In vitro antimicrobial activity of some plant extracts against Arcobacter butzleri and Arcobacter cryaerophilus

Correspondence to: Dr. Yemisi Adesiji, Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology, Ogbomoso, Nigeria. E-mail: drmrsadesiji@yahoo.com Background: There are increasing reports of resistance to current antibiotics employed in treatment of Arcobacter related infections. Materials and Methods: Carica papaya, Vernonia amygdalina, Ocimum gratissimum and Momordica charantia were tested against A. butzleri reference strain (ABSH3-1137) and A. cryaerophilus isolated from pig and chicken using disk diffusion technique. Results: The aqueous extract of C. papaya inhibited the growth of A. butzleri at inhibitory dose (ID) of 0.5 mg and the two A. cryaerophilus at ID of 1 mg while the methanol extract did not inhibit the growth of any strains. The aqueous and methanol extracts of V. amygdalina inhibited the growth of A. cryaerophilus isolated from pig and chicken at ID of 10 mg but did not inhibit A. butzleri. The methanol extracts of O. gratissimum inhibited A. butzleri and A. cryaerophilus pig isolate at ID of 2.5 mg and the chicken isolate at ID of 1 mg. The aqueous extracts of O. gratissimum inhibited A. butzleri, A. cryaerophilus of chicken isolate at ID of 5 mg, and A. cryaerophilus of pig isolate at 2.5 mg. The methanol extracts of M. charantia had ID of 2.5 mg for A. butzleri and A. cryaerophilus pig isolate and ID of 1 mg for A. cryaerophilus chicken isolate while the aqueous extracts had ID of 5 mg for all the three strains tested. Conclusions: The result obtained from this study showed that both aqueous and methanol extracts of four commonly used herbs in Nigeria inhibited the growth of Arcobacter species to a varying degree with the aqueous extract of C. papaya leaves reported in vitro highest antimicrob ial activity and thereby may serve as potential sources for new antimicrob ial agents against Arcobacter.


INTRODUCTION
The genus Arcobacter was described in 1991 as a second genus within the family Campylobacteriaceae to encompass bacteria formerly known as aerotolerant Campylobacter (1).Arcobacter species are considered to be emerging pathogens with A. butzleri, A. cryaerophilus and A. skirrowii being linked to disease in humans (2).Arcobacters have been isolated from diarrhoeal and bactereamic cases of which most common symptoms include watery diarrhoea, abdominal pain, nausea and vomiting (3).They have also been detected in drinking water (4), poultry meat (5), beef and pork (6).In two surveillance studies from Belgium and France, Arcobacter was identified as the fourth most commonly isolated Campylobacter-related organism in diarrhoea illness in humans (7).
The spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases (5), and reports have shown that Arcobacter appears to be resistant to antimicrobial agents typically used in the treatments of diarrhea illnesses caused by Campylobacter species.However, some chemical preservatives, spices and herbs were shown to possess anti-infective properties against some strains of Arcobacter (8).
In view of increasing reports of resistance to current antibiotics employed in treatment of Arcobacter related infections, there is need to develop new or alternative antimicrobial agents effective against Arcobacter.In Nigeria, extracts of Ocimum gratissimum, Morinda lucida, Carica papaya, Phyllanthus amarus and Citrus aurantifolia are among the folkloric herbal remedies that have been scientifically confirmed to possess anti-diarrhoeal, anti-bacterial and antiprotozoan properties (9,10).Most especially, ethanolic preparation of C. papaya and M. lucida has been shown to possess tremendous anti-infective potentials against Nigerian isolates of Helicobacter pylori (9), a related organism to Arcobacter.
Hence this study was undertaken to assess the antimicrobial activity of four prevalent herbal plants in Nigeria, C. papaya, V. amygdalina, O. gratissimum and M. charantia, against 2 species of Arcobacter: A. cryaerophilus and A. butzleri.

Arcobacter test organism
A. butzleri clinical isolate (ABSH3-1137) and 2 field isolates of A. cryaerophilus were obtained from healthy pigs (11) and chickens (12).Three Arcobacter strains in this study were confirmed by multiplex PCR assay that amplified the 401-bp fragment of A. butzleri and the 257-bp fragment of A. cryaer-ophilus (13), and they were maintained in stock cultures at minus/-25 °C in glycerol Arcobacter broths at the Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Nigeria.

Preparation of Arcobacter inoculum
Arcobacter subcultures were first prepared from the stock cultures on Brain Heart Infusion (BHI) agar supplemented with 5% yeasts and 7% sheep blood.BHI agar plates were incubated at 37 °C in microaerophilic atmosphere (1).Arcobacter inoculum was prepared by collecting bacteria colonies from BHI agar plates at the exponential growth phase and diluting in 0.85% saline.The resulting bacteria suspension was then standardized by McFarland nephelometry to 10 5 CFU/ml.

Plant materials and extract preparation
C. papaya (Pawpaw leaf), V. amygdalina (Bitter leaf), O. gratissimum (Basil leaf: efirin) and M. charantia (Coral leaf: ejirin) were obtained from various locations in Osogbo, Nigeria and authenticated by a botanist in Obafemi Awolowo University, Ile-Ife, Nigeria.The leaves of each plant were cut into pieces and ground into powder using a sterile electric blender.Ten (10) grams of the powder were steeped in 100 ml distilled water and another 10 g in 100 ml methanol with thorough shaking at regular intervals at room temperature for a few days.The resulting crude extracts were filtered using Whatman No 1 filter paper (Oxoid, UK) and allowed to dry in an oven to obtain a residue, and each was labeled as the crude extract of an individual plant.For the aqueous extract of each plant, 2.5 mg/ml, 5 mg/ml, 10 mg/ml, 25 mg/ml and 50 mg/ml concentrations were prepared with distilled water in sterile bottles while the similar concentrations of the methanolic extracts were reconstituted with phosphate buffer saline (pH 7.2) to nullify the effect of methanol on the organisms (10).

Preparation of herb disks
Disks for antimicrobial sensitivity tests were prepared by the modification of the Gould and Bowie method (14).Briefly, a 6.25 mm diameter plunger was used to punch Whatman No 1 absorbent filter paper to obtain 6.25 mm diameter paper disks.The disks were labeled, dispensed in screw-capped bottles and sterilized at 40 °C in a hot air oven for 1 hour.
Under aseptic conditions, 1 ml of extracts of each herbal plant at various concentrations (2.5 mg/ml, 5 mg/ml, 10 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml) were added to 10 prepared disks arranged side by side inside a sterile Petri dish for each extract to prepare disks of approximately 0.25 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg and 10 mg, respectively.
The Petri dishes were placed in the hot air oven at 40 °C for 5 minutes to allow the disks to dry.The impregnated disks were then stored in a sterile screw-capped container with a cap tightly screwed, and the container was kept at 4 °C in a refrigerator until use.

Susceptibility testing procedure
Susceptibility testing of the organism to each plant extract was done in triplicate using the modified Kirby Bauer single disk principle (15).The disks were placed on the surface of the agar plate previously seeded with Arcobacter and incubated in microaerophilic environment at 37 °C for 24 hours (16).A sterile disk impregnated with sterile phosphate buffer served as a negative control while standard disks of nalixidic acid (30 μg), cephalothin (30 μg) and erythromycin (15 μg) served as a positive control.The zone diameter of inhibition around the disk was measured with a caliper.Interpretation of the zone diameter of inhibition as sensitive or resistant to nalidixic acid, cephalothin and erythromycin was based on the interpretive criteria of the Clinical and Laboratory Standards Institute (16).

RESULTS
The antimicrobial activity of the four herbal extracts (Table 1) against the Arcobacter species is summarized in Table 2.The aqueous and methanol extracts of V. amygdalina inhibited the growth of A. butzleri and A. cryaerophilus (chicken) but methanol extracts did not inhibit the growth of A. cryaerophilus (pig) at ID of 10 mg.
The aqueous and methanol extracts of V. amygdalina did not inhibit the growth of A. butzleri but inhibited the growth of two A. cryaerophilus at ID of 10 mg.The methanol extracts of O. gratissimum inhibited A. butzleri reference strain and A. cryaerophilus pig isolate at ID of 2.5 mg and the chicken isolate at ID of 1 mg while the aqueous extracts inhibited A. butzleri and A. cryaerophilus chicken isolate at ID of 5 mg, and A. cryaerophilus pig isolate at 2.5 mg.The methanol extracts of M. charantia had ID of 2.5 mg for A. butzleri and A. cryaerophilus pig isolate and ID of 1 mg for A. cryaerophilus chicken isolate while the aqueous extracts had ID of 5 mg for all the three strains.The positive control for this experiment using cephalothin, erythromycin and nalixidic acid is given in Table 3.In general, the methanol extracts showed greater antibacterial activity than the aqueous extracts and the two A. cryaerophilus field isolates from pig and chicken were resistant to nalixidic acid and cephalothin but susceptible to erythromycin.The A. butzleri reference strain (ABSH3-1137) was resistant to cephalothin and erythromycin but susceptible to nalixidic acid.

DISCUSSION
The use of herbal extracts as alternative medicine and natural therapies has been documented for ages.In Nigeria, more than 70% of the populace depend on various forms of plant extracts used as herbal concoctions for treatment of many ailments including infectious diseases (17).The result obtained from this study showed that both aqueous and methanol extracts of four commonly used   herbs in Nigeria inhibited the growth of Arcobacter to a varying degree.The aqueous and methanol extracts of V. amygdalina appeared to have the least antimicrobial potential at an inhibitory dose of 10 mg of the leaf extract although its root has been de monstrated to contain a proven antimicrobial activity on micro-organisms causing gingivitis, despite the fact that methanol extracts are more efficient substances that are related to antimicrobial activity of various herbs (18).The aqueous extract of leaves of C. papaya in this study showed the highest antimicrobial activity while the methanol extract did not have inhibitory activity on any of the three Arcobacter strains.This finding may be attributed to the high phenolic compound in C. papaya leaf which is responsible for its antimicrobial activity (19), and which appears better extracted with water than methanol.C. papaya has been shown to possess several biological functions with its seeds having demonstrable anti-fertility effect in rats (20) and its leaf with anti-H.pylori activity in-vitro (9).Our result is an extension of many biological activities of this herb.Both aqueous and methanol extracts of O. gratissimum exerted relatively mild antimicrobial activity on the Arcobacter strains in the study.O. gratissimum has been shown to possess antioxidant property (21) and its leaf has been demonstrated both in-vitro and in-vivo to inhibit aetiologic agents of diarrhoea (22).The anti-H.pylori activity of O. gratissimum was also demonstrated in the study by Smith et al. (9).The present findings in this study are in agreement with those of others and indicate that O. gratissimum may be a candidate herb that needs to be further studied to characterize the antiinfective ingredients present in the plant.
M. charantia leaf extracts demonstrated moderate antibacterial activity on the Arcobacter strains in this study although there are no previous reports of the antimicrobial property of this herb.However, the plant has been reported to possess immunostimulatory and anti-diabetic activity (24), and our findings revealed that this plant may possess additional anti-infective properties on Arcobacter and probably other related bacteria.
Although the ethanolic acid extraction me thod has been shown to yield more active ingredients from medicinal plants (25), both water and methanol extracted the anti-Arcobacter property of V. amyg dalina, O. gratissimum and M. charantia in this study with methanol extraction appearing more effective.However, aqueous extraction was the only effective method for the anti-Arcobacter property of C. papaya leaf.This sharply contrasts the study of Smith et al. (9), where ethanolic and not aqueous extraction method was effective for anti-H.pylori activity of C. papaya and M. lucida.The A. butzleri reference strain was resistant to erythromycin and cephalothin while the two A. cryaerophilus field isolates demonstrated resistance to nalidixic acid and cephalothin.These three antibiotics are known to be effective against Campylobacter-like organisms, although there have been reports of field isolates of Arcobacter resistant to erythromycin (26).With the demonstration of resistance to erythromycin, nalidixic acid and cephalothin in this study, the need to develop new effective antibacterial agents becomes highly imperative.C. papaya, O. gratissimum and M. charantia possess anti-Arcobacter properties and may serve as potential sources for new antimicrobial agents effective against Arcobacter.

Table 1 .
Herbal extracts used in the study

Table 2 .
Zone inhibition of 4 plant extracts against Arcobacter strains

Table 3 .
Zone of inhibition of Arcobacter using selected antimicrobials