Petra Edquist

The DiversiLab system versus pulsed-field gel electrophoresis: Characterisation of extended spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae

Fast and reliable epidemiological typing methods for identifying outbreaks and epidemic strains of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae are urgently needed. The DiversiLab system (DL) has been proposed for these purposes. We compared DL to pulsed-field gel electrophoresis (PFGE) on a national collection of ESBL-producing Escherichia coli (n=258; of which 226 isolates were typeable with PFGE) and Klebsiella pneumoniae (n=48) isolated in 2007. For E. coli the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was only 19.8% and the probability of two isolates of the same PFGE type having the same DL type was 90.4%. For K. pneumoniae the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was 100% and the probability of two isolates of the same PFGE type having the same DL type was 79%, indicating that for this K. pneumoniae strain collection DL was slightly more discriminatory. Only four of 48 isolates had discordant results with the two methods. In E. coli 42% of the isolates were sequence type 131 and these isolates were related at >95% similarity with DL and at ≥60% similarity with PFGE. In summary, for E. coli DL performed well in identifying isolates related by PFGE, but overestimated the genetic relatedness in the studied collection. This indicates that DL could be a primary screening method for excluding unrelated isolates. Isolates shown to be related must be confirmed with a more discriminatory method. For K. pneumoniae, DL discriminated well but overestimated the diversity of the isolates compared to PFGE, assuming a risk of missing possible genetic relatedness.

Prevalence of faecal ESBL carriage in the community and in a hospital setting in a county of Southern Sweden.

The aim of this study was to investigate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in patients at various hospital wards and in a group of relatively healthy volunteers, in order to obtain greater knowledge on how common these bacterial strains are in hospital settings and in the general community. Participants (n = 427) were enrolled at a University Hospital and at Primary Health Care Units (PHCUs) in Sweden in 2008 and 2010. The participants provided rectal swabs, which were tested for the occurrence of ESBL-producing bacteria. Positive samples were analysed with polymerase chain reaction (PCR) methods for bacterial strain typing and ESBL phylogroups. In 2008, the prevalence was 2.1% (2/96) in PHCU subjects and 1.8% (2/113) in hospital patients. In 2010, the prevalence was 3.0% (3/100) in PHCU subjects and 6.8% (8/118) in hospital patients. The dominating phylogroups were CTX-M-1 and CTX-M-9. All ESBL-positive isolates were Escherichia coli. We found a higher prevalence of ESBL faecal carriage than expected, both in the hospital setting and in the PHCU group.

Epidemiology of extended-spectrum β-lactamase-producing Escherichia coli in Sweden 2007–2011

Extended-spectrum β-lactamase (ESBL) -producing Enterobacteriaceae have been notifiable according to the Swedish Communicable Disease Act since 2007. A major increase in the number of cases has been observed, with 2099 cases in 2007 and 7225 cases in 2012. The majority of the isolates are Escherichia coli. Additionally, Swedish data on the prevalence of ESBL-producing invasive isolates of E. coli are available through EARS-Net, and through biannual point prevalence studies, where molecular characterization of isolates from the entire country is carried out. This paper describes major trends in the Swedish epidemiology of ESBL-producing E. coli in the period 2007-2012. Isolates from the point prevalence studies were subjected to antimicrobial susceptibility testing, ESBL genotyping, pulsed-field gel electrophoresis, multi-locus sequence typing and phylogenetic grouping with PCR. The distribution of sequence types, resistance genes and susceptibility levels were all stable over the three study periods. The dominating resistance gene conferring ESBL was blaCTX -M-15 , found in 54-58% of the isolates. ST131 represented 34-38% of the isolates. Other major sequence types were ST38, ST69, ST405, ST617 and ST648, each representing 2-6% of the isolates. Phylogenetic group B2 was the most common, and was observed in 41-47% of the isolates. However, among ST131 isolates the B2 phylogenetic group represented 90-98% of the isolates. The most important epidemiological difference seen over time was that the median age of infected women decreased from 62 to 52 years (p <0.0001) and infected men from 67 to 64 years. A potential explanation might be the shift towards a higher proportion of community-acquired infections in individuals lacking comorbidities.

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

INTRODUCTION Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBL(M)) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL(M) are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla(AmpC) detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes. MATERIAL AND METHODS Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla(AmpC) real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing. RESULTS AND DISCUSSION The real-time PCR assay was able to detect and sub-classify all acquired bla(AmpC) genes described to date. The assay can be performed in less than 3h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla(AmpC) real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.

Carbapenemase-producing Enterobacteriaceae in Sweden 2007-2013: Experiences from seven years of systematic surveillance and mandatory reporting

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide, and are a major threat to healthcare systems. Recent European data support that many countries have interregional spread of CPE or an endemic situation. In Sweden mandatory laboratory reporting of CPE of both colonisation and infection has been practiced since 2007 and since 2012 also by treating physicians. Between 2007 and 2013, 94 cases of CPE were detected in Sweden, out of which 24 were considered to cause clinical infections (bloodstream infection (n=4), urinary tract infection (n=12), wound infection (n=4), respiratory tract infection (n=2) and catheter related (n=2). The majority were detected in the hospital setting through faecal screening or as probable colonisers in clinical cultures. Travel abroad was observed in the majority of the patients (81%), and among them 84% had been hospitalised. During the study period only two chains of transmissions in Swedish hospitals were reported, involving four patients. Klebsiella pneumoniae was the primarily isolated species (n=57) followed by Escherichia coli (n=29). blaNDM was the predominant carbapenemase gene (n=36), followed by blaOXA-48-group, blaKPC and blaVIM. In 26/94 cases (28%) isolates were categorised as possible XDR (extensively drug-resistant). CPE are increasing in Sweden, but are still at a comparably low level.

A multiresistant clone of Pseudomonas aeruginosa sequence type 773 spreading in a burn unit in Orumieh, Iran

Herein, we describe the phenotypic and genotypic characterization of a multiresistant clone of Pseudomonas aeruginosa disseminating in a burn unit in Orumieh, Iran. A total of 58 isolates of P. aeruginosa were collected during August 2007 and June 2008. Minimum inhibitory concentrations (MICs) of P. aeruginosa isolates were determined against 11 antimicrobial agents by E test. Serotyping, pulsed‐field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were used for studying the clonal relationship among the isolates. Antibiotic susceptibility testing revealed that most of the isolates were multidrug resistant and colistin was the antibiotic with the highest activity. Pseudomonas aeruginosa isolates fell into nine different serotypes, and O10 and O11 were the most common. PFGE analyses showed 12 different genotypes and 68.1% of isolates showed more than 80% similarity, indicating possible clonal relatedness. These isolates were found to belong to the same sequence type, ST773. This sequence type has earlier been reported from China, and a double locus variant of this ST has been found earlier in France in a PER‐1 extended‐spectrum β‐lactamase‐producing P. aeruginosa.

Emergence of vanA Enterococcus faecium in Denmark, 2005-15

Objectives To describe the changing epidemiology of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis in clinical samples in Denmark 2005-15 according to species and van type, and, furthermore, to investigate the genetic relatedness of the clinical E. faecium isolates from 2015. Methods During 2005-14, all clinical VRE isolates were tested for the presence of vanA/B/C genes by PCR. In 2015, all clinical VRE isolates were whole-genome sequenced. From the WGS data, the presence of van genes and MLST STs were extracted in silico . Core-genome MLST (cgMLST) analysis was performed for the vancomycin-resistant E. faecium isolates. Results During 2005-15, 1043 vanA E. faecium , 25 vanB E. faecium , 4 vanA E. faecalis and 28 vanB E. faecalis were detected. The number of VRE was <50 isolates/year until 2012 to > 200 isolates/year in 2013-15. In 2015, 368 vanA E. faecium and 1 vanB E. faecium were detected along with 1 vanA E. faecalis and 1 vanB E. faecalis . cgMLST subdivided the 368 vanA E. faecium isolates into 33 cluster types (CTs), whereas the vanB E. faecium isolate belonged to a different CT. ST203-CT859 was most prevalent (51%), followed by ST80-CT14 (22%), ST117-CT24 (6%), ST80-CT866 (4%) and ST80-CT860 (2%). Comparison with the cgMLST.org database, previous studies and personal communications with neighbouring countries revealed that the novel cluster ST203-CT859 emerged in December 2014 and spread to the south of Sweden and the Faroe Islands during 2015. Conclusions VRE increased in Denmark during 2005-15 due to the emergence of several vanA E. faecium clones.

Large variation in ESBL-producing Escherichia coli carriers in six European countries including Russia

We investigated the faecal carriage prevalence of extended-spectrum β-lactamase production in Escherichia coli (EP-EC) and/or Klebsiella pneumoniae (EP-KP) and risk factors associated with carriage among adult study subjects in Finland, Germany, Latvia, Poland, Russia and Sweden (partner countries). The aim was to get indicative data on the prevalence of ESBL-carriage in specific populations in the region. Faecal samples were collected from four study populations and screened on ChromID-ESBL and ChromID-OXA-48 plates. Positive isolates were further characterised phenotypically. Our results show a large variation in carrier prevalence ranging from 1.6% in Latvia to 23.2% in Russia for EP-EC. For the other partner countries, the prevalence of EP-EC were in increasing numbers, 2.3% for Germany, 4.7% for Finland, 6.6% for Sweden, 8.0% for Poland and 8.1% for all partner countries in total. Carriers of EP-KP were identified only in Finland, Russia and Sweden, and the prevalence was < 2% in each of these countries. No carriers of carbapenemase-producing isolates were identified. This is the first study reporting prevalence of carriers (excluding traveller studies) for Finland, Latvia, Poland and Russia. It contributes with important information regarding the prevalence of EP-EC and EP-KP carriage in regions where studies on carriers are limited.