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IJAMBR 2 (2014) 79-85
ISSN 2053-1818
Microorganisms associated with urine contaminated
soils around lecture theatres in Federal University of
Technology, Akure, Nigeria
E. O. Dada and C. E. Aruwa*
Department of Microbiology, Federal University of Technology, P. M. B. 704, Akure, Nigeria.
Article History
Received 22 October, 2014
Received in revised form 17
November, 2014
Accepted 02 December, 2014
Key words:
Microorganisms,
Urine,
Public health,
Soil,
Lecture halls.
Article Type:
Full Length Research Article
ABSTRACT
Study was carried out to investigate microorganisms in urine contaminated soils
around lecture theatres within Federal University of Technology, Akure. Fortyeight soil samples were collected and analyzed using soil serial dilution and
pour plating techniques. Nutrient Agar was used for bacteria isolation and
subculture while Potato dextrose agar and Warcup method were used for fungi
isolation. Urine contaminated soil samples were collected from Biology
Department Lecture Theatre, University Diploma Lecture Theatre and
Microbiology Lecture Theatre. Bacteria isolated from these soils and their
prevalence include Bacillus species (31%), Corynebacterium species (24%),
Staphylococcus species (17%), Enterobacter cloacae (9%), Micrococcus species
(7%), Klebsiella liquefasciens (4%), Flavobacterium rigense (3%), Acinetobacter
anitratus (3%), and Alcaligenes eutrophus (2%). The predominant bacteria were
Bacilli, and the least was Alcaligenes eutrophus. Fungal isolates were
Aspergillus species (46%), Trichoderma viride (15%), Articulospora inflata (11%),
Varicosporium elodeae (9%), Rhizopus nigricans (7%), Gliocladium deliquescens
(6%), Penicillium notatum (4%) and Beauveria bassiana (2%). Predominant fungi
genus in soils from these sites was Aspergillus. Soil from Microbiology
Department area generally had the highest counts. In this study, the researchers
discussed the public health implications of persistent urination in public soils as
potential source of pollution to man and plants.
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INTRODUCTION
Soil is a natural cultural media for the growth of many
types of organism. The organic and inorganic matter in
the soil determines the soil fertility and aid the
proliferation of various micro flora that play vital roles in
maintaining the nutritional balance of the soil. The topsoil
has the highest concentration of organic matter and
microorganism and it is where most of the Earth’s
biological soil activity occurs. Hence, earth depends on
soil to a great extent, and as human population grows, its
depth, season of the year, state of the cultivation, organic
Corresponding author. E-mail: [email protected].
demand for food from crops increases, thereby making
soil conservation crucial. A few of the consequences of
human activity and carelessness are deforestation, overdevelopment, and pollution from man-made chemicals
and human wastes (Joanne et al., 2008).
Fungi and bacteria in the soil are the primary recyclers
of nutrients. The majority of microbial population is found
in the upper 6-12 inches of soil and the number
decreases with depth (Bridge and Spooner, 2001). The
number and kinds of organisms found in soil depend on
the nature of soil, matter, temperature, moisture, and
aeration. At the present time only about 17% of the
known fungal species can be successfully grown in
culture (Bridge and Spooner, 2001). Culturing fungi from
Int. J. Appl. Microbiol. Biotechnol. Res.
soil isolations will only result in the detection of those
propagules that are able to grow and sporulate on the
isolation medium used, and this greatly reduces the
measure of diversity (Bridge and Spooner, 2001). Various
species of bacteria thrive on different food sources and in
different microenvironments in the soil. No single cultural
environment or isolation media can provide adequate
nutrient and physical conditions satisfactory for the
growth of every viable cell (Elaine, 2009). Hence, each
culture media or environment will only support the growth
of cells that can adapt to its particular nutrient and
physical conditions. A wide variety of soil microflora are
yet be discovery. Hoglund et al., (2002) also opined that
much of the experience with bacteria involves disease.
Urine is the pale yellow fluid produced by the kidneys
and it contain urea, uric acid, minerals, chloride, nitrogen,
sulphur, ammonia, copper, iron, phosphate, sodium,
potassium, manganese, carbolic aid, calcium, salts;
vitamins A, B, C, and E; enzymes, hippuric acid, creatinine, as well as lactose. Other sugars are sometimes
excreted in urine, if their concentration in the body
becomes very high. Urea is abundant in the urine of
humans and other mammals (Drangert, 2000). The pH of
urine range between 4-8.
The bladder and urinary tract are usually sterile. The
urethra however, may contain a few commensals, and
also the perineum which can contaminate urine when it is
being passed out. Some of these commensals are
diphtheroids, enterobacteria, Acinetobacter species and
some skin commensals such as Gram-positive
staphylococci, micrococci, and Gram-positive enterococci. Female urine may be passed out along with some
normal microflora of the vagina (Cheesebrough, 2006).
Proper disposal of human waste is important to avoid
pollution and minimize the possibility of spreading
disease. Some possible effects of indiscriminate urination
are that, it is disgusting, damages property value, impacts
the quality of life for the people that have to live with the
stench, and it spreads diseases (Knuttson and KiddLjunggren, 2000; Hoglund et al., 2002).
This study was carried out within the institution
because of the problem of overstretched facilities, which
contributed to the practice of indiscriminate urination
around lecture theatres. The study was carried out while
the institution was in session.
The research was aimed at isolating and determining
the prevalence/percentage occurrence of microorganisms
isolated from urine contaminated soils and comparing
such with those of control soils. The prevalence of isolated microflora could serve as public health indicators.
MATERIALS AND METHODS
Soil collection was carried out according to Fawole and
Oso (2007). The sites used are the undesignated areas
80
for urine discharge. The experimental site is the soil area
noted for regular urine discharge, while a meter away
(not used for urine discharge) was chosen as the control.
A total of 48 samples were collected from the urine soil
contaminated site (8 samples each from 3 sites) and
control soil site (8 samples each from 3 sites) were
collected from the topsoil (between 5-20 cm),
respectively. Sample collection sites were the Microbiology Lecture Theatre, University Diploma Lecture
Theatre, and Biology Department Lecture Theatre.
Samples from contaminated soils and control area were
collected in separate sterile polythene bags, which were
sealed, respectively and were appropriately labelled
before they were transferred to Microbiology Laboratory
of Federal University of Technology, Akure for immediate
analysis (within 2 hrs of sample collection).
Sample preparation and isolation of bacteria and
fungi
Soil samples were subjected to microbiological analysis
using the method of Olutiola et al. (2001). Pour plating
was done using Nutrient agar (NA-Oxoid) for bacterial,
and Potato Dextrose Agar (PDA-Oxoid) for fungal
isolation. Techniques employed to reduce load and
prevent overcrowding of Petri plates are the soil dilution
for bacteria and; soil dilution and Warcup method for
fungi isolation. Culture media were prepared according to
manufacturer’s specification and sterilization of materials
was done in an autoclave at 121°C for 15 min.
Ten grams (10 g) of each soil sample was diluted in 90
ml of sterile distilled water, followed by 1st to 7th fold serial
dilution (10-3 to 10-7). One-tenth of a millilitre (1/10th ml) of
the 1st and 4th to 7th fold dilutions were plated out in
duplicates on NA-Oxoid and PDA-Oxoid media. NA
plates for bacteria were incubated at 35–37°C for 24 hrs,
and PDA plates for fungi were incubated at 25-27°C for
48–72 hrs. Using the Warcup method, 1 g of each soil
sample was directly plated and overlaid with media
(Nutrient agar and Potato dextrose agar). Bacterial
counts were recorded in colony forming units per ml
(cfu/ml), and fungal counts as spore forming units per
millilitre (sfu/ml–for serial dilution technique) and spore
forming units per gram (sfu/g–for the Warcup method).
Isolation and identification of bacterial and fungal
isolates
Distinct colonies of bacteria were purified by repeated
subculture on the respective isolation media, and
preserved on slants at 4°C according to Olutiola et al.,
(1991). Morphological and biochemical tests to identify
isolates were carried out using the methods of
(Fawole and Oso, 2001) and Bergey’s manual of
Dada and Aruwa
81
systematic bacteriology (Claus and Berkeley, 1986).
Biochemical tests carried out in the conventional method
include the following- Voges-Proskauer and Methyl-Red
test, fermentation of carbohydrate, hydrolysis of starch,
utilization of citrate, hydrolysis of casein, hydrolysis of
gelatin. Fungal isolates were subcultured using the same
isolation media and their identification made possible
using macroscopic and microscopic (stereomicroscope)
fungal features. Fungi were identified using the cottonblue in lactophenol method (Olutiola et al., 2001).
Determination of organism prevalence
Percentage occurrence was obtained by recording the
occurrence of each microorganism after identification
divided by total number of organism isolated. The fraction
obtained was then multiplied by a factor of 100.
RESULTS
Table 1 shows microorganism associated with each of
the soil sample sites. Microbiology Lecture Theatre urine
area had the widest array of bacteria and fungi
associated with it, followed by Biology Lecture Theatre
urine area and then University Diploma Lecture Theatre
area.
Table 2 shows the overall prevalence of microorganisms isolated from the urine contaminated and
control soils. The Bacillus species (31%) were most
prevalent, followed by the corynebacteria, and
staphylococci species. The genus Aspergillus species
(46%) were the most prominent among the fungal
species isolated.
Table 3 shows counts observed for bacteria and fungi
from urine contaminated and control soils, respectively.
Highest counts were observed for soil samples from
Microbiology lecture area (15×104 and 3×106 cfu/ml),
4
followed by the University diploma lecture area (11×10
6
and 2×10 cfu/ml). For the control sites, highest counts
were observed for soil samples from Microbiology control
area (48×104 cfu/ml), followed by Biology Department
lecture area (43×104 cfu/ml) and University diploma
lecture area (13×104 cfu/ml), respectively. Fungal counts
for soil samples from the Microbiology lecture area was
1
highest (17×10 sfu/ml), followed by the University
diploma (13×101 sfu/ml) and Biology (9×101 sfu/ml)
lecture areas. The same trend was observed for the
control areas where bacteria count ranged from 13–
4
1
48×10 cfu/ml, and fungi count ranged from 7–13×10
sfu/ml.
Table 4 shows the fungal counts obtained for Warcup
method. It was aimed at encouraging maximum
expression of all possible fungi present in samples
analysed. Comparatively for all sites, count range was
between 4 and 12 sfu/ml on Potato dextrose agar. The
highest count was obtained in soil from Microbiology
lecture area (11 sfu/g), followed by the Biology
Department lecture area (10 sfu/g), and the University
Diploma lecture area (7 sfu/g).
DISCUSSION
The presence of soil microorganisms isolated from soil
samples in this study is expected. The study has
revealed that soils from public urinals consist of
opportunistic microbial species of importance to human
and public health. Bacterial isolates obtained from
sampled soils in this study are in agreement with the
work of Madigan and Martinko (2005), and
Cheesebrough (2006). The presence of Acinetobacter
anitratus may not only be due to its being widely
distributed in soil, but also could be due to its being part
of the normal skin flora of humans, resulting from
throwing hand washed water from the laboratories unto
such urine polluted soils. This organism can however
cause urinary tract as well as wound infections,
abscesses and meningitis in debilitated humans
according to Cheesebrough (2006). Micrococcus luteus
though found in the sampled soils is also part of the
normal flora of the mammalian skin. This organism is
usually non-pathogenic and usually regarded as
contaminant, and could be considered as an emerging
nosocomial pathogen in immuno-compromised patients
and persons. The Flavobacterium species have been
associated with epidemic situations involving outbreaks
of meningitis, especially in hospitals. The majority of
Corynebacterium species are plant and animal
pathogens, with the exception of Corynebacterim
diphtheriae which is an animal pathogen only and it
causes diphtheria in humans. These observations are in
agreement with Cheesebrough (2006).
Micrococcus is a genus of bacteria and it occurs in a
wide range of environments including water, dust, and
soil. Micrococci have been isolated from human skin,
animal and dairy products, and beer. They are found in
many other places in the environment, including water,
dust, and soil. M. luteus is most common and is found in
nature and in clinical specimens. Micrococci can grow
well in environments with little water or high salt
concentrations. Though not a spore former, Micrococcus
cells can survive for an extended period of time
(Greenblat et al., 2004). Micrococcus is generally thought
to be a saprotrophic or commensal organism, though it
can be an opportunistic pathogen, particularly in hosts
with compromised immune systems, such as HIV
patients (Smith et al., 1999). It can be difficult to identify
Micrococcus as the cause of an infection, since the
organism is normally present in skin microflora, and the
genus is seldom linked to disease. In rare cases, deaths
Int. J. Appl. Microbiol. Biotechnol. Res.
82
Table 1. Microorganisms associated with urine contaminated soil and control soil samples.
Microbiology LA
Bacteria
Bacillus subtilis
Bacillus mycoides
Bacillus brevis
Bacillus sphaericus
Corynebacterium sp.
Microbiology CA
Biology LA
Biology CA
University diploma LA
University diploma CA
Bacillus subtilis
Bacillus mycoides
Bacillus brevis
Bacillus coagulans
Bacillus macerans
Bacillus subtilis
Bacillus coagulans
Bacillus brevis
Corynebacterium sp.
Staphylococcus sp.
Bacillus subtilis
Bacillus brevis
Bacillus sphaericus
Bacillus macerans
Bacillus coagulans
Bacillus subtilis
Bacillus coagulans
Bacillus brevis
Corynebacterium sp.
Staphylococcus albus
Bacillus subtilis
Bacillus brevis
Corynebacterium sp.
Bacillus macerans
Bacillus sphaericus
Staphylococcus sp.
Corynebacterium sp.
Micrococci
Enterobacter cloacae
Corynebacterium sp.
Staphylococcus epidermidis
Corynebacterium sp.
Micrococci
Enterobacter cloacae
Staphylococcus sp.
Klebsiella liquefasciens
Staphylococcus sp.
Micrococci
Enterobacter cloacae
Staphylococcus sp.
Flavobacterium rigense
Micrococci
Enterobacter cloacae
Micrococci
Flavobacterium rigense
Acinetobacter anitratus
Flavobacterium rigense
Flavobacterium rigense
Acinetobacter anitratus
Klebsiella liquefasciens
Alcaligenes eutrophus
Acinetobacter anitratus
Klebsiella liquefasciens
Klebsiella liquefasciens
Aspergillus niger
Gliocladium sp.
Beauveria bassiana
Penicillium sp.
Aspergillus sp.
Trichoderma viride
Articulospora inflata
Varicosporium elodeae
Klebsiella liquefasciens
Alcaligenes eutrophus
Fungi
Aspergillus sp.
Trichoderma viride
Articulospora inflata
Varicosporium elodeae
Gliocladium sp.
Beauveria bassiana
Penicillium sp.
Rhizopus sp.
Aspergillus sp.
Trichoderma viride
Articulospora inflata
Varicosporium elodeae
Gliocladium sp.
Penicillium sp.
Aspergillus sp.
Articulospora inflata
Varicosporium elodeae
Gliocladium sp.
Penicillium sp.
Rhizopus sp.
Aspergillus fumigatus
Aspergillus niger
Articulospora inflata
Gliocladium sp.
Rhizopus sp.
LA, Lecture area; CA, Control area.
of immuno-compromised patients have occurred
from
pulmonary
infections
caused
by
Micrococcus. Micrococci may be involved in other
infections, including recurrent bacteraemia, septic
shock, septic arthritis, endocarditis, meningitis,
and cavitating pneumonia (immuno-suppressed
patients). Micrococcus luteus has been reported
as the causative agent in cases of intracranial
abscesses,
pneumonia,
septic
arthritis,
endocarditis, and meningitis (Bannerman and
Peacock, 2007). Transmission is possible through
contact with contaminated objects and/or surfaces
(Harrison et al., 2003). Transmission via inhalation
of contaminated droplets and/or aerosols may
also be possible. Micrococcus spp. are relatively
susceptible to most antibiotics, including
vancomycin,
penicillin,
gentamicin,
and
clindamycin, which have been successfully
Dada and Aruwa
83
Table 2. Overall percentage prevalence of microorganism isolated from contaminated sites.
Bacteria
Bacillus species
B. sphaericus
B. brevis
B. coagulans
B. macerans
B. mycoides
B. subtilis
Corynebacterium spp.
Staphylococcus species
S. epidermidis
S. simulans
S. albus
Enterobacter cloacae
Micrococcus species
Micrococcus luteus
Micrococcus roseus
Klebsiella liquefasciens
Flavobacterium rigense
Acinetobacter anitratus
Alcaligenes eutrophus
Total
Percentage prevalence (%)
31
24
17
Fungi
Aspergillus species
Aspergillus fumigatus
Aspergillus niger
Trichoderma viride
Articulospora inflata
Varicosporium elodeae
Rhizopus nigricans
Gliocladium deliquescens
Penicillium notatum
Beauveria bassiana
Percentage prevalence (%)
46
15
11
9
7
6
4
2
9
7
4
3
3
2
100
100
Table 3. Bacterial and fungal counts for soil serial dilution technique.
Sites
Biology LA
Biology CA
University diploma LA
University diploma CA
Microbiology LA
Microbiology CA
Bacterial counts (dilution unit – cfu/ml)
10-4
10-6
10-7
10
2
1
43
10
2
11
2
2
13
3
2
15
3
2
48
5
2
Fungal counts (dilution unit – sfu/ml)
10-1
10-5
9
2
7
3
13
6
11
4
17
7
13
7
LA, Lecture area; CA, Control area; cfu, colony-forming unit; sfu, spore-forming unit.
Table 4. Average fungal counts for Warcup method.
Site
Biology LA
Biology CA
University diploma LA
University diploma CA
Microbiology LA
Microbiology CA
Counts on PDA (sfu/g)
10
4.5
6.5
5.5
11
6.5
LA, Lecture area; CA, control area; PDA, potato dextrose
agar; sfu, spore-forming unit.
Int. J. Appl. Microbiol. Biotechnol. Res.
used for treating infections caused by these bacteria
(Bannerman and Peacock, 2007). Likelihood of infection
is low; however, the avoidance of accidental parenteral
inoculation, ingestion, and inhalation of infectious
droplets is essential (Liebl et al. 2002).
Acinetobacter species are widely distributed in nature,
and commonly occur in soil. They can survive on moist
and dry surfaces. In immuno-compromised individuals,
several Acinetobacter species can cause life-threatening
infections. Such species also exhibit a relatively broad
degree of antibiotic resistance. It can cause various other
infections, including skin and wound infections, bacteremia. Epidemiologic evidence indicates Acinetobacter
biofilms play a role in infectious diseases such as
periodontitis, bloodstream infections, and urinary tract
infections (UTIs), because of the bacteria ability to
colonize in-dwelling medical devices (such as catheters).
The ability of Acinetobacter species to adhere to
surfaces, to form biofilms, and to display antibiotic
resistance and gene transfer motivates research into the
factors responsible for their spread (Antunes et al., 2011).
Collectively, the aerobic spore forming genus of
Bacillus species are versatile chemoheterotrophs capable
of respiration using a variety of simple organic
compounds (sugars, amino acids, organic acids). B.
cereus is a pathogen of humans (and other animals),
causing food borne illness (diarrhoeal-type and emetictype
syndromes)
and
opportunistic
infections
(endophtalmia, keratitis, septicemia, meningitis, endocarditis, pneumonia, osteomielitis, urinary infections,
cutaneous infections. It also causes infections in
domestic animals (mastitis and abortion in cattle) (Logan,
2005). Staphylococcus is a genus of round, parasitic
bacteria, commonly found in air and water and on the
skin and upper part of the human pharynx. These
bacteria are known to cause pneumonia and septicemia
as well as boils and kidney and wound infections.
Staphylococcus epidermidis does not usually cause
infection, occurring universally in a harmless symbiotic
relationship. It is usually present on most areas of the
skin, in the nostrils, mouth, external ear, and urethra.
However, S. epidermidis can take advantage of a host
with a suppressed immune system and can aggravate an
existing condition. Following heart surgery, S. epidermidis
may cause endocarditis. Staphylococcus epidermidis
may turn an existing abnormality in the urinary tract into
cystitis (Cheesebrough, 2006).
Low bacterial and fungal counts could be attributed to
factors such as the nature of the soil, organic matter
present, and season (rainy) of the year, and agrees with
the report of Bridge and Spooner (2001). The presence of
high organic matter, practice of frequent urination, regular
hand washing and disposal wastewater from the
laboratory may have accounted for the high counts
obtained from soils from the Microbiology urinal area.
Erosion may have also contributed to the washing away
84
of microorganisms from one area to another. The genus
Aspergillus was most prominent among the fungal
species isolated. Some are known to express aflatoxins,
for example, Aspergillus flavus, and hence, could portend
serious health implications to visitors to these public
urinals. Spores of Aspergillus fumigatus when inhaled
can produce pulmonary infection, allergies, eye, and ear
infection. This fungus has been implicated as being one
of the causes of systemic fungal diseases in humans and
animals, causing acute and chronic respiratory tract
infections. Aspergillosis is an infection of the skin, nasal
sinuses, and lungs or other internal organs caused by
molds of the genus Aspergillus. The disease is
contracted by the inhalation of spores (Cheesebrough,
2006).
The Warcup method performed better than the soil
dilution technique with respect to fungal variety. This may
be due to the direct contact of soil with media without
prior dilution. This is in agreements with Olutiola et al.,
(1991). Higher fungal counts were observed from the
urine contaminated soils compared to counts from control
soils. The pH of urine falls more within the acidic range.
The presence of urine may have affected the urine
contaminated soil pH, lowering it to an acidic state which
favoured fungal growth more than bacterial growth
(Drangert, 2000).
Public urinal soils may become major factors in the
spread of infection especially when adequate sanitary
facilities are not available. Microorganisms from public
urine contaminated soils have potential to cause disease
either as primary or opportunistic pathogens. The stench
from these urine contaminated soils is also nauseating.
Organisms found in the urine contaminated soils may be
attributed to persons with UTIs excreting higher amounts
microflora than apparently healthy individuals. Regular
visits to the public urinals may contribute to increasing
microbial load above threshold levels within the body
systems. This agrees with the report of Hoglund et al.
(2002). This would often result in an infected/diseased
state. Hence, persons visiting these urinals stand the risk
of contracting opportunistic infections / diseases.
Indiscriminate urination in public areas is a common
practice in developing countries. Much research has
been carried out on soil and urine microflora, but not on
microflora associated with urine contaminated soils. This
research aimed basically at providing information and
adding to knowledge, and creating awareness to
discourage the practice.
CONCLUSION AND RECOMMENDATION
Urine contaminated soils are public health hazards
whichare avenues for transmission of infection from one
person to another. Poor hygienic habits, overpopulation
and overstretched facilities encourage indiscriminate
Dada and Aruwa
85
urination in public places. This practice can either
increase or decrease the microflora of urine
contaminated soils. An increase in microbial load in such
soil environments may result in increased probability of
contracting opportunistic infections. Available toilet
facilities within the university fall far below that required to
cater for the ever growing population of students
admitted, and staff employed. Hostels are also
overcrowded; hence the attendant result of damage of
available toilet facilities, and indiscriminate urination
around the hostels. The provision of more and adequate
toilet facilities within the institution cannot be
overemphasized. As a result of poor sanitary habits and
practices of most students, and to prevent overstretching
of toilet facilities, students’ toilets could be separated
from staff toilets. Water closet toilet type and/or mobile
toilets could also be made available. Contents of the
mobile toilets must however, be disposed of at regular
intervals. The importance of public investment in basic
sanitation is incontestable. According to WHO/UNICEF
(2000), the practice and enforcement of basic sanitation
rules would help prevent unnecessary deaths and protect
the health of millions of persons.
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