J. Etienne

Exfoliatin-Producing Strains Define a Fourth agr Specificity Group in Staphylococcus aureus

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.

The Panton-Valentine leukocidin vaccine protects mice against lung and skin infections caused by Staphylococcus aureus USA300.

Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal infections in the community. A large percentage of the community-acquired methicillin-resistant (CA-MRSA) strains in the USA produce Panton-Valentine leukocidin (PVL), which is associated with severe infections. The virulence of the clinical CA-MRSA strain USA300 was compared to that of its isogenic pvl-deleted mutant, and it was shown that PVL contributes to lung and muscle tissue destruction, respectively, in murine necrotizing pneumonia and skin infection models. Mice infected with the USA300 strain developed a dominant anti-PVL response. The PVL subunits were therefore tested as vaccinogens against this isolate, and their vaccine efficacy correlated with both the route of vaccination and infection. These data suggest that PVL is a virulence factor in murine CA-MRSA infections.

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.

Species identification of Legionella via intergenic 16S-23S ribosomal spacer PCR analysis

Species identification of Legionella in routine laboratory testing is hampered by the lack of highly discriminatory phenotypic tests. Amplification polymorphism of the intergenic 16S-23S spacer regions (ISR) has been previously developed for identification of species within the Legionellaceae [Hookey, J.V., Birtles, R.J. & Saunders, N.A. (1995). J Clin Microbiol 33, 2377-2381], but it did not provide enough resolution to distinguish all members of the bluish-white autofluorescent species and the red autofluorescent group of the Legionellaceae. By choosing new primers that target regions 4 (positions 1521-1541 of Escherichia coli 16S rRNA gene) and 6 (positions 114-132 of E.coli 23S rRNA gene) within the rDNA operon close to the 16S-23S intergenic spacer, 34 profiles were determined among the 79 type and reference strains representing 42 species that were tested. Analysis of the RFLP generated after Hinfl restriction digestion of the PCR products further improved the method, allowing complete discrimination among the species and subspecies of Legionella tested. Twenty-three well-identified strains from unrelated origins belonging to seven species gave amplification patterns identical to that of their type strain. The technique was also tested on 80 field isolates that could not be unequivocally assigned to groups by phenotypic methods. Seventy-two per cent (58/80) of these isolates had a profile identical to that of a type strain, while 27% (22/80) may correspond to new taxa since their ISR-PCR profiles did not match any of the known profiles.

Reemergence of Gentamicin-Susceptible Strains of Methicillin-Resistant Staphylococcus aureus in France: a Phylogenetic Approach

ABSTRACT The reemergence of gentamicin-susceptible (Gens) methicillin-resistant Staphylococcus aureus (MRSA) isolates in France between 1992 and 1996 was investigated using a phylogenetic approach (multiprimer randomly amplified polymorphic DNA typing). Eighty-six percent (65 of 85) of the French strains were grouped into one phylogenetic cluster within which all but one Gensstrain were grouped into a subcluster. Thus, the reemergence of Gens MRSA strains in France was likely due to the spread of one specific clone which belonged to a cluster comprising most French gentamicin-resistant (Genr) strains. This suggests that the Gens clone has emerged from a Genr strain of this cluster.

Coagulase deficiency in clinical isolates of Staphylococcus aureus involves both transcriptional and post-transcriptional defects

The molecular basis of the non-expression of coagulase was investigated for 14 coagulase-negative isolates of Staphylococcus aureus obtained from different clinical samples. These isolates had typical S. aureus characteristics such as production of clumping factor, DNAase and protein A, but, with one exception, failed to produce detectable amounts of alpha-haemolysin. All 14 strains had DNA homologous to the coagulase gene (coa), but a coa-specific transcript was found in only seven of them. alpha-Haemolysin mRNA was detected in only eight strains without direct correlation to coa-mRNA expression. Thus, coagulase and alpha-haemolysin deficiencies in S. aureus may involve either transcriptional or post-transcriptional alterations although additional regulatory factors may influence the expression of both genes.

Different growth rates in amoeba of genotypically related environmental and clinical Legionella pneumophila strains isolated from a thermal spa

Two cases of legionellosis occurring 3 years apart were acquired in the same French thermal spa and were apparently due to the same strain of Legionella pneumophila serogroup 1, as shown by genomic macrorestriction analysis. Minor differences between the two isolates were found by random amplification PCR profiling which showed an additional band with one of the isolates. Analysis of 107 L. pneumophila strains isolated from the spa waters by genome macrorestriction failed to identify the infective strain, but a closely related L. pneumophila serogroup 3 strain differing from the clinical isolates by only one band was found. To determine if the clinical L. pneumophila serogroup 1 isolates was better adapted for intracellular multiplication than related serogroup 3 environmental isolates, the growth kinetics of six isolates were determined in co-culture with Acanthamoeba lenticulata. One clinical isolate failed to grow within amoeba, while the other clinical isolate yielded the highest increase in bacterial cell count per amoeba (1,200%) and the environmental isolates gave intermediate values. Genetic analysis of L. pneumophila isolates by DNA macrorestriction does not therefore appear to reflect their growth kinetics within amoeba, and is not sufficiently discriminatory to identify potentially virulent strains.

Evaluation of a Rapid Immunochromatographic Assay for Detection of Streptococcus pneumoniae Antigen in Urine Samples

Now Streptococcus pneumoniae Urinary Antigen Test (Binax, USA), has been recently developed to detect Streptococcus pneumoniae antigen in urine samples [1, 2]. This test detects the pneumococcal C-polysaccharide antigen specific for the pneumococcal cell wall and could facilitate the diagnosis of Streptococcus pneumoniae pneumonia. We performed a multicenter study to evaluate this test in adults admitted to three French hospitals with clinical and radiological evidence of community-acquired pneumonia. From January to May 2000, 91 cases of pneumonia, defined according to published recommendations [3], were included in the study. Bacteriological diagnosis of Streptococcus pneumoniae pneumonia was based on at least one of the following criteria: (i) positive Streptococcus pneumoniae culture (by semiquantitative methods) of a bronchoalveolar lavage specimen (≥104 cfu/ml), a tracheal aspirate obtained by protected brush (≥106 cfu/ml), or a sputum sample (≥107 cfu/ml); (ii) positive Streptococcus pneumoniae culture of pleural fluid or positive latex agglutination on pleural fluid (performed with the Wellcogen Streptococcus pneumoniae test; Murex Diagnostics, France); and/or (iii) blood culture positive for Streptococcus pneumoniae. Urine samples from each patient were tested once for Streptococcus pneumoniae urinary antigen (unconcentrated urine) using the Now Streptococcus pneumoniae Urinary Antigen Test (Binax) and for Legionella urinary antigen (concentrated urine) using the Now Legionella Urinary Antigen Test (Binax). The diagnosis of Streptococcus pneumoniae pneumonia was established in 28 of the 91 total patients by standard bacteriological techniques. The Streptococcus pneumoniae Now test was positive for 23 of these 28 patients (Table 1), giving a sensitivity of 82%. Of the 63 patients who did not have pneumococcal pneumonia, as defined by standard microbiological techniques, 14 were positive in the Streptococcus pneumoniae Now test; thus, the crude specificity value of the test was 77%. One of these false-positive results was observed in a patient with Haemophilus influenzae pneumonia, a finding for which there are several possible explanations. There could be cross-reactivity between C-polysaccharide and Haemophilus influenzae (cross-reactivity between pneumococcal antigens and other species has been reported, though not yet with Haemophilus influenzae [1, 4, 5]). Alternatively, there could have been a mixed infection with two microorganisms. Thirteen positive results were observed in patients with no etiological diagnosis. The absence of a positive Streptococcus pneumoniae culture could be attributable to spontaneous lysis of the bacteria in blood specimens, to an initial antibiotic treatment given to the patients, or to the lack of bronchopulmonary sampling carried out in the patients. A previous study showed that the results of the urine antigen detection test were significantly more likely to be positive among children (age 2–60 months) who were nasopharyngeal carriers of pneumococci [2]. Our study was performed in adults,

Instability of characteristics amongst coagulase- negative staphylococci causing endocarditis

Variation in typing of clinically significant isolates of coagulase-negative staphylococci (CNS) was determined by five typing methods with 143 isolates obtained from 19 patients over periods from 2 days to 1 year. In only one case did all isolates give exactly the same typing pattern by all five tests. No single method, or simple combination, provided a ready means of confirming the relatedness of separate isolates. The most frequently useful tests were antibiotic susceptibility and extrachromosomal DNA banding patterns. However, the results of biotyping, serotyping and phage typing were also helpful in showing the relationship between different isolates from a given patient. In most cases a core pattern varying by the gain or loss of a small number of features, characterised a given patients isolates. In two causes, apparently radical changes in the infecting organism were observed, and confirmed by restriction endonuclease analysis. Care should be taken when successive isolates of CNS show distinct typing differences in deciding their clinical relevance.

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France.

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.