|Year : 2001 | Volume
| Issue : 3 | Page : 132--137
Prevalence, identification and distribution of various species of enterococci isolated from clinical specimens with special reference to urinary tract infection in catheterized patients
PJ Desai, D Pandit, M Mathur, A Gogate
Department of Microbiology, Lokmanya Tilak Municipal Medical College, Sion, Mumbai - 400 022, India
Department of Microbiology, Lokmanya Tilak Municipal Medical College, Sion, Mumbai - 400 022
Various clinical specimens were processed to find the prevalence rate of enterococci and to identify the species of clinical isolates of enterococci. Screening of various clinical specimens revealed that enterococci were prevalent in 22.19% of the total specimens, with Foley«SQ»s catheters and burn wounds to be the major site of isolation. High rate of colonization was noted as opposed to infection. Conventional test scheme proposed by Facklam and Collins were successfully used to speciate enterococcal strains. Seven species of enterococci were identified in the study from a set of 202 cultures, with E.faecalis (49.50%) and E. faecium (35.64%) predominating. E. avium (9.40%), E.hirae (2.47%), E.raffinosus (1.98%) and one isolate each of E.gallinarum and E. casseliflavus were the other members of Enterococcus species identified. Urinary tract infection (UTI) by enterococci due to catherisation was found in 8.92% of the patients and is probably the result of high rate of colonization of Foley«SQ»s catheters and use of broad-spectrum antibiotics.
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Desai P J, Pandit D, Mathur M, Gogate A. Prevalence, identification and distribution of various species of enterococci isolated from clinical specimens with special reference to urinary tract infection in catheterized patients.Indian J Med Microbiol 2001;19:132-137
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Desai P J, Pandit D, Mathur M, Gogate A. Prevalence, identification and distribution of various species of enterococci isolated from clinical specimens with special reference to urinary tract infection in catheterized patients. Indian J Med Microbiol [serial online] 2001 [cited 2020 May 30 ];19:132-137
Available from: http://www.ijmm.org/text.asp?2001/19/3/132/8146
Enterococci, an indigenous flora of the intestinal tract, oral cavity and the genitourinary tract of the humans and animals, are known to be relatively avirulent in healthy individuals, but have become important opportunistic pathogens, especially in hospitalized patients. They belong to group D streptococci as characterized by Lancefield in 1938, whose taxonomy has changed considerably in the last few years.
Recent years have witnessed increased interest in enterococci not only because of their ability to cause serious infections like endocarditis, bacteremia, intra-abdominal and urinary tract infection (UTI), but also because of their increasing resistance to many antimicrobial agents. This emphasizes the need for their identification from the clinical specimens and also differentiate them from other group D streptococci which are generally more sensitive to the antimicrobial agents.
An identification scheme utilizing conventional testing media for recognition of all currently described enterococci species has been proposed by Facklam and Collins. Though majority of the isolates so far discussed are E. faecalis, members of all recently described species have also been found in the clinical specimens. We assessed the prevalence of enterococci in the routine clinical specimens at our institution along with their identification and distribution of species.
In a CDC survey of nosocomial infection, enterococci accounted for 13.9% of urinary tract infection, second only to Escherchia coli as a sole agent of nosocomial urinary tract infection. Since there has been significant increase in the rate of isolation of Enterococcus species from urine specimen, especially from hospitalized patients on indwelling catheter, we studied the rate of colonization of Foley's catheters and its role in nosocomial urinary tract infection due to colonizing enterococci species.
Material and Methods
Various clinical specimens such as burn wound swabs, ascitic fluids, surgical and non-surgical wounds, umbilical stumps, abdominal drain fluids, synovial fluids and Foley's catheters followed by subsequent urine specimens of the same patient, obtained in the Microbiology Department, were processed for the isolation of enterococci, for a period of one year. The specimens were plated on Peizers Enterococcus Selective agar which is a bile-esculin medium, for the isolation of enterococci. The specimens were also plated on blood agar and MacConkey's agar for the isolation of concomitant organisms along with enterococci.
Enterococci were identified on the basis of growth on bile-esculin medium, gram staining i.e. gram positive cocci in pairs and short chains, catalase-negative or pseudocatalase positive, growth in 6.5% NaCI and at pH 9.6. Other tests such as bacitracin resistance, acidification of ribose and positive Voges Proskauer (V.P.) test by Coblentz method were also used for the confirmation of isolates as enterococci.
Identification to species
Enterococcal strains were further identified to the species level by using conventional physiological tests devised by Facklam and Collins3 which are based on carbohydrate fermentation using 1% solution of following sugars: glucose, mannitol, rabinose, raffinose, sorbitol, sucrose, lactose, trehalose and inulin; by pyruvate utilization in 1% pyruvate broth; arginine decarboxylation in Moellers decarboxylase broth; hippurate hydroloysis; motility test; pigment production detected on tryptic soya agar (TSA); gelatin liquefaction; starch hydrolysis using 2% starch and polysaccharide production.5 Ability to produce enzyme phosphatase by entercoccus species was also tested using phenolphthalein-phosphate agar. Haemolysin production was detected in the strains of E. faecalis and E.faecium by culturing the isolates on blood agar using 5% human blood. A single colony isolate was inoculated into 5mL Todd-Hewitt broth and incubated overnight at 37° C which was then added as an inoculum of one drop with the help of pasteur pipette. All tests were incubated at 37° C and read at 24 hours and 7 days.
Urinary Tract Infection (UTI)
Instrumentation has been found to be the major cause of enterococcal urinary tract infection. Although Foley's catheter is not recommended for culturing, in order to correlate between colonization of Foley's catheters due to enterococci and subsequent urinary tract infection, Foley's catheters and urine specimens of the same patients were processed. When the same enterococcal species was isolated from Foley's catheter and urine specimen in a pure culture, and in concentration greater than 10 organism per mL of the specimen, it was considered significant and indicative of UTI. The study group comprised of catheterized patients admitted in intensive care area during the study period.
To compare the Foley's catheter colonization and urinary tract infection due to enterococci in catheterized patients, 100 mid-stream urine specimens from non-catheterised patients were processed as control group. Urine specimens from uncatheterized patients were collected from the same medical intensive care areas from where catheterized urine specimens were collected and control subjects were well matched with respect to their age and sex with the study group.
Statistical data analysis
Chi square test (X2) was used to compare the rate of urinary tract infection due to enterococci in catheterized and non-catheterized (control group) patients. P value less than 0.05 was considered significant.
202 strains of enterococci were isolated from a total of 910 various clinical materials as depicted in [Table:1].
The prevalence rate was 22.19%. The most frequent sources of enterococci were found to be Foley's catheters (48.21%) and burns wounds (29.51%). Among 202 isolates of enterococci, only 38 (18.18%) isolates represented pure cultures of enterococci, whereas others were yielded with other gram-positive or gram-negative organisms of which Pseudomonas aeruginosa, Escherchia coli, Proteus species and Staphylococcus aureus were the most common pathogens.
[Table:2] shows the results of various biochemical tests used to differentiate enterococci isolates. None of the enterococci species hydrolysed starch, produce polysaccharide or enzyme phosphate. Identification of enterococci to the species level was readily achieved by using the battery of biochemicals suggested by Facklam and Collins.
[Table:3] displays the distribution and species identity of enterococcal isolates according to the source. Seven different species of enterococci were encountered within a set of 202 cultures of enterococci viz. E. faecalis, E. faecium, E.avium, E.hirae, E.raffinosus, E.gallinarum and E.casseliflavus. The majority of the isolates were E.faecalis (49.50%), while E.faecium accounted for 35.64%. Only 14.85% of the isolates were identified as members of other species. E.faecalis accounted for greater percentage of isolates from burns wounds (54.36%) and Foley's catheters (40.74%) as compared to other specimens, although the smaller sample size from the latter specimens make it difficult to draw a firm conclusion on E.faecalis distribution. The same is true for E.faecium isolates.
[Table:4] shows the correlation between Foley's catheter colonization and urinary tract infection. Although 48.12% of Foley's catheters were colonized by different Enterococcus species as shown in [Table:4], urinary tract infection due to colonizing strain of enterococci was noted only in 8.92% of the patients. The infection due to colonization was found to be significant by application of Chi square, [X2 = 4.74 (pEscherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus in the list of most prevalent pathogens. During the 12 months of the present study, enterococci were isolated from various clinical specimens with the prevalence rate of 22.19%. The wide variety of infectious materials from which enterococci was isolated was found similar to those obtained in other studies.,
Enterococci isolated from various clinical specimens in this study do not reflect the true incidence of infection caused by this organism, but definitely suggest the increased frequency of their isolation from various clinical materials. More often enterococci were found as one of the isolates in the clinical specimens with polymicrobial etiology which are better established as pathogens and are primary target of subsequent therapy. Enterococci by virtue of being non-invasive organisms, their presence along with organisms like Haemolysin production was found to be variable among the isolates of E.faecalis and E.faecium. With 5% human blood, all the three types of haemolysin production was seen in the strains of both the species. About 58% of the E.faecalis strains and 40% of the E.faecium strains were found to produce-b-haemolysins. a-haemolysin production was seen in 12% of E.faecalis and 18% of E.faecium strains.
Staphyloccocus aureus are suggestive of colonization rather than infective etiological agent. The source of this organism is more often faecal flora. Although wound colonization was high in burn's patients, none had sepsis due to enterococci. Prevalence of enterococci in clinical specimens can thus be attributed to their ability to grow and survive due to selective pressure of antimicrobial agents.
Data in the present study indicates that the battery of tests used will identify 95-100% of the enterococcal species isolated from human source. Almost 16 different tests were used to identify the various species of enterococci and we were able to speciate all 202 isolates of enterococci. Conventional tests proposed by Facklam and Collins, were thus successfully used in this study to identify enterococci to the species level, although Buschelam et al11 and Ruoff et al12 experienced problems with strict adherance to the key tests recommended by Facklam and Collins.3 The key tests involve acid production from mannitol, sorbitol and sorbose and arginine hydrolysis. On the basis of these tests, enterococcal isolates are grouped and then additional tests are used to differentiate into species level. However, key tests followed by differentiation tests may sometimes misidentify the species. Without the addition of tellurite reduction and ribose fermentation test, lactose-negative E.faecalis organisms were miscategorised as E.solitarius by the same workers., Therefore emphasis should be placed not on the limited no.of tests but on larger spectrum of tests in order to recognize species within genus and the key tests are interpreted as a whole. Correct speciation is very important since there is variation in resistance to antibiotics by particular enterococcal species.
Seven species of enterococci were identified in the present study which is in accordance with the other reported studies, 7,12,13 with the exception that they had also identified a strain of biochemical variant of E.faecalis and strains of E.durans and E.mundtii. However, by and large, species collection in a stock have been found to vary in different studies., Bryce et al had identified only three species of enterococci viz. E.faecalis, E.faecium, and E.casseliflavus among 140 strains isolated. Although E.durans have been reported by others,,, we have not come across this species. Our observation on the incidence of various species of enterococci were found similar to other studies,,,, and E.faecalis was the most commonly identified species of enterococci isolated from clinical specimens but markedly differed in the isolation rate of this species, in that they have reported relatively high percentage of E.faecalis isolates. Other species of enterococcus like E.avium, E.hirae, E.raffinosus, E.gallinarum, E.casseliflavus accounted for 14.85% at our institution.
E. faecalis was found to be the predominant isolate from all clinical specimens, followed by E.faecium. Both these species are long known to be significantly associated with the clinical disease hence their isolation is a cause of serious concern., Other species such as E.avium, E.hirae, E.raffinosus, E.gallinarum and E.casseliflavus, though have been isolated from clinical specimens, their clinical significance is still doubtfull., A high incidence of b haemolytic strains of E.faecalis and E.faecium was noted in our study which advocates that all the b-haemolytic streptococci obtained from the clinical specimens should be screened for the presence of enterococci.
Presence of enterococci in urine are well documented to be the cause of urinary catheterization or instrumentation. In the present study, although 48.21% colonization of Foley's catheters by enterococci was found to be high, incidence of UTI due to colonizing strain was found to be 8.92%. Others,, have found relatively high rate of UTI due to the indwelling Foley's catheter. It was difficult to assess the change in the rate of enterococcal UTI in our study as previous data on isolation rate of enterococci was not available. But the high rate of colonization of Foley's catheters and the subsequent therapy with broad spectrum antibiotics may change the situation in near future. A high incidence of colonization by enterococci in the present study can be attributed to the underlying condition, longer hospital stay and increasing use of broad spectrum antibiotics particularly cephalosporins, as the subjects studied were from intensive care areas.
For long time, enterococci was frequently considered to be commensal organism and was ignored when isolated in clinical laboratory. But recently due to its capability of causing variety of infections, especially in hospitalized patients and difference in antimicrobial senstivity of each species to varying antibiotics has led to understanding the importance of identification of Enterococcus to species level. High rate of colonization of Foley's Catheter among hospitalized patients was found to be noteworthy in the present study suggesting that catheterization does play a role in increasing the risk of infection due to enterococci.
We are grateful to Dr. Donald Morrison from Central Public Health Laboratory (CPHL) Colindale, for his assisstance in confirming the species of enterococci of the first 40 isolates.
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