Ole Heltberg

Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay

Rapid molecular typing methods can be a valuable aid in the investigation of suspected outbreaks. We used a semi-automated repetitive sequence-based polymerase chain reaction (Rep-PCR) typing assay and pulsed field gel electrophoresis (PFGE) to investigate the relationship between local Klebsiella pneumoniae (K. pneumoniae) producing extended spectrum β-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of blaTEM, blaSHV, and blaCTX-M, and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and blaCTX-M15, blaSHV-28, and blaTEM-1 positive by PCR. Ten out of 52 were ST16 and tested positive for blaCTX-M15, blaSHV-1, and blaTEM-1. Isolates from previously recognized hospital outbreaks were also ST15 and PCR positive for blaCTX-M15, blaSHV-28, and blaTEM-1, and typed within the main cluster by both Rep-PCR and PFGE. In conclusion, K. pneumoniae ST15 containing blaCTX-M15 and blaSHV-28 constitutes an epidemic clone in the Copenhagen area and this clone can be rapidly recognized by semi-automated Rep-PCR.

Bedside diagnosis of imported malaria using the Binax Now malaria antigen detection test

Malaria may be misdiagnosed in non-endemic countries when the necessary experience for rapid expert microscopy is lacking. Rapid diagnostic tests may improve the diagnosis and may play a role as a bedside diagnostic tool. In a multicentre study we recruited patients suspected of malaria over a period of 14 months. The Binax Now Malaria rapid test was used at the bedside and in the clinical microbiology laboratory. The training of clinical staff was monitored and their experience with the use of the test was recorded. 542 patients were included, 80 of whom had malaria diagnosed by microscopy. The rapid test used at the bedside had a sensitivity of 88% for the detection of P. falciparum compared to 95% when the test was performed in the microbiology laboratory. The risk of technical problems and invalid tests was highest when the test was used at the bedside. The rapid diagnostic test may be useful for the diagnosis of P. falciparum malaria when used by routine laboratory staff, but could lead to misdiagnoses when used at the bedside. Microscopy is still essential in order to identify the few missed diagnoses, to determine the degree of parasitaemia, and to ensure species diagnosis, including mixed infections.

Characterization of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark.

The aim of the study was to investigate the molecular epidemiology of 87 third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec) from bloodstream infections in Denmark from 2009. Sixty-eight of the 87 isolates were extended-spectrum beta-lactamase (ESBL) producers, whereas 17 isolates featured AmpC mutations only (without a coexpressed ESBL enzyme) and 2 isolates were producing CMY-22. The majority (82%) of the ESBL-producing isolates in our study were CTX-M-15 producers and primarily belonged to phylogroup B2 (54.4%) or D (23.5%). Further, one of the two CMY-22-producing isolates belonged to B2, whereas only few of the other AmpCs isolates belonged to B2 and D. Pulsed-field gel electrophoresis revealed that both clonal and nonclonal spread of 3GC-R Ec occurred. ST131 was detected in 50% of ESBL-producing isolates. The remaining ESBL-producing isolates belonged to 17 other sequence types (STs), including several other internationally spreading STs (e.g., ST10, ST69, and ST405). The majority (93%) of the ESBL-producing isolates and one of the CMY-22-producing isolates were multiresistant. In conclusion, 3GC-R in bacteriaemic E. coli in Denmark was mostly due to ESBL production, overexpression of AmpC, and to a lesser extent to plasmid-mediated AmpC. The worldwide disseminated CTX-M-15-ST131 was strongly represented in this collection of Danish, bacteriaemic E. coli isolates.

Pasteurella aerogenes Isolated from Ulcers or Wounds in Humans with Occupational Exposure to Pigs: A Report of 7 Danish Cases

Pasteurella aerogenes is rarely isolated from human specimens. The species is found in the digestive tract of pigs. From 1976 to 1994 7 strains were cultured in Denmark from wounds or ulcers. Five patients were bitten by pigs and 2 patients with ulcers were employed in pig farming. A mixture of bacterial species was often found. All 7 strains of P. aerogenes were susceptible to ampicillin, cephalosporins and ciprofloxacin. Ability to hydrolyse urea, to produce oxidase and catalase, to decarboxylate ornithine and to produce gas from glucose and inability to produce indole was characteristic for P. aerogenes. Most bite wounds were located on the lower lateral part of the thigh. Foul smelling pus and abscess formation was the rule. Incision, drainage and antibiotic treatment were usually necessary.

High rates of reduced susceptibility in the Bacteroides fragilis group isolated from blood cultures: The first national survey in Denmark

The authors acknowledge the critical reading of the manuscript by Dr Edward Chan as well as useful discussion with members from SC’s laboratory. Funding: This work was supported by the Chinese National Key Basic Research and Development (973) Programme [2013CB127200] and the Research Fund for the Control of Infectious Diseases from the Food and Health Bureau, the Government of Hong Kong SAR [ZJE4 to SC]. Competing interests: None declared. Ethical approval: Not required.

Characterization of Carbapenem Nonsusceptible Pseudomonas aeruginosa in Denmark: A Nationwide, Prospective Study

From January 1st 2011 through June 30th 2011, 116 nonreplicate, noncystic fibrosis-related Pseudomonas aeruginosa isolates with reduced carbapenem susceptibility were collected from 12 out of 13 Danish departments of clinical microbiology. The presence of acquired β-lactamases was assessed with combination tablet-diffusion methodology and polymerase chain reaction. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay, and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing acquired β-lactamases were further investigated by serotyping and multi locus sequence typing. Eight isolates produced the metallo-β-lactamase (MBL) VIM-2, and one isolate produced OXA-10 and VEB-1-like extended-spectrum beta-lactamase (ESBL). Phenotypic indications of derepressed AmpC and efflux pump were seen in 56 and 43 isolates, respectively. Overall, the results indicate that mutational factors related to permeability--often combined with derepressed, chromosomal AmpC--is the main factor behind carbapenem nonsusceptibility in Danish P. aeruginosa isolates. The ESBL producer and all the VIM producers belonged to international clones. PFGE revealed that most of the isolates were unrelated, but clonal spread was seen; the 116 isolates distributed in 97 PFGE types, with the largest cluster consisting of 4 isolates (including three isolates from the same hospital with 100% similarity). Thirty-two isolates were pair-wise related, while the remaining isolates were clonally unrelated, as were all nine ESBL/MBL producers.