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STUDIES ON THE INCIDENCE OF ENTEROCOCCUS SPECIES IN URINARY TRACT INFECTIONS, AND THEIR IDENTIFICATION BY A TEST SCHEME
Priya A Miskeen, Lina Deodhar
Department of Microbiology, Bombay Hospital and Medical Research Centre, Mumbai - 400 020.
Out of a total of 4030 urine specimens studied bacteriologically, for a period of one and a half years, 1990 specimens were positive in culture. Enterococci were isolated in pure cultures in clinically significant numbers in 147 specimens (7.38%). Enterococcus faecalis was the most predominant species isolated (87.07%). This was followed by the less prevalent species, Enterococcus faecium (10.88%) and Enterococcus durans (2.14%). An identification system based on the grouping of key reactions of 19 phenotypic characteristics of enterococcus species is described.
INTRODUCTION
The term enterocoque was first used by French microbiologists in 1899, who chose the name to emphasise the intestinal origin of this newly discovered gram positive coccus.1 The name Streptococcus faecalis (faecalis relating to faeces) was coined in 1906, to describe an organism isolated from the blood of a patient with endocarditis.2 In 1964, investigators recognised a second species, Streptococcus faecium as a distinct species of enterococcus.3 During the same period, another species of enterococcus namely Streptococcus durans was reported.4
In 1972, group D streptococci were defined as those that documented presence of Lancefield group D polysaccharide cell - wall antigen. These included enterococcal and non-enterococcal group D streptococci.5
The term enterococci was defined as a streptococcal organism which included S. faecalis, its variants (var. zymogenes and var. liquifaciens), S. faecium and S. durans. It was deemed advisable to retain the use of the term enterococcus because of the difference which exists between the antibiotic therapy in patients with enterococcal and non- enterococcal infections.5
The present study was undertaken with a view at characterising the clinical isolates of the genus enterococcus, recovered from the cases of urinary tract infections (UTI) in a hospital environment, on the basis of their morphological and physiological behaviour.
TABLE 1
Key tests for the identification and speciation of enterococcus speciesEnterococcus Non.Enterococcus bovis Tests faecalis faecium durans equinus Growth in media containing 6.5% NaCl + + + Hydrolysis of NaCl esculin broth + + + Growth in methylene blue milk + + + Tolerance of pH 9.6 + + + Growth at 10oC + + + Hydrolysis of arginine + + + Survival at 60oC for 30 minutes + + + Hydrolysis of sodium pyruvate + Reduction of tetrazolium + Resistance to tellurite + Fermentation of carbohydrates
+ Sorbitol Mannitol + + Sucrose + + + + Arabinose + Glycerol + Screening test for vancomycin resistance. + + + positive reaction; negative reaction
TABLE 2
The E faecalis strains were further sub-speciated by employing the following testsTests E. faecalis var liquifaciens zymogenes Gelatin liquifaction + Beta haemolysis +
MATERIAL AND METHODS
Cases taken up for the study included patients admitted at the Bombay Hospital and MRC, Mumbai. Specimens comprised urine, collected from cases clinically suspected or suffering from a UTI. The studies were carried our for over a period of 18 months, from, October, 1997 to April, 1999.
A clean catch, midstream urine sample was collected in a sterile widemouthed, plastic container and brought to the laboratory within half an hour.
A fixed volume of each, well mixed specimen of urine was streaked across blood agar, MacConkey agar, and bile esculin agar medium. The caliberated loop technique was used. The culture plates were incubated at 37oC for upto 48 hours. Quantitation of the colony numbers was an essen tial parameter for determining presence of clinically significant bacteriuria.
The identification of enterococci from the non-enterococcal species, and subsequently its speciation was done using a test scheme5 as shown in Table 1.
RESULTS
A majority of cases (83, 56.46%) showed beta - haemolysis on blood agar. A total of 42 cases (28.57%) did not show any haemolysis, while, 22 cases (14.97%) gave alpha haemolysis on blood agar. On MacConkey agar medium, these organisms produced pinpoint magenta coloured colonies. Bile esculin agar revealed enterococci as pinpoint, colourless colonies which turned the media brownish black.
For a strain to fit the speciation scheme perfectly, all the reactions had to agree with those listed in Tables 1 and 2.
E. faecalis was the most predominant species isolated (128 cases, 87.0%). This was followed by E. faecium (16 cases, 10.88%) and E. durans (three cases, 2.04%). Amongst E. faecalis strains, E. faecalis var. zymogenes was the most prevalent (96, 75%), followed by E faecalis var. liquifaciens (32, 25%). None of the 96 E. faecalis var. Zymogenes spp could liquify gelatin. A large percentage (83, 86.46%) of this species showed beta haemolysis on blood agar. All the 32 E. faecalis var. liquifaciens strains were capable of liquifying gelatin and showed no haemolysis on blood agar. (10.88%) and E. durans (2.05%). High prevalence rates of E. faecalis and E. faecium were also brought about by the studies of Toala et al who reported figures of 73% and 4.5% for the two species respectively. Moreover, E. durans was isolated in only 1% of cases in their study. [8]
Beta haemolysis on blood agar was exhibited exclusively by strains of E. faecalis. This property of enterococcus faecalis has been mentioned in several reports. [9] Diebel (1869) however, has pointed out that E. faecalis strains show frequent loss of haemolytic activity depending upon the source of blood used in blood agar medium. [5]
It is quite ironical that the less intrinsic virulance of enterococci is adequately compensated for by their intrinsic resistance to antibiotics. E. faecium cultures are more resistant than E. faecalis to several commonly used antimicrobial agents, making the resistant than E. faecalis to several commonly used antimicrobial agents, making the identification of enterococcus species useful for aiding in managing patients with enterococcal infections. [10], [11]
Falklam RR (1972) however, has cautioned clinical laboratory workers against relying on any single test for identifying enterococci. At least the number of tests mentioned here should be performed for the species identification of enterococcus to be of differential value.[12]
REFERENCES
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2.Andrews FW, Horder TJ. A study of the streptococcus pathogenic for man. Lancet 1906; 2 : 708.
3.Deibel RH. The group D streptococcus. Bacteriol Rev 1964; 28 : 33-336.
4.Breed RS, Murray RD, Smith NR. Bergeys manual of determinative bacteriology 7th edition. 1957.
5.Falklam RR. Recognition of group D streptococci species of human origin by biochemical and physiological tests. Appl Microbiol 1972; 23 : 1131-9.
6.Gross PA, Harkavy LM, Barden GE, Flower MF. The epidemiology of nosocomial enterococcal UTI, Am J Med Science 1979; 272 : 75-81.
7.Murry BE. The life and times of enterococcus. Clin Microbiol Rev 1990; 3 : 46-65.
8.Toala P, McDonald A, Wilcox C, Finland M. Susceptibility of group D streptococci (enterococci) to 25 antibiotics in vitro, with special reference to species differences. Am J Med Sci 1969; 258 : 41-430.
9.Moellering RC. Enterococcal infections in patients treated with moxalactam. Rev Infect Dis 1982; 4 (suppl) : 708-10.
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11.Leclereq R, Delrot E, Duval J, Courvalin P. Plasmid mediated resistance to vancomycin and teicoplanin in E. faecium. N Engl J Med 1988; 319 : 157-61.
12.Falklam RR, Collins MD. Identification of enterococcus species isolated from human infections by a conventional test scheme. J Clin Microb 1989; 27 (4) : 731-4.
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