Abstract: Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones.
Abstract: Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.
Abstract: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) infections has changed worldwide. From being strictly nosocomial, MRSA is now frequently found as a community-associated (CA) pathogen. Denmark has been a low-prevalence country for MRSA since the mid-1970s but has in recent years experienced an increasing number of CA-MRSA cases. The aim of this study was to describe the emergence of CA-MRSA infections in Denmark. All Danish MRSA specimens and corresponding clinical data from 1999 to 2006 were investigated. Isolates were analyzed by antibiotic resistance and molecular typing and were assigned to clonal complexes (CC). Clinical data were extracted from discharge summaries and general practitioners' notes, from which assessments of community association were made for all infected cases. CA-MRSA cases constituted 29.4% of all MRSA infections (n = 1,790) and an increasing proportion of the annual numbers of MRSA infections during the study period. CA-MRSA was associated with a young age, skin and soft tissue infections, and non-Danish origin. Transmission between household members was frequently reported. Molecular typing showed >60 circulating clones, where 89.4% of the isolates belonged to five CC (CC80, CC8, CC30, CC5, and CC22), 81.2% carried staphylococcal cassette chromosome mec IV, and 163/244 (69.4%) were positive for Panton-Valentine leukocidin. Clinical and microbiological characteristics indicated that import of MRSA occurs frequently. Resistance to > or =3 antibiotic classes was observed for 48.8% of the isolates. The emergence of CA-MRSA in Denmark was caused by diverse strains, both well-known and new CA-MRSA strains. The results suggest multiple introductions of MRSA as an important source for CA-MRSA infections in Denmark.
Abstract: Infections as a result of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are an issue of increasing global healthcare concern. In Europe, this principally involves strains of multi-locus sequence type clonal complex 80 sequence type 80 with methicillin resistance in a staphylococcal chromosomal cassette (SCCmec) type IV arrangement (CC80:ST80-IV). As with other CA-MRSA strains, CC80:ST80-IV isolates tend to appear uniform when analysed by common molecular typing methods (e.g. pulsed field gel electrophoresis, multi-locus sequence type, SCCmec). To explore whether DNA sequence-based differences exist, we compared the genetic composition of six CC80:ST80-IV isolates of diverse chronological and geographic origin (i.e. Denmark and the Middle East) using an Affymetrix high-density microarray that was previously used to analyse CA-MRSA USA300 isolates. The results revealed a high degree of homology despite the diversity in isolation date and origin, with isolate differences primarily in conserved hypothetical open reading frames and intergenic sequences, but also including regions of known function. This included the confirmed loss of SCCmec recombinase genes in two Danish isolates representing potentially new SCCmec types. Microarray analysis grouped the six isolates into three relatedness pairs, also identified by pulsed field gel electrophoresis, which were consistent with both the clinical and molecular data.
Abstract: In Europe, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have been caused predominantly by isolates belonging to the "European CA-MRSA" clone (sequence type 80, staphylococcal cassette chromosome mec type IV). In this study, the epidemiology of European CA-MRSA was investigated on a nationwide scale, covering the period from 1993 to 2004. Denmark has been a low-prevalence country regarding MRSA since the mid-1970s but has experienced an increase in the number of new MRSA cases in recent years. Our results show that European CA-MRSA contributed to this increase. The isolates primarily caused skin and soft tissue infections (SSTIs) in patients outside hospitals, and transmission between household members was the predominant mode of spread. Although some of the isolates were found in hospitalized patients, nosocomial transmission seemed likely in only one instance, pointing to endogenous infections as an important factor. Compared to the CA-MRSA clone most common in the United States (USA300), the European CA-MRSA clone seems less well adapted to persist in hospital environments. Patients with a recent history of travel or family relation to the Mediterranean or Middle East were highly overrepresented. The epidemiological data indicated that the European CA-MRSA isolates were introduced into Denmark on multiple occasions, paralleled by an increasing level of genetic diversity of the isolates found during the study period. European CA-MRSA has previously been described as a rather uniform clone. However, we found pronounced, diverse pulsed-field gel electrophoresis subtypes, staphylococcal protein A gene (spa) types, and susceptibility patterns.
Abstract: OBJECTIVES: To characterize the epidemiology of Staphylococcus aureus isolates resistant to fusidic acid isolated from patients with skin and soft tissue infections (SSTIs) in France, the UK and Ireland. METHODS: One hundred and thirty-six S. aureus isolates with an MIC of fusidic acid above 1 mg/L were isolated during the EPISA study from patients more than 2 years old attending their general practitioners for SSTIs. All isolates were related to clonal complex by a combination of PFGE, spa typing and multilocus sequence typing. The presence of toxin genes and of the fusB determinant was monitored to characterize each represented clonal complex. RESULTS: Eight different clonal complexes were identified. CC121 constituted the majority of the isolates from Ireland and the UK but was not represented in France. Among the other clonal complexes, CC8 and CC5 were the most common in the three countries, although the number of French isolates was limited. CC121 was the only clonal complex significantly associated with a skin infection, namely impetigo (P < 0.05). Toxin genes were present in CC121 and CC80. The fusB determinant was also detected in the same clonal complexes. Enterotoxins were found in four clonal complexes (CC1, CC5, CC8 and CC22). CONCLUSIONS: The impetigo clone (CC121: ST123) was present in the majority of S. aureus isolates from the UK and Ireland but was not detected in France. This strain was associated with impetigo, exfoliative toxins and the fusB determinant. No other clonal complex appeared to be dominant in other types of skin infections.
Abstract: Rapid detection and typing of methicillin-resistant Staphylococcus aureus are important components of infection control programmes. A protocol is described that enables sequencing of the spa gene fragment directly from a multiplex PCR targeting the clinically relevant mecA, pvl and spa genes, resulting in high-throughput characterisation of S. aureus. Implementation of the method in the Danish national reference laboratory has markedly reduced the use of reagents and the requirement for hands-on time, and has also provided fast typing results. In addition, the method reduces the risk of sample mishandling.
Abstract: Resistance to the antibiotic fusidic acid in European strains of Staphylococcus aureus causing impetigo has increased in recent years. This increase appears to have resulted from clonal expansion of a strain we have designated the epidemic European fusidic acid-resistant impetigo clone (EEFIC), which carries the fusidic acid resistance determinant fusB on its chromosome. To understand better the properties of the EEFIC responsible for its success, we have performed detailed phenotypic and genotypic characterization of this clone. Molecular typing revealed the EEFIC to be ST123, spa type t171, and agr type IV and therefore unrelated to earlier prevalent fusB(+) strains found in the United Kingdom. EEFIC strains exhibited resistance to fusidic acid, penicillin, and, in some cases, erythromycin, which are all used in the treatment of impetigo. PCR analysis of the EEFIC and complete DNA sequencing of the 39.3 Kb plasmid it harbors identified genes encoding several toxins previously implicated in impetigo (exfoliative toxins A and B and EDIN-C). The location of fusB was mapped on the chromosome and found to be associated with a novel 16.6-kb genomic island integrated downstream of groEL. Although this element is related to classical staphylococcal pathogenicity islands, it does not encode any known virulence factors and consequently has been designated SaRI(fusB) (for "S. aureus resistance island carrying fusB").
Abstract: Staphylococcus aureus is a leading cause of bacteraemia. This study analysed temporal trends from 18,702 adult cases of S. aureus bacteraemia in Denmark between 1981 and 2000. After stratification for mode of acquisition, 57% of cases were hospital-acquired (HA), 28% were community-acquired (CA) and 15% were of undetermined acquisition (UA). Incidence rates increased from 18.2 to 30.5 cases/100,000 population. Annual rates increased by 6.4% for CA, by 2.2% for HA and by 3.6% for UA cases, respectively. Case-mortality associated with HA bacteraemia decreased from 36.2% to 20.7% (43% rate reduction, p 0.0001), compared with a decrease from 34.5% to 26.5% (23% rate reduction, p 0.0001) for CA bacteraemia. Following multivariate analysis, age, pneumonia, endocarditis and chronic illness were associated with increased mortality, regardless of the mode of acquisition. Overall, mortality associated with S. aureus bacteraemia declined significantly between 1981 and 2000, but incidence rates doubled, so that the total number of deaths increased. These data emphasise the public health importance of S. aureus bacteraemia and the need for further preventive measures and improved care in order to reduce incidence rates and improve outcomes.
Abstract: BACKGROUND: Staphylococcus aureus is known to be a leading cause of bacteremia in childhood, and is associated with severe morbidity and increased mortality. To determine developments in incidence and mortality rates, as well as risk factors associated with outcome, we analyzed data from 1971 through 2000. METHODS: Nationwide registration of S. aureus bacteremia (SAB) among children and adolescents from birth to 20 years of age was performed. Data on age, sex, source of bacteremia, comorbidity and outcome were extracted from discharge records. Rates were population adjusted and risk factors for death were assessed by multivariate logistic regression analysis. RESULTS: During the 30-year study period, 2648 cases of SAB were reported. Incidence increased from 4.6 to 8.4 cases per 100,000 population and case-mortality rates decreased from 19.6% to 2.5% (P = 0.0001). Incidence in the infant age group (<1 year) were 10- to 17-fold greater compared with that in the other age strata and mortality rate was twice as high. Hospital-acquired infections dominated the infant group, accounting for 73.9%-91.0% versus 39.2%-50.5% in the other age groups. By multivariate analysis, pulmonary infection and endocarditis for all age groups, comorbidity for the older than 1 year, and hospital-acquired infections for the oldest group were independently associated with an increased risk of death. CONCLUSIONS: Mortality rates associated with SAB decreased significantly in the past 3 decades, possibly because of new and improved treatment modalities. However, incidence rates have increased significantly in the same period, underscoring that S. aureus remains an important invasive pathogen.
Abstract: This article describes an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in two institutions for multi-handicapped children in Copenhagen. The aim of the study was to determine whether it was possible to eradicate MRSA in a setting with multi-handicapped children and staff where there was a high degree of physical interaction. This was a prospective interventional uncontrolled cohort study that took place from January 2003 to March 2005. All individuals in close contact with the two institutions and/or in close contact with an MRSA-colonized subject from the outbreak were included in the study: 38 children, 60 staff members and 12 close relatives of colonized subjects. Infection control measures included screening all individuals. When MRSA infection or colonization was found, an attempt was made to eradicate MRSA, staff education was undertaken and attempts were made to determine the route of transmission. Eleven individuals were found to be positive for MRSA (10.0%). All isolates were identical by pulsed-field gel electrophoresis and harboured the staphylococcal cassette chromosome mec (SCCmec) type IV. All colonized and infected individuals were associated with a single room in one of the institutions. MRSA was eradicated from all the colonized and infected subjects. This study shows that it is possible to control an MRSA outbreak in institutions for multi-handicapped children where there is a high degree of physical contact.
Abstract: Cefoxitin is increasingly recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. In this study, 95 mecA-negative S. aureus isolates and a highly genetically diverse collection of mecA-positive S. aureus types (n=50) were used to investigate the influence of technical factors such as disk potency, incubation time, and temperature on Mueller-Hinton agar. The use of cefoxitin MIC testing by Etest for the same purpose was investigated under similar conditions. For disk diffusion, the accuracy was high at both 35 degrees C and 36 degrees C using overnight incubation, while incubation at 30 degrees C or 37 degrees C was associated with slightly lower accuracy. Increasing incubation times from 18 to 24 h did not improve accuracy at either temperature. Cefoxitin Etest MICs for mecA-positive strains were 6 mg/liter or higher, while cefoxitin Etest MICs for mecA-negative strains were <or=4 mg/liter. Our findings suggest that the current CLSI zone diameter breakpoints should be adjusted from resistance (R)<or=19 mm to R<or=21 mm. In conclusion, cefoxitin disk diffusion testing and Etest MIC testing can accurately predict the presence of the mecA gene in S. aureus. Testing can be reliably performed using incubation temperatures of 35 to 36 degrees C and incubation times of 18 to 22 h. We suggest MRSA interpretive criteria of susceptible (S)<or=4 mg/liter and R>4 mg/liter, corresponding to S>or=22 mm and R<or=21 mm for the 30-microg disk and S>or=17 mm and R<or=16 mm for the 10-microg cefoxitin disk. These criteria resulted in only one mecA-positive isolate being misclassified as susceptible.
Abstract: OBJECTIVE: To evaluate cefoxitin 5 and 10 microg discs for detection of methicillin resistance in staphylococci. METHODS: Six hundred and forty-one Staphylococcus aureus (261 mecA-negative and 380 mecA-positive) and 344 coagulase-negative staphylococci (CoNS) (132 mecA-negative and 212 mecA-positive) were investigated. The CoNS represented nine species, Staphylococcus epidermidis being the most frequent (n = 231). All isolates were tested using semi-confluent growth on Iso-Sensitest agar (ISA), and Mueller-Hinton agar (MH) using a 5 and a 10 microg cefoxitin disc and overnight incubation in ambient air at 35-37 degrees C. RESULTS: For S. aureus, both cefoxitin discs performed with high accuracy on both media. The sensitivity and specificity for the following proposed interpretive zone diameters were: ISA 5 microg, R < 14 mm (99.5% and 98.1%); ISA 10 microg, R < 22 mm (99.5% and 98.1%); MH 5 microg, R < 12 mm (99.7% and 98.1%); and MH 10 microg, R < 18 mm (99.5% and 98.9%), respectively. All four variants were superior to oxacillin using the former SRGA methodology. In CoNS, a substantial overlap was seen for all variants. However, by avoiding primary interpretation in the overlapping interval, highly accurate results could be obtained for 81%, 80%, 91% and 97% of the isolates, respectively. CONCLUSION: For S. aureus, cefoxitin 5 and 10 microg discs performed with high accuracy on both ISA and MH using semi-confluent growth and standard incubation conditions. With the introduction of a defined interval in which primary interpretation should be avoided, the method could also be used for CoNS.
Abstract: Strict infection control measures introduced during the 1970s have kept the incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections extremely low in Denmark. Nevertheless, similarly to other countries, MRSA infections began to appear in the community in the late 1990s. A nationwide surveillance program has collected and stored all MRSA isolates since 1988 and, since 1999, clinical information has been also recorded. We used this information and isolates in a detailed epidemiological and molecular analysis of the 81 MRSA infections identified in Denmark in 2001. MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and SCCmec typing. Comparison of the 45 community-onset MRSA (CO-MRSA) infections with the 36 hospital-acquired MRSA (HA-MRSA) infections showed several striking contrasts. Most CO-MRSA were recovered from skin and soft tissue infections caused by isolates carrying the Panton-Valentine leucocidin toxin genes, and the majority (84%) of isolates belonged to a single clonal type, ST80-IV, which has been found in the community in other European countries. Clone ST80-IV could be traced in Denmark back to 1993. ST80-IV was rarely found in HA-MRSA infections, which belonged to a large number of clonal types, including some pandemic MRSA clones. The low number of HA-MRSA infections and the diversity of MRSA clones in Danish hospitals may be the result of successful infection control measures that prevent spread of clones in hospitals. The mechanism of spread of the ST80-IV clone in the Danish community is not known, and new control measures are needed to control further spread of this and other CA-MRSA clones.
Abstract: Fusidic acid-resistant epidemic Staphylococcus aureus strains causing impetigo bullosa have been reported in Scandinavia. We show that these strains form part of a European epidemic clonotype that carries the fusB determinant. In contrast, resistance to fusidic acid in a collection of nonepidemic strains resulted primarily from mutations in fusA.
Abstract: In order to find a disk diffusion method with both high sensitivity and specificity for determination of methicillin resistance primarily for S. aureus but also for coagulase-negative staphylococci we screened several methodological variants using a material of 66 S. aureus comprising of 11 methicillin-susceptible, 18 borderline-resistant, and 37 methicillin-resistant strains. Only four of the combinations studied performed with both high sensitivity and specificity. Two of these, the Columbia agar +4.5% NaCl and Mueller Hinton agar +2% NaCl combined with a 5 microg oxacillin disk, confluent inoculum and 24 h incubation at 35 degrees C were further evaluated using 105 MRSA and 91 mecA-negative S. aureus and 193 clinical isolates of coagulase-negative staphylococci. The Columbia agar +4.5% NaCl performed excellently for both S. aureus and coagulase-negative staphylococci. For Columbia agar +4.5% NaCl using a 5 microg oxacillin disk we suggest an interpretive zone diameter of R < or =15 mm and S > or =16 mm for S. aureus and R < or =24 mm and S >or =26 mm for coagulase-negative staphylococci. The Mueller Hinton agar +2% NaCl performed well for coagulase-negative staphylococci but for S. aureus at least three (3%) very major errors were found, making this method less attractive.
Abstract: OBJECTIVES: To evaluate the performance of a cefoxitin 30 microg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36 degrees C, for detection of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: A total of 457 S. aureus, including 190 MRSA of several defined PFGE types and a number of low-level resistant isolates, were tested with a cefoxitin 30 microg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36 degrees C. This method was compared with the standard SRGA (Swedish Reference Group for Antibiotics) method (oxacillin 1 microg disc on Iso-Sensitest agar supplemented with 5% defibrinated horse blood, confluent growth and 24 h incubation in ambient air at 30 degrees C). RESULTS: The cefoxitin method was excellent, with a sensitivity of 100% and a specificity of 99% using an interpretative zone diameter of S > or = 29 mm and R < 29 mm. Its performance was much better than the SRGA method, which with this collection of difficult strains had a sensitivity of only 78% using the current breakpoint of S > or = 12 mm. CONCLUSION: We suggest that the cefoxitin method should replace that currently recommended by the SRGA for the detection of MRSA, and that it would fit well into BSAC methodology.
Abstract: In the first attempt to establish a quality assurance programme for susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones, 20 strains with different fluoroquinolone susceptibility patterns were distributed by the Supranational Reference Laboratory in Stockholm to the other mycobacterial reference laboratories of the Nordic and Baltic countries. Susceptibility testing to fluoroquinolones was performed according to routine procedures in each laboratory. Results were compared to sequence analysis of the gyrA gene and minimal inhibitory concentration determination. Most laboratories found identical susceptibility patterns. The two resistant strains were correctly identified by all laboratories, but three laboratories each falsely reported one susceptible strain as resistant. These results indicate that the participating laboratories yield reliable results in detection of fluoroquinolone-resistant strains, although the need for a standardised quality assurance programme for drug susceptibility testing for fluoroquinolones is stressed by the strains falsely reported as resistant.
Abstract: The relationship between human immunodeficiency virus type 1 (HIV-1) viral RNA and proviral DNA load in vagina and cervix and that found in the plasma and peripheral blood mononuclear cells (PBMC) was investigated in 28 HIV-1-infected women. Of the patients, 64% had > or = 1 HIV-1 RNA-positive genital sample, while 71% had > or = 1 DNA-positive sample. The higher the cervical HIV load, the more widespread was the virus in the genital tract. A strong correlation was found between viral RNA load in plasma and the genital tract, whereas the association between proviral DNA load in PBMC and the genital tract was less evident. Cervical HIV-1 DNA correlated with a viral RNA load > or = 50,000 copies/mL. Cervical HIV-1 RNA levels ranged from 10% to 100% of the plasma levels. Thus, a continuous transmission risk from untraumatized genital epithelium exists in the majority of HIV-1-infected women at all stages of infection.
