Nicole van den Braak

In Vitro Activities of Ertapenem (MK-0826) against Recent Clinical Bacteria Collected in Europe and Australia

ABSTRACT Ertapenem (MK-0826, L-749,345) is a 1-β-methyl carbapenem with a long serum half-life. Its in vitro activity was determined by broth microdilution against 3,478 bacteria from 12 centers in Europe and Australia, with imipenem, cefepime, ceftriaxone, and piperacillin-tazobactam used as comparators. Ertapenem was the most active agent tested against members of the familyEnterobacteriaceae, with MICs at which 90% of isolates are inhibited (MIC90s) of ≤1 μg/ml for all species. Ertapenem also was more active than imipenem against fastidious gram-negative bacteria and Moraxella spp.; on the other hand, ertapenem was slightly less active than imipenem against streptococci, methicillin-susceptible staphylococci, and anaerobes, but its MIC90s for these groups remained ≤0.5 μg/ml.Acinetobacter spp. and Pseudomonas aeruginosawere also much less susceptible to ertapenem than imipenem, and mostEnterococcus faecalis strains were resistant. Ertapenem resistance, based on a provisional NCCLS MIC breakpoint of ≥16 μg/ml, was seen in only 3 of 1,611 strains of the familyEnterobacteriaceae tested, all of them Enterobacter aerogenes. Resistance was also seen in 2 of 135 anaerobes, comprising 1 Bacteroides fragilis strain and 1Clostridium difficile strain. Ertapenem breakpoints for streptococci have not been established, but an unofficial susceptibility breakpoint of ≤2 μg/ml was adopted for clinical trials to generate corresponding clinical response data for isolates for which MICs were as high as 2 μg/ml. Of 234 Streptococcus pneumoniae strains tested, 2 required ertapenem MICs of 2 μg/ml and one required an MIC of 4 μg/ml, among 67 non-Streptococcus pyogenes, non-Streptococcus pneumoniae streptococci, single isolates required ertapenem MICs of 2 and 16 μg/ml. These streptococci also had diminished susceptibilities to other β-lactams, including imipenem as well as ertapenem. The Etest and disk diffusion gave susceptibility test results in good agreement with those of the broth microdilution method for ertapenem.

Genotypic diversity of Campylobacter lari isolated from mussels and oysters in The Netherlands

In order to gain insight into the epidemiology of Campylobacter lari infection in The Netherlands due to the consumption of raw mussels and oysters, batches of these shellfish were screened for the presence of Campylobacter spp. during a 6 month period in 1993-1994. Apparently, 41 out of 59 batches of mussels and 11 out of 41 batches of oysters were colonized with Campylobacter spp. A subset of the isolates was further characterized by additional phenotypic tests, numerical analysis of electrophoretic protein patterns, and genotyping by random amplification of polymorphic DNA (RAPD). Protein electrophoretic analysis of 39 Campylobacter spp. cultured from 24 batches of mussels and oysters, revealed that all isolates, except two, were C. lari. Two strains with an aberrant protein pattern were identified as C. coli and C. hyointestinalis, respectively. Nalidixic acid susceptible campylobacters (NASC) and urease-positive thermophylic campylobacters (UPTC) did not form separate clusters and should be considered biovars only. Several strains were both urease positive and nalidixic acid susceptible, which represents a new biovar within C. lari. The results of RAPD demonstrated the presence of 3 distinct genetic variants, implying that even within a single batch of shell fish, relatively extensive DNA polymorphisms can be found. It is therefore apparent that this complex group of C. lari is characterized by a high degree of genetic diversity, implying the presence of a heterogenous population of C. lari in crustacean organisms living in marine waters in a restricted area in The Netherlands.

Molecular Evidence for Dissemination of Unique Campylobacter jejuni Clones in Curaçao, Netherlands Antilles

ABSTRACT Campylobacter jejuni isolates (n = 234) associated with gastroenteritis and the Guillain-Barré syndrome (GBS) in the island of Curaçao, Netherlands Antilles, and collected from March 1999 to March 2000 were investigated by a range of molecular typing techniques. Data obtained by pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), automated ribotyping, and sequence analysis of the short variable region of the flagellin gene (flaA) were analyzed separately and in combination. Similar groupings were obtained by all methods, with the data obtained by MLST and AFLP analysis exhibiting the highest degree of congruency. MLST identified 29 sequence types, which were assigned to 10 major clonal complexes. PFGE, AFLP analysis, and ribotyping identified 10, 9, and 8 of these clonal groups, respectively; however, these three techniques permitted subdivision of the clonal groups into more different types. Members of seven clonal groups comprising 107 isolates were obtained from November 1999 to February 2000, and no distinguishing characteristics were identified for two GBS-associated strains. The sequence type 41 (ST-41), ST-508, and ST-657 clonal complexes and their corresponding AFLP types have been rare or absent in the Campylobacter data sets described to date. We conclude that several clonal complexes of C. jejuni are associated with human disease in Curaçao, and some of these have not been reported elsewhere. Furthermore, given the observation that C. jejuni-associated diseases appear to be more severe from November to February, it can be speculated that this may be due to the presence of virulent clones with a limited span of circulation.

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.

PCR-Restriction Fragment Length Polymorphism Analysis of Campylobacter jejuni Genes Involved in Lipooligosaccharide Biosynthesis Identifies Putative Molecular Markers for Guillain-Barré Syndrome

ABSTRACT Molecular mimicry of Campylobacter jejuni lipooligosaccharides (LOS) by gangliosides in peripheral nerve tissue probably triggers the Guillain-Barré syndrome due to the induction of cross-reactive antibodies. PCR-restriction fragment length polymorphism analysis of C. jejuni genes involved in the biosynthesis of LOS demonstrated that specific genes were associated with the expression of ganglioside mimics and the development of neuropathy.

Recent Emergence of an Epidemic Clindamycin-Resistant Clone of Clostridium difficile among Polish Patients with C. difficile-Associated Diarrhea

ABSTRACT Analysis of both the antibiotic resistance and the virulence characteristics of anaerobic human microbial pathogens is important in order to improve our understanding of a number of clinically significant infectious diseases, including Clostridium difficile-associated diarrhea (CDAD). We determined the presence of the clindamycin resistance-associated gene ermB and the ribotype of 33 C. difficile strains isolated from Polish patients suffering from CDAD. While all strains produced cytotoxin B (TcdB), enterotoxin A (TcdA) was produced by a subset of 15 strains only. The results showed that a single ermB-positive, TcdA−B+C. difficile strain with ribotype A has disseminated widely in the two Warsaw hospitals under investigation. Although different strains with the same phenotype were detected, the genotype A strain appeared to be the only one with a clear epidemic character. Apparently, enhanced local spread of CDAD-causing C. difficile may be restricted to a limited number of bacterial genotypes only.

Risk Factors Associated with Campylobacter jejuni Infections in Curaçao, Netherlands Antilles

ABSTRACT A steady increase in the incidence of Guillain-Barré syndrome (GBS) with a seasonal preponderance, almost exclusively related to Campylobacter jejuni, and a rise in the incidence of laboratory-confirmed Campylobacter enteritis have been reported from Curaçao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic C. jejuni. Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curaçao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related C. jejuni isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of Campylobacter infections.