Detection of bloodstream infections (BSIs) is one of the most important tasks performed by the microbiology laboratory due to high rates of patient mortality and morbidity.[1] Critically ill patients in intensive care units (ICUs) are at risk of developing nosocomial BSIs; these occur 2–7 times more often in ICU patients than in ward patients.[2] Thus, timely detection of such infections is crucial in deciding the use of appropriate antimicrobial therapy.

Staphylococci represent the most commonly encountered blood culture isolates. With the spread of both MRSA and MRCoNS in hospitals and in the community, rapid and reliable methods for detection are warranted.[3]

Methicillin resistance (MR) is mediated by low-affinity penicillin-binding protein (PBP2a) encoded by the mecA gene. The heterogonous expression of such resistance in some strains, and the traditional identification of staphylococcal blood culture isolates and overnight incubation to obtain isolated colonies, make the phenotypic testing both difficult and slow.[4] Rapid methods and variation of traditional methods have been developed that allow differentiation of staphylococci in a matter of hours, thus reducing the TAT by more than 24 hours.[5,6]

Many remarkable improvements have been made on traditional identification methods of staphylococcal blood cultures isolates. Automated continuous monitoring blood culture systems marked a significant advance in reducing the detection time. Rapid identification of bacterial pathogen also facilitates earlier therapy.[7]

Direct tube coagulase (DTC) test,[8] species identification, AST,[9] and latex agglutination for detection of PBP2a[10] were evaluated for direct identification of methicillin-resistant staphylococcal isolates in blood cultures, but not in parallel with each other. This prospective study aimed to evaluate such tests directly from positive blood cultures in parallel with each other (on the same day) for identification of MR staphylococcal isolates to compare the workflow for each test and to reduce the TAT. These direct tests were compared with those obtained by standard procedures in order to be incorporated in the practical scheme of our microbiology laboratory. Detection of the mecA gene by PCR was used as the “gold standard” in the present evaluation.

The present study was conducted between December 2006 and May 2007 at Theodor Bilharz Research Institute (TBRI), a 300-bed tertiary hospital in Egypt that provides a full range of medical and surgical care.

A total of 56 blood culture bottles (BCBs) (1 per patient) positive for GPC in clusters (suggesting staphylococci) from unique patients admitted to ICU with suspected bacteremia were included.

Patients with true bacteremia were diagnosed clinically by; a temperature above 38° C, chills, white blood cell count > 12,000 cells/mm³ with shift to the left and initiation of specific therapy (vancomycin).[11]

Evaluation of CoNS bacteremia was based on the following exclusion criterion: At least 1 set of blood cultures and/or a catheter tip culture had to be positive for CoNS to warrant direct testing. Reviewing the clinical chart with the clinician will determine the clinically significant CoNS bacteremia.[12]

The blood specimen was inoculated into BCB (B plus [Aerobic/F]) and incubated in Bactec 9050 (Becton Dickinston).

All direct tests were done on the same day in parallel. Positive BCBs were reincubated at 36° C until all direct tests results were available.

Pure isolates were further stored on glycerol stock at −70° C after overnight enrichment on tryptic soy broth (Oxoid) until tested by PCR assay.

Direct inoculum preparation. Two 3.5-mL gel separator tubes (BD Diagnostic vacutainer) were inoculated with a 7-mL broth of positive BCB. The tubes were centrifuged at 3000 rpm for 10 minutes. The supernatant was removed and bacterial pellet was resuspended in 2 mL normal saline; then the suspension was centrifuged at 3000 rpm for 5 minutes. The washing step was repeated twice. The bacterial pellet was used as the inoculum for performing latex agglutination for detection of PBP2a. As for the cefoxitin DD test, species identification by API-staph Kit and AST assays, the prepared bacterial pellet was added to demineralized water to make a suspension equivalent to 0.5 McFarland standard adjusted by using a nephelometer to make the standard suspension.[8]

Confirmation of identity of S aureus: Staphylococci subcultured on blood agar plates from positive specimens were tested by using latex agglutination test (standard test for DTC) (Staphaurex Plus; Murex Diagnostics).[8]

Staphylococcal species identification by API-Staph Kit. The standard suspension was used to perform the direct species identification according to manufacturer instructions by using API-Staph Kit (bioMérieux).[9]

Cefoxitin disk diffusion (Cefoxitin DD) test. This was performed with 30 mcg cefoxitin disk (Oxoid) which was incorporated together with DAST disks using the standard suspension following the guidelines of CLSI.[16] For S aureus, the current CLSI zone diameter breakpoint of ≤ 19 mm were reported as resistant (R) and those with a zone diameter of ≥ 20 mm were reported as susceptible (S), while for CoNS a zone diameter of ≤ 24 mm is (R) and ≥ 25 mm is (S).

Detection of mecA gene by using PCR was considered to provide the definitive results for both PBP2a LA assay and cefoxitin DD test.

Species identification results with the direct method were grouped into 3 categories: (1) correctly identified at the species level; (2) misidentified, whereby the organisms were incorrectly identified at either the genus or species level; (3) nonidentified. All correctly identified species were evaluated by DAST.

Agreement and discrepancies between DAST and SAST method were classified as follows: agreements, very major errors (false susceptibility), major errors (false resistance), and minor errors (susceptible/resistance vs intermediate susceptibility).[14]

Statistical analysis. This was done using the Epi-Info software for data management and analysis. Sensitivity, specificity, and predictive values of the current assays were calculated against the results of different standard assays.

A total of 56 consecutive blood cultures positive for GPC in clusters from patients who were prospectively diagnosed as nosocomial bacteremia in ICU were evaluated in this study. Characterization of such patients are summarized in Table 1.

Table 1 Characteristics of Patients With Staphylococcal Bacteremia

All staphylococci included were deemed clinically significant by standard culture methods, latex agglutination (Staphaurex Plus), and CoNS species screening criteria, of which 31 (55.4%) were CoNS and 25 (44.6%) were S aureus isolates. Consistency was observed between the latex agglutination and the API identification. The incidence of S aureus and CoNS species after subculture from blood agar is detailed in Table 2.

Table 2 Incidence of Staphylococcal Species Isolated From Blood Cultures (N = 56)

All direct assays were performed prospectively without the knowing of the final identification. The results of such assays were typically available 24 hours (TAT) after a blood culture was signaled positive by Bactec. For PBP2a LA assay, TAT was 20 minutes. Hands-on times (HOTs) were 5 minutes for DTC, 20 minutes for direct inoculum preparation used to perform PBP2a assay, and 23 minutes for cefoxitin DD test, species identification by API-staph Kit, and AST assays.

Performance of the current 4 rapid assays for identification of staphylococcal isolates directly from positive blood cultures are displayed in Table 3. After overnight incubation, 24 of 25 S aureus (MSSA) isolates were correctly identified by DTC test (sensitivity, 96%). The undetected strain showed positive latex agglutination with Staphaurex Plus (standard). No false-positive results were detected after 4 hours or overnight incubation (specificity, 100%). The 56 samples positive for GPC showed concordant results between the direct and standard tests for species identification by API-Staph kit. Only 1 isolate of CoNS species (S hominis, MSCoNS) was misidentified directly from positive blood cultures (sensitivity, 96.8% and specificity, 100%).

Table 3 Performance of Two Rapid Assays to Identify Staphylococci Isolates (N = 56) Directly From Blood Cultures

Methicillin resistance detected by the presence of mecA gene by PCR (Figure) as the gold standard was found in 42 of 56 (75%) – 27 of 31 (87.1%) among CoNS isolates and 15 of 25 (60%) among S aureus isolates.

Figure Agarose gel electrophoresis of the PCR products generated by using the 2 oligonucleotide primers internal to the mecA gene. Lane 1 shows molecular size marker (1000 bp; Promega Corporation; Madison, Wisconsin); lane 2 shows negative control (MSSA); lanes (more ...)

Compared with mecA gene detection by PCR, diagnosis of MR directly from positive blood cultures was achieved by PBP2a LA assay and cefoxitin DD test. Both assays had a sensitivity of 100%. Specificity of such assays was 100% and 75%, respectively, and was 90% and 100% among S aureus and CoNS isolates, respectively. The 2 direct assays yielded 2 false-negative results. For all staphylococcal isolates tested directly, MR was correctly identified (100%) by the above 2 assays (Table 4).

Table 4 Performance of Two Rapid Assays to Detect Methicillin-Resistant Staphylococci Directly From Blood Cultures

The SAST results were available for all 42 MR staphylococcal isolates (MRSA, 15 and MRCoNS, 27) tested, compared directly with standard method as shown in Table 5. Each was assessed with 13 antimicrobial agents, resulting in a total of 195 and 351 isolate-antimicrobial agent combination for MRSA and MRCoNS, respectively. There were complete categorical agreement of 12 (80%) and 17 (63%) for MRSA and MRCoNS, respectively – that is, no very major, major, or minor errors for any antimicrobial agent tested. The overall error rate was 1.11%. Very major errors occurring with MR staphylococcal isolates were chloramphenicol with S aureus (MRSA) (2 errors). For CoNS, S hominis and S haemolyticus (MRCoNS) yielded major and minor errors with teicoplanin and clindamycin, and with penicillin and ciprofloxacin, respectively.

Table 5 Antimicrobial Susceptibility Correlation for Methicillin-Resistant Staphylococcal Isolatesa

Because the primary objective of this study was to incorporate direct tests in parallel with each other as part of the routine identification of MR staphylococci from blood cultures, direct tests were evaluated comparable to routinely applied standard methods.

In the current study, a total of 56 positive blood cultures were identified as staphylococcal isolates by using the standard cultural and identification methods. Most of these cultures (66.1%) were obtained from patients suffering from primary bacteremia due to intravascular catheter; 57.1% came from patients with underlying diseases such as cirrhosis, diabetes mellitus, chronic obstructive pulmonary disease, congestive heart failure, or renal failure. Comparable results have been reported by Aygen and associates[19] and Mathur and colleagues.[20]

The impact of rapid reporting of bacterial pathogens on patient outcomes and hospital costs has been stated previously.[21,22] Murdoch and Greenlees[23] and Sogaard and coworkers[24] demonstrated the high accuracy of direct Gram stain reports that could help in rapid identification of staphylococci in positive BCBs. Findings from the current study support this practice. When blood cultures turned positive, the attending physicians were usually notified immediately about Gram stain findings. Our initial report stated that gram-positive cocci resembling staphylococci have been seen. Such information is useful for both clinician (to initiate antibiotic treatment) and for the laboratory (to start designing a flowchart for dealing with positive blood cultures). Therefore, Gram stain is required.

In our study, the method using diluted blood culture broth on the DTC test is both sensitive (96%) and specific (100%) for identifying S aureus and CoNS (all CoNS strains were isolated from patients diagnosed clinically as having true bacteremia, and the possibility of contamination due to CoNS was nil). Isolates from a single positive blood culture in 24 hours (24-hour TAT) can be achieved when there is a night shift available; negative results were reported in 4 hours. Such assay requires the shortest HOT and the smallest amount of equipment. Comparable results were reported by Varettas and associates[8] and Montgomery and colleagues.[25] However, a lower sensitivity (84.1%) for DTC test using undiluted blood culture broth was reported by Chapin and Musgnug,[5] which may be due to carryover of the inhibitors of coagulation used in blood culture media. The DTC test can be set up at any time without batching samples and creates no additional technical demands for existing laboratory staff. Moreover, it would greatly reduce the numbers of samples that might be tested subsequently for the presence of the mecA gene as an additional guide to early effective therapy of S aureus bacteremia.[8]

The results of direct species identification and DAST including cefoxitin were typically available 19–24 hours after a blood culture was signaled positive by BACTEC compared with 35–48 hours for the results of the standard method of such assays, secondary to overnight incubation required to produce isolated colonies. Both direct assays requires HOT of 23 minutes.

In the present study, the direct use of API-Staph kit yielded a sensitivity of 96.8% and specificity of 100% with 1 false-positive (misidentified) result. Similar performance results (98% sensitivity; 100% specificity) was reported by Speers and coworkers[26] and Chapin and associates[5] using API in conjunction with Bactec 9050 bottles. Ng and colleagues[27] demonstrated that a direct inoculation method provided acceptable genus identification of gram-negative bacilli from positive blood culture vials. However, Cueto and coworkers[14] reported that none of his 50 gram-positive cocci showed concordant identification results using Vitek 2 system. The comparisons between the results of different studies are difficult due to the different blood culture systems and identification methods used.

Mixed microbial and nonstandardized inoculum size were considered to be the main source of errors in both bacterial identification and AST by direct inoculation.[28,29] In the current study, the direct Gram stain enables the determination of blood cultures that may be polymicrobial, which would then be excluded from processing by a rapid method. Moreover, we obtained a standardized inoculum by direct inoculation method using the standard 0.5 McFarland suspension.

DAST of positive blood cultures can allow a laboratory to report susceptibility results to clinicians up to 24 hours sooner than SAST. DAST is not a novel concept and was described with the disk diffusion (DD) method using manual culture system with simple broth medium.[30,31] Two major changes in some microbiology laboratories have altered the interpretations. First, the DD method had been replaced by the automated broth microdilution method. Second, the standard blood culture systems are now continuously monitoring instruments that use high-volume blood culture vials.[32] Direct susceptibility testing via DD assay with blood culture system Bactec was evaluated in the present study, with an overall error rate of 1.11%. Most of such errors were derived from results testing teicoplanin, amikacin, clindamycin, and chloramphenicol. On the contrary, Edelmann and associates[33] reported a higher rate of error (5.3%) by using DAST of blood cultures. They reported that the limitation of the DD assay must be kept in mind and communication with the clinician to insure adequate treatment is necessary. It is possible that the high error rates are due to incorrect identification. This reason may justify excluding misidentified and unidentified organisms from analysis,[34] as per our study. Although we used a prompted algorithm for CoNS testing to prevent AST results for probable contamination, we attributed most of the staphylococcal DAST errors to MRCoNS.

Although current automated systems for direct DD assay and DAST have emerged to provide test results in 2–12 hours, variable results have been reported by Chapin and Musgnug.[10] Therefore, the reliability of such automated methods should be tested by individual laboratories before the method is considered for routine use.[14]

The accurate and early determination of MR is of key importance in the prognosis of BSIs caused by S aureus or CoNS isolates. Because patients with BSIs are often treated empirically, differentiation of methicillin-resistant and susceptible staphylococci isolates from a single blood culture would likely facilitate an appropriate change in therapy and an overall cost reduction.[5] Due to the impact on therapeutic regimens, the detection of the mecA gene is part of the assay. In the current study, MR in staphylococcal isolates from positive blood cultures is relatively high; 60% of S aureus isolates and 87.1% of CoNS isolates were identified by detection of mecA gene by PCR assay (genotypic-based).

Identification of the mecA gene is the most reliable method of detecting MR in staphylococcal isolates, but not all laboratories can include molecular biology techniques in their routine clinical practice. For this reason, phenotypic methods may detect such resistance in a rapid and accurate manner to insure correct antibiotic treatment and to avoid the spread of such resistant isolates in the hospital environment.[35]

The PBP2a LA assay used to detect methicillin-resistant staphylococci directly from positive blood cultures yielded a sensitivity of 100% and specificity of 92.3% compared with the mecA gene by PCR. However, these results contradict those of previous direct PBP2a assay results (18% sensitivity and 100% specificity) that Chapin and Musgnug[9] obtained using Bactec-tested blood cultures. Meanwhile, comparable results have been reported by Montgomery and colleagues[25] using the same modifications; the reported sensitivity was different but specificity was excellent. The importance of continued incubation of positive BCBs and attention to washing of bacterial pellets are important steps in the accuracy of this method. The current assay provides a rapid 20-minute slide agglutination test for detection of PBP2a, and the required HOT was 20 minutes.

Although both PBP2a and cefoxitin DD assays recorded similar sensitivity, negative predictive value, and positive predictive value of 100, 100, and 97.7%, respectively, for detecting MR directly from the current positive blood cultures, the latter is an easy and cheap method. Testing using cefoxitin can be performed; at standard temperature (35°–37° C), addition of 2% NaCl to Mueller-Hinton agar is not recommended[6] but it can be used in our routine susceptibility testing method for screening purposes for detecting MR staphylococci directly from positive blood cultures. However, PBP2a assay requires the shortest TAT.

In conclusion, direct methods can identify MRSA and MRCoNS isolates from blood cultures 24 hours earlier than routine subculture method. Incorporation of direct tests with high sensitivity and shortest TAT into routine processing of positive blood cultures may have a significant impact on rapid and accurate identification and susceptibility testing, which in turn can improve outcomes for the patient and reduce costs for hospitals.

Manal Diab, Medical Microbiology and Immunology, Cairo University; Theodor Bilharz Research Institute, Guiza, Egypt Author's email: manalkandil/at/hotmail.com.

Mervat El-Damarawy, General Medicine and Intensive Care, Cairo University; Theodor Bilharz Research Institute, Guiza, Egypt.

Mouhamed Shemis, Biochemistry, Cairo University; Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Guiza, Egypt.