Food Protection Trends - May/June 2022 - 204

Isolation of Campylobacter
Isolation of Campylobacter from fecal samples was done
according to NF EN ISO 10272-1: 2017 as described
previously (25, 26), with little modification. In brief,
approximately 1 g of feces was suspended in 9 mL of
thioglycolate broth supplemented with Preston Campylobacter
selective supplement (SR0204E, Oxoid, Hampshire, UK).
The sample was then incubated at 42 ± 1°C for 48 ± 2 h under
microaerophilic conditions provided by CampyGen (Oxoid).
After incubation, a loopful of the enriched culture was streaked
onto modified charcoal cefoperazone deoxycholate agar plate
(Oxoid) supplemented with Preston Campylobacter selective
supplement (SR0204E) and incubated for 48 ± 2 h at 42 ±
1°C under microaerobic conditions (CampyGen). Distinct
colonies suspected to be Campylobacter were selected from
each modified charcoal cefoperazone deoxycholate agar plate
and purified using Mueller-Hinton agar (Oxoid) supplemented
with Preston Campylobacter selective supplement (SR0204E)
that also was incubated at 42 ± 1°C for 48 ± 2 h. Pure cultures
were stored at −80°C in Mueller-Hinton broth containing 30%
glycerol until further analysis.
Phenotypic characterization of Campylobacter isolates
Biochemical tests included Gram reactions as described
previously (7), with minor modification: 0.1% aqueous
basic fuchsin was used for counterstaining instead of safranin
because it has been reported that Campylobacter species are
not easily visualized with the safranin counterstain (36). In
addition, we conducted the oxidase test by using commercial
oxidase disks (Hi-Media, New Delhi, India), and a color
change of the disk to deep purple within 10 s was considered
positive (49). C. coli ATCC 33559 and E. coli ATCC 25922
were used as positive and negative controls, respectively.
Catalase and urease tests also were carried out on the isolates.
A hippurate test, to differentiate C. jejuni from other species
of Campylobacter, was done on the isolates as described
previously (19, 28). A discrete colony of presumptive
Campylobacter isolate was inoculated into 0.2 mL of sterile
distilled water in a test tube. A hippurate disc (Hi-Media) was
placed in the suspension and incubated at 37°C for 2 h with
gentle shaking in water bath (SHZ 82 shaker water bath).
After incubation, 0.2 mL of ninhydrin reagent (Global Pharma
Hamburg, Germany) was gently dropped down the side of
the tube to the inoculum, and the sample was reincubated at
37°C for 10 min without shaking. C. jejuni is hippurate positive
and turns the suspension deep purple, indicating the presence
of glycine, which led to hippurate hydrolysis. C. jejuni ATCC
29428 and C. coli ATCC 33559 were used for the test and
technique validations.
An antimicrobial susceptibility test was done according
to Kirby Bauer disk diffusion assay (4) by using MuellerHinton
agar (Oxoid) supplemented with 10% sheep red
blood cells. Nine different antimicrobials (Hi-Media) were
used for the test: ampicillin 10 µg (AMP 10), ceftazidime
204 Food Protection Trends May/June
30 µg (CAZ 30), ciprofloxacin 5 µg (CIP 5), ceftriaxone
30 µg (CTR 30), cefotaxime 30 µg (CTX 30), cefoxitin 30
µg (CX 30), gentamicin 10 µg (GEN 30), nalidixic acid 10
µg (NAL 10), and tetracycline 10 µg (TET 10). Inoculum
of fresh isolate was prepared in 5 mL of sterile distilled
water. The turbidity of the inoculum was adjusted to 0.5
McFarland standard corresponding to 1.8 × 105
CFU/mL.
The inoculum was flooded on 10% blood Mueller-Hinton
agar and allowed to stand for 3 min. The excess inoculum was
then discarded and the plate was drained for approximately
3-5 min. Antimicrobials were then dispensed on the isolateseeded
blood Mueller-Hinton agar by using a disc dispenser
(Oxoid). The plates were incubated at 42°C for 24 h under
microaerophilic conditions. The diameters of the zones of
inhibition were measured and interpreted as resistance,
intermediate, or sensitive according to Clinical and Laboratory
Standards Institute (6).
RESULTS
Of the 350 samples collected from the two abattoirs
sampled in Ilorin, 70 (20%) were biochemically positive for
Campylobacter. The isolation rate of Campylobacter from
female animals (11.71%; n = 41) was higher than that from
males (8.28 %; n = 29); however, there was no statistically
significant difference (P > 0.05) between the rates. Similarly,
the rate of isolation of Campylobacter from bovine species
(12.86%; n = 45) was not significantly different from that
of ovine species (7.14%; n = 25; P > 0.05) (Table 1). The
biochemical characterization revealed that all of the 70 (20%)
isolates were Gram-negative curved rods, glucose and lactose
non fermenters, and urease negative and oxidase positive,
whereas 8 (4%) produced H2
S in the lead acetate paper. The
hippurate hydrolysis test revealed that 7.14% (n = 5) of the
total isolates are presumptively identified as C. jejuni, whereas
92.86% (n = 65) are other species of Campylobacter (Table
2). Different rates of antimicrobial resistance were observed
among the isolates, with all the isolates displaying resistance
to nalidixic acid, whereas they were pan-susceptible to
gentamicin (Table 3). The isolates similarly displayed a higher
rate of resistance to the β-lactam antibiotics: cefotaxime (83%)
and ampicillin (76%). Thirteen percent of isolates (9; 3 C.
jejuni and 6 Campylobacter species) displayed resistance to two
antibiotics, whereas 26% (26; 4 C. jejuni and 22 Campylobacter
species) showed resistance to three antimicrobials. Fifty-three
percent (37; 9 C. jejuni and 28 Campylobacter species) of all the
isolates displayed multidrug resistance phenotypes (Table 4).
DISCUSSION
Campylobacter infection is one of the major causes of
human gastroenteritis, and foods of animal origin have been
incriminated as the major source of human infection (9, 25,
41). Campylobacter species also are associated with abortion
and reproductive failure in different species of animals,
including cattle and sheep (38, 39). Campylobacteriosis
Food Protection Trends - May/June 2022

Table of Contents for the Digital Edition of Food Protection Trends - May/June 2022
