2605. Mixed Subpopulation of Hemolytic and Non-Hemolytic Phenotype in Clinical Staphylococcus aureus Blood Isolates

Background Agr is a key regulator that controls expression of secreted exoproteins and surface protein in Staphylococcus aureus. It has been reported that mixed status of two different phenotypes including agr functional and nonfunctional subpopulations can coexist in vitro and in vivo. However, data on the natural course and clinical implication of the mixed agr status is limited. We thus investigated the frequency and characteristics of the mixed agr in clinical settings. Methods We evaluated isogenic paired MRSA isolates collected from patients with persistent S. aureus bacteremia (SAB) between October 2010 and April 2016, and then prospectively performed surveillance for the presence of mixed agr function in MRSA isolates from patients with SAB between May 2016 and December 2017. The mixed agr status was evaluated by single colony evaluation on sheep blood agar plate containing RN4220 supernatant (β-hemolysin) (Figure 1). Cross-streaking with RN4220 and RNAIII measurement were performed to confirm the agr functionality of each of hemolytic and non-hemolytic colonies, separately. The expression levels of RNAIII, hla, and saeS/saeR were measured by real-time reverse transcription polymerase chain reaction. Results A total of 161 first blood isolates were collected during study period, and 6 isolates (4%) displayed mixed phenotype by single colony test. The mixed hemolytic pattern was observed in 5 out of 52 ST72 isolates (10%) and 1 out of 82 ST5 isolates (1%) (Figure 1). No difference was found in the genotypes between hemolytic and non-hemolytic colonies from each isolate. Of the 6 isolates, three lost mixed hemolytic features in the follow-up blood cultures (Table 1). One ST72 and one ST5 isolate showed agr mixed pattern determined by different RNAIII levels, but remaining four ST72 isolates had mixed hemolytic pattern due to different expression of hla correlated with saeS/saeR expression (Figure 2). Conclusion The mixture of agr function status among the clinical blood isolates of MRSA was rarely observed and isolates displaying heterogeneous hemolytic phenotype were largely due to differential expression of α-hemolysin. Further investigation is needed to unveil the clinical significance of mixture of different hemolytic phenotypes. Disclosures All authors: No reported disclosures.

Modified coagglutination procedure for the serological grouping of streptococci

Cowan I staphylococci coated with antisera to streptococcal groups A, B, C, D, F, and G were used as coagglutination reagents in a modified coagglutination procedure (MCAP). Streptococcal group antigens were extracted with a Streptomyces albus-lysozyme enzyme mixture for 30 min at 55 degrees C and centrifuged, and the supernatant was tested by slide coagglutination. Positive coagglutination reactions occurred within 30 s. The cell pellets from overnight broth cultures and colonies taken directly from sheep blood agar plates were tested and compared with the results of the Lancefield capillary precipitin method. Of the 102 strains of broth-grown cells tested, 100 were grouped by the MCAP and the Lancefield capillary precipitin method. The remaining two isolates were serologically identified only by the MCAP. Of the original 102 strains, 97 were tested by MCAP after extraction of five well-isolated colonies from a sheep blood agar plate. When this latter method was used, 95.9% of the strains were correctly identified. Nonspecific reactions were observed only while testing the MCAP with the direct plate assay. These cross-reactions were remedied promptly by either absorption or dilution of the antisera involved. The MCAP was found to be a rapid and reliable technique for the serological grouping of streptococci.

Improved reliability of the primary plate bacitracin test on throat cultures with sulfamethoxazole-trimethoprim blood agar plates

The primary plate bacitracin differentiation disk susceptibility test identified 85% of group A streptococci from throat cultures on SXT-BA(CO2) plates within 24 h, as compared to only 26% on a conventional sheep blood agar plate.

Gentamicin-blood agar for isolation of Streptococcus pneumoniae from respiratory secretions

Previous studies have suggested that the yield of Streptococcus pneumoniae from respiratory secretions can be increased by using a 5% sheep blood agar plate supplemented with 5 microgram of gentamicin (GBA) per ml. We report our experience with 245 lower respiratory specimens in which this method was compared with 5% sheep blood agar (SBA) alone. Of 35 specimens with growth of S. pneumoniae on either plate, 21 were detected exclusively on SBA, whereas only 3 were detected on GBA alone (P less than 0.01). By subculturing representative alpha-hemolytic colonies from the final 169 specimens, the yield of S. pneumoniae was increased by 27% compared with the number of identifications that could be made directly from the primary culture. Minimal inhibitory concentrations of gentamicin for the last 25 isolates were greater than or equal to 8 microgram/ml. Our results do not substantiate the previous observations that S. pneumoniae from respiratory secretions gives an increased yield in cultures on GBA.

CAMP-disk test for presumptive identification of group B streptococci

The modification of the CAMP test for goup B streptococci involved substituting a paper disk impregnated with partially purified beta-hemolysin for the staphylococcal culture that was the source of beta-hemolysin in the original test. The disk is placed onto a sheep blood agar plate beside the streak of Streptococcus being tested. The plate is then incubated aerobically at 35 degrees C. A positive reaction consists of a lunar-shaped clear zone that appears within 24 h in the dark beta-hemolysin zone surrounding the disk. A double-blind study of 135 randomly coded streptococcal isolates showed complete agreement between the CAMP-disk test and the standard Lancefield precipitin test. All group B streptococci tested had positive reactions, and all strains tested from streptococcal groups A, C, D, and G were negative. The CAMP-disk test is a simple and convenient way to identify presumptively group B streptococci.

THE ROLE OF THE PEDIATRICIAN IN RHEUMATIC FEVER CONTROL

The following breakdown of the cost of a "strep only" throat culture came to my attention too late for inclusion in the above Commentary. This accounting was prepared by a university hospital laboratory director as justification for charging $7.50 for each such culture. The technique employed is the inoculation of a sheep blood agar plate plus two weak bacitracin disks for differentiation of group A from other hemolytic streptococci. See Table in the PDF File Obviously 40 minutes of personnel time (exclusive of professional supervision) is unreasonable. Granting that many other factors require consideration, ranging from the expense of a case of rheumatic fever on the one hand to the fact that the laboratory helps support other facets of hospital operation on the other, this cost is clearly prohibitive. Pediatricians and laboratory directors must find a better way.–

GROUP A STREPTOCOCCAL INFECTIONS IN A CHILDREN'S HOME

School-age children living in a children's home had pharyngeal cultures made on 485 consecutive infirmary admissions during a period of 14 months by inoculating the throat swab on the surface of a sheep blood agar plate. ASO titers were determined for the acute and convalescent phases of 95% of the 321 untreated illnesses associated with negative cultures. A rise in ASO titer occurred in 3.6% of these illnesses. ASO titers were also determined on pairs of sera for 51 of 53 untreated febrile illnesses associated with negative streptococcal cultures in 1962 and a rise in ASO titer occurred for only one of these illnesses (2%). This simple method for pharyngeal cultures is adequate and accurate as a laboratory aid to the practicing physician for the diagnosis of streptococcal pharyngitis in normal children. Bacitracin sensitivity showed an excellent correlation with Group A streptococci. Studies of duplicate swabs indicated that overnight storage of a dry swab at room or refrigerator temperature was associated with the recovery of more than 10 colonies of hemolytic streptococci from 80% to 88% of the swabs whose duplicates had more than 50 colonies.