ARCOBACTER SPECIES AND THEIR RISKS IN SOME MEAT AND FISH WITH A SODIUM ACETATE AND SODIUM CHLORIDE INTERVENTION

ARCOBACTER SPECIES AND THEIR RISKS IN SOME MEAT AND FISH WITH A SODIUM ACETATE AND SODIUM CHLORIDE INTERVENTION

M.A.M. AMMAR and S.H. AL-HABATY

Animal Health Research Institute, Assiut Regional Lab. (AHRI), Egypt

Email: mahmoud2014eg@yahoo.com                                                                   Assiut University web-site: www.aun.edu.eg

INTRODUCTION

Meat is the first-choice of animal protein for human and consumption of meat is continuously increasing worldwide. The annual per capita consumption increased by 2.6 fold in 2000 and will increase by 3.7 fold by 2030 compared to that of 1960s,(Dave and Ghaly, 2011). Also meat and meat products are important sources of vitamins and minerals. They also may contain microorganisms which in certain circumstances and in inappropriate proportions can negatively affect human health. One of the most important aspects relating to some of the potential health problems associated with meat consumption is emerging pathogens, (Tarrant, 1998).

Arcobacters are members of the family Campylobacteraceae and phenotipically similar to Campylobacters. They differ from Campylobacters by their ability to grow under both aerobic conditions and under 30°C. The importance of the genus Arcobacter lies in the fact that some species are considered emerging enteropathogens and potential zoonotic agents, (Kayman, 2012). Presently the genus Arcobacter includes a total of 15 species. Three species of Arcobacter have been recovered from man and animals: A. butzleri, A. cryaerophilus, and A.skirrowii, (On et al., 2002).

There is evidence that livestock animals may be a significant reservoir of Arcobacter spp. (Ho et al., 2006). Also, apart from A. nitrofigilis and other species of Acrobacter have been isolated from various animal diseases including abortion, septicaemia, mastitis, gastritis and enteritis (On et al., 2002).

On fecal samples collected at slaughterhouses in Belgium, Arcobacter spp. was isolated from 44% of porcine, 40% of bovine, and 16% of ovine samples. All three animal-associated Arcobacter spp. were isolated and levels of up to 10³cfu/ g feces were found, (Van Driessche and Houf, 2007). Also, investigations revealed that A.butzleri and A. cryaerophilus are commonly present on slaughter equipment (Houf et al., 2003).Arcobacterspp. were already isolated from a wide range of food of animal origin. The highest prevalence is reported for poultry meat, followed by pork and beef (Houf et al., 2002; Shah et al., 2011).

The prevalence of Arcobacter in shellfish has shown to be relatively high i.e. 100% in clams and 41.1% in mussels (Collado et al., 2009). As a result, it was suggested that shellfish should be considered another source of infection because they have an ability to concentrate bacterial pathogens from water and are often eaten poorly cooked or raw (Collado et al., 2009). However, little is knownabout the presence of Arcobacter in fish or fishmeat.

The exact routes of human infection with Arcobacter are not clear, but probably include manipulation of raw meat, the consumption of undercooked products and cross-contamination, (Milesi, 2010). Clinical symptoms are similar to campylobacteriosis, but a higher frequency of persistent and watery diarrhea has been reported, (Vandenberg et al., 2004). Besides the correlation with gastro-enteritis, Arcobacter has also been implicated in extra-intestinal invasive diseases, (On et al., 1995 and Yan et al., 2000). Most of the reported cases of extra intestinal presentation involved bacteraemia and occurred in immunocompromised patients or those with indwelling devices (Collado and Figueras, 2011).

PCR assays to detect all members of the genus Arcobacter and that are specific for each Arcobacter species have been reported. Based on the knowledge of the Arcobacter nucleic acid composition of the 16S rRNA, and by means of five primers, a PCR product of 401-bp was generated for A. butzleri, 257-bp for A. cryaerophilus and 641-bp for A. skirrowii. Those three species were also identified by the PCR assay developed by ( Kabeya et al., 2003).

Minimizing product contamination and delaying or inhibiting growth of spoilage and pathogenic organisms in the product are major keys for improving fresh meat shelf life and increasing consumer safety. While general cleanliness and proper sanitation are very effective, other means of controlling microbial growth in meat products may be prove useful, (Lee et al.,1997).

Sodium chloride (SC) has been long used as a meat preservative. It is added to meats for its effects on sensory, functional and preservation properties. Sodium chloride inhibits the microbial growth by restriction of the available water (i.e. lowers ^aw) in the meat products. However, its pro-oxidant activity accelerates the development of lipid oxidation in refrigerated meats (Lee et al., 1997). Antioxidative effects of sodium organic salts derived from citric, lactic and acetic acids have already been studied on color and lipid oxidation in n-3 oil fortified ground beef patties (Lee et al., 2005).

Lately, the application of organic acids and their salts have been more considered due to their natural and appropriate antimicrobial properties. Acetic, lactic, propionic and sorbic acids and their salts exert antimicrobial activity. They have been traditionally used as food preservatives (Ray, 1996). Acetates increase the acidity of the environment where they are applied and so obstruct the growth of meat spoilage bacteria, (Dragoev, 2004). Also, it possessed antibacterial activity against some bacterial pathogens (Nanasombat and Chooprang, 2009).

Despite the role of raw meat in transmission of Arcobacter infection to consumers, the eventual presence of Arcobacters in beef at retail in Assiut have seldom been assessed also the distribution of Arcobacter spp. in fish is unknown. Therefore, the aim of this study was to evaluate the prevalence of Arcobacter spp. in raw beef, minced beef and fish, to genotype Arcobacter strains isolated from these sources using PCR andto study their antimicrobial susceptibility and their behavior in the presence of sodium chloride and sodium acetate in growth medium and meat model.

MATERIALS and METHODS

Collection of samples:

A total 75 fresh samples of fish (Oreochromis niloticus), minced meat (250g portions) and beef (250g portions), 25 samples each were purchased from retail shops throughout AssiutCity, Upper Egypt. After purchase, the samples were placed in an ice cooler until they were delivered the laboratory. The examination of samples was as rapid as possible within half an hour. During this period, they were stored at 4–6°C.

Isolation of Arcobacter spp.: (O¨ngo¨r et al., 2004)

In case of fish, 25 g muscle samples were aseptically taken from the left hand side of each fish in the anterior dorsal region. Also, 25 g portion each of beef or minced beef were aseptically sampled. For each the samples were separately macerated in a sterile mortar without diluents. Then one gram sample was aseptically inoculated into 10 ml Brucella broth (Difco, Detroit, MI, USA) with CAT supplement (Cefoperazone, 8 mg /L; Amphotericin, 10 mg /L and Teicoplanin, 4 mg/L, Oxoid, Basingstoke, UK) and mixed thoroughly by vortex. The homogenates was incubated aerobically at 30°C for 48 h. These enriched samples were then plated onto Mueller-Hinton agar (CM337, Oxoid) supplemented with 5% (v/v) lysed horse blood and CAT selective supplement. The plates were incubated aerobically at 30°C for 3 days. Arcobacter-like colonies (round, 2–4 mm grey to whitish) were picked for phenotyping according the standard biochemical tests recommended by (Kayman, 2012). The phenotypic characteristics of Arcobacter species were assessed based on Gram staining; productions of oxidase, catalase, urease, alpha-hemolysis; and growth at different conditions (at 30°C, at 37°C, at 42°C, aerobically, microaerobically, and anaerobically).

Identification of isolated strains by polymerase chain reaction (PCR):

1- Primersequencesused for PCR system:

Specific 16S rDNA fragments for A. butzleri, A. skirrowii as well as for A. cryaerophilus were applied for demonstration and characterization of such strains by using the primers shown in (Table 1).

Table 1: Primer sequences for Arcobacter spp. polymerase chain reaction

2. DNA Extraction using QIA amp kit: (Shahet al.,2009)

After overnight culture on nutrient agar plates, one or two colonies were suspended in 20 ml of sterile distilled water, and the suspension was then heated at 100ºC for 20 minutes. Accurately, 50-200 µl of the culture were placed in Eppendorf tube and the following steps were carried out:

2.1. Equal volume from the lysate (50-200 µl) was added, addition of 20-50µl of proteinase K, then incubation at 56 ºC for 20-30 min.

2.2. The solution was added to the column and centrifuged at 8000 rpm for 1 min. then the filtrate was discarded.

2.3. The sediment was washed using AW1 buffer (200 µl), the column was centrifuged at 8000 rpm / 1 min, and the filtrate was discarded.

2.4. Washing was applied by using the AW2 buffer (200µl), the column was centrifuged at 8000 rpm / 1 min. and the filtrate was discarded.

2.5. The column was placed in a new clean tube then, 25-50 µl from the Elution buffer was added, centrifuged at 8000 rpm/1 min. Then the column was discarded. The filtrate was put in clean tube containing the pure genomic DNA.

3. Amplification reaction of Arcobacter species (Wesley et al., 1995):

PCR reactions were performed in a reaction mixture (50 µl volume) containing 2 µl of lysed bacteria, 5 µl of Gibco BRL 10U PCR bu¡er, 1.5 U of Taq DNA polymerase (Gibco), 0.2 mmol l31 of each deoxyribonucleotide triphosphate, 1.3 mmol l31 MgCl2, 5 Wl of loading bu¡er (4 mM cresol red, 60% sucrose) and 50 pmol of the primers ARCO, BUTZ, CRY1, CRY2, and 25 pmol of primer SKIR.

Accurately, PCR involved 32 cycles of denaturation (94°C for 45sec), primer annealing (61°C, 45 sec) and chain extension (72°C, 30 sec). Amplified products were detected by electrophoresis in 1.5% agarose in 0.5 U Tris- borate, EDTA buffer at 100 volts for 40 min. The PCR products were electrophoresed in 1.5% of agarose gel electrophoresis stained with ethidium bromide and visualized on UV transilluminator.

Antimicrobial susceptibilities of Arcobacter species:

Three different species, Arcobacter cryaerophilus A. skirrowii and A. butzleri isolated in the present study were used. A total of 10 commercially available antibiotic disks (Oxoid Hampshire, UK) were employed. The antibiotics and their concentrations (μg/disk) are shown in the Table (3). The disk-diffusion test was used for the determination of the antimicrobial susceptibility of the Arcobacter isolates as described by (Woods and Washington, 1995). Briefly, the isolates were grown aerobically at 30 °C for 48 h. After cultivation, a suspension of each organism was made in physiological saline and the turbidity of each inoculum was adjusted to McFarland 0.5. Bacteria from each suspension were inoculated onto blood agar that comprised 5% (v/v) defibrinated sheep blood in blood agar base no. 2 (Oxoid CM271) using a sterile cotton-tipped swab. Thereafter, each antibiotic disk was placed onto the agar and the plates were kept at 4 °C for about 20 min in order to allow the antibiotics to diffuse into agar. Incubation of the plates took place aerobically at 30 °C for 48h and the diameter of the inhibition zones was measuredwith calipers. The susceptibility patterns (resistance / sensitivity) of the strains were determined according to previously defined criteria (Woods and Washington, 1995).

Determination The minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) of Sodium chloride (SC) and sodium acetate (SA) against Arcobacter spp.:

Preparation of inoculums: (Elaine, 2005)

Genotypedstrains of A. cryaerophilus A. skirrowii and A. butzleri isolated in the present study were stored at –70°C in Meuller-Hinton broth supplemented with 20% glycerol. Before use, they were subcultured onto 5% bovine blood agar plates and incubated aerobically at 30 °C for 48h. Isolated colonies of each culture were individually inoculated into liquid growth media aerobically at 30 °C for 30 h (the target stationary-phase cells were obtained in this period). Suspensions turbidity was adjusted to match that of 2 McFarland standard to obtain a final concentration of 10⁷ cells/ml of target Arcobacter spp.

The (MIC) and (MLC) of SC and SA were established using the broth dilution method, as described by Jayana et al. (2010).Two-fold serial dilution of SC and SA (0·03%, 0·06%, 0·125%, 0·5%, 1%, 2%, 4% and 8 %, 16 % and 24% (w/v) were prepared separately  using sterile Muller Hinton broth. Each tube was inoculated by 100 µL from the 30 h age culture of target organism to obtain final bacterial concentration of approximately 1 x 10⁷ CFU / ml broth. The tubes together with the control tube (an inoculated and non inoculated tubes contained broth only) were incubated aerobically at 30 °C for 72h. The lowest concentration of the antibacterial that inhibits growth of the organism as detected by lack of visible turbidity was designated the MIC. To determine the MLC, 100 µL from each clear tube (no visible growth) was surface spread over the dry surface of Campylobacter blood-free agar (Oxoid, UK) after 24, 48 and 72 hours of incubation. In each case, the inoculated plates were incubated aerobically at 30 °C for 72h. Growth of the microorganism from any incubation period at a particular concentration indicated that the lethal concentration was not achieved. MLC was defined as the lowest concentration of tested substances that killed the test organism (No growth or survival at the given concentration within 72 h). The mean MIC and MLC was recorded from triple readings in each test.

Behaviar of Arcobacter butzelri in presence of both sodium chloride and sodium acetate in growth medium: (Phillips, 1999)

Sodium chloride (SC) at 3% was combined with (SA) at five levels separately. G1 (3 % SC and 0.01% SA), G2 (3% SC and 0.02% SA), G3 (3 % Sc and 0.03% SA), G4 (3 % SC and 0.04% SA) and G5 (3 % SC and 0.05% SA) were prepared using sterile Muller-Henton broth. Each tube was inoculated by 100 µL from the 30 h age culture of A. butzleri to obtain final bacterial concentration approximately of 1 x 10⁷ CFU / ml broth. The tubes together with the control tubes (an inoculated and non inoculated tubes contained broth only) were incubated aerobically at 30 °C for 72h. One -ml samples from each culture were serially diluted using sterile 0.1% peptone water. Appropriate dilutions were surface spread over the dry surface of Campylobacter blood-free agar (Oxoid, UK) plates and incubated aerobically at 30 °C for 72h. The plates were counted and the counts were expressed as log 10 CFU/ml.

Behaviar of Arcobacter butzelri in presence of both sodium chloride and sodium acetate in minced beef model:

Preparation of minced beef and inoculation:(Elaine,2005)

Fresh beef from the thigh area (Musculus Semimembranosus) were purchased from the local meat retailer for each replicate. The meat were assessed for Arcobacter then were manually cut into trimmings and aseptically coarse grounded with a meat grinder with a 5mm hole diameter strainer and stored at ( –18°C ) for 24h  in order to eliminate background microflora that were likely to confound the results. As needed, portions of ground meat were thawed overnight at 4°C, mixed for homogeneity and divided into two groups. To group A (treated group) SC at level of 3% and SA at 0.05% were added and aseptically mixed. Group B was (the control). Individual 25 g portions of both groups were aseptically weighed and transformed manually into minced meat finger. Calculated count of 10⁷ CFU/g (one ml) portion of A. butzleri suspensions prepared as motioned previously were injected into the center of each finger using a sterile syringe. Inoculated fingers were inserted through a sterile stomacher sac and placed 4 °C.

Counting of survivors:(Phillips, 1999)

Samples were withdrawn at selected intervals (0, 3, 6, 12 and 24h).Treated and control samples were analyzed for Arcobacter count. Approxmately, 225 ml of peptone water was add to each sample, and the sample was stomached (Tekmar model 400, Tekmar, Cincinnati, OH) for 2 min at normal speed. Aliquots of appropriate dilutions were plated on 5% bovine blood agar plates with CAT supplement, which were incubated aerobically at 30 °C for 72h. Colonies were counted and converted to log10 CFU/g counts. All experiments were repeated 3 times and the packaged SPSS program for windows version 12.0.1 was used for statistical analysis according to (SPSS, 2007). Data were expressed as mean ± standard error (SE). Differences between groups were determined by means of a Student "t"-test. Significance level was set at P < 0.05.

RESULTS

Table 2: Prevalence of Arcobacter spp. in meat, minced meat and fish.

Table 3: Susceptibility of Arcobacter spp. to antimicrobial agents.

S: susceptible     R: resistance     I: intermediate

Table 4: MIC and MLC of sodium chloride and of sodium acetate against Arcobacter spp.

Fig. 1: Combined effect of sodium chloride 3% and different concentration of sodium acetate on Arcobacter butzleri in the growth medium.

Fig. 2: Combined effect of 3% sodium chloride and 0.05% of sodium acetate on Arcobacter butzleri in refrigerated minced meat

Fig. 3: Agarose gel electrophoresis of multiplex PCR for characterization of A. cryaerophilus, A.              butzleri and A. skirrowii.

                        M          1         2          3          4        5         6         7         8         9        10         11     12     13

Lane M: 100 bp ladder as molecular size DNA marker.

Lane 1: Control positive for A. cryaerophilus, A. butzleri and A. skirrowii.

Lane 2: Control negative for A. cryaerophilus, A. butzleri and A. skirrowii.

Lanes 7 & 13: Positive A. cryaerophilus strains for 23S rDNA (257 bp).

Lanes 4, 8, 9 & 12: Positive A. butzleri strains for 16S rDNA (401 bp).

Lanes 3, 5, 6, 10 & 11: Positive A. skirrowii strains for 16S rDNA (641 bp).

DISCUSSION

Arcobacter spp. are considered ‘emerging’ pathogens based on the characteristics they share with Campylobacters, potentially extending from morphological similarities to infectious capabilities and transmission routes, (Wesley, 1996). Miller et al. (1998) discussed characteristics of A. butzleri that contribute to its consideration as an‘emerging’ pathogen, and suggested that factors involved in the emergence of Escherichia coli O157:H7 may be shared by A. butzleri.

In this study, only 47% and 15% of raw food samples were found to be contaminated with Arcobacter spp. by phenotyping (PT) and genotyping (GT) methods, respectively (Table 2). Fresh beef samples were the most contaminated within surveyed raw foods in retail shops. By (PT), 52 % contained Arcobacter spp. while (GT) revealed their presence in 20% of samples only. Of the fresh minced beef samples, 48 % and 8% contained Arcobacter spp. by (PT) and (GT), respectively. Also fish acts as a source of Arcobacr spp in this study. As shown in (Table 2), the (PT) procedures detected Arcobacr spp in 40% of samples. By (GT), 16 % were proved to harbor Arcobacr spp. Arcobacters are biochemically inert and have fastidious growth requirements, which make their speciation problematic using standard phenotypic procedures, (On et al., 1995). Vytrasova et al. (2003) stated that biochemical tests alone are not adequate to confirm Arcobacter spp., unless they are followed by PCR assay. The reasons were explained by Milesi, (2010) who mentioned that differentiating of Arcobacter spp. by using phenotypic tests might give erroneous results because of the shortage of clear-cut differentiating tests, a phenomenon which has also been observed in the closely related genus Campylobacter.

However, the PCR assay using primers specific to each Arcobacr spp (Houf et al., 2000), which was employed in the present study, has shortened significantly the time required for the identification of Arcobacters at the species level and also removed the possibility of false positive results due to Campylobacters. The findings commented before explained the low prevalence of Arcobacr spp. using (GT) comparing with (PT) in the present study.

In other studies, detection by molecular methods has shown an incidence of Arcobacr spp ranging between 1.4% (Collado et al., 2009) and 55 % (Vytrasova et al., 2003). A study included detection using culturing and molecular method in parallel reported that 0.7% of the samples positive by culturing, and 1.4% by molecular detection (Collado et al., 2009).

The isolation rate of Arcobacters from meat samples was higher than, 2.2% (Kabeya et al., 2003) and lower than 55.6% (Vytrasova et al., 2003), 22% (Rivas et al., 2004), 34% (Scullion et al., 2006) and 37% (Aydin et al., 2007) which were reported for fresh beef samples. Various factors such as differences in hygienic conditions in each abattoir as well as differences in the sensitivity of sampling and isolation methods used in these studies may have contributed to these variation.

Regarding minced beef samples, our findings (8%) were within the range of 4 – 10% recorded by (Rohder et al., 2007; Nieva-Echevarria et al., 2013), respectively and lower than that (90.9%) of (Abd El Rahman et al., 2012).

From(Table 2), A. butzleri and A.skirrowii, predominated A. cryaerophilus in both fresh beef and fresh minced beef samples. A. cryaerophilus was not detected in minced beef. The predominance of A. butzleri in beef or minced beef was also recorded by (O¨ ngo¨ r et al., 2004). A. cryaerophilus were reported to be less frequent in meat samples including poultry meat, (Houf et al., 2001; Atabay et al., 2002; Kabeya et al., 2003). However, the isolation of Arcobacters from red meat samples, which were collected from retail markets, appears significant when the risk for human health was considered (O¨ngo¨ r et al., 2004).

Arcobacters in cattle have been associated with different syndromes such as mastitis, enteritis and reproduction disorders including abortion and recurrent breeding problems in the herd (Logan et al., 1982; Ho et al., 2006). However, none of those studies have shown an unequivocal relation between the presence of Arcobacters and those pathologies. Moreover, Arcobacters are frequently present in healthy cattle by which they may act as an unnoticed contamination risk during slaughter.

It is commonly assumed that enteric pathogens found on raw meat are mainly derived from faecal origin (Heuvelink et al., 2001). A. butzleri was the predominant species on all carcasses sampling sites, which corresponds with the species distribution in cattle prior to slaughter (O¨ngo¨r et al., 2004). Spreading of Arcobacters between animals may occur in the holding pen prior to slaughter or by the slaughter equipment. Also, crosscontamination between carcasses during slaughter was reported by Van Driessche and Houf (2007).

Arcobacter spp. were isolated from fish muscle, (Table 2). Forty percent and 16% of fish samples contained Arcobacter spp. using (PT) and (GT), respectively with A. skirrowii as the dominant species detectable in 8 % of the samples. The prevalence of A. cryaerophilus and A.butzleri was parallel each (4 %). Patya et al. (2011) recorded the detection of Arcobacr spp. by 17.33 and 21.33% using culture and PCR techniques, respectively. Nonetheless, comparable prevalence was detectable in shellfish (73.3%) and musscles (41.1%) in northern Spain (Collado et al., 2009; Nieva-Echevarria et al., 2013).

 Our data suggests that fish represents an important reservoir for Arcobacter spp. and confirm that genetic diversity of A. butzleri strains is also common among isolates originated from fish. The abundant presence of three Arcobacter species in red meat and fish suggests an important role of contaminated food in the transmission of these bacteria.

In the current study, a total of three isolates of various Arcobacter spp. including A. cryaerophilus, A. skirrowii, and A. butzleri that were isolated from meat and fish samples were tested for their susceptibilities to 10 antibiotics. The results are summarized in table 3. The three Arcobacter spp. tested were resistance to Lincomycin, Vancomycin, Cloxacillin, Cephradine, Novobiocin, Tetracycline and Oxacillin. A. cryaerophilus was the most resistance within tested Arcobacters. It was resistance to 80% of tested antibiotics. A. skirrowii and A. butzleri were parallel in their resistance, 70% each.

A. skirrowii was the most susceptible within Arcobacters. It showed susceptibility to Gentamycin, Ciprofloxacillin and Neomycin. A. butzleri showed only intermediate susceptibility to Neomycin while A. cryaerophilus was the only resistance to Ciprofloxacillin, (Table 3). Gentamycin was the most active antibiotics against the A. cryaerophilus, A. skirrowii and A. butzleri.

Lack of gold-standard sensitivity methods and break points of antibiotics has made the comparison of results of antibiotic resistance patterns more difficult. A. skirrowii was reported to be the most susceptible Arcobacter species, (Houf et al., 2001) which coordinate with our findings. By using E-test method, Otth et al. (2004) reported that all Arcobacter tested isolates were sensitive to Gentamycin. Unver et al. (2012) found that A. cryaerophilus, A. skirrowii and A. butzleri were resistance to Vancomycin and Cloxacillin.

Different results were reported in a recent study that evaluated the resistance to antibiotics of several strains recovered from cattle, beef, milk and water using a disk diffusion method. Only 6.5% of the tested strains showed resistance to Tetracycline, 21.7% to Ciprofloxacine and 26.1% to Gentamycin, (Shah et al., 2011). When considering the results obtained for clinical strains using different methods (Kayman et al., 2012 and Mandisodza et al., 2012), reported that most isolates showed susceptibilities to Ciprofloxacin, Gentamycin and Tetracycline.

In the present study, A. cryaerophilus, A. skirrowii, and A. butzleri isolates were resistant to six or more antibiotics (multi drug resistant). Multi drug resistance (MDR) in Arcobacter spp. has also reported by some other researchers. Son et al. (2010) reported 71.8% of A. butzleri were MDR, whereas Vandenberg et al. (2006) recorded 6.2% of A. butzleri isolates showing MDR resistance to Ampicillin, Erythromycin and Nnalidixic acid.

The increased level of drug resistance, as encountered in this study, is important in terms of both animal and public health. The more popular opinion is that the use of antibiotics, especially in food animals, will lead to the development of antibiotic resistance which in turn can be disseminated through the environment and led to resistant infections in humans, (Angulo, 2003). Because of the similarity in antibioticuse between animals and humans, a serious concern is that onceresistance develops in animals it will soon affect humans, (CDC, 2006).

The incidence of antibiotic susceptibility in Arcobacters varied among species, which suggests that suitable antibiotic(s) should be selected for the treatment of infectious disease(s) and/or when developing selective media for the isolation of a wide range of Arcobacters. Lincomycin, Vancomycin, Tetracycline, Cloxacillin, Cephradine, Novobiocin and Oxacillin, could not be considered as drugs of choice for treatment of Arcobacter borne gastrointestinal disease. Contrary to this, Gentamycin would be the drugs of choice and Neomycin as alternative.

To study the growth and survival characteristics of Arcobacter spp. with a view to identify intervention techniques that would reduce their presence in food products and environments with which they have been associated, the behavior of Arcobacter spp. against SC and SA alone or mixed was studied. The three tested Arcobacters (A. butzleri and A. skirrowii, and A. cryaerophilus) respond to SC and SA environment in the same way. Arcobacters couldn't tolerate 9% salt (SC) concentration in growth medium, (Table, 4). The MIC of the three Arcobacter spp. tested was 8%. In a related study, Elaine, (2005) found that Arcobacter spp. could grow at SC levels up to 5% depending on the species and strain of concern. The growth in nearly similar concentrations of (SC) was recorded for other foodborne pathogens. McClure et al. (1989) observed growth of L. monocytogenes within 72 hours in 10% SC at 25 °C.

Sodium acetate showed bactericidal effect against Arcobacters. When tested against A. butzleri and A. skirrowii, and A. cryaerophilus, the MIC of SA was 6% while the MLC was 9%, (Table, 4). Sodium salts of the low molecular weight organic acids, such as acetic lactic and citric have been used to control microbial growth, improve sensory attributes and extend the shelf life of various food systems. In addition to their effect on food spoilage bacteria, these organic salts were shown to possess antibacterial activities against foodborne pathogens (Blom et al., 1997 and Ehsani et al., 2013). One advantage of SA is that its antibacterial action less affected by the pH of the medium particularly at pH 5.0-6.5. Against Yersinia enterocolitica, SA resulted in MIC of47.80 mg/ml at pH 4.5 while MIC (52.50mg/ml) at pH range from 5.0-6.5, (Nanasombat and Chooprang, 2009). Sodium acetate has also been known to exhibit antilisterial effect, (FDA, 2000).

In food system cold storage alone was not sufficient to reduce Arcobacter risk to an acceptable level. Cold storage was reported to reduce viability of Arcobacter. Freezing reduces the number of Arcobacter by 1 – 2 logs, but freezing alone is not sufficient to reduce risk to an acceptable level, (Hansen and Olsen 2007). Additional steps are needed to insure that the meat and meat products are safe for the consumers. All of these additional steps are combined with meat curing or brining. In the last few years different combinations of common salt and salts of organic acids (acetic, lactic, tartaric or citric) have been made that can be applied to the meat during the curing of brining process, or directly (without curing or brining).

In the present study, the application of SC or SA against Arcobacter spp. in meat system was faced with two problems. First, the both MIC and MLC of SA were higher than the limit (5000mg/kg) recommended for use in meat, (Queensland Government, 2013). The 2^nd is that new trends in minced meat technology works in the direction of SC reduction. To overcome these problems the combined effect of low concentrations of both antimicrobials were tested against A. butzleri in growth medium.

From the results shown in (Fig. 1), the reduction in A. butzleri counts due to combined effect of SC and SA ranged from 0.2 - 2.7 log CFU/ml after 24 h incubation at 30 °C compared with the control (initial count). The highest reduction level in A. butzleri cells was related to combination of 3% SC plus 0.05% SA (G5). There were significant differences (P ˂ 0 .05) between treatments (G1 – G5) compared with control, (Fig.1). Despite the various studies showed the use of SC, singly agonist Arcobacter spp. in broth, there is scarce of literature concerned with the combination effects with SA.

As the formula (3% SC + 0.05% SA) was the most effective treatment against A. butzleri in growth medium, it was chosen for addition in minced beef system. The antibacterial mixture needed 12 h to produce 1 log reduction in the initial A. butzleri count and the reduction was proximate at 24h period, (Fig. 2). By the end of 24 of refrigeration storage, the A. butzleri count was 5.48 CFU/g in antimicrobial mixture treated samples, it reached 7.19 CFU/g in control samples. The difference in A. butzleri   count between treatment and control samples was significant (P ˂ 0 .05) as shown in (Fig. 2).

Sodium salt is GMP (Good Manufacturing Practice) listed with meats, (DJC, 2009). Sodium salt of chloride has a rich history of use in ensuring meat safety before refrigeration, (American Meat Institute, 2010). Besides the antimicrobial properties, SC increases the bind, firmness, cooked yield and taste, in minced meat, (Madril and Sofos, 1985).The use of SC in meat processing at level of 3% was reported. Anbalagan et al. (2013) found that 3% SC treated group was recorded the very low bacterial load in all meats (Chicken, Mutton and Beef) compared to other treated groups. It was found that the pro-oxidant activity of SC accelerates the development of lipid oxidation in refrigerated meats (Lee et al., 1997) but acetates can antagonize that effect. It had been reported that acetates have antioxidant effects and they prevent the occurrence of the undesirable changes in the sensory properties of the products, such as colour, taste, odour, etc., (Gökalp et al., 2004).

The combination of sodium chloride with other antimicrobial agents may have an impact on the overall inhibitory effect. Sallam (2007) reported 1.2 log reductions in the total bacterial count by application of SA, and SC combination on refrigerated sliced salmon. Casey and Condon (2002) found that SC reduced the inhibitory effect of acid pH on the growth of Escherichia coli O157: H7. Tan and Shelef (2002) reported that a combination of SC and sodium lactate was more effective than lactates alone in delaying the onset of meat spoilage and its effects on its color and bacterial counts. Sallam and Samejima (2004) reported the use of sodium chloride in combination with sodium lactate reduced the microbial growth, maintained the chemical quality and extended the shelf life of ground beef during refrigerated storage.

Sodium acetate has proven useful for controlling pathogens in a variety of meat and poultry products. An uncured turkey product was able to remain free of the Clostridium botulinum neurotoxin for over 18 days at 28°C when treated with 6% sodium acetate, (Miller et al., 1992). The use of sodium acetate and diacetate as flavor enhancers should be limited to less than 0.25% by weight of total formulation, (USDA-FSIS, 2000).

Recent studies have shown the effects of sodium acetate combined with other antimicrobial agents at inhibiting L. monocytogenes. Individually, 2.5% sodium lactate and 0.25% sodium acetate both strongly inhibit the growth of L. monocytogenes,(Blom et al., 1997). However, in the same study, a combination of 2.5% sodium lactate, 0.25% sodium acetate, and 2.75% salt completely inhibited the organism, (Blom et al., 1997).

Survival of pathogens in the environment and in food products is governed by a complex array of factors. Several of these factors are inherent in the genotypic composition of the genus and are reflected in the ability to adapt to adverse conditions commonly encountered in their reservoir area (soil, water, animals) or in the environment into which they have been artificially introduced (foods, susceptible unnatural host animals, etc.). These adaptive mechanisms are often transferable between genera, or more commonly species, conferring ‘potential pathogen’ status on ‘newly emerging' microorganisms, (Elaine, 2005).

In our study, the antimicrobial combination infood system was not as effective as in broth when usedat the same concentration. This was in agreementwith Drosinos et al. (2006) who indicated thataddition of MIX 1 (lactic acid, sodium acetate andpotassium sorbate) and MIX 2 (potassium lactateand potassium acetate) prevented the lactic acidbacteria in growth medium, but not in meatproduct. Moreover, meat composition includingprotein and fat and some components thatare cryoprotectants may protect microorganismsfrom destruction. It has been suggested that Arcobacter spp. can survive in food because they can tolerate high sodium chloride concentrations, desiccation, can grow at lower refrigeration temperatures and have the ability to attach to various types of surfaces (Collado and Figueras, 2011).

In conclusion, this study revealed that the fresh meat and fish from the retail market are important source of Arcobacters that may play role in the contamination of the environment and human food chain. Further efforts are needed to investigate cases with diarrheal illness to elucidate the role of A. butzleri in veterinary public health in this geographical area. Such epidemiologic data is important for pre­ventive strategies and control of diarrheal diseases, especially in remote areas where populations share food sources available in only a few local markets. Gentamycin would be drugs of choice and Neomycin as alternative for treatment of Arcobacter borne gastrointestinal infection in this geographical area.

Using organic acid salt (SA) in combination with SC is capable of decreasing the number of viable cells of A. butzleri in fresh minced beef under refrigerated storage, thereby enhancing microbiological safety of minced beef products. However, addition of organic acid salts at concentrations higher than the permissible limits is recommended is in order to eliminate the A. butzleri effectively.

REFERENCES

Abd El Rahman, H.A.; Ahmad, A.M.; Mona, M. Abdelwahab and Salowa, M. Salem (2012): Arcobacter species as newly emerging food-borne pathogen in meat at Ismailia Governorate. SCVMJ, XVII (2): 21- 26.

American Meat Institute "AMI" (2010): Salt Use in Meat and Poultry Products. AMI Fact Sheet. January 2010.  http://www.MeatAMI.com.

Anbalagan, M.; Ganesh Prabu, P.; Krishnaveni, R.E. and Manivannan, S. (2013): Effect of Sodium Chloride (NaCl) on the Bacterial Load in Chicken, Mutton and Beef Meat Samples in Relation to Meat Spoilage. International Journal of Research in Zoology 4(1): 1-5.

Angulo, L. (2003): Public health consequences of use of antimicrobial agents in agriculture. In: Knobler, S.L., Lemon, S.M., Najafi, M., Burroughs, T. (Eds.), Forum on Emerging Infections: The Resistance Phenomenon in Microbes and Infectious Disease Vectors. Implications for Human.Centers for Disease Control and Prevention. 2005. http://www. cdc.gov/narms/(viewed Nov. 2005).

Atabay, H.I.; Bang, D.D.; Aydin, F.; Erdogan, H.M. and Madsen, M. (2002): Discrimination of Arcobacter butzleri isolates by polymerase chain reaction-mediated DNA fingerprinting. Letters of Applied Microbiology 35, 141–145.

Aydin, F.; Gumussoy, K.S.; Atabay, H.I.; Ica, T. and Abay, S. (2007): Prevalence and distribution of Arcobacter species in various sources in Turkey and molecular analysis of isolated strains by ERIC-PCR. J. Appl Microbiol. 103: 27–35.

Blom, H.; Nerbrink, E.; Dainty, R.; Hagtvedt, T.; Borch, E.; Nissen, H. and Nesbakken, T. (1997): Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 4ºC. Int. J. Food Microbiol. 38:71-76.

Casey, P.G. and Condon, S. (2002): Sodium chloride decreases the bactericidal effect of acid pH on Escherichia coli O157: H7. Int. J. Food Microbiology, 76: 199-206.

"CDC" Centers for Disease Control and Prevention (2006): Preliminary Food Net data on the incidence of infection with pathogens transmitted commonly through food -10 states, United States 2005. MMWR Morb. Mortal Wkly Rep; 55: 392-5.

Collado, L.; Guarro, J. and Figueras, M.J. (2009): Prevalence of Arcobacter in meat and shellfish. J. Food Prot. 72: 1102–1106.

Collado, L. and Figueras, M.J. (2011): Taxonomy, epidemiology and clinical relevance of the genus Arcobacter. Clin. Microbiol. Rev., 24: 174-192.

Dave, D. and Ghaly, A.E. (2011): Meat spoilage mechanisms and preservation techniques: A critical review. American Journal of Agricultural and Biological Sciences 6 (4): 486-510

DJC ''Department of Justice Canada'' (2009): Food and Drug Act, Department of Justice Canada. http://laws.justice.gc.ca/en/showtdm/cr/C.R.C.-c.870.

Dragoev, S. (2004): Development of technology in the industry for temperature had on good sensory characteristics of meat cuts that meat and fish. Academic Edition UFT Plovdiv,  259-263.

Drosinos, E.H.; Mataragas, M.; Kampani, A.; Kritikos, D. and Metaxopoulos, I. (2006): Inhibitory effect of organic acid salts on spoilage flora in culture medium and cured cooked meat products under commercial manufacturing conditions. Meat Sci. 73: 75-81.

Ehsani, A.; Jasour, M.S.; Hashemi, M.; Mehryar, L. and Khodayari, M. (2013): Zataria multiflora Boiss essential oil and sodium acetate: how they affect shelf life of vacuum-packaged trout burgers International Journal of Food Science and Technology (1-8) online version doi:10.1111/ijfs.12400.

Elaine, M. D'Sa. (2005): Effect of pH, NaCl Content, and Temperature on Growth and Survival Arcobacter spp. Journal of Food Protection, 8, 1: 18-25.

FDA ''Food and Drug Administration'', U.S. (2000): Food additives for use in meat and poultry products: sodium diacetate, sodium acetate, sodium lactate, and potassium lactate. Fed. Regist. 65:13, 3121-3123.

Gökalp, Y.G.; Kaya, M. and Zorba, Ö. (2004): Et urunleri isleme mühendisligi (besinci baski). Atatürk Universitesi Ziraat Fakültesi Ofset Tesisi, Erzurum.

Hansen, F. and Olsen, K.E.P. (2007): Arcobacter – an emerging food borne pathogen. NMKL Technical Report No. 2, Nordic Committee on Food Analysis.www.nmkl.org.

Heuvelink, A.E.; Roessink, G.L.; Bosboom, K. and De Boer, E. (2001): Zero-tolerance for faecal contamination of carcasses as a tool in the control of O157 VTEC infections. Int J Food Microbiol 66: 13–20.

Ho, H.T.K.; Lipman, L.J.A. and Gaastra, W. (2006): Arcobacter, what is known and unknown about a potential foodborne zoonotic agent!. Vet Microbiol 115: 1–13.

Houf, K.; De Zutter, L.; Verbeke, B.; Van Hoof, J. and Vandamme, P. (2003): Molecular characterization of Arcobacter isolates collected in a poultry slaughterhouse. Food Protection 66: 69-364.

Houf, K.; Devriese, A.L.; Dezutte, L.; Van Hoof, J.V. and Vandamme, P. (2001): Susceptibility of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii to Antimicrobial Agents Used in Selective Media. J. Clin Microbiol. 39(4): 1654–1656.

Houf, K.; De Zutter, L.; Van Hoof, J. and Vandamme, P. (2002): Assessment of the genetic diversity among Arcobacters isolated from poultry products by using two PCR-based typing methods. Applied and Environmental Microbiology 68: 2172–2178.

Houf, K.; Tutenel, A.; De Zutter, L.; Van Hoof, J. and Vandamme, P. (2000): Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS Microbiology Letters 193: 89–94.

Jayana, L.; Prasai, T.; Singh, A. and Yami, K.D. (2010): Study of antimicrobial activity of lime juice against Vibrio cholerae. Scientific World, 8, (8) 44–46.

Kabeya, H.; Kobayashi, Y.; Maruyama, S. and Mikami, T. (2003): One step Polymerase chain reaction-based typing of Arcobacter species. Int. J. Food Microbiol., 81: 163-168.

Kayman, T. (2012): Arcobacter Cinsi: Genel Özellikleri, Epidemiyoloji ve Laboratuvar Tanısı. Türk Mikrobiyol Cem Derg 42(2):     43-50.

Kayman, T.; Abay, S.; Hizlisoy, H.; Atabay, H.I.; Diker, K.S. and Aydin, F. (2012): Emerging pathogen Arcobacter spp. in acute gastroenteritis: molecular identification, antibiotic susceptibilities and genotyping of the isolated arcobacters. J. Med. Microbiol. 61: 1439-44.

Lee, S.K.; Mei, L. and Decker, E.A. (1997): Influence of sodium chloride on antioxidant enzyme activity and lipid oxidation in frozen ground pork. Meat Science. 46: 349–355.

Lee, S.; Decker, E.A. and Faustman, C. (2005): The effects of antioxidant combinations on color and lipid oxidation in n-3 oil fortified ground beef patties. Meat Science, 70(4): 683-689.

Logan, E.F.; Neill, S.D. and Mackie, D.P. (1982): Mastitis in dairy cows associated with an aerotolerant Campylobacter. Vet. Rec. 110: 229–230.

Madril, M.T. and Sofos, J.N. (1985): Antimicrobial and functional effects of six polyphosphates in reduced sodium chloride comminuted meat products. Lebensmittel- Wissenschaft- und -Technologie, 18(5): 316−322.

Mandisodza, O.; Burrows, E. and Nulsen, M. (2012): Arcobacter species in diarrhoeal faeces from humans in New Zealand. N Z Med. J. 125:    40-46.

McClure, P.J.; Roberts, T.A. and Oguru, P.O. (1989): Comparison of the effects of sodium chloride, pH, and temperature on the growth of Listeria monocytogenes on gradient plates and in liquid medium. Lett. Appl. Microbiol., 9: 95-99.

Milesi, S. (2010): Emerging pathogen Arcobacter spp. in food of animal origin. Doctoral Program in Animal Nutrition and Food Safety, Graduate School of Veterinary Science For Animal Health  And Food Safety ersità degli Studi di Milano.

Miller, A.J.; Call, J.E. and Whiting, R.C. (1992): Comparison of organic acid salts for Clostridium botulinum control in an uncured turkey product. J. Food Prot. 56: 958-962.

Miller, A.J.; Smith, J.L. and Buchanan, R.L. (1998): Factors affecting the emergence of new pathogens and research strategies leading to their control. J. Food Saf. 18:243-263

Nanasombat, S. and Chooprang, L. (2009): Control of Pathogenic Bacteria in Raw Pork using Organic Acid Salts in Combination with Freezing and Thawing. Kasetsart J. (Nat. Sci.) 43: 576–583.

Nieva-Echevarria, B.; Martinez-Malaxetxebarria, I.; Girbau, C.; Alonso, R. and Fernandez-Astorga, A. (2013): Prevalence and genetic diversity of Arcobacter in food products in the North of Spain. J. Food Prot. 76: 1447–1450.

O¨ngo¨r, H.; Cetinkaya, B.; Acik, M.N. and Atabay, H.I. (2004): Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey. Letters of Applied Microbiology 38: 344-39.

On, S.L.; Jensen, T.K.; Bille-Hansen, V.; Jorsal, S.E. and Vandamme, P. (2002 :( Prevalence and diversity of Arcobacter spp. isolated from the internal organs of spontaneous porcine abortions in Denmark. Veterinary Microbiology 85, 159–167.

On, S.L.; Stacey, A. and Smyth, J. (1995): Isolation of Arcobacter butzleri from a neonate with bacteremia. J. Infect., 31: 225-227.

Otth, L.; Wilson, T.M.; Cacino, R. and Fernandez, H. (2004): In vitro susceptibility of Arcobacter butzleri to six antimicrobial drugs. Arch. Med. Vet. XXXVI, 2,: 207-210.

Patya, A.; Rathore, R.S.; Mohan, H.V.; Dhama, K. and Kumar, A. (2011): Prevalence of Arcobacter spp. in Humans, Animals and Foods of Animal Origin Including Sea Food from India. Transboundary and Emerging Diseases .58, 5: 402–410.

Phillips, C.A. (1999): The effect of citric acid, lactic acid, sodium citrate and sodium lactate, alone and in combination with nisin on the growth of Arcobacter butzleri. Letters in Applied Microbiology 29: 424–428.

Queensland Government (2013): Food industry fact sheet, Appendix 2. Permitted additives for meat and meat products. www.health. qld.gov.au/foodsafety.

Ray, B. (1996): Fundamental Food Microbiology.CRC Press, New York. 516

Rivas, L.; Fegan, N. and Vanderlinde, P. (2004): Isolation and characterization of Arcobacter butzleri from meat. Int J Food Microbiol 91, 31–41.

Rohder, A.; Kleer, J. and Hildebrandt, G. (2007): Using microbiological analysis by Johnson & Murano and multiplex PCR by Harmon and Wesley for the identification of Arcobacter spp. in fresh poultry and beef sold in retail markets in Berlin. Arch Lebensmittel hygiene 58: 188–191.

Sallam, K.I. (2007): Antimicrobial and antioxidant effects of sodium acetate, sodium lactate and sodium citrate in refrigerated sliced salmon. Food Contr., 18: 566-575.

Sallam, K.I. and Samejima, K. (2004): Microbiological and chemical quality of ground beef treated with sodium lactate and sodium chloride during refrigerated storage. Lebenson Wiss Technol., 37: 865-871.

Scullion, R.; Harrington, C.S. and Madden, R.H. (2006): Prevalence of Arcobacter spp. in raw milk and retail raw meats in Northern Ireland. J. Food Prot 69: 1986–1990.

Shah, A.H.; Saleha, A.A.; Zunita, Z. and Murugaiyah, M. (2011): Arcobacter an emerging threat to animals and animal origin food products. Trends Food Sci. Technol. 22: 225–236.

Shah, D.; Shringi, S.; Besser, T. and Call, D. (2009): Molecular detection of foodborne pathogens, Boca Raton: CRC Press, In Liu, D. (Ed). Taylor & Francis group, Florida, USA, Pp. 369-389.

Son, I.M.D.; Engien, M.E.; Berrang, P.J.; Fedorka, C. and Harrison, M.A. (2010): Antimicrobial resistance of Arcobacter and Campylobacter from broiler carcasses. Int. J. Antimicrob. Agents, 29: 455-451.

SPSS (2007): Sample Power Statistic, SPSS, 12.01 Syntax Reference Guide for SPSS Base. SPSS Inc, 233South Wacker Drive, Chicago, IL.pp111-119.

Tan, W. and Shelef, L.A. (2002): Effects of sodium chloride and lactates on chemical and microbiological changes in refrigerated and frozen fresh ground pork. Meat Sci., 62: 27-32.

Tarrant, P.V. (1998): Some research advance and future priorities in research for the meat industry. Meat Science, 49 (Suppl 1), S1-S16.

Unver, A.; Ataby, H.I.; Şahin, M. and Eleb, Z. (2012): Antimicrobial susceptibilities of various Arcobacter species. Turk. J. Med. Sci. (2013) 43: 548-552.

USDA-FSIS ''United States Department of Agriculture, Food Safety and Inspection Service'' (2000): Food additives for use in meat and poultry products: sodium diacetate, sodium acetate, sodium lactate and potassium lactate: direct final rule. Federal Register 65: 3121–3123.

Van Driessche, E. and Houf, K. (2007): Characterization of the Arcobacter contamination on Belgian pork carcasses and raw retail pork. Int. J. Food Microbiol 118,   20–26

Vandenberg, O.; Dediste, A.; Houf, K.; Ibekwem, S.; Souayah, H.; Cadranel, S.; Douat, N. and Zissis, G. (2004): Arcobacter species in humans. Emerg Infect Dis 10: 1863–1867.

Vytrasova, J.; Pejchalova, M.; Harsova, K. and Binova, S. (2003): Isolation of Arcobacter butzleri and A. cryaerophilus in samples of meats and from meat processing plants by a culture technique and detection by PCR. Folia Microbiol., 48(2): 227-232.

Wesley, I.V. (1995): Oligonucleotide probes for the genus Arcobacter and for Arcobacter butzleri based on 16SrRNA sequence. J. Clin. Microbiol. 77: 1691-1698.

Wesley, I.V. (1996): Helicobacter and Arcobacter species: risks for foods and beverages. J. Food Prot. 59:1127-1132.

Wesley, I.; Schroeder, L.; Baetz, A.; Dewhirst, F. and Paster, B. (1995): Arcobacter species and Arcobacter butzleri species 16 rRNA-based DNA probes. J. Clin. Microbiol., 33:         1691-1698.

Woods, G.L. and Washington, J.A. (1995): Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Murray, P.R.; Baron, E.J.O.; Pfaller, M.A.; Tenover, F.C. and Yolken, R.H., editors. Manual of Clinical Microbiology. WashingtonDC: ASM Press; p.1327–416.

Yan, J.J.; Ko, W.C.; Huang, A.H.; Chen, H.M.; Jin, Y.T. and Wu, J.J. (2000): Arcobacter butzleri bacteremia in a patient with liver cirrhosis. J. Formos. Med. Assoc., 99: 166-169.