Developmental Microbiology and Molecular Biology

ISSN 0976-5867 Volume 1, Number 1 (2010), pp. 107-112

© Research India Publications
http://www.ripublication.com

Study of Coagulase-negative Staphylococci (CNS)

Isolated from Urinary Tract Infected Patients

Zubair Ahmad1, Shipra Agarwal2 and Vishal Kumar Deshwal3*

1
Department of Microbiology,
Teerthankar Mahaveer Dental College & Research Centre, (TMU),
Moradabad (UP), India
2
Department of Biotechnology,
Institute of Foreign Trade & Management (IFTM), Moradabad (UP), India
3
Department of Microbiology,
Doon (PG) Paramedical College, Dehradun (Uttarakhand), India
*Corresponding Author E-mail: vishal_deshwal@rediffmail.com

Abstract

Total 312 samples of UTI suspect patients were screened for present study and
142 samples were UTI positive. On the basis of gram staining and coagulase
test confirmed that 90 strains were Coagulase negative Staphylococci (CNS).
Further, biochemical characterization of isolates confirmed that five types of
CNS strains- Staphylococcus saprophyticus, Staphylococcus epidermidis,
Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus sp.
were isolated from UTI infected patients. Further, results showed that 52 girls
and 38 males were UTI positive and causing organisms were CNS. Study
showed that above age of 60, maximum CNS was present in male and female.
Our result and scientific evidences confirmed that isolated CNS strains were
responsible for UTI in female and male of different age groups.

Introduction
Staphylococcus (from the Greek: staphylē, "bunch of grapes" and κόκκος, kókkos,
"granule") is a genus of Gram-positive bacteria. Under the microscope they appear
round (cocci), and form in grape-like clusters [1]. Genus Staphylococcus comprises
42 validly described species and subspecies of gram positive cocci [2].
Staphylococcus is a ubiquitous microorganism that is responsible for a wide range of
both acute and chronic infection [3].
Coagulase negative staphylococci (CNS) are group of adaptable and opportunistic
pathogens [4]. Among coagulase negative staphylococci (CNS) in UTI the
predominant species was found to be Staphylococcus hominis followed by S.
108 Zubair Ahmad, Shipra Agarwal and Vishal Kumar Deshwal

epidermidis [5]. Ashour and El-sharif [6] mentioned that CNS strains such S.
simulans, S. gallinarum, S. haemolyticus, S. xylosus, S. epidermidis caused UTI in
cancer patients.
Urinary tract infections (UTIs) are serious problems affecting millions of people
each year. UTI in women are common problem. In United States each year,
Physisians write approximately 11.3 million prescriptions for adult women with UTIs
[7]. Significant bacteria is defined as the persistent isolation of 105 colony forming
unit (CFU) of bacteria per ml of clean voided, mid-stream urine specimens plated
within 6h of collection [8].
UTI are associated with multiplication of organism in urinary tract, usually, the
microbial invasion of any of the tissue of the urinary tract of extending from renal
cortex to urethral meatus [9]. Further, it can spread from urethra to urinary bladder
then migrate to kidney or prostrate [10]. The higher prevalence in females as
compared with male is attributable to the shortness of the female urethra and so is
more liable to contamination during sexual intercourse, urethra massage and even
urination with chronic flora that resides in the perineal skin [11]. In addition, urine of
female was found to have more suitable pH and osmatic pressure for the growth of
bacteria than urine from male [12]. Aim of present study that isolation,
characterization of Coagulase-negative staphylococci (CNS) from urinary tract
infected patients.

Materials and methods

Isolation of Staphylococcus from UTI infected patient
For isolation of pathogenic Staphylococcus strains strain urine sample was collected
in a sterilized container from a patient suffered from urinary tract infection. The mid-
stream urine was collected after carefully cleaning the genitalia and mid-stream urine
was collected because the first portion of urine may contain most of contaminants.
Bacterial count is done by SPC (standard plate count) method. 1ml urine sample
mixed in 9 ml sterilized distilled water and this was 10-1dilution of urine sample.
Similarly, urine was diluted up to 10-5. 1ml of each dilution was transferred into
sterilized Petridish (90mm diameter) and separately, poured 14ml of sterilized
medium such as Mannitol salt agar medium, MacConkey agar, Blood agar medium.
Mixed wall and plates were incubated at 37OC for 24-48h.
Staphylococcus strains grow on mannitol salt agar medium while the growth of
most other bacteria will be inhibited due to high salt concentration (7.5%). Strains
produce luxuriant growth with yellow zones around colony. Composition of mannitol
salt agar medium is proteose peptone- 10g; Beef extract- 1g; D-Mannitol- 10g;
Sodium chloride- 75g; Agar- 15g; Phenol red- 0.025g; Distilled water 1000ml; pH
7.4±0.2

Characterization of Coagulase-negative staphylococci (CNS)

Isolated strains were characterized on the basis of gram staining and bio-chemicals
tests. Such tests were done according to Bergey's manual of Determinative
Bacteriology (Holt et al., 1994).
Study of Coagulase-negative Staphylococci (CNS) Isolated 109

Coagulase test
This test is useful in differentiating Coagulase positive and coagulase-negative
staphylococci. It is done by two methods-
i. Slide agglutination test: Dense suspensions of Staphylococci from culture are
made on two ends of clean glass slide. One should be labeled as “test” and the
other as “control”. The control suspension serves to rule out false positivity due
to autoagglutination. The test suspension is treated with a drop of citrated
plasma and mixed well. Agglutination or clumping of cocci within 5-10 seconds
is taken as positive.
ii. Tube coagulase test: Three test tubes are taken and labeled “test”, “negative
control” and “positive control”. Each tube is filled with 0.5 ml of 1 in 10 diluted
rabbit plasma. To the tube labeled test, 0.1 ml of overnight broth culture of test
bacteria is added. To the tube labeled positive control, 0.1 ml of overnight broth
culture of known S. aureus (coagulase positive) is added and to the tube labeled
negative control, 0.1 ml of sterile broth is added. All the tubes are incubated at
37oC and observed up to four hours. Positive result is indicated by gelling of the
plasma, which remains in place even after inverting the tube. If the test remains
negative until four hours at 37oC, the tube is kept at room temperature for
overnight incubation.

Results and discussion

Total 312 samples of suspect of UTI patients were screened for present study. On
Mannitol salt agar medium, total 142 samples showed growth and total bacterial count
was grater than 105. One Staphylococcus colony from each petriplate was select for
further study. Gram staining and cell arrangement confirmed that all isolated strains
were gram positive Staphylococcus. Further, Coagulase test confirmed that total 90
strains were Coagulase negative Staphylococci (CNS). Further, biochemical
characterization of isolates confirmed that five types of CNS strains- Staphylococcus
saprophyticus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus
haemolyticus, Staphylococcus sp. were isolated from UTI infected patients (Table-1).
Similar observations have been mentioned in Bergey’s manual of determinative
bacteriology [13] and Cowan and Steel’s Manual for the identification of medical
bacteria [14].
UTI infected patients were characterized on the basis of sex and age group.
Results showed that 52 females were UTI positive and infected with CNS strains and
only 38 males were CNS positive. Study showed that above age of 60, maximum
CNS was present in male and female but at above 60 Males were more positive as
compared to females. Least percentage of CNS positive was age between 0-15 in both
male and female (Table 2). Similarly, Kolawole et al. [15] reported that 66.67%
females and 33.33% males were UTI positive and also mentioned that least UTI
patients on the basis of age groups. Such informed support the finding that females
were more receptive to UTI as compared to males. As reports suggested that higher
prevalence in females as compared with male are attributable to the shortness of the
female urethra. But other factors including improper cleaning of perineum, the use of
110 Zubair Ahmad, Shipra Agarwal and Vishal Kumar Deshwal

napkins and sanitary towel together with pregnancy and sexual intercourse contribute
to higher incidence of UTIs in various women [16].

Table 1: Relationship of Age-Sex of UTI patient with Coagulase Negative

Staphylococcus (CNS) isolates.

S. No. Age (year) Male Female

Number Percentage Number Percentage
1 0-15 2 5.26 4 7.69
2 15-30 3 7.89 8 15.38
3 30-45 7 18.42 12 23.07
4 45-60 10 26.31 9 17.30
5 Above 60 16 42.10 19 36.53
Total 38 52

Table 2: List of different Coagulase negative staphylococcus (CNS) of different Age-

Sex of UTI patient.

S. No Coagulase negative staphylococcus (CNS) Number Percentage

1 Staphylococcus saprophyticus 32 35.55
2 Staphylococcus epidermidis 44 48.88
3 Staphylococcus hominis 08 8.88
4 Staphylococcus haemolyticus 04 4.44
5 Staphylococcus sp. 02 2.22
Total 90

Above 60year persons were most infected with UTI. There may be reason that
they CNS strain are normal microflora and they can transfer if proper hygienic is not
maintain. So UTI can be caused by combination of one or more factors as discusses
earlier. Our result and scientific evidences confirmed that isolated CNS strains were
responsible for UTI in female and male of different group. Some reported supported
our point that CNS strains are a part of the normal microflora in human [17] and also
mentioned that large, relatively stable reservoirs were identified in the faeces, around
the ear, and in the axilla and nares [18]. So it may probability that such normal flora
can cause the UTI disease. Our results concluded that CNS strains cause UTI disease
and females are more susceptible to UTI as compared to males. Further, Aging factors
and unhygienic condition can also be the factor that old age person were infected with
CNS.
Study of Coagulase-negative Staphylococci (CNS) Isolated 111

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