Pia Littauer

Multiple hospital outbreaks of vanA Enterococcus faecium in Denmark, 2012–13, investigated by WGS, MLST and PFGE

OBJECTIVES In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm. METHODS A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE. RESULTS One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power. CONCLUSIONS This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.

Clonal Spread of Macrolide- and Tetracycline-Resistant [erm(A) tet(O)] emm77 Streptococcus pyogenes Isolates in Italy and Norway

Over the last 15 years, a general increase in macrolide resistance in Streptococcus pyogenes has been observed in many parts of the world (1). In Europe, its prevalence is higher in Mediterranean countries and lower in Scandinavia. In particular, whereas an overall incidence around 43%, the highest rate recorded after a Japanese epidemic in the 1970s (10), was reported in a nationwide survey in Italy (17), the resistance rate remains low (<5%) in Norway (8). An association between erythromycin resistance and cell invasiveness in S. pyogenes has been documented in Italy (3).

Epidemiological factors associated with ESBL- and non ESBL-producing E. coli causing urinary tract infection in general practice

Abstract The purpose of the study was to evaluate how use of antibiotics precedes the presence of ESBL-producing E.coli in general practice. The authors performed a triple-case-control study where three case groups were individually compared to a single control group of uninfected individuals. Urine samples were prospectively collected and retrospective statistical analyses were done. This study included 98 cases with urinary tract infection (UTI) caused by ESBL-producing E. coli, 174 with antibiotic-resistant (non-ESBL) E. coli, 177 with susceptible E. coli and 200 with culture negative urine samples. Case groups had significantly higher use of antibiotics than the control group within 30 days before infection (p < 0.0001). The ESBL group had significantly more hospital admissions than the other case groups (p < 0.05). Hospital admission was an independent risk factor for community onset UTI by ESBL-producing E. coli. Exposure to antibiotics was a risk factor for UTI with E. coli, while prior antibiotic usage was not an indisputable predictor for infection with ESBL-producing E.coli in general practice.

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.

Dissemination and characteristics of a novel plasmid-encoded carbapenem-hydrolyzing class D β-lactamase, OXA-436, found in isolates from four patients at six different hospitals in Denmark

ABSTRACT The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D β-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Fecal carriage of extended-spectrum and AmpC β-lactamase-producing Enterobacteriaceae in surgical patients before and after antibiotic prophylaxis

The impact of antibiotic prophylaxis on fecal carriage of ESBL-/AmpC-/carbapenemase-producing Enterobacteriaceae (CPE) was investigated. Patients admitted for elective surgery or diagnostic procedure in a Department of Surgical Gastroenterology (SG) (n= 450) and Orthopedic Surgery (OS) (n= 300) provided a fecal swab at admission and responded to a questionnaire on possible exposures. SG patients received gentamicin/penicillin G (±metronidazole); OS patients received cefuroxime. Two days after surgery a second swab was taken. From SG patients, 6% of first swabs and 9% of second swabs were positive for ESBL-/AmpC-producers. A similar carriage rate was observed in OS patients (6% and 8%, respectively). No CPE were detected. Escherichia coli was the predominant species and blaCTX-M-15 (29% and 22%) and blaCTX-M-14 (11% and 17%) were the most prevalent ESBL genotypes among SG and OS patients. Two different prophylactic antibiotic regimens had no impact on carriage rates. Previous hospitalization and antimicrobial treatment were associated with carriage for SG patients.

Typing of vancomycin-resistant enterococci with MALDI-TOF mass spectrometry in a nosocomial outbreak setting

OBJECTIVES To investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm). METHODS Fifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme. RESULTS Nine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain. CONCLUSIONS Using visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting.

Septicemia with Streptococcus pseudopneumoniae: report of three cases with an apparent hepatic or bile duct association

Abstract Streptococcus pseudopneumoniae was described in 2004 as a new human pathogen, acknowledged in a range of clinical infections typically associated to the respiratory tract. This report demonstrates that S. pseudopneumoniae has the potential to cause invasive infection. In blood cultures from three patients, growth of an atypical Streptococcus pneumoniae (non-capsular, non-serotypeable, optochin susceptible under ambient atmosphere and bile-intermediately soluble) was recovered. All three patients had a history of a haematological disease (myelodysplastic syndrome and multiple myeloma) and an apparent origin of infection related to the liver or bile duct. All isolates were genome sequenced and subsequently identified as S. pseudopneumoniae by multi-locus sequence analysis (MLSA). Multi-locus sequence typing (MLST) based on the S. pneumoniae scheme revealed unknown sequence types and the antibiogram and resistome revealed no antibiotic resistance.