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Chin Med J (Taipei) 1997;59:259-64.
Section of Bacteriology, Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, R.O.C.
Background. Group A streptococci and enterococci can be differentiated from other streptococci on the basis of their ability to cleave L-pyrrolidonyl-beta-naphthylamide.
Methods. In the present study, the L-pyrrolidonyl-beta-naphthylamide (PYR) test, pigment medium and bile esculin medium have been used to presumptively identify the streptococci. In total, 114 strains of group A streptococci, 350 strains of non-group A streptococci, 202 strains of enterococci and 197 strains of non-enterococci have been tested.
Results. The results of the present investigation show that sensitivities of different test methods are: PYR broth, 99.08%; Murex PYR, 98.48%; bacitracin, 95%; bacitracin and sulfamethoxazole/trimethoprim (SXT), 95%; pigment medium, 99.23%; bile esculin medium, 99.26%. Additionally, specificities of various tests are: PYR broth and Murex PYR, 99.82%; bacitracin, 90.90%; bacitracin and SXT, 98.87%; pigment medium and bile esculin medium, 100%, respectively.
Conclusions. PYR test has been observed to be very easy to use and may hence be considered as a rapid, reliable and cost-effective method for presumptive identification of group A streptococci and enterococci in the clinical laboratory.
[Chin Med J (Taipei) 1997;59:259-64.]
Keywords: enterococci, group A streptococci, PYR test
Received: April 20, 1996.
Accepted: February 4, 1997.
Address reprint requests to: Chi-Hsiang Chen, Section of Bacteriology, Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, No. 8, Section 3, Ting Chow Road, Taipei, Taiwan, R.O.C.
Group A streptococci is known to be a major cause of pharyngitis [1,2] and upper airway infection. It often causes serious wound infection [3] in Burning Centers, then leads to further complications of post. streptococcal glomerulonephritis. Hence, being able to classify this strain of bacteria as early as possible is very important [4].
In 1953, Maxted used the bacitracin sensitivity test [5] for differentiating between group A streptococci and other non-group A streptococci. However, strains belonging to group C and G streptococci caused a positive reaction, indicating that the specificity of this test is not very good [6-9]. In 1967, Gunn took the throat culture, inoculated it directly on a blood agar plate [10], and attached the SXT (23.75 micron/g sulfamethoxazole/ 1.25 micron/g trimethoprim disc, BBL, Cockeysville, MD) to this plate. The accuracy of the test could be greatly enhanced by this method. It is well known that enterococci often causes urogenital infection, wound infection leading to sepsis, or settled in the heart valves and causes subacute endocarditis [11,12]. Moreover, enterococci are highly resistant to drugs, and hence should be diagnosed as early as possible [13]. In 1981, Godsey [14] reported that group A streptococci and enterococci can hydrolyze pyrrolidonyl-beta-naphthylamide (PYR) and shows a prominent cherry red color [14-16]. A commercialized PYR kit provides results in only a few minuets [17-20]. The aim of the present study was to evolve a highly sensitive, specific and simple procedure which could be effectively used in clinical laboratories.
A total of 863 clinical cases from Tri-Service General Hospital during the period from January 1988 to December 1990 were considered for this study. Streptococci isolated from these clinically significant cases were used as testing strains. These bacteria were further differentiated by colony morphology, Gram's stain, catalase test, hemolysis and bacitracin sensitivity test. The microorganisms thus obtained were preserved at -70OC in a tryptic soy broth (Difco Laboratories, Detroit, MI) containing 15 % glycerol.
A solution composed of a mixture of Todd-Hewitt broth (Difco Laboratories) 30 g/l and PYR 0.1 g/l was prepared [13]. The medium was then inoculated with the test microorganisms and cultured at 35OC for 18-24 hours. After incubation, three drops of PYR reagent were added to the cultured medium. The positive reaction yields a cherry red color, while the negative yields an orangish-yellow color. The positive control indicates Streptococcus pyogenes (ATCC 14289) whereas the negative control indicates Streptococcus agalactiae (NCTC 11360).
Murex PYR disc was first put in a Petri dish and rinsed with 5-10 micron l of water [21]. A large amount of test microorganism was then spread on the disc and incubated at room temperature. At the end of two minutes, one drop of color developer was added and the result was noted after another two minutes. A positive reaction yielded a red or pink color while a negative reaction yielded a white or yellowish color. The positive control indicates S. pyogenes (ATCC 14289), whereas the negative control indicates S. agalactiae (NCTC 11360).
The test microorganisms were heavily inoculated over the entire blood agar plate. Taxo A disc (0.004 unit, BBL) was then put on the plate and incubated at 35OC for 18-24 hours [7]. An inhibition zone becomes visible around the disc in case of a positive reaction, whereas, there is none for the negative reaction. The positive control indicates S. pyogenes (ATCC 14289) and the negative control indicates S. agalactiae (NCTC 11360).
The test microorganisms were heavily inoculated over the entire blood agar plate. SXT disc (BBL) was then put on the plate and incubated at 35OC for 18-24 hours [10]. An inhibition zone becomes visible around the disc in case of a positive reaction, whereas none for the negative reaction. The negative control indicates S. pyogenes (ATCC 14289).
The medium containing a mixture of protease peptone No. 3 (Difco Laboratories) 25 g/ l, corn starch 1 g/l and agar 15 g/l was melted [22]. Test microorganisms were inoculated by stabbing them into the bottom of the test tube. They were then incubated at 35OC for 18-24 hours. A positive reaction yielded an orangish-yellow color, while the negative reaction yielded a white color. The positive control indicates S. agalactiae (NCTC 11360), while the negative control indicates S. pyogenes (ATCC 14289).
A medium containing bile esculin medium (Difco Laboratories) 64 g/l was melted [7]. The test microorganisms were inoculated on the slant and incubated at 35OC for 18-24 hours. The slant turned black in case of a positive reaction, whereas no change in color was observed in case of a negative reaction. The positive control indicates Enterococcus faecalis (ATCC 29212) and the negative control indicates S. pyogenes (ATCC 14289).
A medium composed of a mixture of heart infusion broth (Difco Laboratories) 25 g/ l, sodium chloride (sigma, St. Louis, MO) 60 g/l and glucose (sigma) 1 g/l was melted [7]. Test microorganisms were inoculated into the broth and incubated at 35OC for 18-24 hours. The broth became turbid if a positive reaction occurred, whereas it remained clear in case of a negative reaction. The positive control indicates E. faecalis (ATCC 29212), while the negative control indicates S. pyogenes (ATCC 14289).
A latex agglutination method-Streptex (Murex) [23] was used. Microorganisms were inoculated into test tubes containing 0.4 ml enzyme extraction and incubated at 35OC for one hour. One drop of either A, B. C, D, F or G latex, along with one drop of extraction fluid, was added to the card which was then hand shaken for one minute. Positive reaction shows agglutination on the card.
The results of a streptococci diagnosing system using PYR test, pigment medium and bile esculin medium are listed in Table 1. The sensitivity and specificity, along with their positive or negative predictive values and accuracy, are given in Table 2. In total, 114 strains of group A streptococci, 202 strains of enterococci and 7 strains of aerococci were studied in the present investigation. Of those, 321 tested positive in the PYR broth, while 2 tested negative; hence the sensitivity of this test is 99.08 % . For Murex PYR, 319 strains tested as positive while 4 tested negative, yielding a sensitivity of this test as 98.48%. There was, however, one non-grouping streptococci which showed a positive reaction for both PYR broth as well as Murex PYR. The specificity of these tests is hence 99.82%. On combining bacitracin and SxT test, 108 strains of the microorganisms showed a coordinated result, thus yielding a sensitivity of 95%. In the case of non-group A streptococci, 30 strains of group G yielded a positive result for bacitracin test. Additionally, four strains of group C streptococci and one strain belonging to non-grouping streptococci also showed the same result. Hence, the specificity of this test is 90.09% . Furthermore, four strains of the group G streptococci showed different results for the bacitracin test and the SxT test, yielding a test specificity of 98.87%
Group A streptococci and enterococci can secrete a specific enzyme called aminopeptidase which can hydrolyze PYR and release beta-naphthylamide. When acid N,N-dimethylaminocinnamaldehyde is added to the above, a cherry red-colored Schiff base is formed [14,15]. The data obtained in this study showed very good sensitivity and specificity in the results from both PYR broth and Murex PYR for differentiating group A streptococci and enterococci from other streptococci. Similar results have also been reported from other countries [15-20]. Facklam has reported that one strain of group A streptococci [15] shows negative PYR reaction, probably from the lack of M-protein or because the reactivity of aminopeptidase is covered by glycocalyx [24]. However, a similar result was not observed in this laboratory. Facklam has also observed one strain of E. faecium to show negative PYR reaction [15,18]. Furthermore, Panosian [18] has observed that one strain of E. avium showed positive PYR reaction, but a negative PYR disc reaction. However, from the results obtained in here, it was observed that one strain each of E. faecium and E. avium showed negative PYR broth, while two strains each of E. faecium and E. avium showed negative Murex PYR.
Group A streptococci were mainly isolated from the upper respiratory tract and were mixed with normal flora. Many times, the mixed culture masks the appearance of the complete hemolysis [25] by normal flora, and hence interferes with the results [26]. Bellon and Kurzyniski have used throat samples which were directly inoculated on the SXT selective blood agar plate [25,27], attached with a bacitracin disc, which inhibits the growth of viridans streptococci, Staphylococcus epidermidis, Neisseria gonorrhoeae and D, G. F streptococci, hence increasing the isolation rate of the group A streptococci. Petran and Ederer have suggested that the inhibition zone should be about 10 mm in size [6,8] for a positive reading, whereas Pollock and Facklam have suggested that as long as the inhibition zone appears [4,7], the result should be considered as positive or else there will be a 10% false negative rate for the group A streptococci [7]. In general, most researchers and microbiology laboratorians consider the latter as standard. The present data suggests that the bacitracin test is better in terms of sensitivity rather than specificity. Relatively, 5-20% strains of group C and group G streptococci showed false positive bacitracin reaction and were hence misread as group A streptococci [10,15,17]. In clinical samples, occurrence of this kind of streptococci was large in number and the reliability of the bacitracin test was hence questionable [9]. Gunn and Bellon have suggested that if bacitracin tested positive along with SXT resistance, then the strain should belong to group A streptococci [10,25]. In the present study, it was observed that 22% of the strains of group G and 26% of the strains of group C streptococci showed a false positive bacitracin reaction and could hence be mistaken as group A streptococci. Upon combining the bacitracin test and the SXT test, however, only 3% of the strains of group G streptococci showed false positive bacitracin reaction and false negative SXT reaction, and could hence be mistaken as group A streptococci.
Use of pigment medium for identifying group B streptococci has offered very good results [22]. Bacteria are known to form more pigments at the stationary phase and the mid log phase [28]. Few strains of bacteria show weak reaction at the beginning; however, continuous monitoring over a period of time shows an increase in the positive rate [29]. Facklam has reported the effective use of bile esculin medium and 6.5 % NaCl broth for differentiating enterococci [7,30], group D non-enterococci and viridans streptococci. A positive reaction of bile esculin medium is considered to have occurred if half of the medium turns black. However, when there is no change in color or if less than half of the medium turns black, then the reaction would be considered as negative [30].
PYR test provides the laboratory with an innovative method that is easy to use and interpret and requires minimal technical time for accurate determination of group A streptococci and enterococci.
The authors wish to express their thanks to colleagues in the Bacteriology Section, Tri-Service General Hospital.
Copyright: 1997, Chinese Medical Association (Taipei)