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*

1Department of Microbiology, Teerthankar Mahaveer Dental College & Research Centre, (TMU),

Moradabad (UP), India 2Department of Biotechnology,

Institute of Foreign Trade & Management (IFTM), Moradabad (UP), India 3Department 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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