Evaluation of high-resolution melting curve analysis of ligation-mediated real-time PCR, a rapid method for epidemiological typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) pathogens.

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks.

Outbreaks of methicillin-resistant Staphylococcus aureus among staff and dogs in Swedish small animal hospitals.

Methicillin-resistant Staphylococcus aureus (MRSA) was found in a dog for the first time in Sweden in 2006. Between October 2006 and May 2007, MRSA was diagnosed in 7 more dogs that had been treated in 3 different small animal hospitals, located 150-200 km apart, in different counties of Sweden. Screening of the animal hospital staff and environment in these small animal hospitals showed 20 of 152 staff to be positive for MRSA, with rates between 2% and 18% in the different hospitals, while all 128 environmental samples were negative. All MRSA isolates from dogs and staff belonged to spa type t032, were Panton-Valentine leukocidin (PVL)-negative, and had indistinguishable pulsed-field gel electrophoresis patterns, except for 2 isolates with closely related patterns. To our knowledge, this is the first report of multiple outbreaks of MRSA in dogs caused by the same strain within a short time frame, and appearing in a country with low prevalence of MRSA in both humans and dogs. This highlights the importance of infection control programs in animal hospitals and in animal health care. Awareness of MRSA as an occupational risk for veterinary personnel is essential.

Long-term carriage of extended-spectrum beta-lactamase-producing Escherichia coli.

In 2009 we described an outbreak caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in southern Sweden that occurred during 2005-2006. An important finding from the investigation was the long carriage times of the ESBL-producing E. coli in several of the patients, which in some cases exceeded 30 months. Here we report findings from the continued follow-up of bacterial carriage. In September 2010, 5 of the 42 patients still carried the bacteria after a median of 58 months (range 41-59 months), 18 had had repeatedly negative cultures after shedding bacteria for a median of 7.5 months (range 0-39 months), 16 had died while still shedding the bacteria for a median of 9 months (range 0-38 months), and 3 had been lost to follow-up.

Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe.

To estimate the proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans that were sequence type (ST) 398, we surveyed 24 laboratories in 17 countries in Europe in 2007. Livestock-associated MRSA ST398 accounted for only a small proportion of MRSA isolates from humans; most were from the Netherlands, Belgium, Denmark, and Austria.

High-resolution melting-curve analysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.

Imported methicillin-resistant Staphylococcus aureus, Sweden.

Countries such as Sweden that have a low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) offer the opportunity to discern and study transmission of imported cases of MRSA. We analyzed 444 imported cases of MRSA acquisition reported in Sweden during 2000-2003. Risk for MRSA in returning travelers ranged from 0.1 (95% confidence interval [CI] 0.01-0.4) per 1 million travelers to Nordic countries to 59.4 (95% CI 44.5-79.3) per 1 million travelers to North Africa and the Middle East. Most imported cases (246, 55%) were healthcare acquired, but regions with the highest risk for MRSA in travelers showed a correlation with community acquisition (r = 0.81, p = 0.001). Characteristic differences in MRSA strains acquired were dependent on the region from which they originated and whether they were community or healthcare acquired. Knowledge of differences in transmission of MRSA may improve control measures against imported cases.

Multiresistant CTX-M-15 ESBL-producing Escherichia coli in southern Sweden: Description of an outbreak.

An outbreak caused by a multiresistant Escherichia coli producing CTX-M-15 ESBL occurred during autumn 2005 and spring 2006 in Kristianstad, a town in southern Sweden. The outbreak comprised 27 cases and was related to an infectious diseases ward and a neighbouring long-term care facility. Our primary objective was to investigate the epidemiology in order to control the outbreak. In addition, we studied the time of carriage of multiresistant ESBL-producing Escherichia coli by follow-up samples and measured the frequency of carriage of ESBL-producing bacteria in the patient population admitted to the infectious diseases ward during autumn 2006. The outbreak described is one of the first caused by ESBL-producing Escherichia coli in Sweden. The source of the outbreak was not found. Infection control measures were reinforced in the outbreak situation, and epidemiological and microbiological methods, including PFGE typing, were used for analysis. The carriage time of multiresistant Escherichia coli was longer in several of the affected patients than has previously been reported. The longest carriage time to date is 33 months. This demonstrates the risk for new outbreaks unless strict infection control measures are implemented. Among the patients admitted to the ward during autumn 2006, 2.5% carried ESBL-producing enterobacteria.

Epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA): spa typing versus pulsed-field gel electrophoresis.

Molecular methods based on sequencing, such as spa typing, have facilitated epidemiological typing of bacterial isolates compared to the gold standard pulsed-field gel electrophoresis (PFGE), a technically more demanding method. We studied methicillin-resistant Staphylococcus aureus (MRSA) in 4 Swedish counties from 2003 through 2005, and compared spa typing and PFGE results to epidemiological data. Of 280 MRSA isolates, 91 were from sporadic cases and 189 were associated with 35 outbreaks. A total of 50 spa types and 74 PFGE patterns were detected. 60 (21%) of the MRSA isolates carried the Panton-Valentine leukocidin (PVL) genes. 12 of the PVL-positive MRSA were healthcare associated. 25 of the spa types and 31 of the PFGE patterns were associated with outbreaks. In 1 of the outbreaks we found isolates with different but closely related spa types, and in 6 of the outbreaks we observed isolates with different but related PFGE patterns. In this low-endemic setting, with outbreaks limited in time and place, we found spa typing to be a useful tool for epidemiological typing of MRSA, due to its rapidity, accessibility, ease of use, and standardized nomenclature.

Cefuroxime non-susceptibility in multidrug-resistant Klebsiella pneumoniae overexpressing ramA and acrA and expressing ompK35 at reduced levels.

OBJECTIVES: The aims were to study if efflux and down-regulation of porins contribute to cefuroxime resistance in Klebsiella pneumoniae and to co-resistance to unrelated antibiotics. METHODS: Ten cefuroxime-non-susceptible but cefotaxime-susceptible blood culture isolates of K. pneumoniae and one multiply antibiotic-resistant (MAR) laboratory strain (selected by chloramphenicol) were examined. Transcription of the genes acrA, ompK35, ramA, marA and soxS was determined with quantitative RT-PCR. RESULTS: All clinical isolates and the MAR laboratory strain had similar antibiograms with non-susceptibility to cefuroxime, tigecycline, chloramphenicol and nalidixic acid. Phenylalanine arginine beta-naphthylamide (PAbetaN) increased susceptibility to tigecycline, chloramphenicol and nalidixic acid, but not to cefuroxime. Increased acrA transcription and decreased ompK35 transcription was seen in all strains. Increased ramA transcription was seen in all strains except one clinical isolate. CONCLUSIONS: This multidrug-resistant phenotype of K. pneumoniae is associated with increased acrA and ramA transcription and decreased ompK35 transcription. Since the cefuroxime resistance was not reversed by PAbetaN, it was probably attributable to decreased levels of OmpK35, rather than to efflux.

Penicillin-resistant pneumococci in Sweden 1997-2003: increased multiresistance despite stable prevalence and decreased antibiotic use.

Antimicrobial resistance patterns and capsular groups of penicillin-resistant Streptococcus pneumoniae (PRP; MIC penicillin G > or = 0.5 mg/ml) in Sweden between 1997 and 2003 were described, and trends in resistance and antibiotic sales during the same period were compared. The most common serogroups were in descending order 9, 19, 14, 23, and 6. Despite a low and stable annual PRP rate (proportion of PRP out of all pneumococci) of around 2% during the study period, the proportion of PRP resistant to other antibiotics increased. Of all tested PRP isolates, 82% were also resistant to trimethoprim/sulfamethoxazole, 32% had additional resistance to tetracycline, and 26% to erythromycin. Antibiotic sales figures for all studied antibiotic subgroups decreased during the same period. Little correlation was found between antibiotic sales and PRP resistance rates, indicating that there are still other poorly defined factors contributing to the reported resistance levels in the population. However, although PRP strains in Sweden are becoming more commonly resistant to antibiotics other than beta-lactams, the low and further reduced antibiotic sales still might have delayed the development and rapid spread of PRP in the population.

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Sweden 2000-2003, increasing incidence and regional differences.

BACKGROUND: The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) has gradually become more frequent in most countries of the world. Sweden has remained one of few exceptions to the high occurrence of MRSA in many other countries. During the late 1990s, Sweden experienced a large health-care associated outbreak which with resolute efforts was overcome. Subsequently, MRSA was made a notifiable diagnosis in Sweden in 2000. METHODS: From the start of being a notifiable disease in January 2000, the Swedish Institute for Infectious Disease Control (SMI) initiated an active surveillance of MRSA. RESULTS: The number of reported MRSA-cases in Sweden increased from 325 cases in 2000 to 544 in 2003, corresponding to an overall increase in incidence from 3.7 to 6.1 per 100,000 inhabitants. Twenty five per cent of the cases were infected abroad. The domestic cases were predominantly found through cultures taken on clinical indication and the cases infected abroad through screening. There were considerable regional differences in MRSA-incidence and age-distribution of cases. CONCLUSION: The MRSA incidence in Sweden increased over the years 2000-2003. Sweden now poises on the rim of the same development that was seen in the United Kingdom some ten years ago. A quarter of the cases were infected abroad, reflecting that international transmission is now increasingly important in a low-endemic setting. To remain in this favourable situation, stepped up measures will be needed, to identify imported cases, to control domestic outbreaks and to prevent transmission within the health-care sector.

Calibration of fusidic acid disk diffusion susceptibility testing of Staphylococcus areus.

Single strain regression analysis, SRA, was used to calibrate disk diffusion fusidic acid susceptibility testing of Staphylococcus aureus in two laboratories using different standard methods but the same interpretative MIC limits. SRA equation constants were calculated using five different fusidic acid disk contents (1.5, 5, 15, 50, 150 microg). These disks were tested on five separate occasions against quality control strain S. aureus ATCC 29213. The National Committee for Clinical Laboratory Standards (NCCLS) method was employed in Tartu, Estonia (TE) and the Swedish Reference Group for Antibiotics (SRGA) method in Sweden at the Karolinska Hospital (KS). SRA constants obtained were used for calculating zone breakpoints corresponding to MIC breakpoints recommended by the SRGA (S < or = 0.5 mg/L, R > or = 1 mg/L). Zone diameter histograms from KS, performed with a 50 microg disk, and from TE, using a 10 microg disk, showed a clustering of wild type strains around 41 mm and 30 mm, respectively, reflecting differences in methodology. Zone breakpoints calculated from the equations were validated by comparison with the histograms. Breakpoints were also calculated for a suggested lower disk content in Sweden, 10 microg, and validated in tests of clinical isolates and by histogram analysis.

Comparison of daptomycin MIC results by DIN, NCCLS, SFM, and SRGA methods for 297 Gram-positive organisms.

Daptomycin is a novel lipopeptide antibiotic with potent in vitro antibacterial activity against Gram-positive pathogens. For daptomycin minimal inhibitory concentration (MIC) testing, National Committee for Clinical Laboratory Standards (NCCLS) recommends the use of broth containing physiological levels of calcium (50 microg/ml). The daptomycin susceptibility of 297 organisms was determined by NCCLS (Mueller-Hinton (MH) broth), Deutsches Institut für Normung (DIN; isotonic broth), Société Française de Microbiologie (SFM; three batches MH agar), and Swedish Reference Group for Antibiotics (SRGA; PDM agar). All media were supplemented to 50 microg/ml Ca(2+). There was good correlation between DIN and SFM methods (for staphylococci) with NCCLS results. Enterococci MICs using SFM methods were one to three dilutions lower and pneumococci results were one dilution higher than NCCLS. SRGA results were higher than NCCLS by one to four dilutions. Use of isotonic agar is an accepted alternative to isosensitest agar for the DIN method.

Comparison of enterococcal populations in animals, humans, and the environment--a European study.

The objectives of the present study were to generate knowledge of enterococcal populations in the food chain, by studying the population structure (in measures of abundance and diversity) among enterococci in different geographical regions and in different parts of the food chain, as well as the similarities between different enterococcal populations. Altogether, 2868 samples were collected from humans (healthy and hospitalised individuals and clinical isolates), animals (slaughterhouse carcasses and farm animals), and the environment (pig farms, sewage, and surface water) in four European countries-Sweden, Denmark, UK, and Spain. The samples were characterised with regard to presence and numbers of enterococci, and eight (for faecal samples) or 24 (for environmental samples) isolates per sample were phenotyped and preliminarily identified with the PhP-RF system. In total, more than 20,000 isolates were typed. A majority of the samples (77%) showed the presence of presumed enterococci. The diversities of enterococci in environmental samples were generally high, and also faecal samples normally showed presence of more than one enterococcal strain. The most common species found were Enterococcus faecium (33%), E. faecalis (29%), and E. hirae (24%), but different enterococcal populations differed in their species distribution. Clinical isolates, hospitalised patients, and hospital sewage in Sweden showed a clear dominance of E. faecalis (80%, 57%, and 54%, respectively) whereas healthy individuals and urban sewage contained less E. faecalis (39% and 40%, respectively). The species distribution among isolates from slaughterhouses varied between animal species and also between countries, but E. faecalis seemed to be mainly associated with broiler, and E. hirae with cattle and pigs. The results from the study have indicated a simplified method to study the diversity of bacterial populations. Instead of collecting many samples and analysing one or a few isolates per sample, it is possible to collect fewer samples and analyse several isolates per sample. Both approaches yielded similar information on the diversity of the populations. Another useful information was that since samples from hospital sewage, urban sewage, and manure contained enterococcal populations that reflected those in faecal samples of hospitalised patients, healthy humans, and animals, respectively, such samples may be used as pooled faecal samples and may replace cumbersome samplings from many individuals.

Origin and evolution of European community-acquired methicillin-resistant Staphylococcus aureus.

UNLABELLED: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE: With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

Multi-locus variable number of tandem repeat analysis for rapid and accurate typing of virulent multidrug resistant Escherichia coli clones.

One hundred E. coli isolates from Norway (n = 37), Sweden (n = 24), UK (n = 20) and Spain (n = 19), producing CTX-M-type - (n = 84), or SHV-12 (n = 4) extended spectrum ß-lactamases, or the plasmid mediated AmpC, CMY-2 (n = 12), were typed using multi-locus sequence typing (MLST) and multi-locus variable number of tandem repeat analysis (MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence Type Complexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes (MTCs). A strong agreement between the MLST profile and MLVA typing results was observed, in which all ST131-isolates (n = 39) and most of the STC-648 (n = 10), STC-38 (n = 9), STC-10 (n = 9), STC-405 (n = 8) and STC-23 (n = 6) isolates were clustered distinctly into MTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accurate tool for genotyping isolates of globally disseminated virulent multidrug resistant E. coli lineages, including ST131.

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

Fast and reliable epidemiological typing methods for identifying outbreaks and epidemic strains of extended spectrum ß-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.

Interplay of efflux, impermeability, and AmpC activity contributes to cefuroxime resistance in clinical, non-ESBL-producing isolates of Escherichia coli.

Cefuroxime resistance in Escherichia coli strains susceptible to extended-spectrum cephalosporins is not uncommon, but the resistance mechanisms have so far not been elucidated. Therefore, 14 clinical non-extended-spectrum beta-lactamase isolates of E. coli were examined, 11 of which were cefuroxime resistant. Quantitative RT-PCR was used to examine the transcription levels of the genes acrA (encoding AcrA, part of the AcrAB-TolC efflux pump system) and ompF (encoding the porin OmpF). Isoelectric focusing was used for detection of beta-lactamases, and a spectrophotometric assay was used to measure AmpC activity. Among the 11 cefuroxime-resistant isolates, 7 had increased acrA transcription (from 2.4 to 38 times the ATCC strain), 3 isolates had very low ompF transcription levels (

Proposal for common Nordic epidemiological terms and definitions for methicillin-resistant Staphylococcus aureus (MRSA).

The recent increase in the incidence of methicillin-resistant Staphylococcus aureus in all the Nordic countries prompted the Scandinavian Society for Antimicrobial Chemotherapy (SSAC) to create the 'SSAC Working Party on MRSA' with the objective to identify methods to keep the invasive MRSA infections in the Nordic countries below 1%. The lack of common definitions was recognized as a major obstacle for a joint Nordic effort to combat MRSA. The aim of this publication is to present proposals for epidemiological definitions of individual cases, for how to report MRSA frequency per country, and for communication of MRSA strain characteristics between the countries.