CHAPTER - 3 ISOLATION AND SPECIES IDENTIFICATION OF COAGULASE NEGATIVE STAPHYLOCOCCI ISOLATED FROM DIFFERENT SOURCES 3.1. Introduction CNS are widespread in nature and formerly regarded as harmless inhabitants of the skin and mucosal linings are now recognized as major cause of significant clinical infections. Correct identification of CNS species has become important in clinical laboratories, since several species have been recognized as potential pathogens, especially in a nosocomial setting (Layer et al., 2006). CNS are also involved in animal diseases and several studies have shown that domestic animals may serve as a source of transmission of potentially pathogenic microorganisms. This chapter describes the study on species distribution of CNS isolates from different sources. 3.2. Materials and methods The isolates of CNS were obtained from three different sources, comprising clinical samples, human healthy skin and domestic animals. Out of a total 600 isolates, 200 from each of the above sources, 579 isolates were subjected to study. Isolates from clinical samples included 74 from blood cultures, 51 from urine cultures and 68 from other samples. 196 strains of CNS were isolated from normal human healthy skin between an age group of 20-60 both from male (99) and female (97).190 strains of CNS isolated from
56 Chapter 3 domestic animal samples were also studied. They comprised 72 isolates from cow, 58 isolates from cat and 60 isolates from dog. Samples from animals were obtained by swabbing the lateral udder surface and upper flank of the cows and skin of cats and dogs. 3.2.1. Specimen collection The clinical samples were collected from MOSC Medical College, Kolenchery, a tertiary care hospital in Kerala, over a period of 4 years (2005-2009). The collected samples were soon transported to the laboratory for investigation. The details of each sample were carefully recorded in each case, which included cunjunctival swabs, drain tips, catheter tips and I.V.cannulas, the area from which it is collected and details of patients. Using appropriate media the specimens were cultured. The media used for the primary isolation included blood agar (Appendix-1) and trypticase soy agar (TSA) (Appendix-1). The samples were directly streaked on blood agar and trypticase soy agar and the plates were incubated at 37 C for 24 hrs. 3.2.2. Identification of isolates The organisms isolated from these media were identified on the basis of colony characters, morphological and biochemical properties following the guidelines of Kloos and Schleifer (1975). The genus Staphylococcus was differentiated from Micrococcus by the glucose oxidation and fermentation test, resistance to bacitracin (8u/disc) and sensitivity to furazolidone (100µg) as described by Koneman et al (1997) (Details are given in appendix-1).
Isolation and species identification of CNS isolated from different sources 57 3.2.3. Colony characteristics: The colony characteristics of the isolates were studied for the preliminary identification of organisms. The characters considered included colony morphology, size, pattern of hemolysis etc. 3.2.4. Gram staining Gram staining was done as described in appendix-4. 3.2.5. Bacitracin and furazolidone sensitivity Spread a suspension of the bacterium over a plate of Muller-Hinton agar (Appendix-1). Discs containing bacitracin (8u/disc) and furazolidone (100µg/disc) (Himedia) were applied on the agar medium and incubated overnight. An inhibition zone in the range 10-20 mm and 15mm diameter was taken as positive in the case of bacitracin and furazolidone respectively. 3.2.6. Identification of CNS species For species identification within the Staphylococcus genus, the biochemical tests viz phosphatase, urease, ornithine decarboxylase, characterization of haemolysis, nitrate reduction, and resistance to novobiocin, utilization of xylose, sucrose, trehalose, maltose, fructose, lactose, mannose and mannitol were employed. (Details are given in appendix-1) The isolates were grouped as coagulase negative and positive based on the coagulase test. The species identification was done according to Bergey s Manual of Systematic Bacteriology and by the guidelines as described by Kloos and Schleifer (1986).
58 Chapter 3 3.2.7. Coagulase test 3.2.7.1. Slide coagulase test The test was done using human plasma. Plasma was collected by centrifuging fresh EDTA added human blood. Staphylococcal colony was emulsified in a drop of water on a microscope slide with minimum spreading. Similar suspensions of control positive and negative were also included to confirm the proper reactivity of the plasma. Dipped a flamed and cooled straight inoculating wire into the undiluted plasma brought to room temperature, the bacterial suspension on the slide were stirred with this straight wire and examined for clumping within 10 seconds. The absences of clumping or any reaction taking more than 10 seconds to develop were considered as negative. 3.2.7.2. Tube coagulase test A colony of the Staphylococcus under test was emulsified in the diluted plasma (6:1 in saline) and 1 ml volumes of the diluted plasma were taken in small tubes. With each batch of test tubes with known coagulase positive and coagulase negative cultures as controls. A tube of unseeded diluted plasma to confirm that it did not clot spontaneously was also included. The tubes were incubated at 37 C, preferably in a water-bath, for up to 4 h. and examined at 1, 2 and 4h for clot formation by tilting the tube through 90. The negative tubes were left at room temperature over night and re-examined. Any degree of clot formation such as formation of stiff gel or floating clot in the fluid was read as positive. The tube in which the plasma remained wholly liquid or showed only a flocculent or ropy precipitate was read negative.
No. of Isolates Isolation and species identification of CNS isolated from different sources 59 3.3. Statistical analysis Species distributions of CNS samples were analyzed by χ 2 test. The level of significance was set up at p<0.05 for all tests. 3.4. Results The tentative identification of isolated CNS strains revealed the presence of 12 species of coagulase negative staphylococci. Species Distribution of CNS from 3 different sources was compared (Figure 1). Figure 1 Species distribution of CNS from three different sources 70 60 50 Normal Human Clinical Sample Domestic Animal 40 30 20 10 0 S.epidermidis S.saprophyticus S.haemolyticus S.hominis S.lugdunensis S.warneri S.capitis S.xylosus S.simulans S.cohini S.hyicus S.chromogenes Species distribution The biochemical tests are illustrated in plates 1-6. Of the 12 CNS species isolated the predominant were S. epidermidis (27.46%), S. saprophyticus (11.9%), S. haemolyticus (11.7%) and S. hyicus (11.9%). The sources of isolates and species distribution of the CNS samples identified are given in Table 8, 9
60 Chapter 3 and 10. The most predominant species was S. epidermidis in both human healthy skin and clinical samples. Two species i.e., S. lugdunensis and S. xylosus were present only in clinical samples. Species distribution of CNS in male and female were compared. No statistically significant difference (p<0.743) was observed among the species distribution of CNS in male and female (Appendix). The incidence of S. epidermidis in male was more (60%) when compared to female (40%). But in case of S. saprophyticus and S. haemolyticus it was lesser in male compared to female. In domestic animals the predominant species isolated was S. hyicus (11.9%). Presence of two species i.e., S. hyicus and S. chromogenes were observed only in these samples. Comparison of species distribution in these three different sources showed statistically significant difference (p < 0.001) (Appendix-5).The CNS species isolated in a lesser frequency were S. hominis (7.6%), S. warneri (5.2%), S. capitis (4.1%), S. simulans (3.8%) and S. cohini (5.4%). Result of the biochemical tests are shown in the following plates Plate 1: Gram stained smear of coagulase negative staphylococci
Isolation and species identification of CNS isolated from different sources 61 Plate 2: Haemolysis produced by CNS on blood agar Plate 3 Phosphatase test: positive colonies are surrounded by bright yellow medium
62 Chapter 3 Plate: 4 Tube coagulase test showing positive and negative results Plate: 5 Novobiocin sensitivity test
Isolation and species identification of CNS isolated from different sources 63 Plate: 6 Ornithine decarboxylase test showing positive and negative results Table-8 Species distribution of CNS isolates from normal human healthy skin CNS species Total No of Strains Source wise break up Male Female No. % No. % S. epidermidis 60 36 60 24 40 S. saprophyticus 28 12 43 16 57 S. haemolyticus 27 13 48 14 52 S. hominis 19 9 47 10 53 S. warneri 15 8 53 7 47 S. capitis 18 9 50 9 50 S. simulans 15 7 47 8 53 S. cohini 14 5 36 9 64
64 Chapter 3 Table -9 Species distribution of CNS isolates from human clinical samples CNS species Total No of Strains No and % of isolates belonging to each species isolated from different sources Blood (74) Urine (51) Other samples (68) No. % No. % No. % S. epidermidis 69 38 55 12 17 19 28 S. saprophyticus 30 8 27 13 43 9 30 S. haemolyticus 29 9 31 8 28 12 41 S. hominis 14 6 43 nil nil 8 57 S. lugdunensis 12 5 42 nil nil 7 58 S. warneri 6 nil nil 4 67 2 33 S. capitis 6 nil nil 6 100 nil nil S. xylosus 12 4 33 3 25 5 42 S. simulans 7 nil nil 5 71 2 29 S. cohini 8 4 50 nil nil 4 50 Table-10 Species distribution of CNS isolates from domestic animal samples CNS species Total No of Strains No and % of isolates belonging to each species isolated from different sources Cow (72) Cat (58) Dog (60) No. % No. % No. % S. epidermidis 30 8 27 12 40 10 33 S. saprophyticus 11 3 27 6 55 2 18 S. haemolyticus 12 2 17 5 42 5 42 S. hominis 11 nil nil 7 64 4 36 S. warneri 9 4 44 5 56 nil nil S. cohini 9 4 44 nil nil 5 56 S. hyicus 69 33 48 15 22 21 30 S. chromogenes 39 18 46 8 21 13 33
Isolation and species identification of CNS isolated from different sources 65 3.5. Discussion This study documents the diversity in species distribution of CNS samples isolated from three different sources. The present study reiterates that CNS are ubiquitous members of the normal flora and the species are most commonly encountered in clinical specimens as a contaminant (Archer, 1995). In the present study S. epidermidis (27.5%) was the predominant species isolated from human samples. In most human infections, the rank order of the frequencies of the ten predominant species of CNS is similar to the rank order of the cutaneous population density of each species (Kloos et al., 1975), (Sewell, 1984). Thus the most common CNS pathogen, S. epidermidis, typically produces the largest coagulase negative staphylococcus populations of human epidermis. During the last decades S. epidermidis and other CNS have emerged as a major cause of nosocomial infections. These bacteria usually infect immunocompromised patients such as premature newborns and patients with leukemia or other malignant diseases. The predominance of S. epidermidis among CNS species has been reported by earlier workers. According to them the incidence ranged from 60-90% (Hall et al., 1987). S. epidermidis has been reported to cause >80% of the CNS bacteraemias that follow implant surgery, due to its presence in large numbers on human skin (Schumacher-Perdreau, 1991). Bacteraemia caused by CNS is seldom lifethreatening if not treated promptly, although frank sepsis syndrome may occur, especially in immunocompromised patients. However, this simplistic view of CNS opportunism does not entirely explain the emergence of these organisms as significant pathogens in certain types of infections that previously and rarely attributed to them (Quinn et al., 1986) and the predilection of some species, such as S. saprophyticus, for colonizing and infecting specific body sites, such as the urogenital tract (Latham et al., 1983). The next predominant
66 Chapter 3 species isolated were S. saprophyticus in human samples (11.9%) and S. hyicus (11.9%) in domestic animal samples. S.saprophyticus possesses surface properties that allow it to adhere readily to urogenital cells (Colleen, 1979). This characteristic may play a role in its ability to cause urinary tract infections also. S. hyicus was an opportunistic pathogen usually found on pigs and cattle. S. haemolyticus the third predominant species isolated has been reported to play an important role in hospital acquired opportunistic infections related to implanted medical devices (Mack et al., 2006). Two species i.e., S. lugdunensis and S. xylosus isolated at a rate 6.21% and 6.21% were present only in clinical samples. Of these S. lugdunensis has been reported as opportunistic pathogen. There are few reports of infection with S. lugdunensis in newborns. However studies on adult patients have shown that this species is a significant pathogen (Lambe et al., 1990). Majority of the patients with positive growth of CNS had prosthetic and indwelling devices such as intra-vascular lines. This was seen more in patients admitted to the medical wards, intensive care units and the surgical wards. It has also been suggested that the blood isolates of CNS were more likely contaminants than pathogens (Mirrett et al., 2003), (Bodonaik et al., 2004). CNS are important microorganisms indigenous to humans have emerged over recent years as etiologic agents in a series of infections. The predominance of CNS in clinical isolates was in agreement with previously documented reports (Kloos and Bennerman., 1994). In the present study two species S. hyicus (11.9%) and S. chromogenes (6.9%) were the most prevalent species isolated from domestic animal samples. These CNS species are of great interest because they are currently the most commonly isolated microorganisms in cows and are currently considered as emerging pathogens of bovine subclinical mastitis
Isolation and species identification of CNS isolated from different sources 67 (Timms and Schultz, 1987). The incidence of S. hyicus was (48%) more in cows when compared to other domestic animal samples. There were reports that S. hyicus is more pathogenic than the other species belonging to CNS. S. simulans and S. hyicus were the most frequent isolates of CNS in one study (Jorun, 1991) and S. chromogenes and S. hyicus were the most frequent isolates in another study (Nickerson et al., 1995). Several studies have identified S. chromogenes as the most common species associated with subclinical intramammary infections (IMIs) in heifers (Trinidad et al., 1990). Further studies are necessary in order to evaluate the real role of CNS in the etiology of major infections in domestic animals. However the species of CNS isolated in this study were slightly different from those isolated by Tan et al., (2006) and Cuevas et al., (2004). It suggest that the distribution of CNS species is variable and may differ from one country to another (Mohan et al., 2002). Some investigators suggest that the pathogenicity of CNS is explained best by an increased exposure of a host to a high colonization load, which subsequently leads to a greater number of infections (Fleer and Verhoef, 1984). Further studies are required to confirm this correlation.