BIOCHEMICAL CHARACTERIZATION AND ANTIMICROBIAL SUSCEPTIBILITY OF Bacillus cereus ISOLATES FROM SOME RETAILED FOODS IN OGUN STATE, NIGERIA

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December – January, 2019, vol. 9, no. 3
pages: 616-621
Article type: Microbiology of Microbiology
DOI: 10.15414/jmbfs.2019/20.9.3.616-621
Abstract: Food borne disease caused by Bacillus cereus has been a major health issue because of its ability to cause two syndromes: diarrhoeal and emetic which sometimes lead to death. Six hundred (600) samples of some retailed foods: cooked rice, jollof rice, fried rice, meat pie, smoked fish: African chad, Titus, blue whiting, fried meat, smoked hide, carrot, runner beans, cabbage and raw green pea were collected from the eight main markets in Ogun State. Serial dilutions of the samples were carried out and cultured on Mannitol Egg Yolk Polymyxin Agar (MYP) using the spread plate technique. B. cereus was confirmed with standard biochemical methods. Antibiotic susceptibility was performed by the Kirby – Bauer disc diffusion method with nine antibiotics. The mean microbial load in the retailed food was in the range of 1.00 x 104 – 8.92 x 104cfu/g. All the isolates were gram positive rods, catalase and citrate positive. Most of the isolates were motile (97.7%). Two hundred and twenty one (221) isolates were confirmed as B. cereus with biochemical tests. They were 100% sensitive to gentamicin and 100% resistant to penicillin and ampicillin which are β – lactam antibiotics. All the isolates showed resistance to more than two antibiotics. This study has clearly revealed the presence of B. cereus in some retailed foods sold in Ogun State, Nigeria in which all the isolates were resistant to β-lactam antibiotics. Therefore, extreme caution should be taken when handling foods to avoid contamination by B. cereus and prevent any future food-borne outbreak by B. cereus.
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BIOCHEMICAL CHARACTERIZATION AND ANTIMICROBIAL SUSCEPTIBILITY OF Bacillus cereus ISOLATES FROM SOME RETAILED FOODS IN OGUN STATE, NIGERIA


AUTHORS

Titilayo O. Adesetan, Moses O. Efuntoye, Olubukola O. Babalola

ABSTRACT

Food borne disease caused by Bacillus cereus has been a major health issue because of its ability to cause two syndromes: diarrhoeal and emetic which sometimes lead to death. Six hundred (600) samples of some retailed foods: cooked rice, jollof rice, fried rice, meat pie, smoked fish: African chad, Titus, blue whiting, fried meat, smoked hide, carrot, runner beans, cabbage and raw green pea were collected from the eight main markets in Ogun State. Serial dilutions of the samples were carried out and cultured on Mannitol Egg Yolk Polymyxin Agar (MYP) using the spread plate technique. B. cereus was confirmed with standard biochemical methods. Antibiotic susceptibility was performed by the Kirby – Bauer disc diffusion method with nine antibiotics. The mean microbial load in the retailed food was in the range of 1.00 x 104 – 8.92 x 104cfu/g. All the isolates were gram positive rods, catalase and citrate positive. Most of the isolates were motile (97.7%). Two hundred and twenty one (221) isolates were confirmed as B. cereus with biochemical tests. They were 100% sensitive to gentamicin and 100% resistant to penicillin and ampicillin which are β – lactam antibiotics. All the isolates showed resistance to more than two antibiotics. This study has clearly revealed the presence of B. cereus in some retailed foods sold in Ogun State, Nigeria in which all the isolates were resistant to β-lactam antibiotics. Therefore, extreme caution should be taken when handling foods to avoid contamination by B. cereus and prevent any future food-borne outbreak by B. cereus.


KEYWORDS

Antibiotics, Bacillus cereus, biochemical tests, Mannitol Egg Yolk Polymyxin Agar (MYP), retailed foods

INTRODUCTION

Food is essential for man’s sustenance. Microorganisms attack our food for survival thereby causing deterioration and contamination of the food. Therefore, the food we eat can be beneficial to our body and at the same time cause harm when microorganisms in interaction with the food release their metabolites thereby causing illness. One of such bacteria is Bacillus cereus. Members in the B. cereus group are gram positive, ubiquitous rod shaped spore former. They are known to survive virtually in all environments because of their ability to form spores that are resistant to heat and acid. There are seven members in the group namely: B. anthracis, B. cereus sensu stricto, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis and B. cytotoxicus. The first three are the most important within the group (Lapidus et al. 2008; Logan and de Vos, 2009).

The economic situation of some countries has encouraged the consumption of ready-to-eat foods and establishment of more fast foods restaurant, canteens and road side food outlets. These foods are believed to be affordable and easily accessible. Food-borne pathogens may multiply in foods that are not well handled, prepared or stored due to lack of hygiene and poor sanitation (Angelidis et al., 2006; Desai and Varadaraj, 2009).

B. cereus causes two types of poisonings namely: diarrhoeal with abdominal cramp and diarrhoea as the symptom and emetic with nausea and vomiting. Diarrhoeal poisoning is associated with proteins such as meat while starchy foods such as rice and pasta have been linked to the emetic poisoning. It had been reported to cause illnesses (Ghelardi et al., 2002; Martinelli et al., 2013; Zhou et al., 2014) or death (Dierick et al., 2005; Naranjo et al., 2011).

B. cereus can survive an array of stress situations including those found in some foods because of its ability to form spores. It has been isolated from a wide variety of foods, including milk and dairy products (De Jonghe et al., 2008;Arslan et al., 2014), rice and pasta (Rajkovic et al., 2013), infant foods (Organji et al., 2015), spices (Iurlina et al., 2006), meat product (Gueven et al., 2006), seafood (Das et al., 2009), fresh vegetables (Valero et al., 2002), vegetable salad (Valero et al., 2007) and ready to eat foods (Fang et al., 2003; Samapundo et al., 2011). Bacillus infections can be treated with antibiotics such as vancomycin, clindamycin, ciprofloxacin, and gentamicin. Penicillins and ampicillins are not effective (Lindback and Granum, 2006; Chon et al., 2012) because they produce β-lactamase that hydrolyses the β-lactam ring of the antibiotics.

Review of literatures in Nigeria showed that there is scarcity of information on characterization of B. cereus in retailed foods in Ogun State; therefore, there is need to characterize these food pathogens using both morphological and biochemical properties and determine their susceptibility to common antibiotics to forestall any future occurrence of food borne outbreak.

MATERIALS AND METHODS

Sample Collection

Six hundred (600) samples of ready-to-eat foods and vegetables such as cooked white rice (Oryza sativa), jollof rice, fried rice, meat pie, cooked spaghetti, smoked African chad (Etmalosa fimbriata), smoked Titus (Scumber  scumbrus), smoked blue whiting (Micromesistius poutassou), smoked hide, fried meat (Bos taurus), green pea (Pisum sativum), sweet pepper (Capsicum annuum), runner bean (Phaseolus vulgaris), cabbage (Brassica oleracea) and carrot (Daucus carrota)  (Table 1) were collected randomly inside sterile  sampling bags/plastics from food outlets/canteens in the markets namely: Oke-Aje and Ago-Iwoye markets (Ijebu), Sagamu and Ikenne markets (Remo), Kuto and Omida markets (Egba) and Ayetoro and Imeko markets (Yewa) in the four geographical regions in Ogun State, they were sealed to prevent contamination and transported to the laboratory for analysis. Forty samples were collected from each food type. The samples were collected in the morning and it was ensured that the vegetables were fresh ones without any sign of deterioration. The samples were collected between September 2013 and April 2015. The analyses were carried out at the Laboratories of the Department of Microbiology, Olabisi Onabanjo University, Ago – Iwoye, Nigeria and Microbial Biotechnology Laboratory of North – West University, Mafikeng Campus, South Africa.

Table 1 Number of food samples collected from each division

Food groups Food name Food Code Egba Ijebu Remo Yewa Total
Starch Cooked Rice WR 10 10 10 10 40
Jollof Rice JR 10 10 10 10 40
Fried Rice FR 10 11 11 8 40
Meat pie MP 10 10 10 10 40
Spaghetti SG 10 10 10 10 40

 

Protein Fried Meat MT 10 10 10 10 40
Smoked Titus TT 10 10 10 10 40
Smoked African chad SW 10 10 10 10 40
Smoked Blue whiting PN 10 10 10 10 40
Smoked Hide PM 10 10 10 10 40

 

Vegetable Green pea GP 12 12 12 4 40
Sweet pepper SP 10 10 10 10 40
Cabbage CB 11 12 11 6 40
Carrot CR 10 10 10 10 40
Runner Bean RB 10 10 10 10 40
Total 153 155 154 138 600

Sample preparation

Samples of meat, runner beans, carrot, cabbage, smoked hide and sweet pepper were chopped into pieces with sterile knife after which 10g of each sample was added to 100 ml sterile peptone water (1:10 dilution) and diluted up to 10-5.

Isolation of B. cereus

Mannitol Egg Yolk Polymyxin (MYP) agar (Oxoid, UK) plates were inoculated with 0.1ml of appropriate dilution and spread evenly onto surface of each plate with sterile bent glass rod. Plates were incubated at 30C for 18hrs and observed for colonies surrounded by precipitate zone, which indicated that lecithinase was produced (Tallent et al., 2001).

Microbial load

After incubating for 24hrs, the plates with distinct colonies were counted and then multiplied with the dilution factor to get the total bacteria count in colony forming unit per gram (cfu/g).

Confirmation of B. cereus

The colonial morphology of the isolates was first recorded after which the biochemical tests based on Food and Drug Administration (FDA) methods as described by Tallent et al. (2001) and Cheesebrough (2000) were carried out. Briefly, five (5) or more eosin pink, lecithinase-positive colonies from MYP agar (Oxoid, UK) plates were transferred to nutrient agar plates (MAST, Merseyside UK) and incubated for 24 h at 30C. The isolates were gram stained and confirmed using the following tests: glucose fermentation, nitrate reduction, motility test, catalase test, citrate utilization, starch hydrolysis, casein hydrolysis, rhizoid growth, haemolysis and growth at 420C.

Antimicrobial susceptibility test

This test was performed using Kirby-Bauer Disc Diffusion method (Bauer et al. 1966). Nine (9) different antibiotics (Bio-Rad, USA) were employed: penicillin (PEN) 10IU, ciprofloxacin (CIP) 5 µg, tetracycline (TET) 30µg, erythromycin (ERY) 15 µg, gentamicin (GMN) 10 µg, amoxicillin-clavulanic acid (AMC) 30 µg, vancomycin (VAN) 30 µg, ampicillin (AMP) 10 µg and clindamycin (CMN) 2 µg (Oxoid, UK).  The results were interpreted using Clinical Laboratory Standard Institute CLSI (2013) guideline for Gram positive and /or aerobic bacteria.

Statistical Analysis

Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 20.0 (IBM Corp, 2011). Mean values were compared using Analysis of Variance (ANOVA). Results were presented as Mean±Standard deviation. Post hoc test was done using the Student-Newman-Keuls (SNK). p < 0.05 was considered to be statistically significant.

RESULTS

Microbial load of retailed foods

The microbial load in the food samples is presented on Table 2. Food with the highest microbial load was spaghetti from Yewa with mean bacterial load of 8.92 x 104cfu/g, followed by White rice from Yewa with 4.18 x 104cfu/g while the food with the least count was green pea from Ijebu –Ode with mean bacterial load of 1.00 x 104cfu/g. The One Way Analysis of Variance (ANOVA) revealed a significant difference (p ≤ 0.05 in the microbial load of the food samples SP and SW collected from all the locations. There was also a significant difference (p ≤ 0.05) between the locations where the samples were collected.

Table 2 Mean Microbial load in food samples collected from different divisions (x104cfu/g)

Location /

Food code

      Egba       Ijebu        Remo        Yewa
WR 3.42±1.22a 2.82±0.83a 3.92±0.76a 4.18±0.80a
JR 3.70±0.20a 3.00±0.57a 4.90±0.10a 0.00±0.00b
FR 3.10±1.27b 6.85±0.64a 1.20±0.20c 3.10±0.10b
MP 1.90±0.28a 2.03±1.33a 1.60±0.14a 1.60±0.56a
SG 5.28±3.18a 5.68±4.17a 4.08±1.81a 8.92±2.60a
MT 2.78±1.59a 2.00±0.72a 2.70±1.44a 2.62±0.60a
TT 3.48±0.94a 3.64±1.55a 3.00±0.96a 5.23±0.42a
SW 6.03±3.46a 2.86±1.49b 2.38±0.54b 4.70±1.04a
PN 3.65±4.31b 2.90±0.28b 6.50±0.50a 3.10±2.12b
PM 4.00±0.50a 4.37±1.21a 4.35±0.21a 4.70±0.20a
GP 1.35±0.21a 1.00±0.50a 1.38±0.28a 2.00±0.60a
SP 1.57±0.40b 2.00±0.60b 2.18±0.17b 3.90±0.28a
RB 4.04±0.57a 4.10±0.96a 2.88±1.04a 3.96±1.68a
CB 2.77±0.68b 2.53±0.51b 1.70±0.26b 3.30±0.30a
CR 2.00±0.60b 3.70±0.20a 2.65±0.92b 3.40±0.40a

abcMeans (±Standard deviation) in the same row having similar superscripts are not significantly different at p < 0.05. n = 181

WR – White Rice     JR – Jollof Rice    FR – Fried Rice      TT – Smoked Titus    GP – Green Pea      SG – Spaghetti                        RB – Runner Bean      MP – Meat pie           CR–Carrot     MT– Meat    CB – Cabbage       SW – Smoked African chad        SP – Sweet Pepper          PN – Smoked blue whiting     PM – Smoked hide

Colonial morphology of B. cereus

The isolates formed opaque, irregular, smooth/rough, creamish colonies on Nutrient Agar. Some also showed waxy growth while others had short hair-like growth around the colony. Most of the colonial characteristics exhibited were typical of Bacillus cereus.

Gram stain reaction and biochemical characterization

All the isolates were Gram positive rods. Many were in chains while few were single rods. Some have central to sub-terminal spores which do not swell the sporangium. All the isolates were catalase and citrate positive.  95.9% of the isolates were haemolytic, 97.7% were motile, 77.4% and 86.0% hydrolysed starch and casein respectively. They grew well at 420C (Table 3).

Table 3 Biochemical tests pattern for B. cereus isolates from some retailed foods

Pattern/Test Glucose fermentation Motility Rhizoidal growth Haemolysis Nitrate Reduction Catalase Starch hydrolysis Casein hydrolysis Citrate utilization Growth at 420C Egba Ijebu Remo Sagamu
I + + + + + + + + 1 0 0 1
II + + β + + + + + + 32 41 40 31
III + + α + + + + + 1 0 0 0
IV + + β + + + + + 9 6 7 13
V + + α + +    – + + 0 0 1 2
VI + + β + + + + + 7 5 3 7
VII + + + + + + + 1 1 1 1
VIII + + + + + + + 0 1 0 0
IX + + + + + + 0 1 0 0
X + β + + + + + 0 2 0 0
XI + + + + + + 0 1 0 0
XII + + α + + + + + + 0 0 1 0
XIII + + β + + + + 0 0 2 0
XIV + β + + + + + + 0 0 1 0
XV + α + + + + 1 0 0 0
TOTAL 100% 97.7% 100% 95.9% 100% 100% 77.4% 86.0% 100% 100% 52 58 56 55

α alpha haemolysis, β beta haemolysis, + positive  – negative

Occurrence of B. cereus isolates in food samples                                                                                                                                 

The occurrence of B. cereus in the retailed food sample collected from the four geographical divisions in the state is presented on Table 4.The highest occurrence of B. cereus was from food samples from Ijebu while the least was from Egba division.

Table 4 Occurrence of B. cereus in some retailed food sample

Location/

Food code

No of isolates       Egba           Ijebu     Remo          Yewa
WR 33             8            7           8              10
JR 4           1            2           1               –
FR 6           2            2           1               1
SG 34           8            9           7             10
MP 10           2           3           2              3
MT 18           4           6           3              5
SW 27           4           8           8              7
PN 8           2           2           1              3
PM 7           1           3           2              1
TT 16           4           5           4              3
GP 8           2           1           4              1
RB 25           7           5           6              7
CR 5           1           1           2              1
CB 10           3           3           3              1
SP 10           3           1           4              2
221 52 (23.5%) 58 (26.2%) 56 (25.3%)     55 (24.0%)

WR – White Rice     JR – Jollof Rice    FR – Fried Rice      TT – Smoked Titus    GP – Green Pea         SG – Spaghetti                     RB – Runner Bean      MP – Meat pie           CR–Carrot     MT– Meat    CB – Cabbage     SW – Smoked African chad        SP – Sweet Pepper          PN – Smoked blue whiting     PM – Smoked hide

Antimicrobial Susceptibility Test

All the isolates were resistant to penicillin and ampicillin while 99.5% were resistant to amoxicillin-clavulanic acid. They were all susceptible to gentamicin (Table 5).  The resistance pattern of the B. cereus isolates from the retailed food is presented on Table 6. Some of the isolates were resistant to more than one antibiotic. Resistance to penicillin, ampicillin and amoxicillin-clavulanic acid was displayed by 7.7% of the isolates, 22.6% showed resistance to penicillin, amoxicillin-clavulanic acid, erythromycin and ampicillin while 5.4% were resistant to all the antibiotics except gentamicin.

Table 5 Antibiotic Resistance of B. cereus from each location

Class of Antibiotics β-lactam Aminoglycosides β-lactam Lincosamide Macrolides Quinolones Tetracyclines Glycopeptide β-lactam
Antibiotics/

Location

PEN

(10IU)

GMN

(10μg)

AMC

(30μg)

CMN

(2μg)

ERY

(15μg)

CIP

(5μg)

TET

(30μg)

VAN

(30μg)

AMP

(10μg)

EGBA

IJEBU

REMO

YEWA

TOTAL

(%)

52

58

56

55

221

100%

0

0

0

0

0

0%

52

57

56

55

220

99.5%

23

23

25

19

90

40.7%

33

37

45

33

148

67%

19

15

15

17

66

29.9%

17

18

19

13

69

30.3%

25

16

24

21

86

38.9%

52

58

56

55

221

100%

PEN – Penicillin   GMN – Gentamicin    AMC – Amoxicillin-clavulanic acid      CMN – Clindamycin     ERY- Erythromycin    CIP – Ciprofloxacin     TET- Tetracycline    VAN – Vancomycin,    AMP – Ampicillin

I – β-Lactam, II – Aminoglycosides, III – Lincosamide, IV – Macrolides, V – Quinolones, VI – Tetracycline, VII – Glycopeptides

Table 6 Resistance pattern of B. cereus isolates from retailed foods

Resistance pattern Antibiotic class No of isolates Resistance (%)
PEN, AMC, AMP I 17 7.7
PEN, AMC, ERY, AMP I,IV 50 22.6
PEN, AMC, CIP, AMP I,V 7 3.2
PEN, AMC, TET, AMP I,VI 8 3.6
PEN, AMC, VAN, AMP I,VII 4 1.8
PEN, AMC, CMN, AMP I,III 11 5.0
PEN, AMC, ERY, VAN, AMP I,IV,VII 9 4.1
PEN, AMC, CMN, ERY, AMP I,III,IV 14 6.3
PEN, AMC, CMN, VAN, AMP I,III,VII 5 2.3
PEN, AMC, CIP, TET, AMP I,V,VI 5 2.3
PEN, AMC, ERY, CIP, AMP I,IV,V 4 1.8
PEN, AMC, TET, VAN, AMP I,VI,VII 1 0.5
PEN, AMC, ERY, TET, AMP I,IV,VI 3 1.4
PEN, AMC, CMN, ERY, AMP I,III,IV 1 0.5
PEN, CMN, TET, VAN, AMP I,III,VI,VII 1 0.5
PEN, AMC, CMN, TET, AMP I,III,VI 1 0.5
PEN, AMC, CMN, CIP, AMP I,III,V 2 0.9
PEN, AMC, CMN, ERY, TET, AMP I,III,IV,VI 1 0.5
PEN, AMC, CMN, CIP, TET, AMP I,III,V,VI 1 0.5
PEN, AMC, ERY, CIP, VAN, AMP I,IV,V,VII 10 4.5
PEN, AMC, CMN, ERY, CIP, AMP I,III,IV,V 1 0.5
PEN, AMC, CMN, TET, VAN, AMP I,III,VI,VII 2 0.9
PEN, AMC, ERY, CIP, TET, AMP I,IV,V,VI 3 1.4
PEN, AMC, ERY, TET, VAN, AMP I,IV,VI,VII 3 1.4
PEN, AMC, CMN, ERY, VAN, AMP I,III,IV,VII 12 5.4
PEN, AMC, CMN, CIP, VAN, AMP I,III,V,VII 3 1.4
PEN, AMC, CMN, ERY, TET, AMP I,III,IV,VI 3 1.4
PEN, AMC, CIP, TET, VAN, AMP I,V,VI,VII 2 0.9
PEN, AMC, ERY, CIP, TET, VAN, AMP I,IV,V,VI,VII 5 2.3
PEN, AMC, CMN, ERY, TET, VAN, AMP I,III,IV,VI,VII 9 4.1
PEN, AMC, CMN, ERY, CIP, TET, AMP I,III,IV,V,VI 3 1.4
PEN, AMC, CMN, CIP, TET, VAN, AMP I,III,V,VI,VII 3 1.4
PEN, AMC, CMN, ERY, CIP, VAN, AMP I,III,IV,V,VII 5 2.3
PEN, AMC, CMN, ERY, CIP, TET, VAN, AMP I,III,IV,V,VI,VII 12 5.4

                                                                 

PEN – Penicillin   GMN – Gentamicin    AMC – Amoxicillin-clavulanic acid      CMN – Clindamycin   ERY- Erythromycin    CIP – Ciprofloxacin     TET- Tetracycline    VAN – Vancomycin,    AMP – Ampicillin

I – β-Lactam, II – Aminoglycosides, III – Lincosamide, IV – Macrolides, V – Quinolones, VI – Tetracycline, VII – Glycopeptides

Table 7 showed the percentage resistance for each food sample. All the isolates from each food sample showed 100% resistance to penicillin, ampicillin and amoxicillin-clavulanic acid except for isolates from PM which showed 85.7% resistance to amoxicillin-clavulanic acid. Isolates from CB and JR showed 100% resistance to erythromycin. PM isolates showed the highest resistance of 71.4% to tetracycline.

Table 7 Percentage (%) resistance of B. cereus isolates in each food sample

Food code No of

Isolates

PEN

(%)

AMC

(%)

CMN

(%)

ERY

(%)

CIP

(%)

TET

(%)

VAN

(%)

AMP

(%)

WR

SG

SW

MT

RB

TT

CB

SP

CR

FR

GP

JR

PM

PN

MP

33

34

27

18

25

16

10

10

5

6

8

4

7

8

10

 100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

  100

100

100

100

100

100

100

100

100

100

100

100

85.7

100

100

  33.3

41.2

29.6

61.1

32.0

50.0

30.0

60.0

40.0

33.3

37.5

50.0

28.6

25.0

40.0

 81.8

76.4

74.1

22.2

32.0

68.8

100.0

70.0

80.0

33.3

62.5

100.0

85.7

62.5

80.0

 48.5

29.4

33.3

33.3

28.0

12.5

10.0

60.0

20.0

0.0

12.5

0.0

14.3

37.5

30.0

  24.2

35.3

29.6

22.2

40.0

18.8

10.0

40.0

40.0

0.0

37.5

0.0

71.4

25.0

50.0

 57.6

47.1

29.6

38.9

48.0

37.5

10.0

30.0

20.0

33.3

37.5

25.0

42.9

25.0

40.0

 100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

PEN – Penicillin   GMN – Gentamicin    AMC – Amoxicillin-clavulanic acid      CMN – Clindamycin    ERY- Erythromycin    CIP – Ciprofloxacin     TET- Tetracycline    VAN – Vancomycin,    AMP – Ampicillin  WR – White Rice     JR – Jollof Rice    FR – Fried Rice      TT – Smoked Titus    GP – Green Pea     SG – Spaghetti                 RB – Runner Bean      MP – Meat pie           CR–Carrot     MT– Meat    CB – Cabbage       SW – Smoked African chad        SP – Sweet Pepper          PN – Smoked blue whiting     PM – Smoked hide

DISCUSSION

Six hundred (600) different food samples were analysed for the presence of Bacillus cereus.  The microbial load in the retailed food sample was high. Fang et al. (2003) also reported B. cereus load in 18οC ready-to-eat food products in the range of 104 to ≥ 105cfu/g. According to Gilbert et al. (2000) B. cereus count in food greater than 104 – <105 is unsatisfactory while >105 is unacceptable.  There were significant differences in the microbial load of the food samples FR, SW, PN, SP, CB and CR from the different locations at p ≤ 0.05.

Two hundred and twenty one (221) B. cereus isolates were recovered from the retailed foods analyzed. Ninety three point two (93.2%) formed β – haemolysis on sheep blood agar. Six strains were α haemolytic and nine were non-haemolytic. This is in line with the work of Chaves et al., (2011) who reported 81.4% of their B. cereus to be β – haemolytic, six were non haemolytic while seven were α – haemolytic.  Chon et al., (2012) reported that 89% of their strains were haemolytic.  Eighty five point one (86.0%) and seventy seven point four (77.4%) of the strains hydrolysed skim milk, and starch agar.  Chon et al. (2012) reported that 84% and 89% each of their strains hydrolysed lecithinase and starch. They all grew very well at 42C.  This is in line with the findings of Lindbäck and Granum (2006) who reported that B. cereus grows fast at 42C. Detection of B. cereus in retailed foods in this research is in line with the work of Desai and Varadaraj (2009); Al-Abri et al. (2011); Naranjo et al. (2011); Martinelli et al. (2013); Lopez et al. (2015).

The rate of occurrence of B. cereus from the retailed food samples showed that samples from Ijebu division had the highest occurrence while Egba had the least occurrence. Sixteen percent (16%) of B. cereus isolates were recovered from spaghetti. This may be as a result of poor hygiene practised by the food handlers. The food handlers put spaghetti in small transparent plastics buckets instead of hot food containers that can keep them hot. Also, most of them use their bare hands to support the spaghetti when serving it with fork to the consumers. These same hands were used to receive money and handle their food utensils. These can cause cross contamination from the money to the food because currency notes are known to harbour pathogenic microorganisms including Bacillus species (Uneke and Ogbu, 2007; Awe et al., 2010; Orukotan and Yabaya, 2011).

All the B. cereus strains were 100% sensitive to gentamicin. This compared well with the works of Chaves et al. (2011); Chon et al. (2012); Mohammed et al. (2012) and Organji et al. (2015) who reported 100% sensitivity to gentamicin by B. cereus strains recovered from food. Also, 62% of the strains in this study were sensitive to vancomycin, while 38% were resistant to the drug. Ankolekar et al. (2009) reported that 44% of the isolates were resistant to vancomycin, 56% were intermediate strains and none was sensitive to the drug while Organji et al. (2015) reported 100% sensitivity to vancomycin. WR strains showed the highest resistance of 57.6% to the drug.

All the B. cereus strains were 100% resistant to penicillin and ampicillin while 99.5% were resistant to amoxicillin-clavulanic acid. This conforms to the work of Park et al. (2009), Chon et al. (2012) and Savic et al. (2016) who reported that their strains displayed 100% resistance to ampicillin and penicillin. It was reported by Arslan et al. (2014) that B. cereus strains were resistant to ampicillin and penicillin G with the equal rate of 89.7% while 27.6% and 13.8% showed resistance and intermediate resistance respectively to amoxicillin-clavulanic acid. This is in contrast with the result in this work where higher percentage (99.5%) of the strains was resistant to amoxicillin-clavulanic acid. Penicillin, ampicillin and amoxicillin-clavulanic acid are β – lactam antibiotics and resistance to these drugs is as a result of synthesis of β – lactamase by B. cereus.

Sensitivity to ciprofloxacin was 70.1%, 29.0% showed intermediate resistance while 0.9% was completely resistant to the antibiotic. Jawad et al. (2016) reported that 42% of B. cereus strains from fried rice showed resistant to the antibiotic. This result contradicts the work of Luna et al. (2007); Chon et al. (2012); Savic et al. (2016) who reported that B. cereus isolates were 100% sensitive to ciprofloxacin. SP isolates showed 48.5% resistance to the drug.

Resistance to clindamycin was 9.1%; intermediate resistance was 30.7% while 60.2% were sensitive to the drug. This is in contrast to the work of Luna et al. (2007) and Park et al. (2009) who reported that 15% and 72% of B. cereus strains were resistant to clindamycin. Likewise, Arslan et al. (2014) reported 6.9% resistant to the drug while Chaves et al. (2011) recorded ten intermediate strains among B. cereus strains. However, some authors like Ankolekar et al. (2009), Chon et al. (2012), Organji et al. (2015) reported 100% sensitivity to the drug by B. cereus. MT strains showed the highest resistance of 61.1% to clindamycin.

Comparatively, the B. cereus strains in this research were highly resistance to erythromycin. Isolates from CB and JR demonstrated the highest resistance of 100% to erythromycin. Resistance to erythromycin was 38%, Intermediate resistance was 29% while 33% were sensitive to the drug. However, the result of our study is supported by the work of Jawad et al. (2016) who reported that 42% of strains from fried rice were resistant to the drug. Other workers like Park et al. (2009), Chon et al. (2012) and Arslan et al. (2014) had reported 82%, 93.1% and 97% sensitivity to the drug respectively. Although, Ankolekar et al. (2009) reported that 58% of B. cereus strains from rice were resistant to erythromycin. Authors like Organji et al. (2015) and Savic et al. (2016) reported 100% sensitivity to the drug. A higher sensitivity to tetracycline (69%) was obtained in this study compared to 54.8% and 54% sensitivity reported by Mohammed et al. (2012) and Chon et al. (2012) respectively. Ankolekar et al. (2009) reported that 98% of their B. cereus strains were resistant to tetracycline. Park et al. (2009) and Jawad et al. (2016) stated that 85% and 86% of their isolates were sensitive while Savic et al. (2016) reported 100% sensitivity to the drug.  Resistance to the drug was displayed by 71.4% of PM strains while the least resistance was 10.0% by CB isolates. PM is animal hide and the high resistance may be as a result of misuse of antibiotics.

More than 50% of the isolates displayed multiple drug resistances i.e. resistance to three or more antimicrobial classes. The highest resistance was displayed by fourteen strains which were resistant to three classes of antibiotics followed by twelve strains that were resistant to all the antibiotics except gentamicin.

CONCLUSION

This research work has revealed that B. cereus contaminated some retailed foods sold in Ogun State especially the samples from Ijebu division and are multi-resistant to common antibiotics. Therefore extra caution should be taken when handling foods to avoid contamination of the food from the handler and the environment.

Acknowledgement: The authors appreciate the financial support provided by the University management through the Tertiary Education Tax Fund (TETFUND) and the Microbial Biotechnology Laboratory, NWU, South Africa for hosting the first author.

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