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Criteria for susceptibility testing of mecillinam against 533 isolates of Escherichia coli and a further 309 Enterobacteriaceae, according to NCCLS methodology, were determined. Correlation of MIC to inhibition zones was good for all species. For urinary isolates of E. coli, the following agar dilution breakpoints and corresponding interpretive zone diameters seem appropriate:or = 8 mg/L/or = 15 mm for susceptible; 16 mg/L/12-14 mm for intermediate susceptible andor = 32 mg/L/or = 11 mm for resistant. The appearance of isolated colonies within the inhibition zone was sometimes noted with disc diffusion, particularly for non-E. coli Enterobacteriaceae. The relevance of these colonies to clinical (bacteriological) efficacy was determined and the results suggested that they could be ignored when testing urinary E. coli.

Two hundred and ninety-two staphylococci were tested using fusidic acid Neosensitabs ® , a semiconfluent inoculum on Mueller Hinton agar and three blood containing agar media in order to investigate the interpretative zone diameters published by the manufacturer (Rosco). Zone diameters were, as expected, smaller on blood containing agar compared with Mueller Hinton agar. Many susceptible strains were intermediate on Danish Blood agar using the current breakpoints. We suggest that the interpretative zone diameters be changed to S ≥32, R ≤30 for Mueller Hinton agar and S ≥24, R ≤22 on blood containing media.

Objectives: To evaluate the performance of a cefoxitin 30 µg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36°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 µg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36°C. This method was compared with the standard SRGA (Swed- ish Reference Group for Antibiotics) method (oxacillin 1 µg disc on Iso-Sensitest agar supplemented with 5% defibrinated horse blood, confluent growth and 24 h incubation in ambient air at 30°C). Results: The cefoxitin method was excellent, with a sensitivity of 100% and a specificity of 99% using an interpretative zone diameter of S ≥ 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 break- point of S ≥ 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.

We were delighted to see the recent article by Felten et al. on the detection of low-level methicillin-resistant Staphylococcus aureus (MRSA) (1). The study is, to our knowledge, the first to specifically test the MRSA-screen test on low-level MRSA strains. However, Felten et al. concluded that the MRSA-screen test is the best commercially available test for distinguishing very-low-level class 1 MRSA from methicillin-susceptible S. aureus. We would therefore like to point the authors' attention to the EVIGENE MRSA Detection Kit (Statens Serum Institut, Copenhagen, Denmark), which is also a commercially available product for detecting methicillin resistance in staphylococci. This kit is a DNA probe-based hybridization assay for detecting the mecA gene in staphylococci. Each test also detects the nuc gene for differentiating between S. aureus and coagulase-negative staphylococci as well as internal positive and negative controls. The kit uses no amplification steps; thus, the issues of contamination applicable to PCR do not apply to this kit. The kit result, which is obtained in 3.5 h, is visually read as yellow or blank. We have evaluated the EVIGENE MRSA Detection Kit with 275 S. aureus isolates, including 18 borderline-resistant strains; the results showed both the sensitivity and the specificity to be 100% for the entire collection (3). For coagulase-negative staphylococci, 242 strains have been evaluated (128 mecA-positive and 114-mecA negative strains). Of these 242 strains, 5 (2%) produced a positive control signal below the cutoff defined for the kit, thus rendering the five tests invalid. For the remaining 239 strains, the sensitivity and specificity were as for S. aureus: both 100% (2).

The European methicillin-resistant Staphylococcus aureus (MRSA) clone ST80-IV has historically dominated community-associated infections in major parts of Europe and is a lineage strongly linked to skin and soft tissue infections. Here, we report the genome sequence of an ST80-IV representative, 11819-97, isolated from a skin infection in Denmark in 1997.

Sixty-three Staphylococcus aureus isolates with a wide distribution in quantitative beta-lactamase production were tested in vitro against amoxycillin and penicillin in combination with clavulanic acid to establish the influence of total amount of beta-lactamase present on the ability of clavulanic acid to protect against beta-lactamase degradation. The beta-lactamase stability of cefuroxime and dicloxacillin was also evaluated. MIC was determined by agar dilution using Mueller-Hinton agar with both a conventional as well as a 100 times higher inoculum. The strains were tested both with and without induction of the beta-lactamase production. Clavulanic acid was highly effective in protecting against beta-lactamase degradation of both penicillin and amoxycillin. Even when using a high inoculum of strains with induced beta-lactamase production, all strains had MICs below the NCCLS breakpoint of 4/2 mg/l for amoxycillin-clavulanic acid. Both cefuroxime and dicloxacillin were highly stable against staphylococcal beta-lactamase degradation. This study encourages further in vivo evaluation of amoxycillin-clavulanic acid for severe staphylococcal infections.

Objective To compare pulsed-field gel electrophoresis (PFGE) typing and O:K-serotyping of Klebsiella in two different epidemiological settings. Methods One hundred and four bacteremia isolates without known epidemiological relation and 47 isolates from an outbreak in a neonatal intensive care unit (NICU) were K-typed by countercurrent immunoelectrophoresis (CCIE), O-typed by an inhibition enzyme-linked immunosorbent assay method, and typed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. Results Typing data for the 104 bacteremia isolates were compared with regard to typability, number of types, maximum number of isolates per type, and the Discriminative Index (DI). O-typing combined with K-typing (DI 0.98) as O:K-serotyping (DI 0.99) gave a very discriminative typing system, whereas O-typing alone was not very discriminative (DI 0.76). PFGE (DI 1.00) was a more discriminative typing method than O:K-serotyping, as it could subdivide 13/22 O:K-serotypes into smaller groups. Isolates with the same PFGE-type had the same O:K-serotype, indicating that isolates with different O- and/or K-types could be expected to be of different PFGE-types. Typing of the 47 isolates from the outbreak in the NICU showed that 38 isolates belonged to a single clone, and that during an epidemic limited in time and space, differences in the electrophoretic patterns of up to five bands between a parental pattern type and a subtype may be found in the PFGE profiles. Conclusions Both O:K-serotyping and PFGE typing are highly discriminative typing methods. PFGE is the most discriminative method and is excellent for typing outbreaks with few isolates. If large numbers of isolates are to be typed, a more convenient strategy might be first to K- or O:K-serotype isolates followed by PFGE typing of possible identical isolates. Since K- or O:K-serotyping is a definitive typing method, while PFGE typing is a comparative one, PFGE cannot, for the time being, replace O:K-serotyping for surveillance purposes.

Three hundred and eight staphylococci and 43 Streptococcus pyogenes were tested by agar dilution, microbroth dilution, E-test, and disk diffusion using 5, 10 and 50 microg disks, as outlined by the NCCLS, in order to correlate the different MIC methods and to establish tentative species specific interpretive zone diameter breakpoints for fusidic acid. MIC results of the three methods were comparable. For Staphylococcus aureus, using MIC breakpoints ofor = 0.5 mg/L for susceptible andor = 2 mg/L for resistant tentative interpretive zone diameters ofor = 20 mm andor = 21 mm for susceptible andor = 17 mm andor = 18 mm for resistant are suggested for the 5 microg and the 10 microg disk, respectively. The 50 microg disk did not separate susceptible from resistant isolates. For streptococci a uniform MIC distribution of 2-8 mg/L was found.

Moraxella canis was isolated from an infected foot ulcer in a patient suffering from diabetes mellitus with neuropathy. Bacteriological findings and 16S rDNA data are presented.

The problem of methicillin resistant Staphylococcus aureus (MRSA) is increasing worldwide, and the spread of MRSA in the community challenges infection control since it is no longer restricted to hospital settings but involves private homes, places of work and kindergartens [1]. Furthermore, community acquired (CA)-MRSA may circumvent existing hospital infection control, since patients are rarely screened at admission. In the United States, the predominant CA-MRSA is defined by the Center for Disease Control (CDC) as the USA300 (ST8) clone. USA300 primarily causes skin and soft tissue infections (SSTI) in the community [2], but healthcare acquired infections with USA300 are rapidly emerging in the United States [3,4]. Comparison of the Danish collection of MRSA from 1997-2005 with the USA300 reference strain showed that USA300 has been introduced into Denmark on several occasions. Between 2000 and 2005, we identified 44 isolates which in addition to identical pulsed-field gel electrophoresis (PFGE) pattern shared other molecular characteristics with USA300: spa type t008 or closely related variants, Panton-Valentine leukocidin (PVL) positive and Staphylococcal Cassette Chromosome mec (SCCmec) type IVa. The isolates primarily caused SSTI, but cases of invasive infections were also encountered. The number of USA300 has increased several-folds in Denmark from 2003 to 2005 (2, 11 and 28 new cases, respectively) and with the experience from the US in mind, this is of great concern, especially as it is observed in a country with a long reputation for controlling MRSA.

Arcanobacterium haemolyticum has been implicated mainly in non-streptococcal pharyngitis and wound infections. Rarely, it has been reported to cause systemic infection, often in combination with other pathogens. Two cases of systemic and deep-seated infections caused by Arcanobacterium haemolyticum are reported, and the literature is reviewed. Sixteen cases of bacteremia and seven cases of non-bacteremic deep-seated have been published previously. Eight of the bacteremic and two of the non-bacteremic cases occurred in younger, apparently healthy immunocompetent patients. Six patients had infections of the central nervous system. The optimal treatment of infections caused by Arcanobacterium haemolyticum is not known. Although in vitro susceptibility tests have demonstrated tolerance of Arcanobacterium haemolyticum to penicillin, penicillins with or without aminoglycosides have been the most widely used antibiotics, in most cases with success.

Until 1994, methicillin resistant Staphylococcus aureus (MRSA) were isolated from less than 60 patients annually in Denmark. We describe an outbreak of MRSA involving eight patients within a timespan of six weeks. Bacteriophage-typing and DNA typing using pulsed field gel electrophoresis revealed that three different strains of MRSA were involved in the outbreak. This emphasizes the importance of typing MRSA in order to clear up the spread in an outbreak. Of the eight patients four died, and in one case MRSA was thought to be the major cause of death. The expenses of the extra hygienic measures i.e. isolation of patients, medication and bacteriological surveillance were estimated to approximately DKK 80.000 pr. patient. Patients, who have been admitted to or worked at a hospital outside Scandinavia within the last six months, are potentially colonized with MRSA. Specific guidelines for hospital hygiene are necessary to prevent the spread of such strains.

To the Editor: In their article, Collignon et al. (1) present a table comparing absolute numbers and incidence rates of Staphylococcus aureus bacteremia (SAB) in Australia to those of 5 other countries, and state that "some data are available from other countries for comparison" and "only 2 countries, Denmark and England, appeared to have comprehensive collection systems." We would like to add data from the European Antimicrobial Resistance Surveillance System (EARSS) to their table. EARSS is a multinational surveillance system that links national networks by collecting comparable and validated data on the prevalence of antimicrobial resistance of 5 microorganisms, including S. aureus (2). A total of 30 countries participate in EARSS. To ensure representativeness of the data it publishes, EARSS has set criteria, which can be found elsewhere (3). In 2003, through a questionnaire, we collected background information on all hospitals served by laboratories participating in EARSS, including the estimated hospitals' catchment populations. Proportion of the country population covered by EARSS was then calculated by dividing the sum of catchment populations by the total population of the country (4). Catchment populations of hospitals providing single specialty or supra-regional type of care were not counted to avoid overlap with other hospitals within the same country. Only the countries that provided denominator data for at least 60% of the isolates were included to ensure that the sample of hospitals was still representative of the country as a whole. The number of SAB and the incidence of SAB per 100,000 inhabitants in 2003 were calculated from EARSS data, adjusted for population coverage, and are presented in the Table. These are crude estimates of the true number of SAB and should, thus, be interpreted with caution. For example, we assumed that the isolates for which hospital background information was not available did not differ from isolates for which we had this information, and that hospitals that participated in EARSS in 2003 were a representative sample of the countries' hospitals. Additionally, the incidence of SAB was positively correlated with the blood culturing rate (Spearman r = 0.74, p = 0.002), which means that the incidence of SAB is likely to have been underestimated in countries that reported few blood cultures. Although some countries did not report their blood culturing rate, the incidences of SAB in these countries were among the highest and are unlikely to be underestimated. For example, the EARSS data for Denmark and Ireland nicely fitted those presented in Table 4 of the article by Collignon et al. (1). Finally, reporting to the EARSS system greatly improved over the years, which is why this study was performed on the last available year, 2003. Nevertheless, one cannot exclude underreporting of SAB by EARSS participating hospitals since EARSS is a voluntary reporting system. For example, England reported 18,403 SAB cases or an incidence of 37 SAB per 100,000 inhabitants from April 2002 to March 2003 through its mandatory surveillance scheme (5), whereas an estimate for the United Kingdom from the EARSS database would only give 7,800 SAB cases for 2003. However, it is impossible to determine whether this discrepancy was due to poor voluntary reporting of SAB cases, a lower blood culturing rate in EARSS participating hospitals, or a poorly representative sample of the country's hospitals. Data from the United Kingdom were excluded from the present study on the basis of the latter possibility; denominator information for

Staphylococcus aureus ST291 has been reported as a homologue recombinant double locus variant of the livestock associated S. aureus ST398. However, whole genome sequencing show that ST291 is a unique genetic lineage with highly variable content within its accessory genome compared to both human and livestock associated genome sequenced CC398s.

Staphylococcus aureus strains of phage group II have increased in frequency in hospital-acquired infections during recent years. A total of 184 penicillin resistant group II strains from bacteraemia cases in the years 1961-1990 were analysed for the amount of beta-lactamase produced and the location of the beta-lactamase gene. Until 1977 all strains investigated had a chromosomally located beta-lactamase gene, but since then a 21 kb beta-lactamase plasmid has increased in occurrence among group II strains, especially among those strains typable only at high phage concentrations [100 x Routine Test Dilution (RTD) and 1000 x RTD]. In 1990, 84% of the group II strains contained this plasmid. Plasmid-containing strains produced more beta-lactamase than strains without the plasmid. S. aureus strains of the 94,96 complex, which since 1984 have decreased in frequency from 18 to 9% in 1993, have remained high producers of beta-lactamase.

Aerococcus species are Gram-positive cocci having Gram-stain characteristics of staphylococci (i.e., form clusters), but biochemical and growth characteristics resembling streptococci and enterococci (i.e., catalase-negative, alpha-hemolytic growth on blood agar). A case of fatal endocarditis due to Aerococcus urinae is presented. Endocarditis should be suspected and, if confirmed, treated aggressively when A. urinae is recovered from blood cultures.

LEO Pharma, Industriparken 55, DK-2750 Ballerup, Denmark; Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark; Service de Bacteriologie-Hygiene, CHU Pitie-Salpetriere, Paris, France; Hopital Robert Debre, Service Dermatologie, Avenue de General Koening, F-51092 Reims Cedex, France; Department of Clinical Microbiology, Central Pathology Laboratory, St James’s Hospital, Dublin 8, Ireland; Department of Clinical Microbiology, Leeds General Infirmary, Great George Street, Leeds, West Yorkshire LS1 3EX, UK

In the aftermath of a methicillin-resistant Staphylococcus aureus (MRSA) ST22 hospital outbreak, we investigated the prevalence of long-term carriage, the efficacy of MRSA decolonization treatment (DT) and the spread of MRSA to households of patients and healthcare workers (HCWs). Furthermore, we evaluated the efficacy of repeated DT in long-term MRSA carriers. Of 250 index persons (58 HCWs and 192 patients), 102 persons (19 HCWs and 83 patients) and 67 household members agreed to participate. Samples from all 169 persons were taken from the nose, throat, wounds and devices/catheters, and urine samples were additionally taken from index persons. Samples from companion animals (n = 35) were taken from the nostrils and anus. Environmental sites (n = 490) screened were telephone, television remote control, toilet flush handle, favourite chair and skirting board beside the bed. Sixteen (19%) patients and two household members, but no HCWs, were ST22-positive. The throat was the most frequent site of colonization. In a multivariate analysis, chronic disease (p

Objective To study the possibility of reporting results of identification and susceptibility testing of Gram-negative bacilli the same day as bacteremia is detected by using direct inoculation from positive blood cultures (Bactec 9240) into VITEK GNI+ and GNS-GA cards. Methods All blood cultures with Gram-negative enteric bacillus-like morphology on microscopy found to be positive on workdays between 15 June 1999 and 29 February 2000 were included. Identification and susceptibility testing were done by three methods: the direct method using a suspension made by differential centrifugation of positive blood culture broth for inoculation of the VITEK cards; the standard method using an inoculum made from an overnight culture on a solid media; and the routine method (reference method) using conventional testing. Results Of 169 isolates, the direct method resulted in 75% correct identifications, 9% misidentifications and 17% non-identifications. All misidentified isolateswere Escherichia coli , of which 80% were reported as Salmonella arizonae . Five biochemical tests yielded most of the aberrant results; correcting the citrate and malonate reactions in most cases led to correct identification by the VITEK database. Despite a negative H 2 S reaction, 11 E. coli isolates were reported as S. arizonae . Two-thirds (69%) of identifications were reported within 6 h, and 95% of these were correct. The direct susceptibility testing method was assessable for 140 isolates. Correct results were found in 99% of isolate-antimicrobial combinations, and 85% were reported within 6 h. Conclusion The direct VITEK method could correctly report identifications and susceptibility patterns within 6 h, making same-day reporting possible for almost two-thirds (63%) of bacteremic episodes with Gram-negative bacilli. These results could probably be improved by modification of the identification algorithms of the VITEK software.

Objectives Due to their multiple antibiotic resistance properties, methicillin-resistant Staphylococcus aureus (MRSA) are a major public health problem. After the recently described emergence of MRSA in animals, the authors investigated the risk of nasal MRSA carriage among people with professional contact with animals. Methods Nasal swabs and information on animal exposure and known MRSA risk factors were obtained from participants (N=702) at five conferences organized by national veterinary and farmer associations and students at a business school in Denmark. All of the participants were screened by standard microbiological techniques for MRSA detection and characterization. Results MRSA carriage was significantly (P