/24 (MIC 128 pgiml). The carcase isolates considered resistant totalled nine; six of serotype 3/4 and serotypes 25, 30 and 32. Three of the resistant serotype 3/4 isolates were also resistant to erythromycin. The others were from different pig carcases, two of which were con- secutively sampled. Serotypes 25 and 32 were from the same lamb carcase.

Benzyl penicillin. All of the test isolates were considered to be susceptible (MICs 6 0.5

pg/ml), except one (serotype 281, which was clas- sified as intermediate (MIC = 1.0 pg/ml).

Chlorumphenicol. All of the test isolates were considered to be susceptible (MIC < 16 pg/ml).

Discussion

The greater number of Cl. perfringens serotypes isolated from retail minced beef samples than from carcases sampled at a commercial abattoir immediately after slaughter (Smart et ul. 1979), probably reflects the additional diverse micro- bial contamination that can, and usually does, occur during distribution and preparation of meat for retail sale. The value of serotyping in ecological studies is self-evident.

Serotype 3/4 was isolated from marginally more minced beef samples (Table 1) than any other serotype. It was also the most common serotype isolated from beef, pork and lamb car- cases (Smart et al. 1979). The frequency of oc- currence of this serotype therefore might, at least in part, account for the fact that it was incriminated in more food poisoning outbreaks associated with beef, including minced beef, than any other in the UK between 1971 and 1977 (Stringer et al. 1979). However, when CI. per- fvingens serotypes isolated from several sources,

Antibiotic susceptibility of C1. perfringens 139

including several strains of serotype 3/4, were tested in uitro for enterotoxin production, almost all of those isolated from sources other than outbreaks of food poisoning, including car- cases and minced beef were negative (1 72/174) whilst those from outbreaks were mostly posi- tive (56/65; Skjelkvale e t a/. 1979). Apparent loss of enterotoxigenicity was observed in strains isolated from outbreaks of food poisoning (Skjelkvale et a/. 1979). The ease with which Cl. perfringens can be isolated from meat empha- sizes the continuing need for the education of catering staff in the use of procedures that ensure control of its growth and thereby mini- mize the risk of food poisoning.

The antibiotic resistance of Cl. perfringens serotypes has not been studied previously, al- though there have been several studies of mainly clinical isolates which had not been subjected to serological typing. With the exception of serotype 3/4 (Table 3) no serotype or group of serotypes displayed abnormal resistance pat- terns. Three isolates of serotype 3/4 from the same beef carcase were resistant to both eryth- romycin and tetracycline. Rood et al. (1978) in the USA have reported the isolation of C / . per- fringens strains from porcine faeces that were resistant not only to erythromycin and tetra- cycline but also to clindamycin and lincomycin. Such multiple antibiotic resistance was not ob- served in this study.

The antibiotic susceptibilities of the serotyped C1. perfringens isolates tested in this study were generally similar to, or more susceptible than, those of the mainly clinical isolates studied pre- viously (Martin et al. 1972; Mathias et al. 1974; Dornbusch e t al. 1975; Sutter & Finegold 1976; Rood et a/. 1978; Dutta & Devriese 1980). Penicillin-, chloramphenicol- or clindamycin- resistant isolates were not found in this study and although penicillin is the generally accepted antibiotic used in the treatment of clostridial in- fections, i f contraindicated (e.g. in an allergic patient), other effective antibiotics are obviously available. The evidence presented here suggests that meat is not a common source of resistant or multiple resistant C/. perfringens strains.

DUTTA, G.N. & DEVRIESE, L.A. 1980 Susceptibility of Clostridiurn perfringens of animal origin to fifteen antimicrobial agents. Journal of Veterinary Pharma- cology and Therapeutics 3, 227-236.

HEPNER, E. 1980 Food poisoning and salmonella in- fections in England and Wales, 1976-1978. Public Health. London 94.337-349.

LINTON, A.H. 1977 Antibiotic resistance: the present situation reviewed. Veterinary Record 100, 354-360.

MARTIN, W.J., GARDNER, M. & WASHINGTON 11, J.A. 1972 in uitro antimicrobial susceptibility of anaero- bic bacteria isolated from clinical specimens. Anti- microbial Agents and Chemotherapy 1, 148-158.

1974 Susceptibility of clinical isolates of Clostridium perfringens to 13 antibiotics. Canadian Journal of Public Health 65, 64.

ROBERTS, T.A., BRITTON, C.R. & HUDSON, W.R. 1980 The bacteriological quality of minced beef in thc UK. Journal ofHiygiene, Ctrmbridge 85,211-217.

ROOD, J.I., MAHER, E.A., SOMERS, E.B., CAMPOS, E. & DUNCAN, C.L. 1978 Isolation and characterization of multiple antibiotic-resistant Clostridium per- fringens strains from porcine faeces. Anfirnicwhinl Agents and Chemotherapy 13,871-880.

SAPICO, F.L., KWOK, Y., SUTTER, V.L. & FINEGOLD, S.M. 1972 Standardized antimicrobial disc suscepti- bility testing of anaerobic bacteria: in uitro suscepti- bility of Clostridium perfringens to nine antibiotics. Antimicrobial Agents and Chemotherapy 2 , 320--325.

SCHWARTZMAN, J.D., RELLER, L.B. & WANG, W.L. 1977 Susceptibility of Clostridium perfringens Iso- lated from human infections to twenty antibiotics. Antimicrobial Agents and Chemuthrrapy I I , 695- 697.

SKJELKVALE, R., STRINGEK, M.F. & SMART, J.L. 1979 Enterotoxin production by lecithinase-positive and lecithinase-negative Clostridium perjringens isolated from food poisoning outbreaks and other sources. Journal of Applied Bacteriology 47, 329-339.

SMART, J.L., ROBERTS, T.A., STRINGER, M.F. & SHAH, N. 1979 The incidence and serotypes of Clostridium perfringens on beef, pork and lamb carcases. Jour- nal of Applied Bacteriology 46, 377-383.

STANECK, J.L. & WASHINGTON 11, J.A. 1974 Anti- microbial susceptibilities of anaerobic bacteria: recent clinical isolates. Antimicrobial Agents and Chemotherapy 6, 311-315.

STRINGER, M.F. SHAH, N. & GILBERT, R.J. 1979 Sero- logical typing of Clostridium perfringens and its epi- demiological significance in the investigation of food poisoning outbreaks. Proceedings of the Xth international Symposium on Food Microbiology and Hygiene, September 1977, Szczecin, Poland, 2, 189- 196. Academy of Agriculture, Szczecin and Ars Polona, Warsaw.

SIJTTER, V.L. & FINEGOLD, S.M. 1976 Susceptibility of anaerobic bacteria to 23 antimicrobial agents. Anti- microbial Agents and Chemotherapy 10,736752,

MATHIAS, R., GUKWITH, M., RONALD, A. & HOBAN. S.

References

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