Antimicrobial susceptibility testing in European hospitals: report from the ARPAC study.

This observational study describes the antimicrobial susceptibility testing (AST) methods and interpretive criteria used in European hospitals during 2001, focusing specifically on detection of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Of 263 hospitals that took part in the ARPAC study, 192 submitted data on AST. Of these, 89% (n = 170) routinely used a disk-diffusion AST method, 43% (n = 82) used a semi-automated method, and 70% (n = 135) routinely determined MICs. Hospitals in southern Europe were less likely to use disk-diffusion, but were more likely to use a semi-automated method (p <0.001). In total, 173 (90%) interpreted AST results using CLSI breakpoints; 30% of these detected MRSA using unmodified CLSI disk-diffusion methods, while 35% used the unmodified CLSI agar-screening method for MRSA; 41% and 30% adhered to unmodified CLSI methodology for disk-diffusion and agar-screening, respectively, to detect VRE. Some of the modifications made may have greatly reduced the ability of the tests to detect MRSA/VRE. For example, 20% of respondents used excessively high incubation temperatures and 13% used inadequate incubation times to detect MRSA by disk-diffusion, and 28% used Mueller-Hinton agar instead of brain-heart infusion agar in VRE screening plates. The majority of respondents stated that they followed CLSI guidelines, but a high proportion had modified the CLSI methods for detecting MRSA and VRE, which may compromise clinical management and antimicrobial resistance surveillance.

High-dose cefepime as an alternative treatment for infections caused by TEM-24 ESBL-producing Enterobacter aerogenes in severely-ill patients.

This study evaluated retrospectively the efficacy of treatment with cefepime vs. a carbapenem, in combination with amikacin or ciprofloxacin, for seriously-ill patients infected with ESBL-producing Enterobacter aerogenes who were admitted to an intensive care unit. Forty-four episodes of infection were investigated in 43 patients: 21 treated with cefepime; 23 with a carbapenem. The two treatment groups did not differ statistically in terms of age, APACHE II scores, and infection sites, but the average duration of antibiotic exposure was significantly shorter in the cefepime group (8.5 days vs. 11.4 days; p 0.04). Clinical improvement was seen in 62% of patients receiving cefepime vs. 70% of patients receiving a carbapenem (p 0.59). Bacteriological eradication was achieved in 14% of patients receiving cefepime vs. 22% of patients receiving a carbapenem (p 0.76). The 30-day mortality rates related to infection were 33% in the cefepime group and 26% in the carbapenem group (p 0.44). Thus, outcome parameters did not differ significantly between the two groups. Nevertheless, a statistically significant increase in failure to eradicate ESBL-producing E. aerogenes was observed as the MICs of cefepime rose (p 0.017). Pulsed-field gel electrophoresis revealed three distinct clones, but one predominant clone harbouring the bla(TEM-24) gene was associated with most (42/44) of the episodes of infection. It was concluded that cefepime may be an alternative agent for therapy of severe infections caused by TEM-24 ESBL-producing E. aerogenes, although further studies are required to confirm these observations.

[Use of multilocus sequence typing and pulsed-field gel electrophoresis for the study of serogroup B Neisseria meningitidis isolates from Casablanca (Morocco)]. / Etude de souches de Neisseria meningitidis sérogroupe B isolées à Casablanca par multilocus sequence typing et électrophorèse en champ pulsé.

A previous study showed that B:4:P1.15 was the most frequent phenotype of Neisseria meningitidis isolated in Casablanca (Morocco). To determine if there was an epidemic clone, MLST and PFGE were used to compare 13 B:4:P1.15 strains isolated from September 1999 to December 2000. MLST showed 4 Sequence Types (ST): ST-33 was the most frequent ST (9/13 strains) and 4 strains belonged to 3 newly described STs. Twelve stains belonged to ST-32 complex, and one strain presenting a new ST (ST-2502) did not belong to any known ST complex. The analysis by PFGE showed that the strains were subdivided into 7 clusters, and that there was no epidemic clone. MLST is useful for long-term epidemiological studies on N. meningitidis strains from varied geographical origins. PFGE seemed to be well adapted to the comparison of a small number of strains isolated during a short period within a defined community.

Antimicrobial resistance of Acinetobacter spp. in Europe.

Bacteria of the genus Acinetobacter are ubiquitous in nature. These organisms were invariably susceptible to many antibiotics in the 1970s. Since that time, acinetobacters have emerged as multiresistant opportunistic nosocomial pathogens. The taxonomy of the genus Acinetobacter underwent extensive revision in the mid-1980s, and at least 32 named and unnamed species have now been described. Of these, Acinetobacter baumannii and the closely related unnamed genomic species 3 and 13 sensu Tjernberg and Ursing (13TU) are the most relevant clinically. Multiresistant strains of these species causing bacteraemia, pneumonia, meningitis, urinary tract infections and surgical wound infections have been isolated from hospitalised patients worldwide. This review provides an overview of the antimicrobial susceptibilities of Acinetobacter spp. in Europe, as well as the main mechanisms of antimicrobial resistance, and summarises the remaining treatment options for multiresistant Acinetobacter infections.

Antimicrobial susceptibility of nontyphoidal Salmonella isolated from humans in Belgium.

Human nontyphoidal Salmonella infections are the primary cause of foodborne disease in developed countries, resulting in considerable morbidity and occasionally death, especially in immunocompromised patients. Strains of Salmonella that are resistant to antimicrobial agents have become a world-wide health problem. Fluoroquinolones are drugs of choice for treatment of human invasive salmonellosis, and have been useful for the treatment of infections caused by multi-resistant strains. However, strains resistant to ciprofloxacin have been noted. A random sample of 378 Salmonella strains of human origin was collected during 1998. Their susceptibility to 11 antimicrobial agents was determined by the agar dilution method according to NCCLS standards. In total, 38 serotypes were represented of which S. Enteritidis (20.4%), S. Typhimurium (20.4%), S. Hadar (9.0%), S. Brandenburg (7.9%), S. Infantis (7.7%), and S. Virchow (5.3%) were the most common. All strains were susceptible to ceftriaxone and ciprofloxacin. For nalidixic acid the rate of resistance was 19.0%. Of the 72 strains resistant to nalidixic acid, 31 were S. Hadar, and thus 91.2% (31/34) of the S. Hadar isolates showed resistance to nalidixic acid. Most of the S. Hadar strains were also resistant to ampicillin, tetracycline and sulphamethoxazole, and an elevated MIC50 (0.25 microgram/ml) and MIC90 (1 microgram/ml) was observed for ciprofloxacin. The high rate of resistance to nalidixic acid can be a first step towards the development of resistance to ciprofloxacin.

Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium.

Campylobacter spp. are a frequent cause of diarrhoea in man, originating mostly from poultry. It has been suggested that the veterinary use of antibiotics is largely responsible for resistance in human isolates, particularly to quinolones. During a 6 month period from June to December 1998, 677 Campylobacter isolates were obtained from healthy poultry and pigs. Samples were taken at Belgian slaughterhouses. Species identification was performed by biochemical tests, multiplex PCR and SDS-PAGE of whole-cell proteins. The in vitro susceptibility to six antimicrobial drugs was determined by the agar dilution method. Campylobacter jejuni was found more often in poultry than Campylobacter coli (79% C. jejuni versus 21% C. coli). In pigs the situation was reversed (6 versus 94%). Erythromycin resistance was significantly higher (P < 0.05) in C. coli, particularly in C. coli isolated from pigs (67.2%). Alarmingly high rates of resistance to ciprofloxacin were also noted, particularly for C. coli from broilers (62.1%). The latter indicates that resistance of Campylobacter in humans could derive from animals.

Organization of the ribosomal operon 165-235 gene spacer region in representatives of Neisseria gonorrhoeae.

Ribosomal rRNA gene fragments (rDNA) encompassing part of the 16S rDNA, the 16S-23S rDNA spacer region and part of the 23S rDNA of 229 Neisseria gonorrhoeae strains were enzymatically amplified using conserved primers. The fragments of approximately 1200 bp were subjected to restriction analysis with HinfI. This revealed 13 patterns (patterns I-XIII) of which patterns I (78 strains), II (32 strains), III (38 strains) and IV (56 strains) were the most abundant, comprising 89.1% of the strains. The obtained restriction patterns consisted of 3 to 8 bands, ranging in size from 32 to 854 bp. The sum of the obtained bands was about 1200 bp for patterns I, II, III, IV, V, IX, and XIII. However, for patterns VI, VII, VIII, X, XI and XII, the sum of the bands well exceeded the estimated size of approximately 1200 bp. We demonstrated that this results from sequence divergence in the 4 rRNA operons, present in the genome of N. gonorrhoeae, giving rise to patterns that are a combination of several other patterns.

Evaluation of the discriminatory power of typing methods for Neisseria gonorrhoeae.

A panel of 18 strains of Neisseria gonorrhoeae, known to be temporally and geographically diverse, was used to evaluate a number of typing systems, including conventional auxotyping and serotyping and the molecular methods of arbitrarily primed PCR (AP-PCR), amplified ribosomal-DNA restriction analysis (ARDRA), opa typing, and pulsed-field gel electrophoresis (PFGE). The discriminatory power of the different typing methods were determined with a collection of 87 clinical isolates from commercial sex workers in Indonesia, and Simpson's index of diversity was calculated. Of the two traditional techniques, auxotyping and serotyping, the latter gives the highest discriminatory index (DI) (DI, 0.846). The combination of auxotyping and serotyping yields a high DI (DI, 0. 928). D11344- and D8635-primed PCR showed low DIs of 0.608 and 0.622, respectively, but a combination of the two primers had a DI of 0. 849. The combination of serotyping with D11344-primed or D8635-primed PCR resulted in DIs of 0.936 and 0.937, respectively. ARDRA revealed a low DI of 0.743 alone but a DI of 0.955 in combination with serotyping. PFGE using the restriction enzyme BglII and opa typing produced the highest discrimination (DIs, 0.997 and 0. 996, respectively) for isolates of N. gonorrhoeae.

Molecular epidemiology of recent belgian isolates of Neisseria meningitidis serogroup B.

In Belgium an increase in the incidence of meningococcal disease has been noted since the early 1990s. Four hundred twenty clinical strains isolated during the period from 1990 to 1995, along with a set of 30 European reference strains, and 20 Dutch isolates were examined by random-primer and repetitive-motif-based PCR. A subset was investigated by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis. The data were compared with results obtained by serotyping (M. Van Looveren, F. Carion, P. Vandamme, and H. Goossens, Clin. Microbiol. Infect. 4:224-228, 1998). Both phenotypic and molecular epidemiological data suggest that the lineage III of Neisseria meningitidis, first encountered in The Netherlands in about 1980, has been introduced in Belgium. The epidemic clone, as defined by oligonucleotide D8635-primed PCR, encompasses mainly phenotypes B:4:P1.4 and B:nontypeable:P1.4, but strains with several other phenotypes were also encountered. Therefore, serotyping alone would underestimate the prevalence of the epidemic clone.

Antimalarial and cytotoxic potential of four quassinoids from Hannoa chlorantha and Hannoa klaineana, and their structure-activity relationships.

Hannoa chlorantha and Hannoa klaineana (Simaroubaceae) are used in traditional medicine of Central African countries against fevers and malaria. Four stem bark extracts from H. klaineana and four quassinoids from H. chlorantha were examined in vitro against Plasmodium falciparum NF 54. The extracts displayed good activities, while the quassinoids were highly active, with IC50 values well below 1 microgram ml-1, those of chaparrinone and 15-desacetylundulatone being much lower than 0.1 microgram ml-1 (0.037 and 0.047 microgram ml-1, respectively). Chaparrinone is five times more active than 14-hydroxychaparrinone against P. falciparum, indicating that the hydroxyl function at C-14 is unfavourable for antiplasmodial activity. As 14-hydroxychaparrinone has a seven-times higher cytotoxic activity against P-388 cells than chaparrinone, the latter compound has the better antiplasmodial therapeutic index. All four quassinoids were evaluated in vivo in a standard 4-day test as well. 15-Desacetylundulatone was proven to be the most active compound, almost totally suppressing the parasitaemias of OF1 mice for at least 7 days, while both chaparrinone and 14-hydroxychaparrinone were active for at least 4 days. Quassinoids have ED50 values much lower than 50 mg kg-1 body weight day-1 and none of them caused obvious side effects. The keto function at C-2 in 15-desacetylundulatone is apparently of crucial importance for its high activity. 6-alpha-Tigloyloxyglaucarubol was not active at all. Chaparrinone is considered the most interesting of the investigated quassinoids and its in-vivo antimalarial potential will be examined further.

New epidemiological features of meningococcal disease in Belgium.

SCIENTIFIC EDITOR In Belgium, after the epidemic of the early 1970s caused by Neisseria meningitidis B:2b:P1.2 (highest incidences in 1971 and 1972 with 5 cases per 100 000 population per year), the incidence of meningococcal disease fell to normal inter

Larvicidal activity of the naphthylisoquinoline alkaloid dioncophylline A against the malaria vector Anopheles stephensi.

The larvicidal activity of dionocophylline A, a naphthylisoquinoline alkaloid derived from the tropical vine Triphyophyllum peltatum (Dioncophyllaceae), was investigated against the malaria vector Anopheles stephensi. In direct and indirect inhibition assays it was demonstrated that the younger larval stages were very sensitive towards this natural product, with LC50 values below 1 mg/l. Pronounced effects were observed within 4 h of exposure. Aging larvae, however, were less sensitive, while pupae were totally insensitive to the action of dioncophylline A. The transformations from larvae to pupae and from pupae to adult mosquitoes remained unaffected. Therefore, dioncophylline A can be regarded as a promising specific larvicide.

Antiplasmodial activities and cytotoxic effects of aqueous extracts and sesquiterpene lactones from Neurolaena lobata.

Aqueous and lipophilic extracts of Neurolaena lobata (Asteraceae), obtained from Guatemala, were tested against Plasmodium falciparum in vitro. Moreover, sesquiterpene lactones, of the germacranolide and furanoheliangolide type, isolated from N. lobata, were shown to be active against P. falciparum in vitro. In addition to their antiplasmodial activity, their cytotoxic effects on human carcinoma cell lines were evaluated. Structure-activity relationships are discussed.