Géraldine Durand

Detection of New Methicillin-Resistant Staphylococcus aureus Clones Containing the Toxic Shock Syndrome Toxin 1 Gene Responsible for Hospital- and Community-Acquired Infections in France

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) clones harboring the toxic shock syndrome toxin 1 (tst) gene have been detected in France and in Switzerland since 2002. During a passive survey conducted between 2002 and 2003, we collected 103 tst-positive S. aureus isolates from 42 towns in France, of which 27 were resistant to methicillin. The tst-positive MRSA belonged to two clones: a major clone comprising 25 isolates of sequence type (ST) 5 and agr group 2 and a minor clone comprising two isolates of ST30 and agr3. The tst-positive MRSA clones were associated with both hospital-acquired (12 cases) and community-acquired (8 cases) infections. The MRSA clones were mainly isolated from children (overall median age, 3 years). They caused a variety of clinical syndromes, including toxic shock syndrome and suppurative infections. Both clones were found to harbor a type IV staphylococcal chromosomal cassette mec (SCCmec) and to have similar antibiotic resistance profiles (usually resistant to oxacillin, kanamycin, and tobramycin and with intermediate resistance to fusidic acid). The origin of these clones is unclear. The tst-positive agr2 MRSA clone has the same sequence type (ST5) of two pandemic nosocomial MRSA clones, namely, the Pediatric clone and the New York/Japan clone. These findings suggest that all these clones are phylogenetically related. The pulsotype of the tst-positive MRSA clones differed from that of methicillin-sensitive S. aureus (MSSA) clones by a single band involving the SCCmec element. These findings suggest that the tst-positive MRSA clones may have emerged from their respective MSSA counterparts.

Epidemiology of invasive methicillin-resistant Staphylococcus aureus clones collected in France in 2006 and 2007.

ABSTRACT We conducted a prospective multicenter study of methicillin-resistant Staphylococcus aureus (MRSA) isolates, including the first five consecutive clinical isolates, collected between September 2006 and February 2007 in 23 hospitals located throughout France (Fig. 1). The 111 isolates were tested for their antibiotic susceptibility patterns and were extensively characterized by screening for drug resistance and agr alleles, multilocus sequence typing (ST), staphylococcal cassette chromosome mec (SCCmec) typing, spa typing, and PCR profiling of 21 toxin genes. Clones were designated by their ST followed by their SCCmec type (I to VI). The Lyon clone ST8-IV or ST8-IVvariant (n = 77; 69.4%) was widely distributed. Four minor clones were also detected, namely, the “classical” Pediatric clone ST5-IV (n = 9; 8.1%), the “new” Pediatric clone ST5-VI (n = 8; 7.2%), the clone Geraldine ST5-Itruncated (n = 7; 6.3%), and the European clone ST80-IV (n = 4; 3.6%). The six other isolates were related to five rare clones. Relative to that of other European countries, the situation in France is marked by the predominance of a specific major clone and the worrying emergence of minor clones with enhanced virulence and new antibiotic susceptibility profiles.

Virulence determinants in community and hospital meticillin-resistant Staphylococcus aureus

Staphylococcus aureus produces many virulence factors, most of which act in a synergistic and coordinated fashion. Some appear to be specifically associated with certain severe infections and are produced by meticillin-resistant Staphylococcus aureus (MRSA) clones distributed worldwide. Superantigenic exotoxins appear to be major virulence factors in hospital MRSA clones (HA-MRSA), and staphylococcal enterotoxin A (SEA) may be involved in the physiopathology of septic shock. Panton Valentine Leucocidin (PVL) has emerged as a major virulence factor in community-acquired Staphylococcus aureus (CA-MRSA) infections. In particular, the leukotoxic action of PVL is responsible for the high mortality rate associated with necrotizing pneumonia. CA-MRSA can also harbour the toxic shock toxin 1 (TSST-1) and rarely the exfoliative toxin.

Identification of the Capsular Polysaccharides in Staphylococcus aureus Clinical Isolates by PCR and Agglutination Tests

ABSTRACT Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The predominance of two capsular polysaccharides, types 5 and 8, on the surface of clinical isolates led to the development of a conjugate vaccine (StaphVAX) based on capsular polysaccharides types 5 and 8 conjugated to a carrier protein. We have studied the capsular phenotypes and genotypes of 195 isolates representative of all clinical syndromes that encompassed both hospital and community-acquired infections. These isolates were mainly detected in France between January 2001 and December 2004. In this population, most of clinical isolates (87%) expressed either capsular polysaccharide type 5 (42%) or 8 (45%), whereas 13% were nontypeable by the serotyping method with antibodies specific to capsular polysaccharide type 5 or 8. These 26 nontypeable strains were further serotyped and were demonstrated to express the cell wall surface antigen 336, a polyribitol phosphate N-acetylglucosamine, which resembles cell wall teichoic acid. Among methicillin-resistant Staphylococcus aureus (MRSA) strains, we found a predominance of serotype 5 for 64% of strains, whereas MSSA isolates were predominantly capsular serotype 8 (60%). All S. aureus clinical isolates included in the present study have been investigated by PCR method, demonstrating that all isolates carried either the cap5 or the cap8 locus.

Microbial Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Do We Need Guidance for Data Interpretation?

ABSTRACT The integration of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has revolutionized species identification of bacteria, yeasts, and molds. However, beyond straightforward identification, the method has also been suggested to have the potential for subspecies-level or even type-level epidemiological analyses. This minireview explores MALDI-TOF MS-based typing, which has already been performed on many clinically relevant species. We discuss the limits of the methods resolution and we suggest interpretative criteria allowing valid comparison of strain-specific data. We conclude that guidelines for MALDI-TOF MS-based typing can be developed along the same lines as those used for the interpretation of data from pulsed-field gel electrophoresis (PFGE).

Identification and typing of the emerging pathogen Candida auris by matrix-assisted laser desorption ionisation time of flight mass spectrometry.

Candida auris is an emerging antifungal resistant yeast species causing nosocomial and invasive infections, emphasising the need of improved diagnostics and epidemiological typing methods. We show that MALDI‐TOF VITEK‐MS followed by amplified length polymorphisms allows for accurate species identification and subsequent epidemiological characterisation of strains encountered during potential outbreaks.

Detection of Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF Mass Spectrometry

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01–10.24; p = 0.023; CI 95%: 1.20–12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

ABSTRACT The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

Toxin Profiling of Staphylococcus aureus Strains Involved in Varicella Superinfection

ABSTRACT The most common complications of varicella are bacterial skin and soft tissue infections, generally due to Staphylococcus aureus and group A beta-hemolytic streptococci. The aim of this study was to characterize the toxin and antibiotic resistance profiles of S. aureus isolates involved in varicella complications. Between 2002 and 2007, the French Reference Centre for Staphylococci collected 58 S. aureus isolates involved in varicella superinfection. All the isolates were characterized by screening for 12 toxin genes, agr typing, and mecA gene detection; some isolates were also studied by spa typing, multilocus sequence typing (MLST), and resistance profiling. A major toxin gene was detected in 53% (31/58) of the isolates (genes for exfoliative toxins A and B, 17.2%; Panton-Valentine leukocidin gene, 8.6%; toxic shock syndrome toxin 1 gene, 27.6%). Most clinical manifestations were directly compatible with the classical activity of these toxins. Nineteen isolates (33%) were resistant to methicillin, and 12 of these isolates belonged to an emerging agr-2, ST5 clone that harbors the toxic shock syndrome toxin 1 gene. These data should be considered in the management and treatment of patients with varicella complicated by S. aureus superinfection. Antibiotics that decrease toxin production, such as clindamycin, may provide benefit, and their efficacy against bacterial superinfections in children with varicella should be studied.