High Prevalence of Pseudomonas Species in Soil Samples from Ternate Island-Indonesia
Noura, K.M. Salih,
In the present study, Ten soil samples were examined and the pH of the soil was recorded. For bacterial isolation, a sterile nutrient and blood agars were used. Gram stain and biochemical tests were done for identification. A total of 384 genus were isolated, 314 (81.8%) were identified as Pseudomonas species of which 245 (78.0%) were Pseudomonas aeruginosa, 42 (13.4%) were Pseudomonas fluorescens, 13 (4.2%) were Pseudomonas mallei, 10 (3.1%) were Pseudomonas putida and 4 (1.3%) were Pseudomonas syringe and are regarded as pathogenic and harmful to man, animal and plants. This study shows that Pseudomonas aeruginosa had a high adaptation capability to grow in soil samples from Ternate, Indonesia. The rest of the bacterial isolates (18.2%) were identified as follows: 24 samples (6.2%) were Micrococcus, 23 samples (6.0%) were E. coli, 12 samples (3.1%) were Pasteurella and 11 samples (2.9%) were Staphylococcus. Pencillium was also isolated.
Soil samples from Ternate Island, Indonesia has a very high percentage of Pseudomonads,
one of them is Pseudomonas fluorescens nonpathogenic saprophytes that
has a proper role in bio-control and has been applied directly to soils as a
way of preventing the growth or establishment of crop pathogens such as P.
fluorescens strains (CHAO or Pf-5) which induce systemic resistance in the
host plant, (Haas and Defago, 2005). The Pseudomonas
fluorescens produce pyoverdin (fluorescein) pigment, particularly
under conditions of low iron availability. This pigment is a soluble, bluish-green
fluorescent pigment that led to the group's name (Meyer et
al., 2002). Certain Pseudomonas species may also produce additional
types of siderophore, such as pyocyanin by Pseudomonas aeruginosa (Lau
et al., 2004) and thioquinolobactin by Pseudomonas fluorescens
(Matthijs et al., 2007). These bacteria are
generally obligate aerobes; however, some strains can utilize NO3
instead of O2 as an electron acceptor. They have simple nutritional
requirements; they grow well in mineral salts media supplemented with any of
a large number of carbon sources. Some researches seek make use of Pseudomonas
fluorescens to partially or completely degrade pollutants such as styrene,
TNT and polycyclic aromatic hydrocarbons. Several strains of this bacteria also
have the ability to suppress plant diseases by protecting the seeds and roots
from fungal infection (DOE). This ability is due to secondary metabolites produced
by these bacteria such as antibiotics, siderophores and hydrogen cyanide as
well as the ability of these bacteria to rapidly colonize the Rhizosphere and
out-compete some of pathogens (DOE).
The other species of Pseudomonads are the most pathogenic types which
threatens the general health especially the patients that has been hospitalized
for long period of time, the swimmers in common swimming pools, human with compromised
host defense mechanisms, animals and also plants so they should be aware of
its high fatality which is 50%. Pseudomonas bacteria can be found in
many different environments such as soil, water and plant and animal tissue.
The species of these bacteria are 80% of the opportunistic pathogens that affect
humans (Brown, 1975), animals and plants. Pseudomonas
aeruginosa infection is a serious problem in patients hospitalized with
cancer, cystic fibrosis and burns; the case fatality is 50%. Other infections
caused by 5Pseudomonas species include endocarditis, pneumonia and infections
of the urinary tract, central nervous system, wounds, eyes, ears, skin and musculoskeletal
system. Pseudomonas aeruginosa, called the epitome of opportunistic pathogens,
almost never infects uncompromised tissues; however, it can infect practically
any type of tissue if that tissue has some type of compromised defenses (Kenneth,
2004). Pseudomonas mallei affect animals specially horses and cause
a serious disease called melioidosis and Glanders, respectively, Pseudomonas
syringe is a plant pathogen.
Because of their widespread occurrence in nature, the pseudomonads were
observed early in the history of microbiology it means 'false unit', being derived
from the Greek pseudo (ψευδo false) and Monas (μovάς
/μovάςδα a single unit). The term monad was used in
the early history of microbiology to denote single-cell organisms. Gram-negative,
rod-shaped, 0.5-0.8x1-3 μm, non-spore forming and polar-flagella bacteria
(Cornelis, 2008). Some species of these bacteria, such
as Pseudomonas aeruginosa, are opportunistic pathogens that secrete extra
cellular proteases and adhere and invade host tissue (Ryan
and Ray, 2004).
Pseudomonas species normally inhibit in soil, marshes, coastal marine and can be isolated from the skin, throat and stool of healthy persons, the plant and animal tissue. Spread is via contact with fomites or by ingestion of contaminated food and water. Generally, these bacteria can tolerate a variety of physical conditions.
Since, the mid 1980s Pseudomonads have been applied to cereal seeds
or direct to the soil as a way of preventing the growth or establishment of
crop pathogens e.g. Pseudomonas fluorescens which induce systemic resistance
to the plant host or might contend other pathogenic soil microbes (Hass and
Defago, 2005). Pseudomonas chlororaphis produce a phenazine type of antibiotic
which is active against certain fungal plant pathogens (Chin-A-Woeng
et al., 2000). Some species are able to metabolize pollutants in
the environment and as a result can be used as a bioremediation, e.g., Pseudomonas
alcaligenes degrades polycyclic aromatic hydrocarbons (OMahony
et al., 2006), Pseudomonas mendocina which degrades toluene
(Yen et al., 1991), Pseudomonas veronii which
degrades a variety of simple aromatic organic compounds (Nam
et al., 2003; Onaca et al., 2007),
Pseudomonas pseudoalcaligenes which is able to use cyanide as a nitrogen
source (Huertas et al., 2006) and Pseudomonas
putida which has the ability to degrade organic solvents such as toluene
of high capability to convert morphine in aqueous solution into hydromorphone
which is the stronger and somewhat expensive to manufacture drug (Marques
and Ramos, 1993). Among the fluorescent species of Pseudomonads,
Pseudomonas fluorescens is the most important as a biocontrol and bioremediation
and out of many researches done few of them refer to Pseudomonas fluorescens
in relation to the pathogenic strains.
Pseudomonas aeruginosa is common in human with compromised host defense
mechanisms and is the most common pathogen isolated from patients who have been
hospitalized longer than one week (Qarah and Cunha, 2003).
It is common for these bacteria to cause nosocomial infections like pneumonia,
urinary tract infections and bacteraemia (Cornelis, 2008).
Infections caused by Pseudomonas can become complicated and may even
be life threatening. It is both invasive and toxigenic and has three stages
of infection: the first is bacterial attachment and colonization, the second
is local infection and the third is bloodstream dissemination and systemic disease.
The bacteria produce extra cellular proteases that assist adherence and invasion
and are important in the organism's virulence. Pseudomonas frequently
cause out breaks of Pseudomonas dermatitis. This is a self-limiting rash
about two weeks duration, often associated with swimming pools and pool
type saunas and hot tubes. When many people use these facilities, the alkalinity
rises and the chlorine become less effective, at the same time, the concentration
of the nutrients that support the growth of Pseudomonads increases. Hot
water causes hair follicles to dilate, facilitating the entry of bacteria. Competition
of swimmers is often troubled with otitis externa, or swimmers ear, a
pseudomonad infection of the external ear canal leading to eardrum. Pseudumonas
aeroginosa is also a very common and serious opportunistic pathogen in burn
patients causes blue-green bus this color is caused by the bacterial pigments
(pyocyanin) (Roger et al., 1988).
This study carries one objective that is to isolate and characterize the bacteria in the soil samples from Ternate Island, East Indonesia.
MATERIALS AND METHODS
This study was conducted in the School of Bioscience and Biotechnology, Faculty of Science and Technology, UKM, Malaysia during the years 2007-2008.
Soil samples were collected from Indonesia (Ternate Island) from different
areas of grasslands, non-agricultural soil; 50 mL of each sample were ground
and passed through a 0.5 mm sieve and dissolved in 50 mL sterile distilled water,
mixed throughly then left for 2 h then centrifuged at 3000 rpm for 10 min. One
milliliter of the samples was diluted into eight serial tubes of 9 mL distilled
water, pre-inoculation in blood agar plates or eosin-methyl thionine blue agar
or nutrient agar and incubated at 37°C for 24 h. Gram negative bacilli were
identified according to the standard biochemical methods and Bergys Manual
of Determinative Bacteriology Holt et al. (1994)
and Meyer et al. (2002). Especially the presence
of flagella, inability to ferment lactose, the oxidase and the catalase tests,
methyl-red test, glycogen hydrolysis, arginine dihydrolase and some produce
insoluble pigments were also examined. The pH of the soil was determined by
using pH meter (DEITA 320).
RESULTS AND DISCUSSION
The bacterial isolates from Ternate Island soil-Indonesia were identified as follows: 314 isolates (81.8%) were Pseudomonads; 24 isolates (6.2%) were Micrococcus; 23 isolates (6.0%) were E. coli; 12 isolates (3.1%) were Pasteurella and 11 isolates (2.9%) were Staphylococcus (Table 1).
The total of (314) isolates of Pseudomonads were isolated from 10 soil samples, with an average of 31 isolates per sample approximately. A high percentage of Pseudomonas aeruginosa (78.0%) was found within the total of species of Pseudomonads isolated.
Five species of the Pseudomonads were isolated e.g., Pseudomonas aeruginosa (78.0%), Pseudomonas fluorescens (13.4%), Pseudomonas mallei (4.2%), Pseudomonas putida (3.1%) and Pseudomonas syringe (2.9%) (Table 2). The pH of the soil samples varies from 5.2 to 8.7 with an average of 6.9.
The Psedomonas species were isolated in Blood agar (Fig. 1) colonies were white, round, convex, glistening and non-haemolytic and when cultured in Nutrient agar (Fig. 2) the colonies were mucoid, flat, grayish and lobulated, after 24 h of incubation at 37°C.
In the present study a beneficial bacteria to biotechnology and bio-control
in the soil samples from Ternate Island, East Indonesia was isolated. A high
prevalence of Pseudomonas species were found, with a high average when
compared with other genera of bacteria isolated from the same samples.
||The percentage of different Bacterial isolates from Ternate
Island soil samples
||The percentage of different Pseudomonads species in
Ternate Island soil samples
This may be due to moisture and warmness of the soil characteristics. A microbial
richness was also observed such as Micrococcus, E. coli, Pasteurella
Some species of Pseudomonas has been recently used as a bio-control and use as bioremediation which is able to clear the environmental pollution and improve the hygienic measures and partially or completely degrade pollutants such as styrene, TNT and polycyclic aromatic hydrocarbons. Several strains of this bacteria also have the ability to suppress plant diseases by protecting the seeds and roots from fungal infection (Hass and Defago, 2005).
Other species were described as a toxigenic (produce exo and endo-toxins) and
cause 80% of the diseases that affect man, animals and plants. A high percentage
of Pseudomonads were isolated from the soil samples, the reason of this
high prevalence of Pseudomonads is unknown, but we considered that the
physical, chemical or micro-biological characteristics of the grasslands and
non-agricultural soil may affect (Coburn et al.,
The pH does not affect on the presence of Pseudomonas in the soil because
the pH had a broad range (5.2-8.7) with a medium of 6.9 closely related to the
optimum neutral pH for the growth of Pseudomonas. In conclusion, this
study demonstrates the presence of at least 5 species of Pseudomonads, one
of them is Pseudomonas fluorescens which has a very effective role in
bio-control and bioremediation. The other four species are the most pathogenic
ones e.g. Pseudomonas aeruginosa, Pseudomonas mallei, Pseudomonas
putida and Pseudomonas syringe with high percentage. We believe that
this study gives a possible explanation of Pseudomonas related diseases
in Ternate Island and will highlight the steps needed for the spread of pathogenic
strains of Pseudomonas, which can best be controlled by observing proper
isolation procedures, aseptic technique and careful cleaning and monitoring
of all of the instruments in hospitals, the swimming pools for the efficiency
of chlorine. Topical therapy of burn wounds with antibacterial agents such as
mafenide or silver sulfadiazine, has dramatically reduced the incidence of Pseudomonas
aeruginosa (Cross et al., 1980).
Pseudomonas is frequently resistant to many commonly used antibiotics, as penicillin and the majority of related beta-lactam antibiotics, although many strains are susceptible to piperacillin, imipenem, gentamicin, tobramycin, colistin, amikacin, or ciprofloxacin. Resistant forms have developed, making susceptibility testing essential. The combination of gentamicin and carbenicillin is frequently used to treat severe Pseudomonas infections, especially in patients with leukopenia. Several types of vaccines are being tested, but none is currently available for general use.
This study was supported by the University of Bahar Alghazal (UBG), Sudan and Universiti Kebangsaan Malaysia (UKM), Malaysia, Grant No. STGL 003-2008. The authors wish to thank Mr. Ratuah Mohammed the senior technician, for his cooperation and kindness.
1: Brown, M.R.W., 1975. The Role of the Cell Envelope in Resistance of Pseudomonas aeruginosa. John Wiley, Chichester, pp: 71-107.
2: Cornelis, P., 2008. Pseudomonas: Genomics and Molecular Biology. 1st Edn., Caister Academic Press, United Kingdom, ISBN: 978-1-904455-19-6.
3: Chin-A-Woeng, T.F.C., G.V. Bloemberg, I.H.M. Mulders, L.C. Dekkers and J.J. Ben, 2000. Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol. Plant Microbe Interact., 13: 1340-1345.
CrossRef | Direct Link |
4: Haas, D. and G. Defago, 2005. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat. Rev. Microbiol., 3: 307-319.
CrossRef | PubMed | Direct Link |
5: Holt, J.G., N.R. Kreig, P.H.A. Sneath, J.T. Staley and S.T. Williams, 1994. Bergey's Manual of Determinative Bacteriology. 9th Edn., Lippincott Williams and Wilkins, Baltimore, USA., ISBN-13: 9780683006032, Pages: 787.
6: Huertas, M.J., V.M. Luque-Almagro, M. Martinez-Luque, R. Blasco, C. Moreno-Vivián, F. Castillo and M.D. Roldán, 2006. Cyanide metabolism of Pseudomonas pseudoalcaligenes CECT5344: Role of siderophores. Biochem. Soc. Trans., 34: 152-155.
PubMed | Direct Link |
7: Lau, G.W., D.J. Hassett, H. Ran and F. Kong, 2004. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends Mol. Med., 10: 599-606.
8: Marques, S. and J.L. Ramos, 1993. Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways. Mol. Microbiol., 9: 923-929.
9: Matthijs, S., K.A. Tehrani, G. Laus, R.W. Jackson, R.M. Cooper and P. Cornelis, 2007. Tioquinolobactin a Pseudomonas siderophore with antifungal and anti-Pythium activity. Environ. Microbiol., 9: 425-434.
10: Meyer, J.M., V.K. Geoffroy, N. Baida L. Gardan and D. Izard et al., 2002. Siderophores typing: A powerful tool for the identification of fluorescent and non-fluorescent Pseudomonads. Applied Environ. Microbiol., 68: 2745-2753.
PubMed | Direct Link |
11: Nam, I.H., Y.S. Chang, H.B. Hong and Y.E. Lee, 2003. A novel catabolic activity of Pseudomonas veronii in biotransformationof pentachlorophenol. Applied Microbiol. Biotechnol., 62: 284-290.
PubMed | Direct Link |
12: Cross, A.S., J.C. Sadoff, B.H. Iglewski and P.A. Sokol, 1980. Evidence for the role of toxin A in the pathogenesis of infections with Pseudomonas aeruginosa in human. J. Infect. Dis., 142: 538-546.
Direct Link |
13: O’Mahony, M.M., A.D. Dobson, J.D. Barnes and I. Singleton, 2006. The use of ozone in the remediation of polycyclic aromatic hydrocarbon contaminated soil. Chemosphere, 63: 307-314.
14: Onaca, C., M. Kieninger and K.H. Engesser, 2007. Degradation of alkyl methyl ketones by Pseudomonas veronii. J. Bacteriol., 189: 3759-3767.
15: Qarah, S. and B.A. Cunha, 2003. Pseudomonas aeruginosa infection. At E. medicine > Infectious Diseases > Medical Topics. http://emedicine.medscape.com/article/226748-overview.
16: Roger, Y.S., J.L. Ingraham, M.L. Wheelis and P.R. Painter, 1988. General Microbiology. 5th Edn., Macmillans Publishers, UK., ISBN: 0-333-41768-2.
17: Ryan, K.J. and C.G. Ray, 2004. Sherris: Medical Microbiology. 4th Edn., McGraw Hill, UK., ISBN: 0838585299.
18: Kenneth, T., 2004. Textbook of bacteriology. Pseudomonas aeruginosa, University of Wiscons-Madison 53706. Department of Bacteriology.
19: Coburn, J., R.T. Wyatt, B.H. Iglewski and D.M. Gill, 1989. Several GTP-binding proteins, including o24 C-H-ras, are preferred substrates of Pseudomonas aeruginosa exoenzymes. S. J. Biol. Chem., 264: 9004-9004.
20: Yen, K.M., M.R. Karl and L.M. Blatt, 1991. Cloning and characterization of a Pseudomonas mendocina KRI gene cluster encoding toluene-4-monooxygenase. J. Bacteriol., 173: 5315-5327.