In Vitro Study on Commercial Organic Acid Activate WD (WD)Against Four Pathogeni

Jiang Guo-zheng, Li Ji-chang*, Han Zhen-xing, Liu Ting, Wang Yuan, and Cao Hong

1 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China

2 Novus International Trade Limited Company, Beijing 100015, China

Introduction

On large-scale poultry farms, Staphylococcus aureus(S. aureus), Escherichia coli (E. coli), Salmonella pullorum (S. pullorum), and Campylobacter jejuni(C. jejuni)are the most common pathogenic organisms, threatening public health as well as causing a huge economical damage (Otaki, 1995).Consumption of contaminated meat often becomes the source of infection (Petersen et al., 2001).Poultry is initially infected by these bacteria at the farm level. Meanwhile, water as a vehicle plays an important role in the horizontal transmission route on broiler farms. Many studies have shown that some pathogenic bacteria could survive or multiply in natural surface water, public water supplies and in wastewater from slaughterhouses (Chaveerach et al.,2002). Thus, poultries are likely to become infected by these organisms from rearing water sources on the farm. Antimicrobial agents have been widely added into rearing water to prevent bacterium infection.Unfortunately, with the increase in resistance of many organisms to the currently used antimicrobials and many side effects (Ricke, 2003), there is a need to look for alternatives.

Short-chain organic acids (SCA)have been widely used in preserving food products in western European countries with no residue problems. Their bactericidal effects can also prevent spoilage and kill potential pathogenic organisms (Thomson and Hinton, 1997). However, the bactericidal activities of SCA as a disinfectant in rearing water on farm are rarely reported. Thus, increasing the hygiene barrier by reducing the level of infection in poultries by decontaminated water could be one of the best methods for preventing poultries from bacterium infection. The objective of this work was to investigate the antibacterial activity of commercial organic acid Activate WD (WD)against four pathogenic bacteria in a mixture of water and feed at different pH levels, and the result will provide an experimental basis for being used in clinic.

Materials and Methods

Organic acid

Commercial organic acid WD was provided kindly by Novus International Trade Co., Ltd.

Bacterial strains

Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 43889, Salmonella pullorum ATCC 9120 and Campylobacter jejuni ATCC 33296 were purchased from China Institute of Veterinary Drugs Control.

C. jejuni was cultured in Brucella broth media at 37℃ for 24-48 h under microaerophilic conditions by using a GasPak (Becton Dickinson Co, MD 21030).The other three pathogen strains were cultured in MH media for 24 h at 37℃. Prior to experimentation, all strains were subcultured onto Brucella agar or MH agar plate. One colony was transferred into Brucella broth or MH broth and was incubated at 37℃ overnight. One milliliter of each Brucella or MH broth was transferred into 9 mL of the physiological salt solution.The result was a stock solution, having approximately 106CFU of bacteria/mL, for use in this experiment.

Determination of minimal inhibitory concentration (MIC)and minimal bactericidal concentration (MBC)

The MIC and MBC were determined by double broth dilution method. The final concentration of WD was 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%,0.078%, 0.039%, and 0.02%, resppectively.

Feed preparation

A commercial broiler feed in the form of granules was used. It was on the ground and sterilized by hot-dry sterilization for 3 h at 160℃. A 0.25 g feed sample was put into a 50 mL centrifuge tube containing 10 mL of tap water. This mixture of feed and water was sterilized at 121℃ for 15 min.

Effect of WD against bacterium populations in water

To test the bactericidal potential of organic acid WD against four pathogenic bacteria, different amounts of WD were put into tubes containing 10 mL prepared water to reach a pH of 3.0, 4.0, and 5.0, measured with an electric pH meter. The control water contained no organic acid, and its pH was approximately 6.8. One milliliter of each pathogenic bacterium from the stock solution was transferred into 10 mL of prepared water with pH at 3.0, 4.0, and 5.0, as well as into a tube with control water. Tubes were kept in a water bath at 37℃until the end of experiment. The survival of bacteria was determined by a direct plate count method of 10-fold dilutions of bacteria on Brucella agar or MH agar plates. Samples were taken at 0, 0.5, 1, 4, 8, and 24 h after adding the bacteria to the organic acid. The experiments were done in triplicate. C. jejuni cultures were kept under microaerophilic conditions by using a GasPak at 37℃ for 42 h. The other bacteria were incubated at 37℃ for 24 h in an incubator. Typical colonies were counted and expressed as log10colonyforming units per milliliter.

Statistical analysis

Data was expressed as means±SD. Differences in bacterial counts among treatments were determined by ANOVA with SPSS software (SPSS Science, Chicago,USA). A value of P<0.05 was considered statistically signif i cant.

Results

The MIC and MBC of WD against bacteria

The MICs and MBCs of WD were: S. aureus: MIC 0.3125%, MBC 0.625%, E. coli: MIC 0.3125%,MBC 0.625%, S. pullorum: MIC 0.3125%, MBC 0.625%, and C. jejuni: MIC 0.3125%, MBC 0.625%,respectively.

Effect of WD on bacterium viability

There were no great differences among bacteria, but there were signif i cant pH differences. At pH 3.0 with WD, the survival of S. aureus, E. coli, S. pullorum,and C. jejuni rapidly declined from the beginning doses of 4.38±0.25, 4.47±0.42, 4.36±0.26, and 4.47±0.16 Log10cfu • mL-1, respectively to below their detection limits within 0.5 h of incubation (Fig. 1).

Fig. 1 Survival of four pathogenic bacteria after being exposed WD at different pH levels and times at 37℃

At pH 4.0 and 5.0, the bactericidal effects of WD against S. aureus, E. coli, and S. pullorum populations gave reductions of 4.53±0.22, 4.33±0.35, and 4.41±0.15 and 4.72±0.24, 4.48±0.41, and 4.56±0.34 Log10cfu • mL-1at 0.5 h of incubation time, respectively. At pH 4.0, 5.0, and 8 h incubation, bacterium numbers were below the detection limits and remained during 24-h test period, whereas for C. jejuni,bacterium numbers declined bellow the detection limits at 4 h of incubation. Four pathogenic bacteria survival in the control samples well compared with the treated samples. Meanwhile, the bacterium populations increased over time except C. jejuni.

Discussion

In the present experiment, the MIC and MBC of WD against four pathogenic bacteria were 0.3125%,0.625%, respectively. These results indicated that WD had signif i cant antibacterial activity against both gramnegative and gram-positive bacteria. The inhibitory mechanism of SCA against bacteria is not clear yet.Investigators have reported that growth inhibition of acids on bacteria is caused by two components. One is the specif i c inhibition of an unidentif i ed metabolic function by undissociated acid, whereas the other is diffusion of undissociated acid into the cell, which releases a proton that acidif i es the cytoplasm (Zhang et al., 2011). We compared the bactericidal effect of WD against four pathogenic bacteria at different pH levels. Our results demonstrated that at low pH levels (3.0)all the bacteria rapidly died. C. jejuni died after 4 h at pH 4.0, 5.0, the other bacteria took 8 h of incubation with WD to a nondetectable level, which indicated that WD could kill C. jejuni more quickly.The reduction rate at pH 4.0 was much higher than that at pH 5.0. These results conformed that there was a signif i cant positive correlation between the germicidal eff i cacy and amounts of WD. In general, rearing water at broiler farms is in-tensively contaminated with dirty organic matter such as feed, feces, bedding, or soil (Chaveerach et al., 2004), where will become a good survival place for pathogenic agents (Gibbens et al., 2001). Therefore, pathogenic reinfection would certainly occur. The use of organic acids in rearing water systems for broilers could reduce cross-infection via rearing water on farm level.

Conclusions

WD had significant bactericidal effects in water, and could be used on farms. Further research, especially animal infection experiments, is necessary to specify the exact effect of acidif i ed water on transmission of bacteria among chickens at farm level.

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