Antibiotic Resistance of Lactic Acid Bacteria Isolated from a Fermented Fish Product, Pla-chom
Lactic Acid Bacteria (LAB) have been isolated from various fermented food products in Thailand. However, the antimicrobial susceptibility of the microbes in fermented food products are a matter of concern in several countries. The aim of this study was to determine the antimicrobial susceptibility of LAB strains isolated from the Thai traditional fermented small fish product, pla-chom. The concentrations of LAB were also studied. The antimicrobial susceptibility and resistance of 10 representative LAB strains to 8 antibiotics were investigated using the disk diffusion method. The antibiotics used for the tests included penicillin, ampicillin, erythromycin, tetracycline, vancomycin, streptomycin, sulfamethoxazole-trimethoprim and metronidazole. The average concentration of LAB, when the product was fermented at 30°C for 3 days, was 2.5x106 CFU g-1 of product. The inhibition zone diameters of all antibiotics were between 0 and 30 mm for all the LAB strains isolated and tested. All 10 LAB isolates were resistant to vancomycin, streptomycin, sulfamethoxazole-trimethoprim and metronidazole. The LAB strains were highly resistant to tetracycline (7 isolates), to penicillin (6 isolates), but showed low resistance to ampicillin (2 isolates). All 10 LAB isolates were sensitive to erythromycin. Three LAB isolates were sensitive to all four antibiotics (penicillin, ampicillin, erythromycin and tetracycline), which are commonly used in the treatment of bacterial infections in humans and animals. These three LAB isolates could possibly be used as starter cultures for this fermented product.
Received: August 05, 2011;
Accepted: November 23, 2011;
Published: January 06, 2012
Thai traditional fermented foods are produced by natural fermentation of various
foods, included fishery products (nam-pla, ka-pi, bu-du),
fermented fish (pla-ra, pla-som, pla-chao, som-fak,
pla-chom), meat products (nham, sai-krog-prieo, mum)
and plant products (naw-mai-dong, phak-gard-dong, miang,
khao-mak, khanom-jeen) (Tanasupawat, 2009).
Pla-chom is most popular in the north-eastern part of Thailand. It is
produced from small raw fish mixed with ground roasted gelatinous rice (khaao
khuaa), salt and garlic. Pla-chom is typically fermented at room
temperature between 28-30°C for 3 days.
Lactic Acid Bacteria (LAB) are a large group of beneficial bacteria that produce
lactic acid as an end product of food fermentation. Currently, there is concern
about the possible spread of antibiotic resistance from antibiotics used for
the inhibition of pathogenic microorganisms, or from beneficial microbes occurring
naturally in foods. LAB from fermented products may act as a reservoir of antibiotic
resistance genes that could be transferred to pathogenic bacteria or normal
flora in the gastrointestinal tract of humans and animals (Florez
et al., 2005). Antibiotic resistance of LAB is comprised of two characteristics:
(1) natural or intrinsic resistance, being nontransmissible; (2) acquired resistance,
usually caused from bacterial mutation and possibly carrying plasmid encoding
of antibiotic resistance genes and which may potentially be transmissible to
other bacteria (Courvalin, 2006). There is little information
about the antibiotic susceptibility of commensal bacteria such as LAB isolated
from fermented food products and no data exist about antibiotic susceptibility
in LAB from the fermented small fish product, pla-chom. The present study
investigated the antibiotic susceptibility profiles of LAB isolated from pla-chom
MATERIALS AND METHODS
Sample collection and isolation of Lactic Acid Bacteria (LAB): In February
2011, twenty samples of the fermented fish product, pla-chom were collected
from local markets in the area of amphur Muang, Khon Kaen province. Pla-chom
is made from small raw fish. Ten kilograms of fish were mixed with garlic, salt
and ground gelatinous rice and then fermented at 30°C for 3 days. Ingredient
proportions of the pla-chom product are shown in Table
1. Samples were kept in air-tight plastic bags at 4°C. To find the volume
LAB, the samples were divided into 5 g portions that were dissolved into 45
mL of Maximum Recovery Diluent solvent (Oxoid Inc., Hampshire, UK) and agitated
for 30 sec. Serial tenfold dilutions from the homogenate were made according
to ISO-6887-1 (1999) and plated in De Man Rogosa and
Sharpe (MRS) agar (De Man et al., 1960), with
modification by adding 0.4% (w/v) CaCO3 using the pour plate method.
Incubation was carried out aerobically for 2 days at 37°C as described in
ISO-15214 (1998). Bacterial colonies were enumerated
in terms of their growth (30-300 colonies) by the appearance of the surrounding
acid-produced clear zone on the plate. Bacterial concentrations were calculated
(volume of bacterial colonies x dilution factor) and expressed as colony- forming
units per gram of sample (CFU g-1). The different morphologies of
bacterial colonies from each sample were gathered and stored in a modified freezing
medium (Criterion tryptic soy broth, 0.6% yeast extract and 20% glycerol)
at -70°C for further testing.
Cell morphologies of bacteria were checked using Gram staining. The isolates
were tested for a catalase reaction. Rod and coccal cells of bacteria determined
to be catalase negative and Gram positive were characterized as LAB (Salminen
et al., 2004) and were selected for further testing.
Antimicrobial susceptibility test: The representative LAB isolates were
further tested for antimicrobial susceptibility by the disk diffusion method
(Bauer et al., 1966).
|| Ingredients used in pla-chom processing
Eight antibiotics commonly used in treating human or animal infections (European
Food Safety Authority, 2008) and from different antibiotics classes were
chosen for the test. They were penicillin G (10 μg), ampicillin (10 μg),
erythromycin (15 μg), tetracycline (30 μg), vancomycin (30 μg),
streptomycin (10 μg), sulfamethoxazole-trimethoprim (25 μg)
and metronidazole (50 μg). All antibiotic disks (diameter = 6 mm) were
obtained from Oxoid. The antimicrobial susceptibility test was similar to that
described by Charteris et al. (1998). Briefly,
each LAB isolate was inoculated with 108 CFU (turbidity of 0.5 Mac-Farland
standard) at 37°C in MRS broth and incubated anaerobically for 18 h. A sterile
cotton swab was dipped in each culture solution and swabbed in three directions
on a Mueller-Hinton agar plate. All antibiotic disks were seeded in the plates
and incubated anaerobically at 37°C for 48 h. The diameters of antibiotic
inhibition zones were measured using a ruler under a colony-counter apparatus
(Weiss-Gallenkamp, UK) and expressed in mm which included the diameter of the
antibiotic disk. Antimicrobial susceptibility was interpreted according to the
cut-off levels proposed by Charteris et al. (1998),
with strains considered resistant if inhibition zone diameters were equal to
or smaller than 19 mm for penicillin G and ampicillin, 14 mm for vancomycin
and tetracycline and 13 mm for erythromycin. Inhibition zone diameters equal
to or smaller than 8 mm for streptomycin, sulfamethoxazole-trimethoprim
and metronidazole were considered to indicate resistance, according to the cut-off
levels (with minimal modifications) of Reddy et al.
(2007). All antibiotics were tested in duplicate.
RESULTS AND DISCUSSION
Lactic acid bacteria isolation: Lactic acid bacteria were successfully
isolated from pla-chom using a selective medium of MRS agar modified
by adding 0.4% (w/v) CaCO3. A clear zone appeared surrounding the
bacterial colonies, caused by a reaction to the coloniesacid production.
The colonies were confirmed to be LAB by cell morphology, Gram staining and
catalase reaction. Previous researchers had attempted to develop a selective
medium for lactobacilli (Jackson et al., 2002).
In this study, a selective medium (MRS agar) was developed to easily isolate
LAB. Characteristics of 14 bacterial isolates and concentrations of 10 representative
LAB strains are shown in Table 2 and 3,
|| Characteristics of 14 bacterial isolates from the fermented
fish product, pla-chom
|+: Positive reaction, -: Negative reaction, *: Isolates were
excluded from the basic properties of LAB
|| Concentration of 10, representative LAB strains isolated
from the pla-chom
|1count with triplicate plates, 2numerous
||Interpretative zone diameters (mm) of 8 antibiotics used on
10 representative LAB strains selected from the fermented fish product,
|Susceptibility expressed as: (R): Resistant, (MS): Moderately
susceptible, (S): Sensitive. P: Penicillin, AMP: Ampicillin, E: Erythromycin,
TE: Showed pinpoint colonies within the inhibition zone tetracycline, VA:
Vancomycin, S: Streptomycin, SXT: Sulfamethoxazole-trimethoprim and
Antimicrobial susceptibility: Lactic acid bacteria such as Lactobacillus
sp., L. farciminis and L. pentosus have been isolated from pla-chom
(Tanasupawat et al., 1998) but no data exist
about their antibiotic susceptibility. In this study, the results of antimicrobial
susceptibility are shown in Table 4. All LAB isolates showed
intrinsic mechanisms of resistance to vancomycin, sulfamethoxazole-trimethoprim,
metronidazole and aminoglycoside antibiotics (streptomycin). Although LAB strains
are species-dependent for intrinsic resistance to sulfamethoxazole-trimethoprim
(Klare et al., 2007), all LAB strains in our
study showed such resistance to the drug, which may derive from certain antagonistic
components such as p-aminobenzoic acid (PABA) and thymidine (Turnidge
and Bell, 2005). The intrinsic resistance to vancomycin demonstrated by
the LAB strains in this study was likely due to the presence of D-alanine ligase,
an enzyme that can inactivate vancomycin (Elisha and Courvalin,
1995). Some LAB strains in this study showed acquired resistance to penicillin,
ampicillin and tetracycline. These strains may have received their antibiotic
resistance by spreading from bacteria had survived antibacterial treatment of
animal infections. LAB in this study showed lower resistance to penicillin (60%)
than in the study by Savadogo et al. (2010) who
reported that LAB strains isolated from fermented milk in Burkina Faso, Africa,
showed 78.94% penicillin resistance. Lactobacillus spp. isolated from
chicken feces have been reported to have 36.37% tetracycline resistance (Sornplang
and Leelavatcharamas, 2010). In addition, there was an isolate (PC1) showed
pinpoint colonies within the inhibition zone with tetracycline. These characteristics
demonstrated the tendency of the bacteria to mutate and develop to be a resistance
to antibiotics (Kipp et al., 2004). In this study,
LAB isolated from pla-chom was highly resistant to tetracycline (70%).
Three selected LAB isolates (PC3, PC8, PC10) were intrinsically resistant to
vancomycin, sulfamethoxazole-trimethoprim, metronidazole and streptomycin
but were sensitive to all four antibiotics (penicillin, ampicillin, erythromycin
and tetracycline), commonly used in the treatment of bacterial infections in
humans and animals. Therefore, these three LAB isolates can be safely applied
and could possibly be used as starter cultures in this fermented fish product.
Although, lactic acid bacteria are a large group of bacteria isolated from traditional fermented small fish product (pla-chom) in present study, there are only some strains that are sensitive to antibiotics commonly used in animals. The selection of these bacterial strains used for the starter culture in food fermentation is an alternative that will give consumers confidence and not to eat products containing lactic acid bacteria to be the reservoirs transferred an antibiotic resistance genes to other bacteria. Further research, should be tested for sensitivity to the antibiotics in the other traditional Thai fermented foods that from animal origins.
The authors would like to thank the Department of Veterinary Public Health, Faculty of Veterinary Medicine, Khon Kaen University, Thailand, for use of their bacterial laboratory facility. Appreciation is also expressed to Associate Professor Kriengsak Poonsuk for valuable advice on lactic acid bacteria isolation techniques.
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