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Resume Materiel et methodes Les concentrations minimales inhibitrices de nine antibiotiques ont ete determinees par une methode de reference (norme M11-A5 du National Committee for Clinical and Laboratory Standards) sur 376 souches de bacteries anaerobies strictes. Les souches se repartissaient ainsi : 254 Bacteroides du groupe fragilis (dont 143 B. fragilis ), 122 autres bacteries a Gram negatif ( Bacteroides spp. , Prevotella, Fusobacterium, Porphyromonas, Suterella, Desulfomonas, Veillonella ). Resultats Chez B. fragilis (groupe) les taux de resistance sont les suivants : amoxicilline–acide clavulanique 2,8 %, ticarcilline 27,5 %, ticarcilline–acide clavulanique 1,9 %, piperacilline–tazobactam 1,9 %, cefoxitine 6,2 %, imipeneme 0,8 %, clindamycine 28,3 %. Considerant les etudes multicentriques anterieures, la resistance a l’imipeneme reste faible en 2003 et n’est observee que chez B. fragilis . La resistance a la clindamycine se maintient et avoisine les 25 %. Aucune resistance au metronidazole n’est constatee, mais une sensibilite diminuee est observee chez B. fragilis , B. merdae et Prevotella , soit chez 4,3 % des anaerobies a Gram negatif. Discussion Cette etude confirme la forte resistance des anaerobies a Gram negatif a la clindamycine, les bonnes activites de l’imipeneme, des associations β-lactamines–inhibiteur de β-lactamase et du metronidazole. Cependant, la sensibilite diminuee au metronidazole semble augmenter.

Resume Des etudes taxonomiques recentes ont demontre l’heterogeneite de l’espece Burkholderia cepacia (ex Pseudomonas cepacia), et on parle desormais de complexe cepacia, constitue a ce jour de neuf especes. Il s’agit de bacilles a Gram negatif non fermentants, presents dans l’environnement naturel et se comportant comme des pathogenes opportunistes de l’homme. Les Burkholderia du complexe cepacia sont essentiellement responsables de colonisations pulmonaires dans la mucoviscidose, ou elles sont particulierement redoutees en raison de leur impact clinique defavorable et du potentiel epidemique de certaines souches. Leur prevalence actuelle dans la mucoviscidose en France est voisine de 3 %, avec une tres large predominance des especes B. cenocepacia et B. multivorans, representant respectivement 46 et 47 % des isolats francais. Ces bacteries sont episodiquement rapportees dans des infections nosocomiales en dehors de la mucoviscidose, survenant souvent sous forme de cas groupes a partir d’une source commune contaminee. Les options therapeutiques sont limitees en raison d’une resistance naturelle a la ticarcilline, aux aminoglycosides et a la colistine, et de frequentes resistances acquises. La piperacilline, la ceftazidime, le meropeneme (contrairement a l’imipeneme), et la temocilline sont les molecules les plus actives. Des synergies ont ete observees in vitro avec des associations doubles ou triples d’antibiotiques, mais aussi des antagonismes, et actuellement aucune association therapeutique n’est reellement validee en clinique. De nombreux travaux recents ont permis une meilleure comprehension de l’epidemiologie, de la physiopathologie et des mecanismes de resistance de ces pathogenes emergents, qui sont passes en revue dans cette synthese.

We report three cases of delivery and postpartum bacteremia due to unusual anaerobic bacteria in healthy young women. Leptotrichia amnionii bacteremia occurred during delivery in two mothers and was associated with fetal distress during labor. Conversely, Sneathia sanguinegens bacteremia occurred postpartum, 2 days after delivery, without consequence for the neonate.

Gram-negative bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community. Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity. The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene wbiI . Homologues of wbiI are found in polysaccharide biosynthesis gene clusters in other bacteria. Primers to a conserved region of the Bcc wbiI gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested. Sequencing and restriction enzyme digestion of this wbiI PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates. In five of nine instances, Bcc isolates of a single serotype had a single wbiI restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple wbiI RFLP types. Species determination of the Bcc isolates revealed no obvious correlation between wbiI RFLP type and species. There was also no apparent correlation between wbiI RFLP type and the ability of a single Bcc isolate to infect an individual with CF. However three of five Bcc outbreaks involved isolates with the same wbiI RFLP type, indicating that wbiI RFLP typing may be a useful tool to help track Bcc outbreaks.

Resume Objectif. – Nous avons voulu evaluer la monotherapie par amoxicilline-acide clavulanique (AMC), aux posologies humaines, sur un modele de bacteriemie a E. coli secreteur de penicillinase de niveau intermediaire chez le microporc Yucatan. Methodes. – Nous avons developpe un modele experimental de bacteriemie chez 15 microporcs Yucatan : a j1h0, injection I.V. de 4,8 × 10 9 UFC d’ E. coli de phenotype intermediaire. Neuf d’entre eux ont ete traites par AMC (1g–0,1g, 3 × jour en intraveineux pendant une semaine). Cette dose correspond a 2–0,2 g chez l’homme, avec une pharmacocinetique comparable dans les 2 especes. Le traitement a ete debute 24 h apres l’injection de l’inoculum bacterien et poursuivi pendant 7 jours. A j5, les donnees des animaux traites ont ete comparees a celles des 6 non traites. Resultats. – Durant les 24 premieres heures, avant l’antibiotherapie, les microporcs ont presente un sepsis severe avec hemocultures positives (15/15), fievre (39,3 a j1h7 vs 37,9°C a j1h0), apathie, anorexie, leucopenie (2,3 × 10 3 a j1h1 vs 12,1 × 10 3 leucocytes/mm 3 a j1h0) suivie d’ hyperleucocytose (23,9 × 10 3 a j2 vs 12,1 × 10 3 leucocytes/mm 3 a j1h0) et thrombopenie (81 × 10 3 a j2 vs 325 × 10 3 plaquettes/mm 3 a j1h0). Dans le groupe non traite, 5 hemocultures sur 6 sont restees positives jusqu’a j5. Dans le groupe traite, 8 hemocultures sur 9 ont ete negatives a j5 avec une amelioration clinique rapide. Commentaires. – Le protocole therapeutique utilise est efficace dans notre modele, prefigurant une possible efficacite humaine.

Male king penguins are able to conserve food in their stomach for several weeks during their incubation fast. Since a bacterial inhibition could be involved in the process, we investigated the bacterial population of their stomach. Two types of bird were compared, according to whether they conserved (Co) or digested (Dg) food during fasting. Total bacteria and culturable bacteria were studied. In both groups, the food contained a high number of apparently viable bacteria (≥107/ml), but few could be cultured by standard techniques. Culturable bacteria originated mainly from the environment or the upper part of the digestive tract. Neither prevailing bacteria from seawater and fishes nor enteric bacteria cultured. The gastric temperature was around 38°C and the pH of Co bird samples was >4, both conditions favourable for bacterial growth. Morphological characteristics of stressed bacteria (sporulated, deformed or dead cells) were more often found in samples from Co compared to Dg birds. Altogether, these results support the idea that there might be factors inhibiting bacterial growth in king penguins conserving food in their stomach.

Resume Le microbiologiste contribue a l’amelioration de la qualite de l’antibiotherapie en collaborant avec les cliniciens par une interface indispensable. Il doit agir par ses recommandations sur les indications precises des examens bacteriologiques ameliorant la qualite des prelevements, sur l’opportunite de leur realisation et aux bonnes conditions de leur transport. Le prelevement doit etre accompagne d’informations cliniques necessaires a la bonne orientation de l’analyse. Des procedures ecrites doivent constituer un guide. Les performances des actes bacteriologiques doivent etre ameliorees par des methodes rapides, certaines automatisables et par l’apport des moyens moleculaires de diagnostic comme l’amplification genique ou les bio-puces. L’antibiogramme apparaitra sous la forme de valeurs parametriques pour les infections graves, le rendu SIR etant suffisant dans les autres cas. Cet antibiogramme est l’objet d’une lecture interpretative. Completes par la notification des mecanismes enzymatiques de resistance, son compte rendu et son expression doivent etres clairs. Le microbiologiste peut induire la prise en consideration des parametres pharmacocinetiques et pharmacodynamiques en vue d’une optimisation de l’antibiotherapie. La mesure et la surveillance des concentrations seriques des antibiotiques deviennent comparables aux CMI et aux CMB. Une contribution sera apportee par l’etude des pouvoirs bactericides du serum dans les infections graves. En etudiant les actions synergiques en cinetique de bactericide des associations d’antibiotiques sur des souches resistantes, il mettra en evidence les meilleurs couples. Par la surveillance des resistances emergentes aux antibiotiques en indiquant la distribution des especes isolees, le taux de prevalence ou d’incidence et les caracteristiques des bacteries multiresistantes, il apporte sa contribution.

Objectives – Ceftriaxone is a semi-synthetic cephalosporin with a high level of protein binding to albumin. The authors wanted to determine whether the bound fraction was actively bactericidal. Material and method – The bactericidal activity of 3 ceftriaxone concentrations (5, 50, and 200 mg/L) in a brain-heart broth with and without human serum albumin, and in human serum, was evaluated by the time-kill technique on 3 S. pneumoniae strains with varying degree of susceptibility to penicillin G. Results – The low concentration (5 mg/L) exhibits a slightly better bactericidal activity on the penicillin G susceptible strain without albumin than with albumin, in the earlier contact phase. A similar bactericidal activity occurs in both media at the 6th hour and up to the late phase of bactericidal activity. Concerning the strains with an intermediate or total resistance to penicillin G, the bactericidal activity is identical in the three media. This implies that bound ceftriaxone has a bactericidal activity. Only the 5 mg/L concentration doesn't seem to exhibit any activity on the strain with decreased susceptibility. This may be explained by the existence of two binding-sites of ceftriaxone on serum albumin: a high-affinity, low-capacity site, and a low-affinity, high-capacity site. At low concentrations, only the high-affinity sites are bound and thus, the bound ceftriaxone is inactive. At high concentrations, the part of ceftriaxone mainly bound to the low-affinity sites is much more labile, and may thus interact with the PBPs having a higher affinity for ceftriaxone. Conclusion – This study demonstrates that bound ceftriaxone has a bactericidal activity against S. pneumoniae.

Objectives – Ceftriaxone is a semi-synthetic cephalosporin with a high level of protein binding to albumin. The authors wanted to determine whether the bound fraction was actively bactericidal. Material and method – The bactericidal activity of 3 ceftriaxone concentrations (5, 50, and 200 mg/L) in a brain-heart broth with and without human serum albumin, and in human serum, was evaluated by the time-kill technique on 3 S. pneumoniae strains with varying degree of susceptibility to penicillin G. Results – The low concentration (5 mg/L) exhibits a slightly better bactericidal activity on the penicillin G susceptible strain without albumin than with albumin, in the earlier contact phase. A similar bactericidal activity occurs in both media at the 6 th hour and up to the late phase of bactericidal activity. Concerning the strains with an intermediate or total resistance to penicillin G, the bactericidal activity is identical in the three media. This implies that bound ceftriaxone has a bactericidal activity. Only the 5 mg/L concentration doesn't seem to exhibit any activity on the strain with decreased susceptibility. This may be explained by the existence of two binding-sites of ceftriaxone on serum albumin: a high-affinity, low-capacity site, and a low-affinity, high-capacity site. At low concentrations, only the high-affinity sites are bound and thus, the bound ceftriaxone is inactive. At high concentrations, the part of ceftriaxone mainly bound to the low-affinity sites is much more labile, and may thus interact with the PBPs having a higher affinity for ceftriaxone. Conclusion – This study demonstrates that bound ceftriaxone has a bactericidal activity against S. pneumoniae.

Resume Objectifs De juillet 1997 a juin 1998, 15 laboratoires de microbiologie, organises en observatoire regional et repartis sur l'ensemble de l'Alsace, ont participe a un recrutement exhaustif des pneumocoques isoles en situation d'infection dans le but d'etudier leur sensibilite aux antibiotiques. Methode Les souches ont ete isolees de prelevements diagnostiques (prelevements pulmonaires, hemocultures, liquides cephalorachidiens, prelevements ORL). Resultats L'observatoire a recrute 599 souches dont 32,9 % sont des pneumocoques de sensibilite diminuee a la penicilline G (PSDP: concentrations minimales inhibitrices CMI > 0,06 mg/L). Parmi ces PSDP, dix souches presentent une resistance de haut niveau (CMI > 1 mg/L), trois sont resistantes a l'amoxicilline (CMI > 2 mg/L) et aucune au cefotaxime. La prevalence des PSDP est plus elevee chez l'enfant que chez l'adulte (45,3 % contre 28,2 %) et dans les pus d'oreille (59,4 %). Les PSDP sont frequemment resistants a l'erythromycine (76,6 %), au cotrimoxazole (65,2 %) et a la fosfomycine (18,4 %). Les serotypes 23, 14 et 9 sont les plus frequents: 29,1 %, 26,1 % et 17,9 % des souches respectivement. Conclusion Cette enquete a permis de preciser, pour la premiere fois, l'impact exact de la resistance de Streptococcus pneumoniae a la penicilline G et aux antibiotiques dans la region Alsace. La prevalence des PSDP est l'une des plus faibles observees en France la meme annee avec une proportion tres restreinte de souches resistantes a la penicilline G et aux autres β-lactamines, γ compris dans les pus d'oreille de l'enfant.

Resume En bacteriologie et virologie les techniques rapides doivent reduire le delai de reponse pour le clinicien et permettre l'instauration precoce d'un traitement adapte. Les systemes par culture faisant appel a de nouveaux substrats d'enzymes specifiques identifient les bacteries et detectent leur antibioresistance en quelques heures. Leurs performances sont dependantes de la richesse et de l'actualisation des banques de donnees. Les methodes de biologie moleculaire permettent l'identification de bacteries et de virus difficilement ou non cultivables (HCV) en deux a trois jours, ainsi que la detection de resistances aux agents antimicrobiens de Mycobacterium tuberculosis . La commercialisation de kits d'amplification genique ne doit pas faire oublier le controle de qualite ni les problemes de contamination et d'inhibiteurs. Les tests de detection d'antigenes peuvent apporter un resultat en 15 a 20 minutes. Leur realisation delocalisee necessite neanmoins un environnement adapte en raison des risques de contamination, ainsi qu'une reflexion quant a leur opportunite et leurs performances.

Since Lyme arthritis was first described in the United States, it has now been reported in many countries of Europe. However, very few strains of the causative bacterium, Borrelia burgdorferi , have been isolated from synovial samples. For this reason, different molecular direct typing methods were developed recently to assess which species could be involved in Lyme arthritis in Europe. We developed a simple oligonucleotide typing method with PCR fragments from the flagellin gene of B. burgdorferi sensu lato, which is able to differentiate seven different Borrelia species. Among 10 consecutive PCR-positive patients with Lyme arthritis from the northeastern France, two species were identified in synovial samples: B. burgdorferi sensu stricto in 9 cases and B. garinii in 1 case. Conversely, all B. burgdorferi sensu lato species detected in 10 consecutive PCR-positive biopsies from a second set of patients with erythema migrans from the same geographical area were identified as either B. afzelii or B. garinii ( P < 0.001). These results indicate that B. burgdorferi sensu stricto is the principal but not the only Borrelia species involved in Lyme arthritis in northeastern France.

Resume La reussite des examens bacteriologiques pour la recherche des bacteries anaerobies strictes depend tres etroitement des conditions de prelevement et de leur transport. II est imperatif de les respecter afin de pouvoir apporter une documentation precise dans les infections ou ces bacteries sont impliquees. Les imperatifs generaux a respecter pour cette recherche sont: ne pas contaminer le prelevement par la flore endogene (peau et muqueuses hebergent naturellement des bacteries anaerobies) ; eviter tout contact avec l'oxygene qui engendre une mortalite rapide des anaerobies et par la suite, favorise l'emergence a la culture des bacteries aerobies associees. Les conditions particulieres pour les prelevements exigent que des dispositifs specifiques soient utilises: toute collection purulente doit etre ponctionnee a la seringue, en evitant les bulles d'air et les contaminations de surface, puis introduite dans un flacon adapte. L'utilisation de la seringue elle-meme comme vehicule n'est qu'une demarche par defaut. Lorsque le site infecte n'est pas ponctionnable il faut, apres une decontamination de surface, utiliser une curette pour obtenir un prelevement profond qui sera place dans un milieu de transport ou a defaut, prelever vigoureusement en profondeur avec un ecouvillon d'alginate de calcium, place ensuite dans un milieu de transport. Les prelevements solides issus de biopsies ou les pieces anatomiques peuvent etre deposees dans un tube sterile dispose dans un sachet plastique scelle contenant un melange reducteur ; le sang et les liquides internes peuvent etre injectes directement et transportes dans les flacons d'hemoculture anaerobie. L'acheminement des prelevements vers le laboratoire doit etre rapide et effectue a la temperature de 20 °C, la refrigeration a 4 °C etant nefaste. Un bon prelevement et des conditions de transport adaptes permettront une mise en evidence correcte des bacteries anaerobies strictes lorsque les cultures seront ensuite realisees selon un processus d'anaerobiose continue.

From 1990 to 1996, routine screening for whooping cough identified 399 patients with a calmodulin-dependent adenylate cyclase-positive test result and yielded 69 Bordetella pertussis isolates. None of the patients were fully vaccinated, and most were less than 6 months old. Analysis of total DNA by pulsed-field gel electrophoresis (PFGE) after Xba I, Spe I, or Dra I macrorestriction yielded 19, 15, and 5 different patterns, respectively, whereas ribotyping failed to demonstrate any strain polymorphism. Discrimination among the isolates was improved by combining the PFGE profiles. Some patterns were more frequent, but the corresponding patients were not clearly epidemiologically related. The patterns for two strains obtained during a 3-month period from patients who were neighbors differed by the length of a single DNA fragment. These data strongly suggest that one type of isolate is widely spread throughout the world and is carried by individuals other than patients who develop a true illness.

Site-directed mutagenesis was performed on genes encoding HlgA and HlgC, two of the three proteins expressed from the staphylococcal y-hemolysin locus, which originate two pore-forming toxins (HlgA + HlgB, HlgC + HlgB). As related proteins, HlgA and HlgC were found to bind first to cell membranes. Amino acid substitutions concerned residues that would predictably disrupt a 13 amino acid conserved beta-sheet of the Chou and Fasman secondary structure prediction. The mutation of a threonin into an aspartic acid residue from HlgA (T28D) and from HlgC (T30D) that would break this predicted N-terminal structure lowered dramatically the biological activities on purely lipidic vesicles, erythrocytes and polymorphonuclear cells. The change in secondary structure was confirmed by Fourier Transformed Infrared spectroscopy. The binding of mutated and native proteins at the same kind of sites onto polymorphonuclear cells was evidenced with flow cytometry and fluorescein-labelled anti-class S antibodies or wild type HlgA or HlgC. However, the subsequent binding of fluorescein-labelled HlgB to membrane-bound mutated HlgA or HlgC complexes was inhibited. In conclusion, the first binding of class S components is essential for the subsequent binding of class F components, and a predicted beta-sheet seems to be at least one of the functional domains involved.

Endocytosis of fluorescently labelled cells of Legionella pneumophila (L. pneumophila) by free-living Acanthamoeba palestinensis (A. palestinensis) has been studied using flow cytometry. L. pneumophila cells were labelled with CM-DiI, a lipophilic fluorescent probe under conditions that did not modify viability. Coculturing the bacteria with amoebae was accompanied by rapid endocytosis; after 5 min, 90% of the amoebae had internalized bacteria. This percentage remained unchanged during further coculture, but the number of bacteria ingested per amoeba increased. Moreover, the number of ingested bacteria was found to be dependent on the size of the amoeba. The validity of the internalization analyzed by flow cytometry was confirmed by observation using epifluorescence and phase contrast microscopy. CM-DiI labelling associated with flow cytometry provides a very valuable technique for the determination of bacteria endocytosis by free-living amoeba. Cytometry 27:269–274, 1997. © 1997 Wiley-Liss, Inc.

Resume L'objectif de l'etude etait de suivre l'evolution de la sensibilite a la ceftazidime, au cefotaxime, a l'imipeneme et a la ticarcilline, de souches bacteriennes appartenant a des especes porteuses de mecanismes de resistance acquise a ces antibiotiques: Klebsiella, Enterobacter, Serratia, Pseudomonas aeruginosa, Burkholderia (Pseudomonas) cepacia, Stenotrophomonas (Xanthomonas) maltophilia, Acinetobacter baumannii. De 1989 a 1993 (1994 pour P. aeruginosa), toutes les souches non repetitives appartenant a ces especes etaient recueillies le premier mois de chaque trimestre dans les laboratoires de bacteriologie de 12 CHU francais. La determination de la sensibilite aux antibiotiques etait realisee suivant la methode classique de diffusion en gelose et interpretee suivant les recommandations du Comite de l'Antibiogramme de la Societe Francaise de Microbiologie. Sept mille souches, en moyenne, ont ete annuellement etudiees. Ces souches ont ete isolees a partir de prelevements d'origine variee, en particulier a partir des urines et de prelevements respiratoires. Les activites in vitro de la ceftazidime et du cefotaxime sur les enterobacteries, exprimees en pourcentages de souches resistantes, sont demeurees relativement stables ces dernieres annees. Il est a noter que la frequence des souches de Klebsiella pneumoniae resistantes a la ceftazidime et au cefotaxime, apres avoir atteint un pic a 24 % en 1991, est retombee a 18 % en 1993. La frequence des souches resistantes a la ticarcilline chez Enterobacter cloacae et Serratia marcescens a egalement diminue. Quant a P. aeruginosa, la frequence des souches resistantes a la ceftazidime est demeuree a peu pres stable dans l'ensemble, de 7 % en 1989 a 8 % en 1994. Il en est de meme de la resistance a la ticarcilline (30–40 % de souches ticarcilline-R), tandis que la frequence des souches resistantes a l'imipeneme est passee de 12 % en 1989 a 18 % en 1994. Par ailleurs, concernant les phenotypes de resistance, alors que la frequence des souches sensibles a ces trois antibiotiques reste stable (≈ 60 %), on observe parallelement une augmentation de la frequence de la resistance isolee a l'imipeneme, passant de 4,6 % en 1992 a 8,1 % en 1994 et une diminution de la resistance isolee a la ticarcilline (16,3 % en 1994). Pour A. baumannii, la frequence de la resistance a la ceftazidime et au cefotaxime a augmente, celle a la ticarcilline a diminue et celle a l'imipeneme est demeuree stable, inferieure a 1 %. Pour les autres especes bacteriennes, l'activite de la ceftazidime est demeuree stable sur B. cepacia : 2 a 10 % de souches resistantes, sur K. oxytoca : 1 a 3 % et sur S. maltophilia : 70 a 80 %. L'interet de la surveillance reguliere de la sensibilite des principales especes bacteriennes pathogenes aux antibiotiques est de donner des bases objectives a l'antibiotherapie probabiliste et de permettre une bonne economie de l'antibiotherapie. Celle-ci sera facilitee par l'etude de la nature et de l'evolution des mecanismes de resistance en cause.

The potential for aerobic biotransformation of the herbicide atrazine was investigated in groundwater microcosms under laboratory conditions. Fresh groundwater samples were collected from an agricultural area (Southern Alsace, France) where corn crops have been cultivated for at least ten years. Water microcosms were incubated at 20°C and the biodegradation of atrazine (2.104 mg l−1) was followed weekly over 180 days. After a lag period of 15 to 44 days, catabolism occurred rapidly and total degradation of the added pesticide occurred in 21 to 70 days depending on the physico-chemical composition of the groundwater. Re-application of the pesticide to a sample revealed an induction phenomenon with an increase in rate of the catabolism and disappearance of the initial lag-phase. A combination of enrichment culture techniques and plating on selective atrazine-agar have permitted the isolation of several aerobic Gram negative bacteria able to biodegrade the herbicide in a mixed culture of two or three species.

The 16S rRNA genes (rDNA) of 50 strains belonging to 26 different coryneform bacterial species and genomospecies and of the type strain of Rhodococcus equi were enzymatically amplified. Amplified rDNA restriction analysis (ARDRA) with the enzymes Alu l, Cfo l and Rsa l was carried out. The combination of the ARDRA patterns obtained after restriction with these three different enzymes enabled the differentiation between the following species: Corynebacterium accolens (number of strains = 2), C. afermentans subsp. afermentans (2), C. afermentans subsp. lipophilum (2), C. amycolatum (3), CDC coryneform group ANF-1-like (1), CDC coryneform group ANF-3-like (1), C. cystitidis (1), C. diphtheriae (4), C. jeikeium (3), C. macginleyi (2), C. minutissimum (1), C. pilosum (1), C. pseudotuberculosis (2), C. renale (2), C. striatum (2), C. urealyticum (3), C. xerosis (1), CDC coryneform groups B-1 (2), B-3 (2), F-1, genomospecies 1 and 2 (6), G, genomospecies 1 (1) and G, genomospecies 2 (2). The following strains or species could not be differentiated from each other; C. pseudodiphtheriticum (2) from C. propinquum (former CDC coryneform group ANF-3) (2), CDC coryneform group F-1, genomospecies 1 (4) from genomospecies 2 (2) and C. jeikeium genomospecies A (1) from genomospecies C (2). ARDRA may represent a possible alternative for identification of coryneforms, since this technique enabled the identification of most coryneforms tested and since DNA extraction ( i.e. cell lysis by boiling), amplification, restriction and electrophoresis can be carried out within 8 hours. This might allow quick identification of C. diphtheriae and other possible pathogens of the genus Corynebacterium .

We studied 12 coryneform isolates having similar biochemical profiles which did not permit their assignment to any recognized taxa. Human semen was the source for seven of these strains, whereas the other strains were isolated from urethra, urine, and blood specimens of adult male patients. These bacteria were found in significant quantities (10(4) to 10(5) CFU/ml) in semen specimens from infertile male patients with the diagnosis of prostatitis. These strains had characteristics of the genus Corynebacterium, such as 60 mol% G + C in the DNA and corynemycolic acids, meso-diaminopimelic acid, arabinose, and galactose in the cell wall. Quantitative DNA-DNA hybridizations (S1 nuclease procedure) and phylogenies based on comparisons of almost-complete small-subunit ribosomal DNA sequences confirmed that these strains constitute a single new species within the genus Corynebacterium. All 12 strains showed similar phenotypic features, i.e., good growth on sheep blood agar in contrast with poor growth on the same medium supplemented with 1% Tween 80, a positive CAMP test in the presence of Staphylococcus aureus, glucose and sucrose fermentation, and the presence of beta-glucuronidase. Some strains reduced nitrate and hydrolyzed urea or esculin. These features allowed us to distinguish these strains from members of any other coryneform taxon, and the proposed name is Corynebacterium seminale with strain IBS B12915 (CIP 104297) as the type strain. The description and delineation of these strains as a new species should be useful for further studies, including evaluations of their prevalence among the normal flora and their clinical implications.

The genus Gordona was proposed by Tsukamura in 1971 (M. Tsukamura, J. Gen. Microbiol. 68:15-24,1971) and was revived in 1988 by Stackebrandt et al. (E. Stackebrandt, J. Smida, and M. D. Collins, J. Gen. Appl. Microbiol. 34:341-348, 1988) to accommodate Rhodococcus species containing mycolic acids with 48 to 66 carbon atoms and MK-9(H2) as the predominant menaquinone type. On the basis of mycolic acid composition as determined by high-performance liquid chromatography, DNA-DNA similarity data (S1 nuclease procedure), and the rRNA gene restriction patterns of the type strains, we propose that Rhodococcus chubuensis Tsukamura 1982 is a subjective synonym of Gordona sputi (Tsukamura 1978) Stackebrandt 1989 comb. nov. We found that R. chubuensis type strain ATCC 33609, and G. sputi type strain ATCC 29627 exhibit identical mycolic acid profiles, 97% DNA relatedness (difference in thermal denaturation midpoints, 1.0°C), and similar ribotypes after PvuII endonuclease digestion. Thus, as defined by the type strains and considering the priority of the name G. sputi, these two species should be regarded as a single species named Gordona sputi; an emended description of this taxon is given.

A 33 basepair oligonucleotide probe, designed from the sequence of the Clostridium difficile toxin B gene, was evaluated for its ability to detect toxigenic Clostridium difficile directly in stool samples, without culture or DNA isolation. Two different labelling techniques were investigated: radiolabelling and digoxigenin-labelling. One hundred ninety-six stools were tested, with a good correlation (96%) obtained between the oligonucleotide probe and the gold standard, the cytotoxicity tissue culture assay. The sensitivity and specificity were 83% and 100%, respectively. In parallel, a new commercially available enzyme immunoassay for the detection of Clostridium difficile toxin A in stool specimens was investigated. In 162 samples tested, a sensitivity of 80% and a specificity of 98% were obtained.

Chemotaxonomic and genomic studies were performed on seven Corynebacterium group ANF-3 strains isolated from human sources. All these strains possess cell wall component type IV (meso-diaminopimelic acid, arabinose and galactose), corynemycolic acids and a G+C content of DNA of 57 to 59 mol%. These results confirm that they can be placed in the genus Corynebacterium. Six of these strains were found to constitute a tight hybridization group distinct from named Corynebacterium species or related organisms. This genomic group constitutes a new species which can be identified within the genus Corynebacterium by ribotyping or phenotypic tests and for which the name Corynebacterium propinquum is proposed. The type strain is strain B 77159 (= Collection of the Institut Pasteur CIP 103792).

Several natural ecosystems, including soil and water microcosms, have been explored for their potential to biodegrade atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-1,3,5-triazine). Subculturing soil or water samples with several applications of the herbicide leads to an enhancement of atrazine catabolism correlating with a decrease in the number of bacterial species. A combination of enrichment culture techniques, the imposition of carbon limitation, and plating on selective atrazine-agar plates have permitted the isolation of several bacteria able to biodegrade atrazine from different polluted or unpolluted ecosystems. One of the selected bacteria, originating from an agricultural soil, has been identified as Acinetobacter calcoaceticus, which alone in pure culture can degrade atrazine. From another agricultural soil Pseudomonas alcaligenes associated with an Agrobactenum sp. was selected. A garden soil treated only once with the herbicide was found to contain an association of five different bacteria that together degrade atrazine. Finally, a Pseudomonas putida/Xanthomonas maltophilia pair has been isolated from an aquatic ecosystem. In consortium, in coculture, or in pure culture with well-defined experimental parameters, the induced bacteria degrade added atrazine in 18 h.

A rapid and sensitive dot blot assay using nonradioactive labeled oligonucleotides as probes, was developed for detection of genes coding for staphylococcal enterotoxins (SEs) A, B, C, D, E and toxic shock syndrome toxin-1 (TSST-1). A specific probe selected and synthesized for each of the six toxin genes, was 3′-labeled by Dig-11-dUTP and dATP tailing. Hybridization conditions were optimized from the manufacturer conditions so that sensitivity and specificity of the digoxigenin assay were equivalent to those obtained when using radioactive methods. Such a nonradioactive genotypic assay, suitable for epidemiological studies, is therefore of particular interest for all biological laboratories interested in staphylococcal enterotoxins and TSST-1 detection.

Toxin B from Clostridium difficile is cytopathic in vitro for various types of cells, including polymorphonuclear cells, lymphocytes, and monocytes. Since intestine lamina propria is rich in macrophages, we studied the effect of toxin B on human monocytes and on human macrophages generated in vitro by long-term culture of purified circulating blood monocytes. Upon addition of toxin B, human monocytes exhibited few modifications whereas macrophages adopted a stellate morphology, with rounding up of the perikaryon. Toxin B made microfilaments of actin disappear and induced an important reorganization of vimentin and a redistribution of tubulin. Membrane area increased by approximately 16%. Toxin B did not affect the viability of human mononuclear phagocytes and did not exert any significant lytic effect. It profoundly altered the phagocytic function of macrophages. When activated by gamma interferon in the presence of toxin B, monocytes were more cytotoxic for U-937 target cells than control monocytes activated in absence of toxin. Finally, the combined treatment of monocytes with gamma interferon and toxin B increased significantly the secretion of tumor necrosis factor alpha, whereas toxin B alone was unable to induce tumor necrosis factor production. These results suggest that morphological and functional alterations induced in human mononuclear phagocytes by toxin B from C. difficile are due to the disorganization of the cytoskeleton and the resulting impairment of the membrane traffic equilibrium.

Ofloxacin penetration into heart tissue (valve and myocardium), mediastinal fat, and sternal bone marrow was the object of a prospective nonrandomized study. Thirty-six patients undergoing mitral and/or aortic valve replacement were included. Patients were divided into two groups of 18 patients each. Group 1 patients were administered a single 400-mg intravenous dose of ofloxacin over a 30-min period upon anesthesia (n = 6) or at 1 h (n = 6) or 6 h (n = 6) prior to surgery. Group 2 patients received a 200-mg oral dose of ofloxacin every 12 h during the 48 h preceding surgery. In this group, the final dose of ofloxacin was administered 3 h (n = 9) or 8 h (n = 9) before anesthesia. Plasma and tissue ofloxacin concentrations were assayed by high-pressure liquid chromatography. In group 1 patients, the peak level in plasma was 15.9 +/- 2.5 micrograms/ml. Peak ofloxacin levels in tissue were reached by hour 1 and were 8.89 +/- 2.16 micrograms/g in myocardium and 5 +/- 0.75 micrograms/g in heart valves. A significant decrease in ofloxacin levels in heart valve tissue and sternal bone marrow was observed after hour 3. Nevertheless, ofloxacin myocardial, heart valve, and sternal bone marrow levels remained higher than the MICs for the usually susceptible pathogens for at least 3 h. In group 2 patients, myocardial levels were long lasting (6.46 +/- 1.92 micrograms/g [4 to 8 h] and 5.92 +/- 0.95 micrograms/g [8 to 12 h]) and remained higher than those observed in the other tissues over the entire study period. A progressive but insignificant decrease in ofloxacin heart valve levels was observed (from 2.46 +/- 0.40 micrograms/g [4 to 8 h] to 1.57 +/- 0.22 micrograms/g [8 to 12 h]). In both groups, concentration in mediastinal fat were lower and tended to decrease with time. These were 1.83 +/- 0.61 micrograms/g with the first hour and 0.85 +/- 0.43 micrograms/g between hours 8 and 12 in group 1 and 1.74 +/- 0.52 micrograms/g between hours 4 and 8 and 0.67 +/- 0.11 micrograms/g between hours 8 and 12 in group 2. In conclusion, satisfactory diffusion of ofloxacin into heart tissue seems to favor use of the drug in the treatment of bacterial endocarditis due to susceptible pathogens. Furthermore, the progressively decreasing concentrations observed in heart valve and sternal bone marrow and the poor levels achieved in mediastinal fat suggest the need for renewing injection 3 h following initial infusion if the drug is used as an antibiotic prophylactic agent during cardiovascular surgery.

Twenty-one Corynebacterium group D2 (“C. urealyticum” strains were found to constitute a tight DNA hybridization group distinct from named Corynebacterium species. The strains of Corynebacterium group D2 had cell wall component type IV, short chain mycolic acids and G+C content of DNA of 65–66 mol %. Corynebacterium group D2 constitutes a genomic species which can be identified by phenotypic tests.

The seabird tick Ixodes uriae (White) is common in subantarctic areas and is likely to be a vector of pathogenic agents. This paper reports on the prevalence of tick-infested adult king penguins (Aptenodytes patagonicus) and on antibodies to the Lyme disease agent (Borrelia burgdorferi sensu lato) in adult and chick penguins. Fifteen percent of the adults of the colony studied were tick infested. B. burgdorferi antibodies were detected in 14% of tick-infested adults' and in 6% of chicks' sera. The percentage of tick-infested adults differed significantly according to location in the colony, the highest density occurring on the upper slopes of the colony. The potential role of king penguins as B. burgdorferi reservoirs and the effect of this agent on king penguin populations remain unknown. The consequences for human health are discussed.

The BBL Crystal MRSA ID test (Becton Dickinson) was applied directly to blood culture vials containing clusters of gram-positive cocci. The sensitivity and specificity of the test were 84 and 100% and 54 and 100% for vials containing Staphylococcus aureus and coagulase-negative staphylococci, respectively. This test is a reliable method for direct detection of methicillin resistance in positive blood culture vials when S. aureus is identified in parallel by rapid identification procedures.

A one-step reverse transcriptase PCR (RT-PCR) method for detection of Borrelia burgdorferi mRNA in infected C3H mice is described. This simple procedure, less prone to nucleic acid cross-contamination than the standard method, was found to be 10-fold more sensitive than a classical two-step RT-PCR assay. By using one-step RT-PCR, flagellin mRNAs were detected in synovial and heart tissues from all seven infected mice tested.

Resume La reaction d'hybridation entre molecules d'acides nucleiques represente le fondement de l'utilisation de sondes d'ADN pour l'identification ou la detection de bacteries. Cette methode a l'avantage de permettre le diagnostic de bacteries difficiles a identifier par les methodes traditionnelles, toutefois ces bacteries doivent au prealable avoir ete cultivees sur milieux bacteriologiques. Elle permet egalement de detecter des genes codant des facteurs de virulence particuliers produits par certaines bacteries. Enfin, elle permet theoriquement de reperer des bacteries directement dans un produit pathologique, sans avoir a passer par l'etape de la culture classique. Malheureusement, cette methode etant tres specifique, elle ne permet de rechercher ou d'identifier que les especes pour lesquelles le biologiste possede des sondes. De plus, cette technique ne permet pas de determiner la sensibilite d'une bacterie a tous les antibiotiques. C'est pourquoi, a moins de rechercher specifiquement un germe defini dans un prelevement, cette methode ne dispense pas de la culture classique. Beaucoup de trousses diagnostiques actuelles utilisent des radioelements (125I), ont un prix de revient encore eleve et ont encore une sensibilite inferieure a celle de la culture lorsqu'il s'agit d'identifier une bacterie directement dans un produit biologique. On espere realiser des progres grâce aux techniques d'amplification de genes qui permettront d'augmenter la sensibilite de la methode.

Using an agar reference method (Norma M11-A5, National Committee for Clinical and Laboratory Standards) the minimal inhibitory concentrations of nine antibiotics were determined for 376 anaerobic strains. The following strains were investigated: 254 Bacteroides fragilis group (including 143 B. fragilis), 122 other gram-negative anaerobes (Bacteroides spp., Prevotella, Fusobacterium, Porphyromonas, Suterella, Desulfomonas, Veillonella).In the B. fragilis group resistance rates were: coamoxyclav 2.8%, ticarcillin 27.5%, ticarcillin-clavulanic acid 1.9%, piperacillin-tazobactam 1.9%, cefoxitin 6.2%, imipenem 0.8%, clindamycin 28.3%, respectively. Based on previous studies, resistance to imipenem remained low in 2003 and was only observed for B. fragilis. Resistance to clindamycin was maintained around 25%. No metronidazole resistance was observed, but decreased susceptibility was found for B. fragilis, B. merdae and Prevotella, as in 4.3% of gram-negative anaerobes.This study confirms the high resistance rate of gram-negative anaerobes to clindamycin, the efficient activity of imipenem, beta-lactam/beta-lactamase inhibitor combinations and metronidazole. However, reduced metronidazole susceptibility seems to be increasing.

We evaluated the incidence of Streptococcus pseudopneumoniae in clinical isolates by phenotypic methods and DNA-DNA hybridization. The pathogenic role of this organism was investigated with the mouse peritonitis/sepsis model. Our results show a low incidence (1/120 pneumococcal isolates) and a potential pathogenic effect for S. pseudopneumoniae .

The automated MagNA Pure DNA extraction method for Chlamydia trachomatis was compared with the manual Cobas Amplicor protocol using 100 µL of input sample volume from 964 specimens. Agreement between protocols was 96.1%. The automated extraction method had a sensitivity of 99% and a specificity of 100%. Amplification inhibition observed after manual preparation of samples (3.8%) was not apparent following automated extraction. Using 200 µL of sample in the automated extraction process lowered the detection limit without raising the inhibition rate. Furthermore, the automated extraction method halved the hands-on time required for the procedure.

Two Aeromonas strains, IBS S6874T and IBS S6652, were isolated from the faeces of two healthy monkeys (Macaca fascicularis) from Mauritius that were kept in quarantine in the Centre for Primatology, Strasbourg, France. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two isolates formed an unknown genetic lineage within the genus Aeromonas. The two isolates had nearly identical sequences (0·1 % nucleotide substitution) that were related closely to those of recognized Aeromonas species (1·7–3·5 % nucleotide substitution). DNA–DNA hybridization showed that strains IBS S6874T and IBS S6652 had high DNA–DNA similarity (89 %) to each other and a low level of DNA–DNA similarity to closely related taxa (18 % relatedness to Aeromonas trota and 16 % relatedness to Aeromonas schubertii). Phenotypically, the two monkey isolates differed from most previously described mesophilic Aeromonas species by their lack of haemolysis on sheep-blood agar and inability to produce indole, gas from glucose or acid from mannitol. They differed from the most closely related species, A. schubertii, by their ability to produce acid from d-cellobiose and d-sucrose and by their pyrazinamidase activity. The name Aeromonas simiae sp. nov. is proposed for these isolates; strain IBS S6874T (=CIP 107798T=CCUG 47378T) is the type strain.

Staphylococcus aureus bacteraemia (SAB) may be complicated by secondary metastatic infection such as endocarditis, osteomyelitis or septic arthritis. This cohort study aimed to assess the prognostic value of sustained bacteraemia for outcomes related to Staphylococcus aureus bacteraemia.The study took place in three tertiary-care, university-affiliated hospitals. Patients were prospectively included if they agreed to participate and if the following data were available: (a). surveillance blood culture taken between 24 and 48 h after commencement of effective antibiotic therapy; (b). appropriate investigations (at least a TTE) performed as suggested by the infectious diseases consult service. Patients with sustained bacteraemia defined as persistent positive blood cultures more than 24 h after commencement of effective antibiotic therapy were compared to patients for whom the surveillance blood culture was negative.One hundred and four patients were enrolled, including 51 patients diagnosed with sustained bacteraemia. Sustained bacteraemia was significantly associated with a higher frequency of secondary metastatic infection (p0.001) and with a higher frequency of CRP100 mg/l. Frequency of acute complications due to infection, septic shock and death due to bacteraemia was higher for patients with sustained bacteraemia but this difference was not statistically significant. Using a Cox model, the risk for death associated with sustained SAB, controlling for Index of comorbidity and age (categorised asoror=70 years), was 1.2 (95% CI: (0.5, 3); p0.05).In conclusion, surveillance blood cultures, especially performed on effective antibiotic therapy, may be a simple and cost-effective way to select a population at risk for secondary metastatic infection from SAB.

Aeromonas caviae, an ubiquitous aquatic organism, has long been considered to be of low pathogenicity, and its virulence mechanisms are still not clearly understood. Twenty-eight A. caviae isolates of clinical origin, most often monomicrobic, were identified in our university hospital over a four year period. Patients, mostly immunocompromised, were: eight diarrhoeal infants, 13 diarrhoeal adults, seven bacteraemic adults. Adults were frequently suffering from underlying intestinal malignancy, hepatobiliary disease, gastrectomy. Virulence factors were investigated. Adherence, studied by use of tissue culture HEp-2 cells, and staining of characteristic lateral flagella, were observed in diarrhoeal strains. Extracellular hemolytic activity was tested on rabbit erythrocytes suspensions at 25 and 37 degrees C. One blood culture isolate showed an important hemolytic activity at 25 degrees C, but none at 37 degrees C. Treatment with furin activated the aerolysin precursor and resulted in significant hemolysis at 37 degrees C, and fluid accumulation in rabbit ileal loops similar to that of A. hydrophila as control. The presence of the hemolysin gene was confirmed in this strain by PCR. In conclusion, A. caviae was shown to be a pathogen isolated from diarrhoea and bacteraemia in immunocompromised patients with malignancies and low gastric acidity as favouring factors. Virulence including the ability to adhere to cells and the production of lateral flagella was observed in diarrhoeal strains. The expression and the production of extracellular hemolytic activity and enterotoxicity at 37 degrees C depended on the activation of the pore forming toxin aerolysin precursor by furin. In vivo the protoxin is probably processed to its mature form by host proteases.

In this report, we review two cases of brain infection due to Dialister pneumosintes in previously healthy patients. The bacterium was isolated from the first patient by blood culture and directly from a brain abscess in the second patient. In both cases, the infection was suspected to be of nasopharyngeal or dental origin. The patients had favorable outcomes following surgical debridement and antibiotic treatment. After in vitro amplification and partial sequencing of the 16S rRNA gene, two strains were classified as D. pneumosintes . However, traditional biochemical tests were not sufficient to identify the bacteria. In addition to causing periodontal and opportunistic infections, D. pneumosintes, contained in mixed flora, may behave as a clinically important pathogen, especially in the brain. In addition to phenotypic characterization, 16S rRNA partial sequencing was used to identify D. pneumosintes definitively.

Objective The purpose of this study was to investigate and characterize in vitro the post-β-lactamase inhibitor effect (PLIE) of clavulanic acid against two β-lactamase-producing species of bacteria. Methods The PLIE was investigated against one strain of Klebsiella pneumoniae and one strain of Haemophilus influenzae. A stationary-phase inoculum of about 10 7 colony-forming units per mL of each bacterium was pre-exposed for 2 h to clavulanic acid, either alone or in combination with amoxicillin at various concentrations. After pre-exposure, the dilution required to remove the β-lactamase inhibitor was 1:100 or 1:1000 according to the bacterial species and their susceptibilities to clavulanic acid. Bacteria were counted hourly after drug removal, on solid agar medium. Results Control cultures exposed to amoxicillin alone after dilution, showed a delay in growth, which may be inherent to the time required to synthesize sufficient β-lactamase after the dilution steps. Control experiments clearly distinguished the post-antibiotic effect and the growth delay from the PLIE. Conclusion The PLIE could be one of several factors explaining why β-lactam/β-lactamase inhibitor combinations remain effective throughout the dosing interval, even if a few hours after in vivo administration, serum concentrations of β-lactamase inhibitor fall below levels that are active in vitro.

The Yucatan micropig has been used to develop an experimental model of chronic bacteremia. This animal exhibits clinical and biological characteristics that are close to those in humans, and the pharmacokinetic behaviours of many classes of drugs in this model are similar to those in man. Six adult female were intravenously inoculated with a mean Escherichia coli inoculum of 5.1 x 10(9) bacteria. During five days of spontaneous evolution, the medical follow-up includes biological, clinical and bacteriological parameters. A systemic inflammatory syndrome, a sepsis, an organ insufficiency and positive blood cultures mimic the human disease. In all animals there is an adynamia, a lack of motor coordination, an anorexia, a tachypnea, a fever, a leuconeutropenia followed by an hyperleucocytosis, an anemia, a thrombopenia, an acute tubulonephritis and an elevated sedimentation rate. In some cases, there is an increase of the C reactive protein, in others, an increase of IL-6 and IL-8. At day five, all animals are alive, and five micropigs have positive blood cultures. This chronic, reproducible model is thus suitable for further antibacterial treatments evaluations.

Lyme arthritis is caused in Europe by three main pathogenic species of Borrelia burgdorferi sensu lato: Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. Because few synovial samples have yet been analysed by species-specific DNA amplification methods, further studies are needed to define the spectra of clinical manifestations associated with these different species. Two cases of treatment resistant Lyme arthritis are reported here, in which DNA amplification of the flagellin gene followed by dot-blot hybridisation in the synovial fluid identified B garinii as the causative agent. Clinical and biological data did not differ from the usual descriptions of Lyme arthritis, but as the recently reported molecular mimicry between OspA and hLFA1 is not applicable to B garinii, the pathogenesis of the present cases remains unclear. Future studies should aim at assessing the role of B garinii in European Lyme arthritis and its possible pathogenic and therapeutic consequences.

Six E. coli, whose phenotypes of resistance were different, were tested in vitro in order to evaluate a regrowth delay, the post beta-lactamases inhibitor effect (PLIE). This PLIE was investigated after a brief incubation in contact with clavulanic acid (CA) alone or associated with amoxicillin (AMX). After removal of the drugs used during the pre-exposure phase, the bacteria were incubated with AMX at different concentrations. The PLIE was shown not to be in association with any other regrowth delay (post-antibiotic effect or effect inherent to the technical procedures used). A PLIE was evaluated on the five intermediary or high-level beta-lactamases-producing strains. Generally, the duration of the PLIE was prolonged after the CA alone pre-exposure phase and could reach values up to 22 hours. The concentrations of AMX added in cultures previously exposed to sufficient CA concentrations were related to an extended PLIE.

The in vitro activity of fusidic acid was evaluated against 242 strains of streptococci isolated from skin and soft tissue infections during a prospective multicentre study. Nearly 90% of strains were isolated from dermatology, emergency and medicine units. Groups A, B, C and G streptococci represented, respectively, 41.9, 20.6, 4.4 and 27.8% of the strains. The activity of fusidic acid was dependent on the media used. MICs were generally one dilution lower with heart infusion agar than with Mueller-Hinton agar supplemented with 5% horse blood (MIC(90) for the whole streptococcal population = 8 mg/L and 16 mg/L, respectively). The distribution of MICs was unimodal and only two strains displayed MICs of fusidic acid >/= 64 mg/L. In both media, fusidic acid was moderately active against streptococci. However, antibiotic concentrations obtained in the skin exceed the MIC(90) of fusidic acid for streptococci, possibly explaining its clinical efficacy in the treatment of common cutaneous infections.

In vivo, serum concentrations of beta-lactamase inhibitors measured during the last part of the dosing interval are below the levels associated with in vitro activity. Nevertheless, beta-lactam plus beta-lactamase inhibitor combinations remain active in vivo throughout the dosing interval. One of the many reasons for this contradiction may be the PLIE. The PLIE can be evaluated only in the light of the postantibiotic effect (PAE). Also, accurate determination of the PLIE requires a careful investigation of all bacterial regrowth delays (BRDs) inherent to the technical procedures used. The purpose of the study reported herein was to determine the true in vitro PLIE of clavulanic acid (CA) against two beta-lactamase-producing strains, a Klebsiella pneumoniae strain (amoxicillin [AMX] MIC256 mg/L; CA MIC = 64 mg/L; and AMX + CA MIX = 4 mg/L) and a Haemophilus influenzae strain (AMX MIC = 32 mg/L; CA32 mg/L; AMX-CA = 1 mg/L). For each strain, a stationary phase inoculum of 10(7) was preexposed for 2 h to either CA alone or CA + AMX in various concentrations. Dilution to 10(-2) or 10(-3) was performed to eliminate the CA and/or AMX after the preexposition phase. Hourly bacterial counts were done between 0 and 8 h and after 24 h. Control cultures exposed to AMX after dilution showed a growth delay possibly ascribable to the time needed for bacteria to produce a large enough amount of beta-lactamases. Control experiments were done to unequivocally differentiate PLIE from PAE and BRD. The true PLIE values thus obtained ranged from 0 to 4.5 h for K. pneumoniae and from 0 to 15 h for H. influenzae. For both strains, a PLIE was demonstrated after exposure to CA alone.

The newly developed Hemofast MRSA system for Staphylococcus aureus identification and methicillin-resistant staphylococci (MRS) detection in blood culture broths was evaluated in 106 Bactec broths containing grapelike clusters of Gram-positive cocci. All 26 S. aureus positive broths were correctly identified by Hemofast MRSA within two hours, and 81% were identified within one hour. Sensitivity and specificity were both 100%. Accuracy of MRS detection was 100%, i.e., no discrepancies versus the agar diffusion method were found. When the 28 broths containing coagulase negative staphylococci (CNS) were tested using an inoculum ten fold larger than for S. aureus testing, a number of discrepancy were recorded. For 49 other broths containing CNS, accuracy was 98.5% when the test was interpreted based on the growth rate of the strain, according to the manufacturer's instructions. One broth containing S. epidermidis strain susceptible yielded a false result with Hemofast MRSA, indicating that it probably contained a contaminant. The other discrepancies occurred with specimens containing mixed populations with CNS or a Micrococcus strain. Most (85%) results were available within 4 hrs 30 min, irrespective of the S. aureus or CNS strains. Avoiding the isolation step on agar, Hemofast MRSA saves 24 to 48 hours, thus allowing earlier antistaphylococcal treatment.