Polyclonal spread and outbreaks with ESBL positive gentamicin resistant Klebsiella spp. in the region Kennemerland, The Netherlands.

OBJECTIVE: The objective of this study was to analyze the transmission dynamics of ESBL positive Klebsiella spp. with an additional resistance towards gentamicin (ESBL-G) in a Dutch region of 650,000 inhabitants in 2012. METHODS: All patient related ESBL-G isolates isolated in 2012 were genotyped using both Amplification Fragment Length Polymorphism (AFLP) and High-throughput MultiLocus Sequence Typing (HiMLST). HiMLST was used to analyze the presence of (unidentified) clusters of ESBL-G positive patients. Furthermore, all consecutive ESBL-G isolates within patients were studied in order to evaluate the intra-patient variation of antibiotic phenotypes. RESULTS: There were 38 ESBL-G isolates, which were classified into 18 different sequence types (STs) and into 21 different AFLP types. Within the STs, four clusters were detected from which two were unknown resulting in a transmission index of 0.27. An analysis of consecutive ESBL-G isolates (with similar STs) within patients showed that for 68.8% of the patients at least one isolate had a different consecutive antibiotic phenotype. CONCLUSION: The transmission of ESBL-G in the region Kennemerland in 2012 was polyclonal with several outbreaks (with a high level of epidemiological linkage). Furthermore, clustering by antibiotic phenotype characterization seems to be an inadequate approach in this setting. The routine practice of molecular typing of collected ESBL-G isolates may help to detect transmission in an early stage, which opens the possibility of a rapid response.

Aerococcus urinae: severe and fatal bloodstream infections and endocarditis.

Aerococcus urinae is a pathogen that rarely causes severe or fatal infections. We describe four cases of severe A. urinae bloodstream infections. All patients had underlying urologic conditions. Urine cultures, however, were negative.

Clostridium difficile ribotype 027, toxinotype III, the Netherlands.

Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A-negative C. difficile PCR ribotype 017 toxinotype VIII strain. The outbreaks are difficult to control.

Intercenter reproducibility of binary typing for Staphylococcus aureus.

The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains.