Identification of methicillin-resistant isolates of Staphylococcus aureus and coagulase-negative staphylococci responsible for bloodstream infections with the Phoenix™ system

doi:10.1016/j.diagmicrobio.2003.11.004

Diagnostic Microbiology and Infectious Disease

Volume 48, Issue 4, April 2004, Pages 221-227

https://doi.org/10.1016/j.diagmicrobio.2003.11.004 Get rights and content

Abstract

We evaluated the reliability of the new Phoenix™ system (Becton Dickinson Microbiology Systems, Sparks, Md.) in species-level identification and detection of oxacillin (methicillin) resistance among 493 staphylococcal isolates (Staphylococcus aureus, n = 223; coagulase-negative staphylococci, CoNS, n = 270) recovered from patients with bacteremia. Identification results were concordant with those of the ID 32 STAPH system (bioMérieux, Marcy l'Etoile, France) for 100% of S. aureus (223/223) and 97.4% (263/270) of CoNS isolates. For S. aureus isolates, Phoenix oxacillin-susceptibility results fully concurred with those of mecA polymerase chain reaction (PCR) (reference method): 96 mecA-positive isolates identified as resistant, 127 mecA-negative strains as susceptible. Two of the 210 mecA-positive CoNS isolates were misclassified as susceptible by the Phoenix™ (sensitivity 99%, positive predictive value 97.6%). Five of 60 mecA-negative CoNS isolates were classified as resistant by the Phoenix™ (specificity 91.7%; negative predictive value 96.5%). The Phoenix™ system can provide accurate and reliable identification of methicillin-resistant staphylococci responsible for bloodstream infections.

Introduction

Methicillin-resistant staphylococci are an increasingly important cause of hospital- and community-acquired blood stream infections throughout the world (Herold et al., 1998, Marshall et al., 1998, Pfaller et al., 1999, Diekema et al., 2001, Almer, et al., 2002). These strains carry the mecA gene, which encodes a modified penicillin-binding protein (PBP2a) that is responsible for resistance to β-lactam antibiotics (Chambers, 1997, Livermore, 2000). Detection of this gene is considered the most reliable method for identifying methicillin-resistant staphylococci (Archer and Pennell, 1990, Chambers, 1997, Bekkaoui et al., 1999, Cloney et al., 1999), but it is too complicated for routine use in most microbiology laboratories.

Methicillin resistance can also be detected by a variety of phenotypic methods (Baron, 1995, Cormican et al., 1996, Mulder, 1996, Kohner et al., 1999, Tenover et al., 1999, Arbique et al., 2001, Sakoulas et al., 2001, Yamazumi et al., 2001a, Yamazumi et al., 2001b, National Committee for Clinical Laboratory Standards, 2002). Although culture-based methods are generally reliable, false-negatives can occur, particularly with clinical isolates that show hetero-resistant phenotype (Tomasz et al., 1991, Archer and Climo, 1994, Chambers, 1997, Felten et al., 2002). Problems have also been encountered in distinguishing methicillin-resistant staphylococci, which harbor the mecA gene, from the so-called borderline oxacillin-resistant strains that lack mecA (Tomasz et al., 1989, Liu et al., 1990, Chambers, 1997, Horstkotte et al., 2002) Nevertheless, phenotypic methods (automated or manual) are still the most widely used approach for identification and antimicrobial susceptibility testing of blood-stream isolates.

We evaluated the new automated Phoenix™ system (Becton Dickinson Microbiology Systems, Sparks, MD) for the identification and detection of methicillin (oxacillin) resistance of staphylococci isolated from blood cultures.

Section snippets

Bacterial strains, study design and definitions

The strains evaluated in this study were selected from a consecutive series of staphylococcal isolates recovered from blood cultures between June 2000 and November 2002 in the Microbiology Laboratory of the Catholic University of the Sacred Heart in Rome. We tested 493 clinically relevant nonduplicate staphylococcal isolates from patients who met the Centers for Disease Control criteria for diagnosis of bacteremia (Garner et al., 1988). The definition of coagulase-negative staphylococci (CoNS)

Comparison methods

Of the 493 blood-stream staphylococcal isolates examined, the most common species was S. aureus (n = 223 isolates) followed by S. epidermidis (n = 200 isolates). Other CoNS species included S. haemolyticus (n = 25 isolates), S. hominis (n = 18 isolates), S. capitis (n = 6 isolates), S. simulans (n = 6 isolates), S. warneri (n = 6 isolates), S. lugdunensis (n = 5 isolates), Staphylococcus schleiferi (n = 2 isolates), and S. xylosus (n = 2 isolates). Most of the strains (total: 419/493; 85%) came

Conclusions

Accurate identification of staphylococcal isolates responsible for bloodstream infections and reliable detection of methicillin resistance are essential for diagnosis, effective treatment, hospital infection control and epidemiologic surveillance (Archer, 1995, Kim et al., 2000, Benjamin et al., 2001). Automated systems can furnish this information rapidly, and for this reason they are increasingly used in clinical microbiology laboratories.

In the present study, we evaluated the performance of

Acknowledgements

This work was partially supported by grants from the Italian Ministry for the University and Scientific Research (ex MURST 2002-2003). We thank Marian Kent for editorial assistance.

References (46)

J. Arbique et al.
Comparison of Velogene Rapid MRSA Identification Assay, Denka MRSA-screen Assay and BBL Crystal MRSA ID System for rapid Identification of methicillin-resistant Staphylococcus aureus
Diagn Microbiol Infect Dis
(2001)
F. Bekkaoui et al.
Rapid detection of mecA gene in methicillin resistant staphylococci using a colorimetric cycling probe technology
Diagn Microbiol Infect Dis
(1999)
L. Cloney et al.
Rapid detection of mecA in methicillin resistant Staphylococcus aureus using cycling probe technology
Mol Cell Probes
(1999)
M.G. Cormican et al.
Phenotypic detection of mecA-positive staphylococcal bloodstream isolateshigh accuracy of simple diffusion test
Diagn Microbiol Infect Dis
(1996)
J.S. Garner et al.
CDC definition for nosocomial infections
Am J Infect Control
(1988)
D.M. Livermore
Antibiotic resistance in staphylococci
Internat J Antimicrob Agents
(2000)
S.A. Marshall et al.
Staphylococcus aureus and coagulase-negative staphylococci from blood stream infectionsfrequency of occurrence, antimicrobial susceptibility, and molecular (mecA) characterization of oxacillin resistance in the SCOPE program
Diagn Microbiol Infect Dis
(1998)
M.A. Pfaller et al.
Survey of blood stream infections attributable to gram-positive coccifrequency of occurrence and antimicrobial susceptibility of isolates collected in 1997 in the United States, Canada, and Latin America from the SENTRY Antimicrobial Surveillance Program. SENTRY Participants Group
Diagn Microbiol Infect Dis
(1999)
E. Sturenburg et al.
Comparison of BDPhoenix and VITEK2 automated antimicrobial susceptibility test systems for extended-spectrum β-lactamase detection in Escherichia coli and Klebsiella species clinical isolates
Diagn Microbiol Infect Dis
(2003)
Archer, G. L. (1995). Staphylococcus epidermidis and other coagulase-negative staphylococci, In G. L. Mandell, J. E....

G.L. Archer et al.

Antimicrobial susceptibility of coagulase-negative staphylococci

Antimicrob Agents Chemother

(1994)

G.L. Archer et al.

Detection of methicillin resistance in staphylococci by using a DNA probe

Antimicrob Agents Chemother

(1990)

Almer, L. S., Shortridge, V. D., Nilius, A. M., Beyer, J. M., Soni, N. B., B. E., Bui, M. H., Stone, G. G., & Flamm, R....

J. Barenfanger et al.

Clinical and financial benefits of rapid bacterial identifications and antimicrobial susceptibility testing

J Clin Microbiol

(1999)

E.J. Baron

Genetic aspects of methicillin resistance in Staphylococcus aureus and methods used for its detection in clinical laboratories in the United States

J Chemother

(1995)

D.K. Benjamin et al.

Bacteremia, central catheters and neonateswhen to pull the line

Pediatrics

(2001)

D.F.J. Brown

Detection of methicillin/oxacillin resistance in staphylococci

J Antimicrob Chemother

(2001)

H.F. Chambers

Methicillin resistance in staphylococcimolecular and biochemical basis and clinical implications

Clin Microbiol Rev

(1997)

P.E. Coudron et al.

Evaluation of laboratory tests for detection of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

J Clin Microbiol

(1986)

D.J. Diekema et al.

Survey of infections due to Staphylococcus speciesfrequency of occurrence, and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe and Western Pacific region for the SENTRY antimicrobial surveillance program, 1997-1999

Clin Infect Dis

(2001)

G.V. Doern et al.

Clinical impact of in vitro susceptibility testing and bacterial identification

J Clin Microbiol

(1994)

A. Endimiani et al.

Identification and antimicrobial susceptibility testing of clinical isolates of non fermenting gram-negative bacteria by the Phoenix automated microbiology system

New Microbiol

(2002)

A.M. Fahr et al.

Two center collaborative evaluation of the performance of the B.D. Phoenix Automated Microbiology system for identification and antimicrobial susceptibility testing of Enterococcus and Staphylococcus spp

J Clin Microbiol

(2003)

Cited by (25)

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis
2019, Journal of Dairy Science
Staphylococcus species, categorized into Staphylococcus aureus and non-aureus staphylococci (NAS), are frequent causes of mastitis in dairy cattle around the world. Current treatments using antimicrobials are under increasing scrutiny due to rising prevalence of multi-drug resistance in S. aureus. Objectives of this study were to determine: (1) genetic diversity of Staphylococcus species isolated from clinical mastitis in cows from large Chinese dairy farms; and (2) prevalence and distribution of antimicrobial resistance genes (ARG) in these isolates. Staphylococcus aureus (n = 96) were isolated from 26 herds located in 12 provinces of China, whereas NAS (n = 112) were isolated from 59 herds located in 18 provinces of China. The NAS were identified at the species level using a partial 16S rRNA sequencing method, whereas random amplification of polymorphic DNA (RAPD) PCR was done to determine genetic relationships of isolates. Finally, PCR was used to detect resistance and biofilm formation genes. Staphylococcus chromogenes (33%) was the most common NAS species, followed by Staphylococcus sciuri (17%) and Staphylococcus epidermidis (8%). Staphylococcus aureus was grouped in 12 genotypes, of which 2 types represented 56% of isolates. Staphylococcus chromogenes (n = 37) clustered into 8 RAPD types, with 2 prevalent types containing 73% of isolates. The most prevalent ARG in S. aureus isolates was blaZ (95%), followed by tetM (33%), tetK (31%), ermT (26%), and aacA-aphD (23%). The mecA and vanA were detected in 16 and 4% of isolates, respectively. In NAS, blaZ (100%), mecA (73%), tetK (79%), tetM (96%), mphC (63%), and msrA (54%) were frequently detected. Antimicrobial resistance genes mecA, tetK, tetL, tetM, dfrG, ermB, msrA, mphC, aadD, and aphA3 were more commonly detected in NAS than in S. aureus. Biofilm formation genes (icaA and icaD) were frequently detected in staphylococci isolated from bovine clinical mastitis. The existence of predominant RAPD types in S. aureus and S. chromogenes isolates across Chinese dairy farms indicated that specific genotypes had disseminated within herds and become more udder-adapted. High prevalence of ARG, especially in NAS, highlighted the risk of selection of multi-drug resistant staphylococci with potential as a reservoir of ARG.
Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to rpoB gene sequencing for species identification of bloodstream infection staphylococcal isolates
2011, Clinical Microbiology and Infection
Citation Excerpt :
We studied a collection of 450 nonrepetitive staphylococcal isolates (Table 1) recovered from blood specimens received by our Institution from January 2007 to September 2009. Most of the CoNS isolates were from cases of bacteraemia as previously defined [13]. PCR amplification and sequencing of the partial rpoB gene from the study isolates were performed using primers 2491F and 3241R [14] and, when appropriate, additional primers specifically designed to identify all staphylococcal subspecies [15].
As a result of variable expression of biochemical characters, misidentification by conventional phenotypic means often occurs with clinical isolates belonging to Staphylococcus species. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of 450 blood isolates of the most relevant staphylococcal species, using sequence analysis of the rpoB gene as the reference method. A correct species identification by MALDI-TOF was obtained in 99.3% (447/450), with only three isolates being misidentified. In addition, MALDI-TOF correctly identified all the staphylococcal subspecies studied, including Staphylococcus capitis subsp. capitis and subsp. urealyticus, Staphylococcus cohnii subsp. urealyticus, Staphylococcus hominis subsp. novobiosepticus and subsp. hominis, Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus schleiferi subsp. schleiferi and Staphylococcus sciurisubsp. sciuri. Thus, MALDI-TOF MS-based species identification of staphylococci can be routinely achieved without any substantial costs for consumables or the time needed for labour-intensive DNA sequence analysis.
Identification of staphylococci by Phoenix: Validation of a new protocol and comparison with Vitek 2
2010, Diagnostic Microbiology and Infectious Disease
Citation Excerpt :
Additionally, the present report demonstrated that the 0.25 McFarland protocol produced low discrimination results exclusively among CoNS isolates, which were more common among the other CoNS isolates (17.8%) than S. epidermidis (4%). Most probably, the lower correct identification rates observed among CoNS isolates in all studies using molecular testing as comparator (Delmas et al., 2008; Heikens et al., 2005; Kim et al., 2008; Layer et al., 2006), including the present report, in comparison to studies using phenotypic assays (Brigante et al., 2008; Carroll et al., 2006; Fahr et al., 2003; Funke and Funke-Kissling, 2005; Spanu et al., 2004; Wallet et al., 2005) are associated with the almost exclusive application of API ID32 Staph by the latter. Of note, this difference was more pronounced among studies evaluating Phoenix than Vitek 2.
Because of their frequent isolation in the routine laboratory and their increasing clinical significance, fast and accurate species identification of staphylococci may be required; this can only be achieved by automated systems. A total of 147 clinical isolates (52 Staphylococcus aureus, 50 Staphylococcus epidermidis, and 45 other coagulase-negative staphylococci [CoNS]) were first identified by molecular methodology and then comparatively tested by Vitek 2 (new colorimetric identification card) and Phoenix using the novel 0.25 McFarland and the standard 0.50 McFarland inoculum protocols. All S. aureus isolates were accurately identified. Vitek 2 identified correctly all S. epidermidis and 93.3% of the other CoNS, whereas the respective rates were 86% and 82.2% for Phoenix's standard and 92% and 82.2% for the novel protocol. It appears that both systems provide excellent identification of S. aureus, but Vitek 2 recognizes CoNS species more accurately than Phoenix. The 0.25 McFarland protocol does not improve system performance.
Detection of mecA-mediated resistance using reference and commercial testing methods in a collection of Staphylococcus aureus expressing borderline oxacillin MICs
2007, Diagnostic Microbiology and Infectious Disease
Citation Excerpt :
In many laboratories, oxacillin susceptibility methods are still used for S. aureus either because this drug is on most commercial susceptibility testing systems or laboratorians may not have confidence in using the cefoxitin disk test alone. The oxacillin methods evaluated in this study perform satisfactorily when the isolates tested are a normal population of methicillin-susceptible and methicillin-resistant S. aureus (Sakoulas et al., 2001; Spanu et al., 2004; Swenson et al., 2005; Yamazumi et al., 2001), although there have not been recent studies of the MicroScan system. With this challenge set of isolates, the only oxacillin methods that achieved sensitivity >90% were DD (91.1%) and the VITEK 2 system (91.1%), although both methods lacked specificity (58.9% and 75%, respectively).
Phenotypic methods for detecting mecA-mediated resistance in Staphylococcus aureus include both oxacillin and cefoxitin susceptibility tests; many laboratories perform multiple tests. Conflicting oxacillin and cefoxitin susceptibility results are most likely to occur for isolates that either have reduced susceptibility to oxacillin by a non–mecA-mediated mechanism or are mecA positive but are very heteroresistant. To understand the performance of oxacillin and cefoxitin tests for such isolates, we tested 135 S. aureus isolates using either cefoxitin or oxacillin and compared the results with mecA polymerase chain reaction. These strains either expressed borderline oxacillin MICs (1–4 μg/mL) and had undetermined mecA status or were mecA positive but were not detected by oxacillin broth microdilution (BMD) or disk diffusion (DD) in original testing. For 24-h readings, performance of cefoxitin tests (sensitivity/specificity) were DD (99/100), Etest using ≤6 μg/mL as susceptible (99/98), and Phoenix MIC using ≤4 μg/mL as susceptible (98/100). Using 6 μg/mL of cefoxitin as a screen test in both BMD and agar dilution also worked well (98/98—100). Sensitivity/specificity of oxacillin methods were oxacillin agar screen (BBL: 80/86; Remel, Lenexa, KS: 85/50), DD (91/59), BMD (85/88), MicroScan (89/96), VITEK Legacy (82/93), VITEK 2 (91/73), and Phoenix, (67/96). These results suggest that a cefoxitin test can be used alone to predict mecA-mediated resistance in S. aureus.
The cefoxitin disk test - what a clinical microbiologist needs to know
2007, Clinical Microbiology Newsletter
Accurate prediction of mecA- mediated resistance in Staphylococcus spp. has been a challenge for the clinical microbiologist ever since the discovery of methicillin resistance. In 2004, a cefoxitin disk diffusion test was recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) for prediction of mecA- mediated resistance in both Staphylococcus aureus and coagulase-negative staphylococci because of its high sensitivity and specificity compared to PCR tests for mecA. It is also much easier to read that the oxacillin disk diffusion test, not requiring close scrutiny of the zone of inhibition or 24 h of incubation. The test is used as a surrogate for oxacillin testing. In other words, when cefoxitin is tested, the results are applied to oxacillin and oxacillin is reported. For resistant strains, the results are applied not only to oxacillin, but also to all other β-lactam agents, which must also be reported as resistant if they are reported. There has been some confusion among clinical microbiologists about when and how the test should be used. We answer some of the questions that have been raised about the test.
Evaluation of the BD Phoenix Automated Microbiology System SMIC/ID panel for identification and antimicrobial susceptibility testing of Streptococcus spp
2005, Diagnostic Microbiology and Infectious Disease
The BD Phoenix Automated Microbiology System SMIC/ID panel was evaluated for identification and antimicrobial susceptibility testing (AST) of various streptococci. A group of 97 consecutive clinical isolates of Streptococcus pneumoniae, 23 Streptococcus pyogenes, 24 Streptococcus agalactiae, and 34 viridans streptococci were collected and comparisons made with routine manual methods used in the clinical microbiology laboratory. Overall, in 162 (91%) of 178 isolates, Phoenix identification results demonstrated agreement. For AST results for the 162 isolates that demonstrated identification concordance, the overall essential agreement rate was 98.5%; the category agreement was 94.9%; and the very major error, major error, and minor error rates were 0%, 0.15%, and 5.8%, respectively. Although relatively high minor error rates were observed with S. pneumoniae and β-lactams, 79.2% of the 77 minor errors were the result of a single log₂ dilution difference. The Phoenix SMIC/ID panel performed favorably and demonstrated the advantages of automation and simple methodology.

View all citing articles on Scopus

View full text

BacteriologyIdentification of methicillin-resistant isolates of Staphylococcus aureus and coagulase-negative staphylococci responsible for bloodstream infections with the Phoenix™ system

Abstract

Introduction

Section snippets

Bacterial strains, study design and definitions

Comparison methods

Conclusions

Acknowledgements

Diagn Microbiol Infect Dis

Diagn Microbiol Infect Dis

Mol Cell Probes

Diagn Microbiol Infect Dis

Am J Infect Control

Internat J Antimicrob Agents

Diagn Microbiol Infect Dis

Diagn Microbiol Infect Dis

Diagn Microbiol Infect Dis

Antimicrobial susceptibility of coagulase-negative staphylococci

Antimicrob Agents Chemother

Detection of methicillin resistance in staphylococci by using a DNA probe

Antimicrob Agents Chemother

Clinical and financial benefits of rapid bacterial identifications and antimicrobial susceptibility testing

J Clin Microbiol

Genetic aspects of methicillin resistance in Staphylococcus aureus and methods used for its detection in clinical laboratories in the United States

J Chemother

Bacteremia, central catheters and neonateswhen to pull the line

Pediatrics

Detection of methicillin/oxacillin resistance in staphylococci

J Antimicrob Chemother

Methicillin resistance in staphylococcimolecular and biochemical basis and clinical implications

Clin Microbiol Rev

Evaluation of laboratory tests for detection of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

J Clin Microbiol

Survey of infections due to Staphylococcus speciesfrequency of occurrence, and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe and Western Pacific region for the SENTRY antimicrobial surveillance program, 1997-1999

Clin Infect Dis

Clinical impact of in vitro susceptibility testing and bacterial identification

J Clin Microbiol

Identification and antimicrobial susceptibility testing of clinical isolates of non fermenting gram-negative bacteria by the Phoenix automated microbiology system

New Microbiol

Two center collaborative evaluation of the performance of the B.D. Phoenix Automated Microbiology system for identification and antimicrobial susceptibility testing of Enterococcus and Staphylococcus spp

J Clin Microbiol

Bacteriology
Identification of methicillin-resistant isolates of Staphylococcus aureus and coagulase-negative staphylococci responsible for bloodstream infections with the Phoenix™ system