Antimicrobial susceptibility of Campylobacter jejuni and Campylobacter fetus subsp. fetus to eight cephalosporins with special reference to species differentiation.

Abstract

Agar dilution antimicrobial susceptibility testing showed that Campylobacter jejuni was significantly more resistant than Campylobacter fetus subsp. fetus (intestinalis) to cephalosporin C, cephaloridine, cephalothin, cefazolin, and cefamandole. No species differences in susceptibility were noted with cephalexin, cefotaxime, and cefoxitin. Rapid species differentiation on the basis of an antibiogram could be achieved with the disk diffusion method. C jejuni failed to produce a zone of inhibition around a 30-microgram cephalothin disk but produced a significant zone around a 30-microgram nalidixic acid disk. C. fetus subsp. fetus (intestinalis) produced exactly the reverse pattern.

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Vol. 18, No. 6
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1980, p. 948-951
0066-4804/80/12-0948/04$02.00/0
Antimicrobial Susceptibility of Campylobacterjejuni and
Campylobacter fetus subsp. fetus to Eight Cephalosporins with
Special Reference to Species Differentiation
M. A. KARMALI, S. DE GRANDIS, AND P. C. FLEMING*
Department ofBacteriology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
Agar dilution antimicrobial susceptibility testing showed that Campylobacter
jejuni was significantly more resistant than Campylobacter fetus subsp. fetus
(intestinalis) to cephalosporin C, cephaloridine, cephalothin, cefazolin, and cefa-
mandole. No species differences in susceptibility were noted with cephalexin,
cefotaxime, and cefoxitin. Rapid species differentiation on the basis ofan antibio-
gram could be achieved with the disk diffusion method. C.jejuni failed to produce
a zone of inhibition around a30-,ug cephalothin disk but produced a significant
zone around a 30-,ug nalidixic acid disk. C. fetus subsp. fetus (intestinalis)
produced exactly the reverse pattern.
The genus Campylobacter contains a number
of species and subspecies, only two of which are
known to be associated with human disease (3).
Campylobacterjejuni (7), corresponding to the
C. jejuni-Campylobacter coli group described
by Veron and Chatelain (14) and to Campylo-
bacter fetus subsp. jejuni described by Smibert
(11), has become established as amajor bacterial
cause of diarrhea (3, 8, 10). C. fetus subsp. fetus
(Veron and Chatelain) is an opportunistic path-
ogen that sometimes causes septicemic illness in
compromised hosts (3) and recently has occa-
sionally been isolated from stools of patients
with diarrhea (2, 6). It should be noted that the
term C. fetus subsp. fetus (Veron and Chatelain)
as used in this report is not synonymous with
the term C. fetus subsp. fetus as used by Smibert
(11), but corresponds instead to the term C. fetus
subsp. intestinalis as used by the latter author.
To avoid confusion, C. fetus subsp. fetus (Veron
and Chatelain) will be referred to in this com-
munication as C. fetus subsp. fetus (intestinalis).
The differentiation of C. jejuni from C. fetus
subsp. fetus (intestinalis) in clinical laboratories
remains unsatisfactory. The principle criteria for
differentiating the two species (11, 14) are the
ability of strains to grow at different tempera-
tures and susceptibility of strains to nalidixic
acid. C. fetus subsp. fetus (intestinalis) grows at
25 and 370C but not at 420C and is resistant to
40 ,ug of nalidixic acid per ml, whereas C. jejuni
fails to grow at 250C, grows at 370C, grows better
at 420C, and is inhibited by 40 ,ug of nalidixic
acid per ml. Recently, Smibert (12) reported
that some strains of C. fetus subsp. fetus (intes-
tinalis) are also able to grow at 420C, and Van-
hoof et al. (13) found that about 5% of C. jejuni
strains are resistant to nalidixic acid. There is,
therefore, clearly a need for extending the cri-
teria for differentiating the two species so that
their role in disease can be more accurately
defined.
Our preliminary investigations (M. A. Kar-
mali, A. K. Alien, and P. C. Fleming, Can. J.
Public Health 71:204, 1980) showed that cata-
lase-positive campylobacters could be differen-
tiated morphologically on the basis of size. We
also noted (Karmali et al., Can. J. Public Health
71:204, 1980) that species differences in antimi-
crobial susceptibility occurredwith certain ceph-
alosporins. Smibert (personal communication)
also noted species differences in susceptibility to
cephalothin.
The purpose of this study was to compare the
susceptibility patterns of C. jejuni and C. fetus
subsp. fetus (intestinalis) to seven cephalospo-
rins and cefoxitin to determine which agents
showed species differences in susceptibility pat-
terns and to evaluate a disk diffusion method for
rapidly differentiating the two species in average
clinical laboratories on the basis of an antibio-
gram.
MATERIALS AND MEMODS
Bacteria. The bacteria used in this study included
60 strains of C. jejuni and 12 strains of C. fetus subsp.
fetus (intestinalis). The 12 strains of C. fetus subsp.
fetus (intestinalis) were eight human blood culture
isolates obtained from N. Chalvardjian, S. McDonald,
M. Tischler, J. L. Whitby, W. J. Martin, and J. Righter,
one isolate obtained from A. J. Winter, and three
reference strains obtained from the American Type
Culture Collection (ATCC) and designated ATCC
15296, ATCC 25936, and ATCC 27374. The 60 strains
of C. jejuni were 59 human fecal isolates and 1 refer-
ence strain (ATCC 29428). The identification to spe-
cies of the strains was based on methods outlined by
948

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CAMPYLOBACTER ANTIMICROBIAL SUSCEPTIBILITY
949
Veron and Chatelain (14) and Smibert (11). All strains
were small, strictly microaerophilic, gram-negative
bacteria which had a curved, S-shaped, or spiral mor-
phology and a rapid, darting, corkscrew-like motility.
All strains were catalase and oxidase positive. Strains
which grew at 37 and 42°C but not at 25°C and were
inhibited by40,ugof nalidixic acid per ml were consid-
ered to be C.jejuni. Strains which grew at 25 and 37°C
but not at 42°C, grew in the presence of 1% glycine,
and were resistant to 40
were considered to be C. fetus subsp. fetus (intestin-
alis).
Agar
dilution
antimicrobial
tests. Before testing, each strain was subcultured for
two to three passages onto blood agar (BA) plates,
which were then incubated for 48 h in a carbon dioxide
incubator (CO2 tension, 7%) set at 36°C. The BA used
consisted of Columbia blood agar base (GIBCO Diag-
nostics) with 7% defibrinated horse blood. Antimicro-
bial susceptibility tests were carried out by the agar
dilution method. Suspensions of 48-h BA cultures of
each strain were made in Penassay broth (Difco anti-
biotic medium no. 3) to a density approximating a
McFarland no. 2 turbidity standard and introduced
into the wells of a Steers replicator apparatus. A 5-pl
volume of each suspension, corresponding to a final
inoculum size of approximately 106 viable organisms,
was then inoculated onto the antibiotic media by
means of the replicator head. The medium used for
antibiotic susceptibility testing was diagnostic sensitiv-
ity test agar (Oxoid) containing 5% lysed horse blood.
The inoculated plates were incubated at 36°C in the
carbon dioxide incubator and read after 48 h. The
endpoint was taken as complete inhibition of growth.
It should be noted that the majority ofcampylobacter
strains adapted well to growth in the carbon dioxide
incubator and grew satisfactorily on non-antibiotic-
containing control plates. A few strains of C. jejuni
which did not adapt well to the carbon dioxide incu-
bator were rejected from the study. The antibiotics
tested were cephalosporin C (reference no. CT 738,
Glaxo Laboratories), cephaloridine (Ceporan, Glaxo
Laboratories), cephalothin sodium B.P. (Ceporacin,
Glaxo Laboratories), cephalexin (Glaxo Laboratories),
cefamandole nafate (Mandol, Eli Lilly& Co.), cefazolin
sodium (Kefzol, Eli Lilly & Co.), cefotaxime (HR 756,
pg of nalidixic acid per ml
susceptibility
Roussel Laboratories), and cefoxitin sodium (Mefoxin,
Merck Sharp & Dohme).
Differential disk diffusion test. All strains were
subcultured onto BA plates, which were then incu-
bated for 48 h at 37°C under reduced oxygen tension.
The reduced oxygen tension was obtained by evacu-
ating two-thirds ofthe air from an anaerobic jar (with-
out catalyst) and replacing the evacuated air with a
carbon dioxide-hydrogen mixture. Suspensions ofeach
culture were made in Penassay broth (Difco antibiotic
medium no. 3) to a density approximating a Mc-
Farland no. 2 turbidity standard and inoculated onto
a BA plate with a swab by the method of Bauer et al.
1). A 30-pug nalidixic acid disk and a 30-ug cephalothin
disk (Sensi-disc Sensitivity Discs, Becton, Dickinson
& Co., Ltd., Mississauga, Canada) were placed on the
surface of the inoculated BA plates, which were then
incubated at 37°C under reduced oxygen tension and
examined after 48 h.
RESULTS
inhibitory The
minimum
concentrations
(MICs) of seven cephalosporins and cefoxitin for
the organisms tested are shown in Table 1. C.
jejuni was significantly more resistant to ceph-
aloridine, cephalothin, and cefazolin than was C.
fetus subsp. fetus (intestinalis). A significant but
less marked species difference in susceptibility
occurred with cephalosporin C and cefamandole,
but no such difference occurred with cephalexin,
cefotaxime, or cefoxitin.
Of the 60 strains of C. jejuni, 59 produced no
zone of inhibition around the30-,ug cephalothin
disk (Fig. 1). The reference strain of C. jejuni
(ATCC 29428) produced a very narrow zone of
inhibition that was barely visible. In contrast, all
strains of C. fetus subsp. fetus (intestinalis) pro-
duced significant zones of inhibition (Fig. 2)
around the cephalothin disks, with zone diame-
ters ranging from 16 to 25 mm (mean, 21 mm).
None of the C. fetus subsp. fetus (intestinalis)
strains produced any zone around a 30-,ug nali-
dixic acid disk (Fig. 2), whereas all strains of C.
TABLE 1. MICs ofeight cephalosporins for C. fetus subsp. fetus and C. jejunia
C. fetus subsp. fetus
Antibiotic
MIC range (pug/ml)
Cephalosporin C
'0.5-2
Cephaloridine
'0.5-1
Cephalothin
16-32
Cefazolin
8-32
Cefamandole
16-64
Cephalexin
32-128
Cefotaxime
4-16
Cefoxitin
32-64
C. jejuni
MIC90 (pg/mi)
1
'0.5
32
32
64
128
16
64
MIC range (ug/ml)
4-32
8-128
128-.512
128-a512
128-'512
16-'-512
2-32
32-'512
MIC90 (pg/ml)
16
64
_512
'512
-512
'512
8
256
aThere were 12 strains of C. fetus subsp. fetus (intestinalis) and 60 strains of C. jejuni. Antibiotic
susceptibility tests were carried out by the agar dilution method with diagnostic sensitivity test agar (Oxoid)
containing 5% lysed horse blood. Inoculum size, =106 organisms. MlC9o, Minimum concentration (,ug/ml)
required to inhibit 90% of the strains.
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950
KARMALI, DEGRANDIS, AND FLEMING
FIG. 1. Forty-eight-hour BA culture of C. jejuni
showing a significant zone ofinhibition around a 30-
pg nalidixic acid disk but no zone around a 30-pg
cephalothin disk.
jejuni produced significant zones (Fig. 1) ranging
from 21 to 38 mm (mean, 31 mm) in diameter.
DISCUSSION
Our findings show that C. jejuni and C. fetus
subsp. fetus (intestinalis) can be reliably differ-
entiated from each other on the basis of their
susceptibilities to cephalosporin C, cephalori-
dine, cephalothin, cefazolin, and cefamandole.
These findings are in keeping with Smibert's
(personal communication) observations but con-
tradict the findings ofChow et al. (5) and Butzler
(3). The disk diffusion method is satisfactory for
testing single isolates in average diagnostic lab-
oratories. Both the30-[Lgcephalothin disk and
the 30-,ugnalidixic acid disk are eminently suit-
able for this purpose because the results are
based on a zone-no-zone phenomenon, so that
actual zone sizes need not be measured.
From the therapeutic viewpoint, none of the
compounds tested showed very good activity
against the two species except for cephaloridine,
which on theoretical grounds at least could be
used to treat infections caused by C. fetus subsp.
fetus (intestinalis). However, good alternative
antibiotics such as erythromycin and gentamicin
are already known to be highly active and effec-
tive in treating campylobacter infections (4, 9).
Butzler's selective medium (4) for isolating
FIG. 2. Forty-eight-hour BA culture of C. fetus subsp. fetus (intestinalis) showing a significant zone of
inhibition around a 30-pg cephalothin disk but no zone around a 30-pg nalidixic acid disk.
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CAMPYLOBACTER ANTIMICROBIAL SUSCEPTIBILITY
951
campylobacters from stools contains 15jigof
cefazolin per ml. Although all our strains of C.
jejuni had MICs greatly in excess ofthis concen-
tration, it should be noted that the MICs of
cefazolin for our strains of C. fetus subsp. fetus
(intestinalis) ranged from only 8 to 32 ,ug/ml,
indicating that Butzler's medium may not be
reliable for the isolation of the latter species.
In conclusion, susceptibility to certain cepha-
losporins provides a valuable additional charac-
teristic for differentiating C.jejuni from C. fetus
subsp. fetus
(intestinalis). This observation
should be of particular value in classifying
strains that react aberrantly in established dif-
ferential tests. The reasons for the species dif-
ferences in susceptibility to certain cephalospo-
rins are not known and remain to be investi-
gated.
ACKNOWLEDGMENT
This work was supported by the Medical Research Council
of Canada (grant no. MA 7063).
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