Evaluation of BMPA, MWY, GVPC and BCYE media for the isolation of Legionella species from respiratory samples.

Culture media performance is a critical factor in the isolation of Legionellae from respiratory samples. We showed that BMPA and MWY media yielded significantly higher isolation rates than GVPC and BCYE media in regard to performance with samples that harbored low Legionella inocula and high contamination levels.

molecular typing of Legionella pneumophila strains isolated from environment in Morocco.

Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most legionellosis outbreaks are linked to contaminated hot water systems or cooling towers. The aim of this study was to determine the genetic diversity of (n= 55) environmental strains of L. pneumophila recovered from the hot water distribution systems of 16 establishments in seven Moroccan towns during the period 2009-2011. Thirteen chromosomal restriction patterns determined by Pulsed field gel electrophoresis were detected. The strains of L. pneumophila serogroup1 exhibited in 6/13 different PFGE patterns, while the strains of L. pneumophila serogroups 2-14 showed 7/13 PFGE patterns. The PFGE showed the existence of various patterns in Morocco, The pattern -XI- have tree similar profiles with the endemic L. pneumophila Paris's strain. This technique also allowed to conclude that the same pulsotype was found for many strains isolated from different establishments. Moreover, different pulsolypes were found for strains isolated from the same establishment. These results showed that PFGE analysis is a powerful tool to reveal the clonal nature and genetic differences among L. pneumophila strains.

[Molecular comparison of Legionella pneumophila serogroup 1 isolated in Tunisia]. / Comparaison moléculaire de Legionella pneumophila sérogroupe 1 isolées en Tunisie.

Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.

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.

Emergence and spread in French hospitals of methicillin-resistant Staphylococcus aureus with increasing susceptibility to gentamicin and other antibiotics.

Oxacillin (methicillin) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is associated with an increased incidence of resistance to other antibiotics, which has increased since it was first reported in 1969. In 1992 a new phenotype of MRSA arose in France; this was characterized by a heterogeneous expression of resistance to oxacillin and susceptibility to various antibiotics, including gentamicin but also tetracycline, minocycline, lincomycin, pristinamycin, co-trimoxazole, rifampin, and fusidic acid. In French hospitals a longitudinal nationwide surveillance of antibiotic resistance in S. aureus has allowed for the detection of changes in antibiotic susceptibility profiles. Seven French clinical laboratories (six from the mainland and one from the West Indies) reported the results of susceptibility testing of 57,347 S. aureus strains isolated in their institutes between 1992 and 1998. Over a 7-year period the incidence of isolation of gentamicin-susceptible MRSA (GS-MRSA) strains has steadily increased to represent, in 1998, 46.8 to 94.4% of the MRSA strains, irrespective of the overall incidence of MRSA. Two predominant types recognized by pulsed-field gel electrophoresis (PFGE) accounted for the majority of the GS-MRSA in different mainland hospitals, both differing from the predominant type observed in the French West Indies. Some GS-MRSA and gentamicin-resistant MRSA (GR-MRSA) strains had closely related PFGE profiles, and hybridization studies confirmed the lack in GS-MRSA of the aac6'-aph2" gene, which confers resistance to all aminoglycosides, with conservation of the ant4' gene, which confers resistance to kanamycin, tobramycin, and amikacin. Thus, it is likely that certain GS-MRSA strains could have emerged from GR-MRSA strains by excision or deletion of the aac6'-aph2" gene.

Single clonal origin of a high proportion of Legionella pneumophila serogroup 1 isolates from patients and the environment in the area of Paris, France, over a 10-year period.

Arbitrarily primed PCR with three primers and pulsed-field gel electrophoresis were used to characterize a set of 75 clinical Legionella pneumophila serogroup 1 isolates, with no apparent epidemiological link, obtained from 24 hospitals in Paris, France, from 1987 to 1997. Unexpectedly, 25 clinical isolates from 15 hospitals had an identical profile (termed type A) by both methods. The same profile was subsequently found in 16 of 64 randomly selected environmental L. pneumophila serogroup 1 isolates from 15 different sites in the Paris area. There was no evidence of geographic clustering or a peak incidence of type A isolation. Type A has not been found in France outside the Paris area, suggesting that a particular type of L. pneumophila serogroup 1 is specifically present in the Paris water distribution network.

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.

Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study.

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.

Comparative analysis of infrequent-restriction-site PCR and pulsed-field gel electrophoresis for epidemiological typing of Legionella pneumophila serogroup 1 strains.

Two methods were compared for the analysis of 48 unrelated and epidemiologically related Legionella pneumophila serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for XbaI and PstI restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with SfiI. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of L. pneumophila from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, > or = 0.996) tool for typing of Legionella. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.

Chronic bacteraemia due to Staphylococcus epidermidis after bone marrow transplantation.

A chronic bacteraemia due to Staphylococcus epidermidis occurred in a patient undergoing allogeneic bone marrow transplantation. Forty-two S. epidermidis isolates were obtained from blood cultures over a period of 5 months. Isolates were separated into three groups by SmaI macrorestriction characterisation with pulsed-field gel electrophoresis (PFE-1, one isolate; PFE-2, 32 isolates; PFE-3, nine isolates). Differences were detected in antimicrobial susceptibility patterns among isolates belonging to group PFE-2. The two strains, PFE-2 and PFE-3, were both responsible for the chronic bacteraemia and were isolated during different admissions to the hospital. A central venous catheter was the portal of entry for PFE-2. DNA macro-restriction analysis with pulsed-field gel electrophoresis was helpful in the epidemiological investigation of this S. epidermidis chronic bacteraemia.

Clonal study of enterotoxin-B producing strains of Staphylococcus aureus.

Sixty-nine Staphylococcus aureus strains, 39 of which produced staphylococcal enterotoxin B (SEB+) and 14 of which were associated with toxic shock (TS+), were studied using the following markers: serotyping, phage typing, antibiotyping, ribotyping, zymotyping and pulsed-field electrophoresis typing. Analysis of the results showed that the enterotoxin B producing strains were derived from at least three clones: the first two consisted of methicillin-susceptible strains, while the third included the methicillin-resistant (MRSA) strains. TS+ strains of nongenital origin appeared to be distributed between the three clones, with no specific characters.

Clinical isolates of Staphylococcus lugdunensis and S. schleiferi: bacteriological characteristics and susceptibility to antimicrobial agents.

The bacteriological characteristics and susceptibility to antimicrobial agents of 108 clinical isolates of Staphylococcus lugdunensis and Staphylococcus schleiferi are described. Fifty out of 108 isolates were considered to be responsible for 16 documented infections, including some severe infections (endocarditis, bacteraemia, osteitis). A number of bacteriological characteristics enabled the identification of these species in the clinical microbiology laboratory: the absence of coagulase and protein A, and the presence of a fibrinogen affinity factor and thermonuclease along with other biochemical characteristics (ornithine and arginine decarboxylases, carbohydrate acidification, novobiocin susceptibility) differentiated these new species from other staphylococci; however, they did not possess virulence markers such as toxins or haemagglutinin, but were haemolytic. In this series, almost all isolates were susceptible to 22 antibiotics and 4 antiseptics representative of the main groups of antimicrobial agents. More information is needed on the ecology and epidemiology of these new opportunistic pathogens.

[In vitro activity of roxithromycin compared to 5 other macrolides against staphylococci]. / Activité in vitro de la roxithromycine comparée à celle de cinq autres macrolides sur les staphylocoques.

This study aimed to compare the bacteriostatic activity of roxithromycin (RU) to those of erythromycin (ERY), troleandomycin (TAO), spiramycin (SPI), josamycin (JOS) and midekamycin (MID) against staphylococci strains. 239 strains of hospital origin were analysed: S. aureus (139), coagulase negative staphylococci (CNS) (100). The MIC were determined by the agar dilution method. The modal MIC, the MIC 50 and 90 observed for the both groups of strains are given according to species and antibiotics. This study gives the opportunity to classify the 6 antibiotics in a decreasing order considering their antistaphylococcal activity: RU = ERY, TAO = SPI, JOS = MID. No difference was noticeable between S. aureus and CNS strains.

[Treatment with erythromycin of experimental legionellosis in guinea pigs infected by aerosol]. / Traitement par l'érythromycine de la légionellose expérimentale du cobaye infecté par aérosol.

The infectious strain L. pneumophila serogroup 1 Philadelphia (ATCC 33152) was cultured on charcoal dialysed yeast extract agar medium (CDYE agar) which produces more virulent strains than those grown on classical agar media. The aerosol was dispersed in a depression chamber by means of a nebuliser and the density was controlled by a density probe. Male albinos Dunkin-Hartley guinea pigs weighing 250-300 g were exposed for 30 minutes to an aerosol dose of 1 LD50 (10(3) viable organisms) and 10 LD50 (10(4) viable organisms). Erythromycin lactobionate (Abbott) was administered subcutaneously 18 hours after the infection, at dosages of 270 mg/kg/day for 4 days in the animals treated with 1 LD50 and for 6 or 7 days in the animals treated with 10 LD50. The guinea pigs were observed for 9 days (weight, rectal temperature; serological and bacteriological tests (cardiac blood, lungs, spleen) and erythromycin assays (serum, lungs) were performed and compared in the treated animals, the non-treated infected control animals and the control animals which only received erythromycin. The percentage survival in the treated guinea pigs after inhalation of 1 LD50 and 10 LD50 (2 tests) were 100%, 75% and 87.5% respectively. Three weeks after treatment, the survivors had antibody titres from 32 to 1,024; the bacteriological cultures and erythromycin assays were negative. In this study, an improvement in the treatment of experimental Legionnaires' disease was observed in comparison with previous experiments. The increased dosage and duration and the early initiation of treatment resulted in survival rates of 75%.(ABSTRACT TRUNCATED AT 250 WORDS)

[Influence of technical factors in the determination of minimal inhibitory concentration by microdilution]. / Influence de facteurs techniques dans la détermination des concentrations minimales inhibitrices par microdilution.

Minimal inhibitory concentrations (MICs) of a new quinolone, norfloxacin, as determined by agar dilution are greater than those found using a liquid dilution micromethod. We report herein an analysis of the various parameters possibly involved in this discrepancy: Mueller-Hinton and BioMérieux, glass or plastic, small or large inoculum, and comparative volumes in which the inoculum (IN) and antibiotic (AB) are presented (1 ml IN + 1 ml AB or 1.5 microliter IN + 50 microliter AB or 50 microliter IN + 50 microliter AB). Volume of the inoculum suspension had a bearing on the results obtained with all three reference strains tested. Norfloxacin MICs for S. aureus 7625 and P. aeruginosa 76110 increased commensurately with the ratio of inoculum volume to antibiotic volume, and vice versa. In contrast, no significant variation was found for E. coli 7624. To evaluate the frequency of this effect, we tested 40 antibiotics on the reference strains, and several antibiotics on savage strains (16 Enterobacteriaceae, 40 Staphylococcus, 12 Pseudomonas and 17 P. aeruginosa). The significance of inoculum and antibiotic volumes was corroborated for some antibiotics. Results most consistent with the reference method were obtained with 50 microliter inoculum and 50 microliter antibiotic solution.

[Multicenter study of the antibacterial activity of a new cephalosporin: CM 40874]. / Etude multicentrique de l'activité antibactérienne d'une nouvelle céphalosporine: le CM 40874.

Minimal inhibitory concentrations (MIC) of CM were evaluated on 2 548 bacterial strains isolated in 8 hospitals. CM demonstrated high activity on Enterobacteriaceae, the MIC being less than or equal to 0.125 micrograms/ml for 71% of the 1 362 strains tested, less than or equal to 1 for 99.6%, and less than or equal to 4 for 99.9%. Mode MIC varies little among the different groups of Enterobacteriaceae (from 0.06 to 0.12 micrograms/ml), with the exception of Serratia sp. (mode MIC : 0.25) and Klebsiella oxytoca (mode MIC : 0.03). Most of Enterobacter, Serratia, and Citrobacter sp. strains not inhibited by cefotaxime are readily inhibited by CM at the same concentrations than susceptible strains. CM has less activity on P. aeruginosa (MIC 2-32 micrograms/ml) and Acinetobacter sp. (MIC 8-128). Staphylococci (MIC 32) and Enterococci are not susceptible. Variable activity is found against other Streptococci. CM inhibits Haemophilus sp. at MICs of 0.12 to 0.5 micrograms/ml and Gonococci at MICs of 0.03 to 0.5 (whether the strains produce beta-lactamase or not). Meningococci have a mode MIC of 0.03 micrograms/ml (range 0.008 to 0.25). Thus, CM 40874 is a new third generation cephalosporin with high activity on Enterobacteriaceae, including those strains not susceptible to cefotaxime and good activity on Haemophilus sp. and Neisseria sp. This additional activity is probably supported by enhanced resistance to enzymatic inactivation by beta-lactamases.

[In vitro effect of rosoxacin on hospital bacteria. Results of a multicenter study]. / Activité in vitro de la rosoxacine sur les bactéries hospitalières. Résultats d'une étude multicentrique.

This work reports a multicenter study of antibacterial activity of rosoxacin, a new antibacterial agent of the quinolone group. Enterobacteriaceae are very sensitive to rosoxacin (modal MIC 0,5 micrograms/ml); resistant strains are observed in all species, but more often among Serratia and Citrobacter. Pseudomonas aeruginosa is less sensitive with a maximum number of strains between 2 and 8 micrograms/ml, so it is nearly for Acinetobacter. Haemophilus are very sensitive, having MIC of 0,03 and 0,06 micrograms/ml. The spectrum of rosoxacin includes Gram positive bacteria, since the MIC of Staphylococci are similar to Enterobacteriaceae; Enterococci are less sensitive, the major part of the strains being inhibited by 4 and 8 micrograms/ml.

[In vitro antibacterial activity of cefmenoxime (SCE 1365)]. / Activité antibactérienne in vitro de la cefménoxime (SCE 1365).

617 clinical isolates were tested, 592 of which were from hospital source. The minimal inhibitory concentrations of cefmenoxime were determined by a microtiter dilution method, using Mueller-Hinton broth. The results obtained give a 92% agreement with the reference agar-dilution method. Cefmenoxime shows a potent activity against Enterobacteriaceae (n = 420): the modal MIC is less than or equal to 0,03 mg/l, and 90% of them are inhibited by 1 mg/l. Some isolates require a higher concentration, above 32 mg/l: Enterobacter (8%), indole-positive Proteus (13%), Serratia (3%), Citrobacter (3%). Pseudomonas (n = 71) and Acinetobacter (n = 62) appear to be less susceptible than Enterobacteriaceae. The modal MIC is 16 mg/l and the concentration inhibiting 90% of the isolates is above 32 mg/l. The modal MIC of cefmenoxime against Staphylococcus aureus (n = 64) is 1 mg/l, and 90% of the strains belonging to this species are inhibited by 32 mg/l.