Fifty five (n=55) isolates of Escherichia coli isolated from ducks in Penang, Malaysia were examined for their susceptibility to eleven different antibiotics and assayed for the presence of plasmid DNAs. All the 55 Escherichia coli isolates were resistant (100%) to vancomycin. Higher resistance (= 60) occurred for tetracycline 51 (92.7%), ampicillin 40 (72.7%), streptomycin 37 (67.3%), and sulfamethoxazole-trimethophrim 37 (67.3%). No and low resistance was observed for nitrofurantoin (0%) and gentamicin (1.8%), respectively. The isolates also showed some intermediate resistances to all antibiotics examined except for vancomycin. The 55 Escherichia coli isolates exhibited 23 different antibiotic resistant patterns with MAR index ranging from 0.09-0.82. Majority of the Escherichia coli isolates exhibited resistant pattern of VA-C-OFX-SXT-TE-AMP-NA-KF and VA-S-C-OFX-SXT-TE-AMP-NA-KF with MAR index of 0.73 and 0.82, respectively. The smallest plasmid DNA size was 1.2 kb and the largest plasmid DNA size was 81.5 kb. 51 (93%) of the duck Escherichia coli isolates harbored plasmids. The was no direct correlation between plasmid DNA sizes and antibiotic resistant among the duck Escherichia coli isolates. Thus, the antibiotic resistant of the Escherichia coli isolates could mostly be mediated by chromosomes instead of plasmids. This study also suggests that the use of antibiotics in duck farming in Penang, Malaysia needs to be controlled to prevent the spread of multiple antibiotic resistant Escherichia coli isolates.
Natural preservatives having the great antioxidant and antimicrobial activity have been utilized in the food industry for many years. In the present study, the effect of of two brands of commercial Assam green tea infusion (represented by A and B) and 0.02% BHA/BHT on microbial growth, anti-lipid oxidation and color change were investigated in cooked beef. The green tea concentration has influenced to the results. It was found that A and B at the concentration of 250 mg/mL significantly reduced the population of Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and E. coli in the cooked beef to an undetectable level within 2 days of storage at 4oC. A and B also exhibited higher anti-lipid oxidation activity compared to 0.02% BHA/BHT, and control. Assam green tea infusions in cooked beef significantly increased ∆ L*
value and decreased ∆ a* and ∆ b* value (p ≤ 0.05). These indicate that Assam green tea infusion might be a potential candidate as a natural preservative for beef and other types of food.
In Malaysia, the aquaculture industry, particularly the production of freshwater aquaculture fish, is growing rapidly. Nevertheless, the illegal use of banned antimicrobial agents such as chloramphenicol in aquaculture has become a major concern in relation to the safety of consumers and also the development of drug-resistant strains in bacteria. Driven by those factors, the main intention of this study was to determine the prevalence and types of chloramphenicol resistance genes in E. coli isolated from aquaculture and other environmental waters. The respective chloramphenicol-resistance genes in the isolates were detected by multiplex PCR with four sense primers C-1, C-2, C-3, C-4 and one antisense primer C-R for targeting cat I, cat II, cat III and cat IV genes, respectively. Out of 27 E. coli isolated, 19 were resistant to chloramphenicol. Cat I, cat II, cat III and cat IV genes were detected in 19, 13, 10, and 6 of the E. coli isolates, respectively. The results of this study revealed that chloramphenicol-resistance E. coli is present in aquaculture and environmental waters, in the study area. This finding suggested that although banned, there could be illegal usage of chloramphenicol antibiotic in local aquaculture. The bacteria in aquaculture may have spread to other environmental water through disposal of aquaculture waste water to other environments.
Antibacterial effect of modified sago starch-alginate edible film incorporating lemongrass oil at various concentrations was studied. Edible films were prepared from a mixture of modified sago starch and alginate. Lemongrass oil (0.1 - 0.4%, v/w) and glycerol (0 and 20%, w/w) were incorporated in the films to act as natural antimicrobial agent and plasticizer, respectively. The films were characterized for antibacterial activity against food pathogenic bacteria such as Escherichia coli O157:H7, Salmonella Enteritidis and Staphylococcus aureus. The edible film exhibited antibacterial activity against Escherichia coli O157:H7 and Salmonella Enteritidis by using agar diffusion assay method. For films tested against Escherichia coli O157:H7, the zone of inhibition increased significantly (p < 0.05) with addition of lemongrass oil at all levels both in the presence and absence of glycerol. The films also significantly (p < 0.05) inhibited the growth of Salmonella enteritidis only with 0.4% lemongrass oil (in the presence and absence of glycerol). However, the films containing lemongrass oil did not show any inhibition effect on Staphylococcus aureus.
Escherichia coli and Escherichia coli O157 were identified from “selom” (Oenanthe stolonifera), “pegaga” (Centella asiatica), beef, chicken, lamb, buffalo, “ulam Raja” (Cosmos caudatus) and “tenggek burung” (Euodia redlevi). The bacteria were recovered using chromagenic agar. Isolated Escherichia coli and Escherichia coli 0157 were further characterized by plasmid profiling and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The virulence genes of the isolates (VT1, VT2, LT, ST, eaeA, inV) that produces pathogenic Escherichia coli and 16S rRNA gene were screened by a multiplex PCR assay. The plasmid profiling analysis showed that out of 176 isolates, only 103 isolates contained plasmids. ERIC-PCR analysis generated amplified products in the range of ~150 bp to > 1000 bp categorizing isolates into a total of 52 different profiles. Multiplex PCR showed that 20 (32.3%) of the isolates carried eaeA gene, 6 (9.7%) isolates possessed inV genes, only 1 (1.6%) have VT2 genes and 1 (1.6%) as well carried VT1 genes, 2 (3.2%) of the isolates harboured LT genes, and only 1 (1.6%) isolate possessed ST genes. There were no correlation between plasmid, ERIC-PCR and virulence genes profiles.
The antibacterial activity of solvent-extracted oil of noni (Morinda citrifolia L.), spinach (Spinacia oleracea L.), lady’s finger (Abelmoschus esculentus (L.) Moench), bitter gourd (Momordica charantia Linn.), and mustard (Brassica nigra L.) seed oils, and coconut (Cocos nucifera L.) oil, palm (Elaeis guineensis L.) mesocarp in hydrolyzed and unhydrolyzed form were determined in order to explore their potential usage as antibacterial agent. The hydrolysis process that was catalyzed by immobilized lipase of Rhizomucor miehei (RMIM) showed highest hydrolytic activity with 1.0 ml of added water volume except bitter gourd seed oil and palm mesocarp oil which has maximum hydrolytic activity with added water volume of 5 ml and 2.5 ml respectively. Before hydrolysis, all oil samples did not show inhibition ring zones (IRZ) on any of the tested bacteria strains (Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7). Hydrolyzed lady’s finger and bitter gourd seed oil showed IRZ on all tested bacteria strains; hydrolyzed mustard seed oil on S. typhimurium and L. monocytogenes; hydrolyzed spinach seed oil and coconut oil on L. monocytogenes; hydrolyzed noni seed oil and palm mesocarp oil did not exhibit IRZ on any of the tested bacteria strains. Most of the hydrolyzed oil exhibit an inhibition activity that was different from their respective dominant fatty acids except noni seed oil and palm mesocarp oil.
Abstract: Ampicillin-sulbactam combination is the most frequently prescribed antibiotic in diabetic foot ulcers. We conducted a retrospective study to evaluate the antibiotic sensitivity of bacteria isolated to this antibiotic. In 33 patients with diabetic foot ulcer (September 2008-March 2009), 67% were culture positive in which Citrobacter spp accounted for 36% of these isolates. The rest isolated included Pseudomonas aeruginosa (22%), Proteus spp (18%), Acinetobacter spp (9%), Klebsiella pneumoniae (5%), Escherichia coli (5%) and Staphylococcus aureus (5%). These isolates were more likely to be ampicillin-resistant (n=18) than were ampicillin-sensitive isolates (n=4). Ampicillin resistance has raised our concern about current practice of prescribing ampicillin/ sulbactam as monotherapy for majority of our patients with such ulcers.
Urinary tract infections frequently affect pregnant mothers. This problem causes significant morbidity and healthcare expenditure. Three common clinical manifestations of UTIs in pregnancy are: asymptomatic bacteriuria, acute cystitis and acute pyelonephritis. Escherichia coli remains the most frequent organism isolated in UTIs. All pregnant mothers should be screened for UTIs in pregnancy and antibiotics should be commenced without delay. Urine culture and sensitivity is the gold standard in diagnosing UTIs. Without treatment, asymptomatic bacteriuria in pregnancy is associated with preterm delivery, intrauterine growth retardation, low birth weight, maternal hypertension, pre-eclampsia and anaemia. Acute pyelonephritis can lead to maternal sepsis. Recurrent UTIs in pregnancy require prophylactic antibiotic treatment.
Background: The public health issue of consuming groundwater is a major concern because people often extract groundwater directly from the aquifers either through wells or boreholes without treating it with any form of filtration system or chlorine disinfection. Based on the Malaysian National Drinking Water guidelines the current study was designed to provide a better understanding on the variable factors that are influencing the quality of well-water in an urbanised village in Malaysia. Well water quality assessment of heavy metals, chemicals, microbial and physical parameters were carried out for Sungai Buloh Village in the Klang Valley to ensure it was safe for human consumption.
Materials and Methods: Water samples were collected from wells at four sites (Sites A,B,C,D), a river and a tap inside a house in Sungai Buloh village. Soil was sampled from the riverbed and area surrounding the wells. Samples were collected every two months over a one year duration from all sites. The water samples were processed and examined for viruses, coliforms and protozoa as well as for heavy metal contaminants.
Results: The turbidity and colour ranged in the average of 0.57-0.13 Nephelometric Turbidity (NTU) and 4.16-5.00 Total Conjunctive Use (TCU) respectively for all sites except Site C. At Site C the turbidity level was 2.56 ± 1.38 NTU. The well-water was polluted with coliforms (1.2 to 2.4 x 103 CFU/100 ml) in all sites, E. coli (0.12 - 4 x 102 CFU/100 ml CFU/ 100 ml) and Cryptosporidium oocysts (0.4 cysts/100 ml). All the heavy metals and chemical parameters were within the Malaysian Guidelines’ limits except manganese. The average pH ranged from 5.44 - 6.62 and the temperature was 28 ºC.
Conclusion: In summary, the well water at Sungai Buloh is considered unsafe for consumption due to pollution. Therefore the major thrust will be to provide better quality of drinking water to the residents of the village.
The objective of this study was to evaluate the antimicrobial potential of methanol, acetone and distilled water stem
bark extracts from Canarium odontophyllum against Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 6633,
Escherichia coli ATCC 25932, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii strain sensitive, Candida
albicans ATCC 64677, Candida glabrata ATCC 90028, Aspergillus niger and Fusarium solani M2781. The extracts from
C. odontophyllum stem bark from 3.125 mg/ml to 25 mg/ml were screened against the tested microorganisms using disc
diffusion method. The Minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the
extracts against susceptible organisms were determined using microbroth dilution method and streak-plate technique,
respectively. From the antibacterial screening assay, the growth of S. aureus, B. cereus and A. baumannii were inhibited
by methanol extract whereas the acetone extract was capable of inhibiting all the tested microorganisms except E.coli,
F. solani and A. niger. The lowest MIC value for methanol extract was against A. baumannii (0.195 mg/ml) whereas
its MBC value was twice its MIC value (0.391 mg/ml), indicating that methanol extract was bacteriostatic against A.
baumannii. While for acetone extract, S. aureus showed bactericidal effect with equal MIC and MBC values at 0.195 mg/
ml. In conclusion, stem bark of C. odontophyllum has the potential to be the source of antibacterial agent and can be
exploited as an alternative phytoantimicrobial.
SnO₂ nanoparticle production using thermal treatment with tin(II) chloride dihydrate and polyvinylpyrrolidone capping agent precursor materials for calcination was investigated. Samples were analyzed using X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), energy dispersive X-ray (EDX), transmission electron microscopy (TEM), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), diffuse UV-vis reflectance spectra, photoluminescence (PL) spectra and the electron spin resonance (ESR). XRD analysis found tetragonal crystalline structures in the SnO₂ nanoparticles generated through calcination. EDX and FT-IR spectroscopy phase analysis verified the derivation of the Sn and O in the SnO₂ nanoparticle samples from the precursor materials. An average nanoparticle size of 4–15.5 nm was achieved by increasing calcination temperature from 500 °C to 800 °C, as confirmed through TEM. The valence state and surface composition of the resulting nanoparticle were analyzed using XPS. Diffuse UV-vis reflectance spectra were used to evaluate the optical energy gap using the Kubelka-Munk equation. Greater calcination temperature resulted in the energy band gap falling from 3.90 eV to 3.64 eV. PL spectra indicated a positive relationship between particle size and photoluminescence. Magnetic features were investigated through ESR, which revealed the presence of unpaired electrons. The magnetic field resonance decreases along with an increase of the g-factor value as the calcination temperature increased from 500 °C to 800 °C. Finally, Escherichia coli ATCC 25922 Gram (–ve) and Bacillus subtilis UPMC 1175 Gram (+ve) were used for in vitro evaluation of the tin oxide nanoparticle’s antibacterial activity. This work indicated that the zone of inhibition of 22 mm has good antibacterial activity toward the Gram-positive B. subtilis UPMC 1175.
Antibacterial activity of different types of P. odorata leaf extracts was evaluated in combination with
standard antibiotics. Persicaria. odorata leaves were extracted with n-hexane (n-hex), dichloromethane
(DCM) and methanol (MeOH). Each extract was applied on vancomycin (30µg), erythromycin (15µg) and
gentamicin (10µg) discs, respectively. Disk diffusion method was used to evaluate the synergistic activity of
each combination on Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes,
Streptococcus pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, and Escherichia coli. Minimum
inhibitory concentration (MIC) and gas chromatography mass spectrometry (GCMS) analysis was performed on
the active extract. Synergistic effects seen were mainly from the n-hex+antibiotics combinations, mainly on
the Gram-positive bacteria (7 additive, 5 antagonistic), with MIC range from 50 µg/ml to 100 µg/ml, as well
as Gram-negative bacteria (2 additive, 2 indifferent, 5 antagonistic). In particular, synergism showed by the
combination of n-hex+van were all additive against the susceptible bacteria. DCM extract combination
showed synergistic effects on three Gram-positive species (S. aureus, S. epidermidis, S. pyogenes).
Meanwhile, MeOH+antibiotics combination showed significant additive synergistic effects (p
Multidrug-resistant organisms (MDROs) such as multidrug-resistant (MDR) Acinetobacter baumannii and Escherichia coli are important pathogens associated with nosocomial infections in both human and animal health care facilities. Surfaces of inanimate objects in health care facilities can serve as sources of infection. However, studies on prevalence of these pathogens in veterinary settings are lacking in the country. Therefore, the objectives of this study were to determine the occurrence of A. baumannii and E. coli and the occurrence of MDR isolates on surfaces of inanimate objects in veterinary health care facilities in Klang Valley, Malaysia. In this study, swab samples were taken from 65 surfaces of inanimate objects that included door knobs, examination tables, labcoats, stethoscopes and weighing scales. The swab samples were cultured and all isolates were subjected to antibiotic susceptibility test. The study revealed that the occurrence of A. baumannii was 9.23% and 5 out of 6 (83.33%) A. baumannii isolates were classified as MDR. However, no E. coli was isolated. In conclusion, surfaces of inanimate objects can be a source of MDR A. baumannii in veterinary health care facilities that is of animal and public health concern.
Escherichia coli O157:H7 is a major food-borne pathogen that has resulted in numerous
outbreaks around the world. Widespread distribution of the organism in various ecological
niches impedes the control measures. This study aimed to detect and quantify E. coli O157:H7
in beef sold in wet markets and hypermarkets in Malaysia and to determine the risk of E. coli
O157:H7 infection linked to consumption of beef. The rfbO157 and flicH7 primers targeted on
somatic antigen (O157) and flagellar antigen (H7) respectively of E. coli O157:H7 was used for
the MPN-PCR method. A total of 99 beef samples were collected from local wet markets and
hypermarkets. The highest E. coli O157:H7 contamination rate was observed in beef samples
collected from wet markets (89.50%), whereas the contamination rate in hyper market A and B
were compratively low (35.35 and 20% respectively). However, the microbial load was highest
in the beef samples from hypermarket A (1100 MPN/g) while E. coli O157:H7 bacterial load
in beef samples from hypermarket B and wet market ranged from 3 to 93 MPN/g and 3 to 240
MPN/g, respectively. Using the Quantitative Microbial Risk Assessment (QMRA) approach
the risk was estimated incorporating the findings of the prevalence study and predictions
based on home storage, cooking and consumption patterns. Three different exposure pathways
were investigated to estimate the risk associated with contaminated beef and Monte Carlo
simulation was used to determine the level of uncertainty. The developed model predicated that
consumption of contaminated beef can be accountable for 1.83E+06 E. coli O157:H7 cases per
year in Malaysia. The reliability of the model, data gaps and further research needs, is discussed.
Through continuous improvement Quantitative Microbial Risk Assessment provides valuable
insight into controlling and prevention strategies.
Foodborne illness is recognized as an emerging infectious disease. The incidence of foodborne
infections is common and the majority cases are undiagnosed or unreported. Apart from some
diarrhea or minor gastrointestinal problem, some foodborne pathogenic microbes may cause
death, particularly to those people with weakened immune system. In this study, we have
developed a new fermented papaya beverage using symbiotic culture of yeast and acetic acid
bacteria under controlled biofermentation process. An in-vitro assessment of fermented papaya
beverage against few foodborne pathogenic microorganism was conducted to determine
its minimum bactericidal concentration (MBC>99). Three types of foodborne pathogen:
Escherichia coli O157, Salmonella enterica serovar Typhimurium ATCC 53648, Salmonella
enterica serovar Enteritidis (isolated from infectious chicken) were selected. From minimum
bactericidal concentration (MBC>99) assay, both fermented papaya pulp and leaves beverages
have shown 100% killing rate against three selected foodborne pathogenic microbes. Inversely,
non-fermented papaya pulp and leaves beverages indicated no inhibition at all. In fact, further
dilution of fermented papaya pulp and leaves beverages demonstrated different degree of
MBC>99 and brix value, but the pH value remained less than 3.5. These findings indicated
the combination of soluble solid compounds presents in both fermented papaya beverage and
product acidity play an important role in the inhibition of pathogenic microorganisms. The
preliminary promising results of this work have shown that the great potential of fermented
papaya beverages as a preventive measure to reduce the incidence of foodborne illness.
Silver nanoparticles (AgNPs) used in this study were synthesized using pu-erh tea leaves extract with particle size of 4.06 nm. The antibacterial activity of green synthesized AgNPs against a diverse range of Gram-negative foodborne pathogens was determined using disk diffusion method, resazurin microtitre-plate assay (minimum inhibitory concentration, MIC), and minimum bactericidal concentration test (MBC). The MIC and MBC of AgNPs against Escherichia coli, Klebsiella pneumoniae, Salmonella Typhimurium, and Salmonella Enteritidis were 7.8, 3.9, 3.9, 3.9 and 7.8, 3.9, 7.8, 3.9 μg/mL, respectively. Time-kill curves were used to evaluate the concentration between MIC and bactericidal activity of AgNPs at concentrations ranging from 0×MIC to 8×MIC. The killing activity of AgNPs was fast acting against all the Gram-negative bacteria tested; the reduction in the number of CFU mL-1 was >3 Log10 units (99.9%) in 1-2 h. This study indicates that AgNPs exhibit a strong antimicrobial activity and thus might be developed as a new type of antimicrobial agents for the treatment of bacterial infection including multidrug resistant bacterial infection.
To gain insight into the microbiological safety of food products routinely traded across international borders in Southeast Asian countries, beef imported from Malaysia to southern Thailand was examined for contamination with Escherichia coli O157 and its subsequent spread into the imported areas. We screened 31 samples exported from Malaysia and 36 domestic Thai samples. Isolation methods including an O157 antigen-targeted immunomagnetic separation technique, screening on CHROMagar O157 medium, and serotype confirmation of E. coli isolates by specific agglutination tests were employed. Fourteen strains of E. coli O157:H7 were isolated from eight Malaysian samples (25.8%) and six strains from four Thai samples (11.1%). These strains were of the stx1-stx2+eae+ genotype except one Malaysian strain which was of the stx1- stx2- eae+ genotype. All 19 O157:H7 strains possessing the stx2 gene produced little or no Stx2 (reversed passive latex agglutination titer ≤ 4). Of the 19 strains, five Malaysian (38.5%) and two Thai (33.3%) strains exhibited resistance to a set of antibiotics. Finally, the results of two DNA fingerprinting
analyses (O157 IS-printing targeted to IS629 and pulsed-field gel electrophoresis, PFGE) of the O157:H7 strains possessing the stx2 gene, indicated that the Malaysian and Thai strains are closely related. Therefore, E. coli O157:H7 might be transferred from Malaysia to southern Thailand through beef trade.
Detection of enterotoxin by targeting entFM and hblA genes in Bacillus cereus BC1 strain inoculated into ready to eat food (RTF) and drink samples using polymerase chain reaction (PCR) was conducted. The B. cereus BC1 strain was confirmed as a Bacillus diarrhoeal enterotoxin (BDE) when tested by a commercially available Enzyme-linked immunosorbent assay-BDE immunoassay (ELISA-BDE immunoassay, TECRA). In the specificity study, both enterotoxin genes were detected on chromosomal DNA of B. cereus BC1 strain by showing a specific band of 1269 bp (entFM) and 874 bp (hblA), respectively. However, none of the target genes were detected for the other 15 genomic DNA bacteria (B. cereus (ATCC 11779), B. subtilis (ATCC 6633), Campylobacter jejuni (ATCC 29428), C. coli (Jabatan Kimia Malaysia, JKM), Clostridium perfringen (ATCC 13124), Enterobacter sakazaki (ATCC 51329), Escherichia coli (ATCC 43888), E. coli (ATCC 11735), Legionella pneumophila (ATCC 33152), Listeria monocytogenes (ATCC 35967), Salmonella typhi (IMR), S. enteritidis (ATCC 13076), S. typhimurium (ATCC 14028), Shigella flexeneri (ATCC 12022) and Vibrio cholerae bengal (Institute Medical Research (IMR), Malaysia) examined. The detection limit of both genes was 0.1 ng of genomic DNA. Thus, in the presence study it is evidence that the PCR analysis targeting enterotoxin of entFM and hblA genes are suitable and useful in detecting enterotoxic B. cereus in RTFs and drinks contaminated sample.
Edible medicinal plants are often used in the treatment of various ailments and spice in traditional food preparation. In this study, 45 of tropical edible medicinal plants extracts from Indonesia, Malaysia, and Thailand were screened for their antimicrobial activity against five standard microorganisms for food preservative namely Aspergillus niger, Candida albicans, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The methanol extracts of Piper nigrum L. seed, Piper cubeba L. seed, and the root of Ligusticum acutilobum Siebold and Zucc. showed antimicrobial activity against five species of standard microorganisms. Among them, P. cubeba L. extract demonstrated the most susceptible against all tested microorganisms. Minimal inhibitory concentration (MIC) and minimal bactericidal or fungicidal concentration (MBC or MFC) were performed by the broth microdilution techniques as described by the Clinical and Laboratory Standard Institute. MIC values of P. cubeba L. extract to A. niger, C. albicans, E. coli, P. aeruginosa and S. aureus were 12.8, 1.6, 3.2, 6.4, and 1.6 mg/ml, respectively. P. cubeba extract killed A. niger, C. albicans, E. coli, P. aeruginosa and S. aureus with MBC values of 25.6, 3.2, 6.4, 12.8, and 3.2 mg/ml, respectively. The potent antimicrobial activity of P. cubeba L. extract may support its use for natural food preservative.
Two cocoa bean fermentation methods (spontaneous fermentation and the use of starter culture) for 7 days fermentation were compared in terms of safety and quality fermented beans. Candida sp. was used as a starter culture in this study. The safety of the fermented cocoa beans were measured by the growth colonies of pathogenic microorganisms namely Bacillus cereus, Escherichia coli, Salmonella sp., Staphylococcus aureus, and Pseudomonas sp., on Bacillus cereus agar, eosin-methylene blue (EMB) agar, xylose lysine deoxycholate (XLD) agar, Baird-Parker agar (BPA), and Pseudomonas agar, respectively. B. cereus, E. coli and Salmonella sp. were early present in both fermentations. Candida sp.-fermentation showed detection of B. cereus at 5.34 log10 CFU/g and absence after 24 hours of fermentation while in spontaneous-fermentation B. cereus was too few to count. Moreover, the log10 E. coli number in Candida sp.-fermentation and spontaneous-fermentation were reduced from 5.72 to 3.66 and from 7.15 to 4.46 on day 1 to day 3, respectively. There were no presences of pathogenic microorganisms on day 5 and day 7 for both fermentations. In term of quality, proximate analysis of spontaneous-fermentation resulted that the content of moisture, ash, fat, crude protein, crude fibre and carbohydrate was 56.47%, 2.32%, 3.17%, 7.02%, 28.14% and 2.88%, meanwhile for the Candida sp.-fermentation was 53.96%, 2.19%, 3.44%, 8.25%, 25.46% and 6.70%, respectively. This study showed that both fermentations are considered to be safe and there is no significant difference in proximate value in fermented cocoa beans from spontaneous-fermentation and Candida sp.-fermentation.