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Microbiological Characteristics, Presumptive Identification, and Antibiotic Susceptibilities of Staphylococcus lugdunensis

American Society for Microbiology
Journal of Clinical Microbiology
Authors:
Thean Yen Tan at Changi General Hospital
Thean Yen Tan
Siew Yong Ng
Siew Yong Ng
Siew Yong Ng
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Jie He
Jie He
Jie He
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Abstract

This study validated abbreviated methods for the presumptive identification of Staphylococcus lugdunensis and studied the antibiotic susceptibilities of 106 isolates. The combination of positive responses to ornithine and pyrrolidonyl arylamidase identified all S. lugdunensis isolates. Resistance to penicillin and methicillin was detected in 27 and 5% of isolates, respectively.
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JOURNAL OF CLINICAL MICROBIOLOGY, July 2008, p. 2393–2395 Vol. 46, No. 7
0095-1137/08/$08.00⫹0 doi:10.1128/JCM.00740-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Microbiological Characteristics, Presumptive Identification, and
Antibiotic Susceptibilities of Staphylococcus lugdunensis
䌤
Thean Yen Tan,
1
* Siew Yong Ng,
1
and Jie He
2
Division of Laboratory Medicine, Changi General Hospital, Singapore,
1
and Department of Pathology,
Singapore General Hospital, Singapore
2
Received 17 April 2008/Accepted 5 May 2008
This study validated abbreviated methods for the presumptive identification of Staphylococcus lugdunensis
and studied the antibiotic susceptibilities of 106 isolates. The combination of positive responses to ornithine
and pyrrolidonyl arylamidase identified all S. lugdunensis isolates. Resistance to penicillin and methicillin was
detected in 27 and 5% of isolates, respectively.
Staphylococcus lugdunensis, a coagulase-negative staphylo-
coccus, has clinical characteristics that resemble those of the
coagulase-positive Staphylococcus aureus. Infections attributed
to S. lugdunensis include infective endocarditis (19), bactere-
mia, meningitis (6), bone and joint infections (7), and soft-
tissue infections (16). S. lugdunensis generally is susceptible to
anti-staphylococcal antibiotics (18), but increasing penicillin
resistance has been reported (9, 13). Meanwhile, oxacillin sus-
ceptibility breakpoints for S. lugdunensis were changed in 2005
(3), and oxacillin disc breakpoints were revised in 2005 and
subsequently replaced by cefoxitin disc breakpoints in 2006 (2).
The current reference method for the identification of coagu-
lase-negative staphylococci (1) is labor-intensive. Screening for
S. lugdunensis by detecting clumping factor (15, 21) or using a
limited number of biochemical tests (5, 12, 15) has been pro-
posed, but these methods have been evaluated only against
small numbers of S. lugdunensis isolates.
The aims of this study were to evaluate the use of simple
screening strategies for the identification of S. lugdunensis,to
comprehensively describe the microbiological characteristics
of S. lugdunensis, and to evaluate the antibiotic susceptibility of
clinical isolates.
The accuracy of three abbreviated identification protocols
and two commercial identification kits (ID 32 Staph and Vitek
ID-GP; both from bioMe´rieux, France) was evaluated against
a collection of coagulase-negative staphylococci that were iso-
lated from clinical samples. Isolates from the collection were
identified using a panel of 30 phenotypic and biochemical tests
(1) and included Staphylococcus capitis (n ⫽7), Staphylococcus
caprae (n⫽5), Staphylococcus cohnii (n⫽2), Staphylococcus
epidermidis (n⫽14), Staphylococcus haemolyticus (n⫽24),
Staphylococcus hominis (n⫽3), Staphylococcus lugdunensis
(n⫽46), Staphylococcus saprophyticus (n⫽2), and Staphylo-
coccus warneri (n⫽3). The identification of 10 isolates (be-
longing to S. haemolyticus,S. cohnii,S. epidermidis, and S.
lugdunensis) was further confirmed by sequencing a 457-bp
sequence of the 16S rRNA gene (8). For the three abbreviated
identification protocols, isolates were tested for the presence
of pyrrolidonyl arylamidase (PYR) (Oxoid, United Kingdom),
ornithine decarboxylase (BioMedia, Malaysia), and urease
(BioMedia, Malaysia); trehalose utilization; alkaline phos-
phatase activity; and mannose utilization (the last three sub-
stances were from Rosco, Denmark). In the first identification
protocol, isolates that were positive for both PYR and orni-
thine decarboxylase were identified as S. lugdunensis.Inthe
second identification protocol, isolates that were positive for
PYR, ornithine decarboxylase, and mannose utilization were
identified as S. lugdunensis. The third identification protocol
identified an isolate as S. lugdunensis if it was positive for
trehalose and ornithine decarboxylase and negative for alka-
line phosphatase (12). The first and second identification pro-
tocols correctly identified all 46 isolates of S. lugdunensis (sen-
sitivity, 100%), while the third testing protocol identified 42
isolates of S. lugdunensis (sensitivity, 91%). None of the other
staphylococcal species was misidentified as S. lugdunensis by
the three protocols (specificity, 100%). ID 32 Staph correctly
identified 45 (98%) isolates of S. lugdunensis, and Vitek ID-GP
correctly identified 43 (93%) isolates of S. lugdunensis.
The 46 strains of S. lugdunensis were subcultured onto tryp-
ticase-soy agar plates with 5% sheep blood and incubated in
ambient atmosphere at 35°C for 3 days. Colonial morphology
was observed after 24, 48, and 72 h of incubation. Colonies
were 1 mm in diameter after 24 h of incubation and increased
to 3 mm after 48 h at 35°C. Forty (87%) isolates showed a
narrow border of beta-hemolysis after 24 h of incubation. After
48 h of incubation, 39 (85%) isolates showed significant beta-
hemolytic activity, and 4 isolates (9%) showed slight beta-
hemolysis. Colonies at 24 h typically were opaque white, with a
glossy sheen (n⫽40; 87%). At 48 and 72 h, 13 isolates (28%)
developed a yellow-white hue resembling that of S. aureus.S.
lugdunensis isolates had a characteristic sweet, hay-like odor
resembling that of the screwpine leaf, and this was prominent
in 38 (79%) isolates after 48 h of incubation.
The detection of clumping factor/protein A using three com-
mercial latex agglutination kits, Pastorex Staph (Bio-Rad,
France), Staphaurex, and BactiStaph (both from Remel,
United States), was performed on overnight cultures. Rapid
and clear agglutination was classified as a positive result, while
slower and less distinct agglutination was classified as a weak
* Corresponding author. Mailing address: Division of Laboratory
Medicine, Changi General Hospital, 2 Simei Street 3, Singapore,
529889. Phone: 65-68504934. Fax: 65-64269507. E-mail: thean_yen_tan
@cgh.com.sg.
䌤
Published ahead of print on 14 May 2008.
2393
positive result. The interpretation of a positive agglutination
test was always made with reference to the manufacturer’s
instructions. All isolates were tested for the presence of free
coagulase using a rabbit plasma tube coagulase test method.
The three latex agglutination kits showed different perfor-
mance characteristics. BactiStaph and Pastorex Staph showed
strongly positive agglutination for 49 and 42% of S. lugdunensis
isolates, respectively, while strongly positive agglutination was
present in only 9% of isolates that were tested by Staphaurex.
For both positive and weak-positive agglutination reactions,
the test kit positivity was 73, 76, and 17% for BactiStaph,
Pastorex Staph, and Staphaurex, respectively. All isolates were
negative for free coagulase.
Antibiotic susceptibility testing was performed on a separate
collection of 106 isolates of S. lugdunensis that were collected
prospectively from clinical specimens from 2004 to 2006 and
identified using the first identification protocol. Antibiotic sus-
ceptibilities were performed by disk diffusion and were inter-
preted using CLSI guidelines (4), except for oxacillin, for which
the last applicable guidelines from 2005 were applied (3).
MICs of penicillin and oxacillin were determined by agar di-
lution. All isolates were screened for the mecA gene (20), and
mecA-positive isolates were tested for the presence of pbp2⬘by
latex agglutination (Oxoid, United Kingdom) using previously
described methods (10). The species identification of all mecA-
positive S. lugdunensis isolates was confirmed by 16S rRNA
gene sequencing. The antibiotic susceptibilities of 106 clinical
isolates are listed in Table 1. Twenty-nine isolates (27%) were
resistant to penicillin by agar dilution. The modal oxacillin
MIC was 1 ␮g/ml (Table 2), which is higher than those for most
other staphylococcal species but similar to that for S. sapro-
phyticus and S. cohnii (11, 13). The oxacillin MICs for five
isolates were ⱖ4␮g/ml, and all were positive for both mecA
and pbp2⬘. Current cefoxitin disk breakpoints accurately de-
tected mecA-positive isolates, as opposed to oxacillin disk
breakpoints (susceptibility breakpoint, ⱕ13 mm; resistance
breakpoint, ⱖ10 mm), which had poor sensitivity (80%) and
specificity (65%).
This study comprehensively examined the use of abbreviated
protocols for the presumptive identification of S. lugdunensis.
Our results show that the use of clumping factor may not be a
reliable screening method to identify this organism, as results
appear to be kit dependent. The two-test protocol that utilized
ornithine decarboxylase and PYR accurately identified all of
our S. lugdunensis strains. The suggested addition of mannose
utilization to differentiate S. haemolyticus from S. lugdunensis
(15) was not required to improve the specificity of this proto-
col. Of the commercial kits, the ID 32 Staph kit performed
marginally better than the Vitek ID-GP card.
S. lugdunensis generally has been characterized as being
susceptible in vitro to most antibiotics. Early studies reported
penicillin resistance rates of ⬍4% (14), while more recent
studies report penicillin resistance rates of 12 to 15% (9, 13).
Only one methicillin-resistant isolate has been reported (17).
Over a quarter of clinical isolates of S. lugdunensis in our
population were resistant to penicillin, while methicillin resis-
tance was present in 5% of study isolates. However, other than
that for tetracycline, resistance to non-beta-lactam antibiotics
was low. Current CLSI breakpoints for oxacillin MIC and
cefoxitin disc testing clearly differentiate mecA-positive strains
of S. lugdunensis. The latex detection of pbp2 also is a viable
alternative.
This study validates a simple screening strategy to differen-
tiate S. lugdunensis from other coagulase-negative staphylo-
cocci. This will improve the recognition of clinical disease and
the surveillance of antibiotic resistance in S. lugdunensis.
This study was funded by a grant from the National Medical Re-
search Council.
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on disc susceptibility testing
a
Antibiotic No. of isolates
tested
No. (%)
sensitive
No. (%)
resistant
Cefoxitin 106 101 (95.3) 5 (4.7)
Ciprofloxacin 106 105 (99.1) 1 (0.9)
Clindamycin 106 104 (98.2) 2 (1.8)
Cotrimoxazole 106 106 (100)
Erythromycin 106 104 (98.2) 2 (1.8)
Penicillin 106 77 (72.6) 29 (27.4)
Tetracycline 106 94 (88.7) 12 (11.3)
a
The interpretative criteria are from reference 4.
TABLE 2. No. of strains positive or negative for the mecA gene
according to oxacillin MICs
a
mecA status No. of strains with oxacillin MIC (␮g/ml) of:
0.125 0.25 0.5 0.75 1 2 16 ⱖ32
Negative 2 13 53 2 29 2
Positive 4 1
a
Methicillin resistance is defined as an oxacillin MIC of ⱖ4␮g/ml.
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VOL. 46, 2008 NOTES 2395
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... The worldwide prevalence of b-lactamase-producing, blaZpositive, penicillin-resistant S. lugdunensis isolates is 7% to 40% (16). Oxacillin resistance mediated by penicillin-binding protein (PBP2A) encoded by mecA is uncommon (17)(18)(19)(20)(21). ...
... In our local review on susceptibility testing for S. lugdunensis, we detected penicillin resistance and blaZ positivity rates of 25% among 200 isolates, which correlates with previously published rates from Denmark (3), Sweden (13,31), and Singapore (19). (32) and 40% from 60 isolates in Spain, respectively (26). ...
... We acknowledge that the applicability and generalizability of our oxacillin data are limited by the small subset of 40 isolates for which mecA PCR was performed and by the low rate of oxacillin resistance of 1% at our institution. Rates of oxacillin resistance above 8% have been reported elsewhere (15,19,31,33). Detection of oxacillin resistance using the Vitek 2 oxacillin MIC, Vitek 2 cefoxitin screen, and mecA PCR are thought to have sensitivities and specificities of above 95% in coagulase-negative staphylococci. ...
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Evaluation of penicillin and oxacillin susceptibility testing was conducted on two hundred Staphylococcus lugdunensis isolates. Disc diffusion with penicillin 1 IU (P1, EUCAST) and penicillin 10 IU (P10, CLSI) was compared with nitrocefin discs (Cefinase®) and automated broth microdilution (Vitek2®). Oxacillin susceptibility was extrapolated from cefoxitin 30μg disc diffusion (FOX) and compared with Vitek2®. Reference methods were blaZ and mecA PCR. Penicillin zone diameter and zone edge correlated with blaZ in all except two P10 susceptible isolates (VME; very major error) and one P1 resistant isolate (ME). One hundred and forty-eight isolates were blaZ -negative of which one hundred and forty-six and one hundred and forty-nine isolates were susceptible by P1 and P10 respectively. One hundred and twenty-seven isolates were penicillin susceptible by Vitek2®. Vitek2® overcalled resistance in twenty-one blaZ -negative, twenty P1 and twenty-two P10 susceptible isolates (Vitek2® ME rate, 14.2%). Two mecA -positive isolates were oxacillin resistant by FOX and Vitek2® (categorical agreement). However, eighteen FOX susceptible, mecA -negative isolates tested resistant by Vitek2®. In conclusion, Vitek2® over-estimated penicillin and oxacillin resistance compared with disc diffusion and PCR. Disc diffusion with zone edge interpretation was more accurate and specific than automated broth microdilution for S. lugdunensis in our study.
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... Strains isolated worldwide from hospitalized patients mainly belong to CC1 and CC3 [6][7][8]. Even if some methicillin resistant clones are circulating in Asia [8][9][10], this species currently remains highly sensitive to antibiotics [3]. Predominance of some CCs could then be explained by the low genetic diversity of the species [11] or by an increased ability of these CCs to survive in the hospital environment by biofilm formation [1] and/or resistance to antiseptics. ...
... The only MRSL strain in our study showed reduced susceptibility to both CHX and BAC. MRSL, more prevalent in Asia [9,10,40], could represent a major health issue worldwide. Analysis of a larger number of MRSL strains would therefore be necessary to track such a putative link between methicillin resistance and reduced susceptibility to antiseptics. ...
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... Two major MRSL strains of sequence type (ST) 3 and ST6 have been reported in previous studies (16). ST6 strains carry SCCmec II more frequently, and represent endemic clones in hospitals (17). ...
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... It is mainly associated with the lower parts of the human body but can also be found in the nasal cavity, causing a wide range of infections, including severe ones with high mortality rates [2][3][4]. Moreover, S. lugdunensis infections may be underestimated since a positive result, as with S. aureus, may be identified with routinely used agglutination tests [5]. The invasive infections are also detected at an insufficient level since coagulase negative staphylococci (CoNS) are often discarded as contaminants. ...
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... The majority of S. lugdunensis isolates, for instance, demonstrate penicillin susceptibility due to the lack of !-lactamase production, unlike other Staphylococcus spp. (Thean, Siew and He, 2008). In PJI, regimes with multiple antibiotic combinations are usually needed because of the gradual build-up of resistance by bacteria (Zimmerli, Trampuz and Ochsner, 2004). 2 ...
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  • Apr 2023
  • Fed Pract
Background: Staphylococcus lugdunensis (S lugdunensis) is a species of coagulase-negative Staphylococcus and a constituent of human skin flora. S lugdunensis has gained notoriety for its virulence, which resembles Staphylococcus aureus (S aureus). S lugdunensis is now recognized as an important nosocomial pathogen and cause of prosthetic device infections, including vascular catheter infections. Case presentation: A 60-year-old man with a history of uncontrolled type 2 diabetes mellitus and end-stage renal disease on home hemodialysis via arteriovenous fistula (AVF) presented to the emergency department for evaluation of subacute progressive low back pain. Initial laboratory tests were notable for elevated inflammatory markers. Magnetic resonance imaging with contrast of the thoracic and lumbar spine revealed abnormal marrow edema in the T11-T12 vertebrae with abnormal fluid signal in the T11-T12 disc space. Cultures grew methicillin-sensitive S lugdunensis. The patient's antibiotic regimen was narrowed to IV oxacillin. He was transitioned to IV cefazolin dosed 3 times weekly after hemodialysis and an outpatient dialysis center. Conclusions: Treatment of bacteremia caused by S lugdunensis or S aureus should be managed with prompt initiation of IV antistaphylococcal therapy, a thorough evaluation for the source of bacteremia as well as metastatic complications, and consultation with an infectious disease specialist. This case highlights AVF as a potential source for infection even without localized signs of infection. The buttonhole method of AVF cannulation was thought to be a major contributor to the development and persistence of our patient's bacteremia. This risk should be discussed with patients using a shared decision-making approach when developing a dialysis treatment plan.
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Necrotizing Fasciitis Due to Staphylococcus lugdunensis: A Case Report and Review of Literature
Article
  • Apr 2023
Necrotizing fasciitis is a complicated infection requiring surgical intervention, antimicrobial therapy, and extensive supportive care. Necrotizing fasciitis presents with a few key clinical features and surgical findings, even though it can be a monomicrobial or polymicrobial infection. Timely medical and surgical management is crucial to prevent mortality and achieve positive clinical outcomes. Although initial treatment includes broad-spectrum antibiotics, therapy is often targeted based on isolated cultures and susceptibilities. First identified in 1988, Staphylococcus lugdunensis has been isolated as a pathogen in an increasing number of invasive infections. We describe a rare case of necrotizing fasciitis associated with S. lugdunensis and review the current clinical and microbiological literature.
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Staphylococcus lugdunensis Uses the Agr Regulatory System to Resist Killing by Host Innate Immune Effectors
Article
Full-text available
Coagulase-negative staphylococci (CoNS) are frequently commensal bacteria that rarely cause disease in mammals. Staphylococcus lugdunensis is an exceptional CoNS that causes disease in humans similar to virulent Staphylococcus aureus, but the factors that enhance the virulence of this bacterium remain ill defined. Here, we used random transposon insertion mutagenesis to identify the agr quorum sensing system as a regulator of hemolysins in S. lugdunensis. Using RNA sequencing (RNA-seq), we revealed that agr regulates dozens of genes, including hemolytic S. lugdunensis synergistic hemolysins (SLUSH) peptides and the protease lugdulysin. A murine bacteremia model was used to show that mice infected systemically with wild-type S. lugdunensis do not show overt signs of disease despite there being high numbers of bacteria in the livers and kidneys of mice. Moreover, proliferation of the agr mutant in these organs was no different from that of the wild-type strain, leaving the role of the SLUSH peptides and the metalloprotease lugdulysin in pathogenesis still unclear. Nonetheless, the tropism of S. lugdunensis for humans led us to investigate the role of virulence factors in other ways. We show that agr-regulated effectors, but not SLUSH or lugdulysin alone, are important for S. lugdunensis survival in whole human blood. Moreover, we demonstrate that Agr contributes to survival of S. lugdunensis during encounters with murine and primary human macrophages. These findings demonstrate that, in S. lugdunensis, Agr regulates expression of virulence factors and is required for resistance to host innate antimicrobial defenses. This study therefore provides insight into strategies that this Staphylococcus species uses to cause disease.
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Etude des facteurs d'opportunisme chez Staphyloccocus lugdunensis
Thesis
  • Nov 2021
Mon projet de thèse portant sur l’étude des facteurs d’opportunisme chez Staphylococcus lugdunensis avait pour but de mieux comprendre comment cette bactérie commensale peut être responsable d’in- fections graves. La première partie de nos travaux a consisté en la construction chez S. lugdunensis de mutants ΔagrA et ΔARNIII, codant des régulateurs potentiellement impliqués dans la virulence, afin de les caractériser phénotypiquement et d’identifier les membres des régulons par des analyses transcripto- mique et protéomique globales. Nos résultats ont mis en évidence que AgrA est le régulateur majeur du système Agr chez S. lugdunensis et qu’il contrôle l’expression de gènes lui permettant d’être plus virulent, de s’adapter aux stress environnementaux (notamment les stress osmotique et oxydatif), d’induire la formation de biofilm ainsi que la synthèse des pigments caroténoïdes. La seconde partie de nos travaux a consisté à évaluer l’impact de la limitation en fer, qui représente un stress rencontré par les bactéries pendant l’infection, sur l’expression de la pathogénicité de S. lugdunensis. Des approches phénotypique, transcriptomique et protéomique ont été entreprises et la méthode d’évaluation de la pathogénicité de S. lugdunensis en modèle d’infection de larves de Galleria mellonella a été mise au point. Nos données ont révélé que la carence en fer chez S. lugdunensis est un stress qui favorise sa capacité à former du biofilm tandis qu’il diminue sa résistance au stress oxydatif ainsi que sa virulence. De plus, en réponse à la condition de limitation en fer, S. lugdunensis est capable de mettre en place plusieurs mécanismes d’acquisition du fer, ce qui lui apporte vraisemblablement un avantage lors de l’infection et peut expliquer en partie le caractère opportuniste de ce pathogène.
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Rapid Detection of mecA-Positive andmecA-Negative Coagulase-Negative Staphylococci by an Anti-Penicillin Binding Protein 2a Slide Latex Agglutination Test
Article
Full-text available
A rapid slide latex agglutination (LA) test, MRSA-Screen (Denka Seiken Co., Niigata, Japan), which detects PBP 2a, was tested for its ability to differentiate between mecA -positive and -negative coagulase-negative staphylococci. A total of 463 isolates from 13 species were included in the study. The mecA gene was detected by PCR, and the oxacillin MIC was determined by the agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). The LA test was performed with oxacillin-induced isolates. The true-positive and true-negative results were defined on the basis of the presence or the absence of the mecA gene. By PCR, 251 isolates were mecA positive and 212 were mecA negative. The sensitivities, specificities, and positive and negative predictive values for the LA test compared to the NCCLS breakpoint for oxacillin resistance (≥0.5 mg/liter) were as follows: for the LA test, 100, 99.5, 99.6, and 100%, respectively; for the NCCLS breakpoint, 100, 60.8, 75.1, and 100%, respectively. One hundred twenty-five mecA -positive isolates were also tested by the LA test without induction of PBP 2a; only 72 (57.6%) gave a positive result and required 3 to 15 min for reaction. With induction, all 251 isolates were positive within 3 min. The LA test was reliable in classifying mecA -negative isolates, but it classified isolates for which the oxacillin MIC was ≥0.5 mg/liter as oxacillin susceptible. For the reliable detection of oxacillin resistance by the MRSA-Screen in coagulase-negative staphylococci, induction of the mecA gene appears to be necessary.
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Staphylococcus lugdunensis: Report of a Case of Peritonitis and an Easy-To-Perform Screening Strategy
Article
Full-text available
  • Mar 1998
We report on a severe case of peritonitis due to Staphylococcus lugdunensis. The clinical course resembled an infection due to S. aureus more than one due to other coagulase-negative staphylococci. Therefore, we strongly recommend identification and propose an easy-to-perform procedure for screening of this pathogen.
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Diagnostics of Neisseriaceae and Moraxellaceae by Ribosomal DNA Sequencing: Ribosomal Differentiation of Medical Microorganisms
Article
Full-text available
Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.
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Specific detection of methicillin-resistant Staphylococcus aureus by multiplex PCR
Article
Full-text available
  • Dec 1995
In Staphylococcus aureus, mecA and femA are the genetic determinants of methicillin resistance. By using a multiplex PCR strategy, 310- and 686-bp regions of the mecA and femA genes, respectively, were coamplified to identify susceptible (lacking mecA) and resistant (mecA+) staphylococci and to differentiate S. aureus (femA+) from coagulase-negative staphylococci (lacking femA). A third staphylococcal genomic sequence, corresponding to IS431 and spanning 444 bp, was used as a PCR control. One hundred sixty-five staphylococcal strains were tested. All 72 methicillin-resistant strains were found to be mecA+, and 92 of the 93 susceptible isolates lacked mecA. Only one coagulase-negative Staphylococcus isolate carrying the mecA gene was highly susceptible to oxacillin. The femA determinant was a unique feature of S. aureus; it was found in 100% of the S. aureus strains tested but was undetectable in all of the coagulase-negative staphylococci tested. The possibility of directly detecting the mecA and femA genes in blood samples was also investigated. After two amplification steps, a sensitivity of 50 microorganisms per ml of freshly collected spiked blood was achieved. In conclusion, coamplification of mecA and femA determinants proved to be very reliable both for rapid detection of methicillin resistance and differential diagnosis between S. aureus and other staphylococci. This technique, which can be successfully performed with blood samples, could be a useful tool in the diagnosis and treatment monitoring of staphylococcal infections.
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Correlation of Oxacillin MIC with mecA Gene Carriage in Coagulase-Negative Staphylococci
Article
Full-text available
The National Committee for Clinical Laboratory Standards has recently changed the oxacillin breakpoint from >/=4 mg/liter to >/=0. 5 mg/liter to detect methicillin-resistant coagulase-negative staphylococci (CoNS) because the previous breakpoint lacked sensitivity. To determine the correlation between the new oxacillin breakpoint and the presence of the mecA gene, 493 CoNS of 11 species were tested. The presence of the mecA gene was determined by PCR, and oxacillin susceptibility was determined by the agar dilution method with Mueller-Hinton agar containing 2% NaCl and oxacillin (0. 125 to 4.0 mg/liter). The new breakpoint correctly classified all CoNS strains with mecA as methicillin resistant and strains of Staphylococcus epidermidis, S. haemolyticus, and S. hominis without mecA as methicillin susceptible. The breakpoint of >/=0.5 mg/liter was not specific for S. cohnii, S. lugdunensis, S. saprophyticus, S. warneri, and S. xylosus, in that it categorized 70 of 74 strains of these species without mecA (94.6%) as methicillin resistant. The results of this study indicate that the new oxacillin breakpoint accurately identifies strains of CoNS with mecA but is not specific for strains of certain species of CoNS without mecA.
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Rapid Detection of mecA-Positive and mecA-Negative Coagulase-Negative Staphylococci by an Anti-Penicillin Binding Protein 2a Slide Latex Agglutination Test
Article
Full-text available
  • Jul 2000
A rapid slide latex agglutination (LA) test, MRSA-Screen (Denka Seiken Co., Niigata, Japan), which detects PBP 2a, was tested for its ability to differentiate between mecA-positive and -negative coagulase-negative staphylococci. A total of 463 isolates from 13 species were included in the study. The mecA gene was detected by PCR, and the oxacillin MIC was determined by the agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). The LA test was performed with oxacillin-induced isolates. The true-positive and true-negative results were defined on the basis of the presence or the absence of the mecA gene. By PCR, 251 isolates were mecA positive and 212 were mecA negative. The sensitivities, specificities, and positive and negative predictive values for the LA test compared to the NCCLS breakpoint for oxacillin resistance (>/=0.5 mg/liter) were as follows: for the LA test, 100, 99.5, 99.6, and 100%, respectively; for the NCCLS breakpoint, 100, 60.8, 75.1, and 100%, respectively. One hundred twenty-five mecA-positive isolates were also tested by the LA test without induction of PBP 2a; only 72 (57.6%) gave a positive result and required 3 to 15 min for reaction. With induction, all 251 isolates were positive within 3 min. The LA test was reliable in classifying mecA-negative isolates, but it classified isolates for which the oxacillin MIC was >/=0.5 mg/liter as oxacillin susceptible. For the reliable detection of oxacillin resistance by the MRSA-Screen in coagulase-negative staphylococci, induction of the mecA gene appears to be necessary.
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Detection of clumping factor-positive Staphylococcus lugdunensis by Staphaurex Plus (R)
Article
  • Dec 1997
  • J MICROBIOL METH
Staphaurex Plus® (Murex Diagnostics), a rapid agglutination test for detecting Staphylococcus aureus, was combined with the free coagulase test to search for the coagulase-negative species Staphylococcus lugdunensis and for S. aureus. Staphaurex Plus® detected 29 S. lugdunensis and 1602 S. aureus in our routine laboratory over 6 months. Furthermore, 10 out of 902 coagulase-negative staphylococci other than S. lugdunensis reacted with Staphaurex Plus®. The sensitivity and specificity for detecting S. aureus were 99.7% and 95.7%, respectively. Clumping factor-positive S. lugdunensis can bind fibrinogen coated on the latex particles of Staphaurex Plus®. If this agglutination was considered true positive, the specificity for S. aureus was 98.9%. We conclude that Staphaurex Plus® is a highly sensitive and specific agglutination test for S. aureus and simultaneously detects clumping factor-positive S. lugdunensis.
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Rapid and economical methods for species identification of clinically significant coagulase-negative Staphylococci
Article
  • Jun 1995
Four methods for the species identification of coagulase-negative staphylococci in the medical microbiology laboratory were compared with 444 consecutive isolates. The methods included (i) the reference method based on growth tests, (ii) API ID 32 Staph (bioMérieux), (iii) Staph-Zym (Rosco), and (iv) a rapid 4-h method developed in our laboratory (UZA method). The last method is based on the detection within 4 h of enzymatic activity of heavy bacterial suspensions in three substrate solutions (nongrowth tests). For 16.5% of the isolates some supplementary growth tests read after 24 h had to be added to the enzyme data for satisfactory identification. The reference method failed to identify four isolates. Of the 440 isolates identified by the reference method, API ID 32 Staph, Staph-Zym, and the UZA method correctly identified 419 (95.2%), 429 (97.5%), and 430 (97.7%) and misidentified 8 (1.8%), 4 (0.9%), and 1 (0.2%), respectively. Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis, S. schleiferi, and S. capitis were identified with an accuracy of 98 to 100% by all the systems tested. S. capitis subsp. ureolyticus was not recognized by the API ID 32 system because the biochemical profiles for it are not yet included in the corresponding database. Whereas API ID 32 identified all 13 S. warneri isolates, both Staph-Zym and the UZA method missed 2 of these. S. hominis was identified with the least accuracy by the API ID 32 system (26 of 39 isolates), whereas the UZA and Staph-Zym methods identified 36 of the isolates belonging to this species. The UZA method did not identify any of the S. cohnii, S. xylosus, S. lentus, and S. sciuri strains, since it included no discriminatory tests for these species, because they are extremely rarely found in humans. Of all 440 isolates tested, the UZA method failed to identify 9 and misidentified 1 other. Eighty-one percent of the isolates were identified within 4 h and 97.7% were identified after 24 h, at considerably less expense than by the API ID 32 Staph and Staph-Zym methods.
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