Advance Publication by J-STAGE

Japanese Journal of Infectious Diseases

High rates of contamination of poultry meat products with

drug resistant Campylobacter in Metro Manila, Philippines

Patrick Wilson N. Lim, Daphne C. Tiam-Lee, Phyllis Anne P. Paclibare,

Ma. Socorro Edden P. Subejano, Juvy Ann S. Cabero-Palma, and Gil M. Penuliar

Received: July 7, 2016. Accepted: September 23, 2016

Published online: October 31, 2016
DOI: 10.7883/[Link].2016.309

Advance Publication articles have been accepted by JJID but have not been copyedited
or formatted for publication.
 High rates of contamination of poultry meat products with

drug resistant Campylobacter in Metro Manila, Philippines

Patrick Wilson N. Lim1, Daphne C. Tiam-Lee1, Phyllis Anne P. Paclibare2,

Ma. Socorro Edden P. Subejano2,1, Juvy Ann S. Cabero-Palma,1 Gil M. Penuliar1,2*

t
ip
cr
1
Institute of Biology, College of Science

us
University of the Philippines, Diliman, Quezon City 1101 Philippines;

2
Natural Sciences Research Institute
an
University of the Philippines, Diliman, Quezon City 1101 Philippines
M
ed

*Correspondence: Gil M. Penuliar, Institute of Biology, College of Science, University of

pt

the Philippines, Diliman, Quezon City 1101, Philippines

ce

Telephone: +632-981-8500 local 3732, Email Address: [Link]@[Link]

Ac

Keywords: Campylobacter, chicken, prevalence, drug resistance

Running title: Drug resistant Campylobacter in Metro Manila

1
SUMMARY

A total of 265 chicken parts were collected from 15 wet markets and 15 supermarkets in

Metro Manila, Philippines. Campylobacter spp. was isolated on modified charcoal

cefoperazone deoxycholate agar plates and identified through biochemical tests and PCR

amplification of genus and species specific genes. Antimicrobial resistance profiles were

t
determined following Clinical and Laboratory Standards Institute protocols. Two hundred

ip
seven (78%) Campylobacter spp. were isolated. C. jejuni and C. coli were detected from 170

cr
(64%) and 32 (12%) of the samples, respectively. Liver and skin samples had the greatest

level of contamination. Most of the isolates were resistant to clindamycin (98.6%),

us
erythromycin (98.6%), nalidixic acid (98.1%), tetracycline (94.2%), gentamicin (65.2%), and
an
chloramphenicol (52.6%). The results indicated that poultry meat sold in markets in Metro

Manila is contaminated with drug resistant Campylobacter.

M
ed
pt
ce
Ac

2
 Campylobacter is the most common cause of acute bacterial gastroenteritis worldwide

and is responsible for 400–500 million cases of diarrhea annually (1). The prevalence rate of

Campylobacter infections have increased in the last two decades, and in some parts of the

world, have exceeded those of Salmonella (2). Two species, C. jejuni and C. coli, are

responsible for most human infections, and when antimicrobial therapy is indicated,

macrolides remain the frontline agents for treating Campylobacter cases. (3). The recent

t
ip
emergence of antimicrobial resistance in Campylobacter, however, has underscored the

importance of antimicrobial susceptibility testing in guiding therapy and monitoring of

cr
resistance. Common sources of Campylobacter contamination include pigs, cattle, cats, and

us
dogs, but the major risk factor for infection in humans is the consumption of undercooked

poultry and the handling of raw poultry (4). The prevalence of Campylobacter among broiler
an
flocks varies among countries, ranging from 5% to more than 90% (5). In the National

Capital Region of the Philippines, Metro Manila, no previous study has been conducted to
M

assess the levels of Campylobacter contamination in poultry products sold in public markets,

hence, the objectives of the study were to determine the prevalence and antimicrobial
ed

resistance profiles of Campylobacter isolated from poultry meat sold in wet markets and
pt

supermarkets.
ce

Chicken samples (n=265), including breast meat (n=41), caeca (n=41), gizzard (n=42),

intestines (n=19), liver (n=38), skin (n=42), and thigh (n=42) were collected from 15 wet
Ac

markets and 15 supermarkets in Metro Manila February to August 2014. Methods outlined in

the US FDA Bacteriological Analytical Manual (6) were used to isolate Campylobacter.

Briefly, 25 g of sample was mixed with 100 mL Bolton broth supplemented with antibiotics

and 5% lysed horse blood. Pre-enrichment was performed at 37oC for 4 h, followed by

enrichment at 42oC for 20 to 44 h, under microaerophilic conditions. Presumptive

identification was based on cultural characteristics, Gram-stain, catalase, oxidase tests, and

3
hippurate hydrolysis. Primer pair MD16S1Upper/MD16S2Lower (7) were used to identify

Campylobacter spp., while primer-pairs QCjmapANF/QCjmapANR (7) and Col3-

F/MDCol2-R (8) were used to identify C. jejuni and C. coli, respectively. Broth microdilution

assay was performed following the guidelines and breakpoints described in the Clinical and

Laboratory Standards Institute document M45-P (9). The antimicrobials used were

chloramphenicol, clindamycin, erythromycin, gentamicin, nalidixic acid, and tetracycline.

t
ip
High rates of contamination was observed in the samples collected (Table 1). The

cr
over-all prevalence of Campylobacter was 78.1%. Most of the isolates were identified as C.

jejuni (64.2%) and C. coli (12.1%), and more Campylobacter were detected in wet markets

us
(84.6%) compared to supermarkets (70.5%). All chicken parts were contaminated with
an
Campylobacter (Table 2). Liver samples had the highest rate of contamination (97.4%),

followed by the intestines (89.5%), skin (88.1%) and gizzard (85.7%), while breast samples
M

had the least rate of contamination (43.9%). It is likely that contamination of the chicken

products started in the poultry farms, and continued in the slaughterhouses. Additional
ed

contamination may have occurred in wet markets, due to poor sanitary and handling

conditions, which may explain the higher rates of contamination. The prevalence reported
pt

here is higher compared to those reported by Magistrado et al. (98/135) in 2001 (10) for
ce

samples collected in Laguna, and in the work of Sison et al (57/120) in 2014 (11) for samples

collected in Nueva Ecijia. Differences in collection sites, sample processing, and

Ac

identification methods between this and previous studies may account for some of the

variations in prevalence observed, but reports in other countries have shown prevalence rates

as high as 100% (12).

High rates of antimicrobial resistance were also observed (Table 3). Nearly all of the

isolates were resistant to clindamycin (98.6%), erythromycin (98.6%), nalidixic acid (98.1%),

and tetracycline (94.2%), while resistance to chloramphenicol (52.7%) and gentamicin

4
(65.2%) were not as prevalent. The antibiotic resistance profiles of the isolates were

independent of chicken product. However, a significant association was found between the

antibiotic resistance profiles and the type of market the isolate came from. Following the

proposed classification of Magiorakos (13), cases of extensively drug-resistant

Campylobacter were most common (n=202), followed by cases of multi-drug resistance

(n=4), and cases of single drug-resistance (n=1), with no isolate displaying susceptibility to

t
ip
all antibiotics tested. These findings indicate that raw chicken products sold in Metro Manila

are contaminated with primarily extensively drug-resistant Campylobacter species. In the

cr
management of campylobacteriosis, fluid therapy is recommended. Antimicrobial treatment

us
is only prescribed to patients in severe cases and in the immunocompromised (3). The results

of the study exemplify the conclusion of Engberg et al. (14) that the established treatment of
an
campylobacteriosis via macrolides and quinolones may not be effective, and that

aminoglycosides may be the most viable course of treatment in human cases, when infection
M

is due to poultry products in Metro Manila.

ed

Our data show that raw chicken products may serve as vehicles for transmitting food-

borne diseases. The presence of drug resistant Campylobacter in the samples tested is also a
pt

public health concern. Further monitoring of chicken products is suggested since the level

and type of contamination represents a significant risk to consumers, unless food safety
ce

management strategies and pathogen reduction performance standards are implemented

Ac

strictly in key production points to lower pathogen carriage in public markets.

Acknowledgments

This work was supported by the Natural Sciences Research Institute of the University of the

Philippines Diliman, through project code BIO-13-2-01.

5
Conflict of interest

None to declare

t
ip
cr
us
an
M
ed
pt
ce
Ac

6
REFERENCES

1. Friedman CR, Neimann J, Wegener HC, et al. Epidemiology of Campylobacter jejuni

infections in the United States and other industrialized nations. In: Nachamkin I, Blaser

MJ, editors. Campylobacter. Washingto DC: ASM Press; 2000. p. 121-138.

2. Atanassova V, Ring C. Prevalence of Campylobacter spp. in poultry and poultry meat

t
in Germany. Int J Food Microbiol. 1999;51:187-90.

ip
3. Blaser B, Engberg J. Clinical aspects of Campylobacter jejuni and Campylobacter coli

cr
infections. In: Nachamkin I, Blaser MJ, editors. Campylobacter. Washingto DC: ASM

Press; 2000. p. 99-121.

4.

us
Wilson DJ, Gabriel E, Leatherbarrow AJ, et al. Tracing the source of
an
campylobacteriosis. PLoS Genet. 2008;4:e1000203.

5. Newell DG, Fearnley C. Sources of Campylobacter colonization in broiler chickens.

M

Appl Environ Microbiol. 2003;69:4343-51.

6. Bacteriological Analytical Manual, 8th Edition, Revision A, 1998 Chapter 7. Available

ed

at

<[Link]
pt

> Accessed June 10, 2016).

ce

7. Inglis GD, Kalischuk LD. Direct quantification of Campylobacter jejuni and

Campylobacter lanienae in feces of cattle by real-time quantitative PCR. Appl Environ

Ac

Microbiol. 2004;70:2296-306.

8. Denis M, Refrégier-Petton J, Laisney MJ, et al. Campylobacter contamination in

French chicken production from farm to consumers. Use of a PCR assay for detection

and identification of Campylobacter jejuni and Camp. coli. J Appl Microbiol.

2001;91:255-67.

7
9. Hindler JA, Humphries RM, Richter SS, et al. Methods for Antimicrobial Dilution and

Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. In: Clinical

and Laboratory Standards Institute. Wayne, PA; 2015. p. 7-8.

10. Magistrado PA, Garcia MM, Raymundo AK. Isolation and polymerase chain reaction-

based detection of Campylobacter jejuni and Campylobacter coli from poultry in the

Philippines. Int J Food Microbiol. 2001;70:197-206.

t
ip
11. Sison FB, Chaisowwong W, Alter T, et al. Loads and antimicrobial resistance of

Campylobacter spp. on fresh chicken meat in Nueva Ecija, Philippines. Poult Sci.

cr
2014;93:1270-1273.

us
12. Shih DY. Isolation and identification of enteropathogenic Campylobacter spp. from

chicken samples in Taipei. J Food Prot. 2000;63:304-308.

an
13. Magiorakos AP, Srinivasan A, Carey RB, et al. Multidrug-resistant, extensively drug-

resistant and pandrug-resistant bacteria: an international expert proposal for interim

M

standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18:268-281.

14. Engberg J, Aarestrup FM, Taylor DE, et al. Quinolone and macrolide resistance in
ed

Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates.
pt

Emerg Infect Dis. 2001;7:24-34.

ce
Ac

8
Table 1. Prevalence of C. jejuni, C. coli, and Campylobacter spp. from samples of retail

poultry collected from wet markets and supermarkets

Market Type No. of C. jejuni C. coli Campylobacter spp. Total

samples n (%) n (%) n (%)
Wet market 143 93 (65.0) 25 (17.5) 3 (2.1) 121 (84.6)
Supermarket 122 77 (63.1) 7 (5.7) 2 (1.6) 86 (70.5)

t
Total 265 170 (64.2) 32 (12.1) 5 (1.9) 207 (78.1)

ip
cr
us
an
M
ed
pt
ce
Ac

1
 Table 2. Prevalence of Campylobacter in chicken parts collected

Chicken Part No. of samples No. of positive samples (%)

Breast 41 18 (43.9)
Caeca 41 31 (75.6)
Gizzard 42 36 (85.7)
Intestines 19 17 (89.5)
Liver 38 37 (97.4)

t
Skin 42 37 (88.1)

ip
Thigh 42 31 (73.8)
Total 265 207 (78.1)

cr
us
an
M
ed
pt
ce
Ac

2
Table 3. Antimicrobial susceptibility profiles of the Campylobacter isolates

Susceptible Intermediate Resistant

Antibiotic
Breakpoint [n, (%)] Breakpoint [n, (%)] Breakpoint [n, (%)]
Chloramphenicol 8 µg/mL 84 (40.6) 16 µg/mL 14 (6.8) 32 µg/mL 109 (52.7)
Clindamycin 2 µg/mL 3 (1.4) 4 µg/mL 0 (0.0) 8 µg/mL 204 (98.6)
Erythromycin 8 µg/mL 2 (1.0) 16 µg/mL 1 (0.5) 32 µg/mL 204 (98.6)
Gentamicin 2 µg/mL 71 (34.3) 4 µg/mL 1 (0.5) 8 µg/mL 135 (65.2)

t
Nalidixic acid 16 µg/mL 3 (1.4) 32 µg/mL 1 (0.5) 64 µg/mL 203 (98.1)

ip
Tetracycline 4 µg/mL 4 (2.0) 8 µg/mL 8 (3.9) 16 µg/mL 195 (94.2)

cr
us
an
M
ed
pt
ce
Ac