Methicillin-resistant Staphylococcus aureus with mecC: a description of 45 human cases in southern Sweden.

In 2011, a novel mecA gene homologue, mecC, was reported in isolates from both humans and dairy cattle. The epidemiology of mecC methicillin-resistant Staphylococcus aureus (MRSA) in humans is not yet well known. In this retrospective study, we present the epidemiology of human clinical cases with mecC MRSA detected in the southern part of Sweden during the period 2005-2014. A total of 45 patients with an isolate positive for mecC MRSA were included in the study. Twenty-six isolates were found before 2012 and were retrospectively tested for mecC. Nineteen isolates were detected in 2012-2014 through routine testing. Culture results, resistance patterns, Panton-Valentine leukocidin (PVL) genes, and spa types were collected from the Clinical Microbiology Laboratory. Epidemiological data were received from the database at the Regional Centre for Communicable Disease Control and the patient's medical files. The majority of the patients with mecC MRSA were of Swedish origin, had underlying diseases, and lived in rural areas. The median age was 60 years. Of the mecC MRSA, 76 % belonged to spa types t373 and t843. The median minimum inhibitory concentration (MIC) value for oxacillin was 16 mg/L (1-64 mg/L) and only one isolate was resistant to other classes of antibiotics. The most common type of infection was skin and soft tissue infections, most often in an existing skin lesion. The patients with mecC MRSA were colonized for a short time and gave rise to few secondary cases. mecC MRSA in our region appears to have a domestic origin and mainly affects patients with underlying diseases or patients with an existing skin lesion. Our data indicate that it could be a poor colonizer.

Epidemiology of MRSA in southern Sweden: strong relation to foreign country of origin, health care abroad and foreign travel.

All notified MRSA cases in Skåne County have been followed since 2000. We have investigated the MRSA epidemiology over time, method of acquisition, whether some spa types are more prone to spread, and/or cause more infections, and the connection between spa type and country of acquisition/origin. All cases between 2000 and 2010 were included. Infection or colonization and the presence of PVL genes were noted. The spa types of the index cases were correlated with community or healthcare acquisition, proportion of MRSA-positive household contacts, country of origin of families and country of acquisition of MRSA. The number of cases increased from 31 in 2000 to 315 in 2010. Most cases were community-acquired and the median age was 30 years. Thirty-two per cent of the MRSA cases were found because of a clinical infection. Of the household contacts 35 % were MRSA-positive. Only 24 % of the MRSA cases were both of Swedish origin and had contracted MRSA in Sweden. An association between spa type and certain regions of acquisition/origin was noted. Spa types t044, t002 and t008 were the most predominant strains. PVL-positive spa types t008, t019 and t044 caused more skin infections than the other spa types. Our results support screening for MRSA in patients with health care contacts abroad, culturing of patients with skin infections contracted outside Sweden and performing contact tracing among household members. Knowledge of spa type might give guidance in the process of contact tracing. Eradication treatment of MRSA spa types causing more skin infections may be warranted.

Unusual increase of psittacosis in southern Sweden linked to wild bird exposure, January to April 2013.

Free-living wild birds worldwide act as reservoir for Chlamydia psittaci, but the risk of transmission to humans through contact with wild birds has not been widely documented. From 12 January to April 9 2013, a total of 25 cases of psittacosis were detected in southern Sweden, about a threefold increase compared with the mean of the previous 10 years. A matched case-control study investigating both domestic and wild bird exposure showed that cases were more likely than controls to have cleaned wild bird feeders or been exposed to wild bird droppings in other ways (OR: 10.1; 95% CI: 2.1-47.9). We recommend precautionary measures such as wetting bird feeders before cleaning them, to reduce the risk of transmission of C. psittaci when in contact with bird droppings. Furthermore, C. psittaci should be considered for inclusion in laboratory diagnostic routines when analysing samples from patients with atypical pneumonia, since our findings suggest that psittacosis is underdiagnosed.

Clinical and microbiological features of bacteraemia with Aerococcus urinae.

Aerococcus urinae is a Gram-positive bacterium that can cause invasive infection, including infectious endocarditis (IE), mainly in older men. A. urinae is often misclassified in routine diagnostic laboratories. Through searches in the laboratory databases we identify 16 isolates of A. urinae causing bacteraemia during a 6-year period in southern Sweden, indicating that bacteraemia with A. urinae occurs in at least three cases per million inhabitants per year. The identity of isolates was confirmed by sequencing of the 16S rRNA genes and antibiotic susceptibility testing identified two ciprofloxacin-resistant isolates. A. urinae was the only significant pathogen isolated in all cases. Fifteen of the 16 patients were male, 15/16 were more than 70 years old, and 12/16 had underlying urological conditions. Though a urinary tract focus was suspected in the majority of cases, the bacterium was rarely found in urinary samples. Nine patients fulfilled the criteria for severe sepsis and an additional four fulfilled the criteria for sepsis. Only one fatality was recorded. Patients were treated mainly with beta-lactam antibiotics but fluoroquinolones and clindamycin were also used. Three cases of IE were diagnosed and these were complicated by spondylodiscitis in one case and by septic embolization to the brain in one case. An increased awareness of A. urinae is crucial to establishing its role as an important pathogen in older men with urinary tract disease.

Evaluating the usefulness of spa typing, in comparison with pulsed-field gel electrophoresis, for epidemiological typing of methicillin-resistant Staphylococcus aureus in a low-prevalence region in Sweden 2000-2004.

The usefulness of spa typing was evaluated in relation to pulsed-field gel electrophoresis (PFGE), as a tool for epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) in a low-prevalence region in southern Sweden. Bacterial isolates from 216 MRSA cases, newly identified in 2000-2004, were studied. The isolates were obtained from infected patients (31%), and from colonized individuals found by screening (69%). In total, 49 spa types and 73 PFGE patterns were identified. The discriminatory power of spa typing was lower (94.9 +/- 1.8%) than that of PFGE (97.3 +/- 1.2%). For two spa types (t002 and t008) the Panton-Valentine leukocidin results added useful discriminatory information. The most common spa types were t044 (n = 31; four PFGE patterns), t002 (n = 24; 10 PFGE patterns), t067 (n = 12; four PFGE patterns), t050 (n = 12; one PFGE pattern), and t324 (n = 11; one PFGE pattern). Epidemiological investigations identified 91 single cases and 39 transmission chains, each involving two to 13 cases. All the transmission chains were held together both by spa and PFGE typing. Among the 91 single-case isolates, 33 spa types and 50 PFGE patterns were unique (matchless) at the time of identification. The low prevalence of MRSA, the low number of outbreaks, and the wide spectrum of strains due to frequent acquisitions abroad (49% of the cases), makes spa typing a useful complement to epidemiological investigations in our setting. However, we still recommend the continued use of PFGE for further discrimination of isolates with identical spa types when epidemiological data can not exclude possible transmission.

Disk with high oxacillin content discriminates between methicillin-resistant and borderline methicillin-susceptible Staphylococcus aureus strains in disk diffusion assays using a low salt concentration.

A separation between mecA+ strains of Staphylococcus aureus and strains lacking mecA was achieved by the disk diffusion assay and the agar dilution method, utilizing disks containing 5 microg of oxacillin and inocula of approximately 5 x 10(5) CFU/spot, respectively, provided that agar with 0 to 0.5% NaCl and incubation at 30 degrees C were employed. The 5-microg oxacillin disks clearly discriminated between borderline methicillin-susceptible and mecA+ strains. The oxacillin MICs were more affected by the inoculum density and salt concentration than were the methicillin MICs, and oxacillin MICs of 4 to 16 microg/ml were obtained for strains lacking mecA. Significantly higher levels of beta-lactamase production and reduced oxacillin susceptibilities were recorded for strains lacking mecA, in particular strains of phage group V, when agar with >/=2% NaCl was used than when agar with 0 to 0.5% NaCl was employed. The results indicate that the borderline methicillin-susceptible phenotype is a salt-dependent in vitro phenomenon of questionable clinical relevance.

Antibiotic-resistant strains of Enterococcus isolated from Swedish and Danish retailed chicken and pork.

Two hundred and seventy-seven Enterococcus isolates representing the predominating enterococcal flora of retailed chicken and pork were identified by phenotypical features, and by random amplified polymorphic DNA. The resistance to nine different antibiotics was determined. Enterococcus faecium was the most frequently occurring species in both Swedish and Danish chicken. Enterococcus faecalis and unidentified groups of Enterococcus dominated in pork. Seventy-three per cent of the Enterococcus isolates from Swedish chicken were resistant to one or more of the tested antibiotics. The corresponding values for Swedish pork, Danish chicken and Danish pork were 9%, 55% and 14%, respectively. Tetracycline resistance was most frequent in isolates from Danish pork and Swedish chicken, while erythromycin resistance was most frequent in Danish chicken. Strains with acquired vancomycin resistance, mainly Ent. faecium, were found only on Danish chicken, except for one isolate from Danish pork. All vancomycin-resistant isolates contained the vanA gene. Vancomycin resistance could be transferred to a vancomycin-sensitive Ent. faecium strain from four of nine tested donor strains.

Identification of clinically important species of Enterococcus within 1 day with randomly amplified polymorphic DNA (RAPD).

The use of randomly amplified polymorphic DNA (RAPD) for rapid, reliable, and easily interpreted identification of enterococci was evaluated. Nineteen type strains of Enterococcus, 12 reference strains, and 114 clinical isolates of Enterococcus were analyzed. Discrimination was obtained between most type strains, the exceptions being Ent. casseliflavus and Ent. flavescens, which had relatively similar RAPD-profiles. Ent. faecalis and Ent. faecium were readily separated, and Ent. gallinarum and Ent. durans could also be identified. Extracts to be used in the polymerase chain reaction (PCR) were prepared directly from agar plate colonies, which made it possible to complete the identification procedure in one day. RAPD was proved to be a fast and reliable method for identification of most Enterococcus spp. of clinical significance.

In vitro anti-staphylococcal activity of heparinized biomaterials bonded with combinations of rifampicin.

Biomaterial implants in various human body tissues are highly susceptible to bacterial colonization. We report here on the coating of heparinized biomaterials with heparin binding extracellular matrix proteins giving special regard to the efficient adsorption and slow release of antibiotics. Heparin was partially degraded and the resulting fragments were covalently end-point attached to 0.5 cm long silicone biomaterial surface. Collagen type I was immobilized on the heparinized biomaterials and then cross-linked with acyl-azide or carbodiimide. Finally, the resulting biosurfaces were exposed to antibiotics, i.e. rifampicin in combination with cefuroxime, fusidic acid, ofloxacin or vancomycin, respectively. The antibiotic bonded biomaterials were evaluated for their anti-staphylococcal activity after elution in NaCl, serum or blood by measuring the zones of inhibition for S. epidermidis strain RP12. Furthermore, we examined the in-vitro colonization resistance to S. epidermidis RP12 for these combinations of rifampicin-bonded biomaterials by an ATP bioluminescence assay. The ATP measurements showed that initially adherent bacteria were eradicated from the polymer surface, for at least 24 or 48 h (fusidic acid > cefuroxime > vancomycin > ofloxacin). The anti-staphylococcal activity of rifampicin-fusidic acid bonded heparinized biomaterials seems of sufficient duration and efficacy to merit testing in an animal model.

Identification of mecA-related oxacillin resistance in staphylococci by the E test and the broth microdilution method.

A set of 165 strains of different staphylococcal species, 67 Staphylococcus aureus, 71 novobiocin-sensitive coagulase-negative staphylococci (CNS) and 27 novobiocin-resistant CNS was used. The oxacillin and methicillin MICs were recorded after 24 and 42 h of incubation at 35 degrees C and at 30 degrees C. Significantly higher MICs were recorded at 30 degrees C compared with 35 degrees C. While a poor discrimination between mecA-positive and mecA-negative strains was obtained with methicillin, the oxacillin MICs enabled identification of resistant strains under certain conditions. The distribution of MICs differed between the three groups of species. Separation of uninduced mecA-positive (> or = 4.0 mg oxacillin/L) and mecA-negative (< or = 2.0 mg oxacillin/L) strains of S. aureus was only achieved with the E test and after 42 h of incubation. Oxacillin-induction yielded higher MICs for mecA-positive strains of S. aureus, and a separation from mecA-negative strains was achieved with the E test after 24 h and with the broth microdilution method after 42 h. Separation of mecA-positive and mecA-negative strains of novobiocin-sensitive CNS required agar supplemented with 5% blood, incubation of MIC trays and E test for 42 h, and species-specific oxacillin MIC breakpoints (S < or = 0.5 mg/L and R > or = 1.0 mg/L). The mecA-positive and mecA-negative strains of novobiocin-resistant CNS were clearly separated after 24 h of incubation by either method.

Species-specific identification of methicillin resistance in staphylococci.

The ability to identify methicillin-resistant staphylococci by the disc diffusion method was evaluated using discs containing oxacillin (1, 5 and 10 micrograms), methicillin (10 micrograms) and cephalexin (30 micrograms). Strains of Staphylococcus aureus (67 strains) and coagulase-negative staphylococci (72 novobiocin-sensitive and 27 novobiocin-resistant strains) were studied using two inoculum densities (10(6) cfu/ml and 10(8) cfu/ml). Inhibitory zones were recorded after 18, 24 and 42 hours of incubation. A mecA-specific application of the polymerase chain reaction was used as a reference method. The inoculum of 10(8) cfu/ml and incubation for 24 hours were optimal for the identification of methicillin-resistant strains. However, one single disc was not sufficient for the identification of methicillin resistance in the different staphylococcal species. The mecA-positive strains of Staphylococcus aureus and novobiocin-resistant coagulase-negative species were clearly separated from the mecA-negative strains when the 5 micrograms oxacillin disc was used, whereas the 1 microgram oxacillin disc was optimal for the identification of the mecA-positive novobiocin-sensitive coagulase-negative strains.

Serum and tissue protein binding and cell surface properties of Staphylococcus lugdunensis.

Eleven strains of Staphylococcus lugdunensis from different clinical sources were investigated for their ability to bind 125I-labelled collagen (Cn) type I and IV, fibronectin (Fn), vitronectin (Vn), laminin (Lm), fibrinogen (Fg), thrombospondin, plasminogen (glu- and lys-form) and human IgG. All the strains bound these proteins, although a higher degree of binding was obtained for Cn types I and IV and IgG with mean values of 36%, 32% and 26% binding, respectively. In tests with proteins immobilised on latex beads in a particle agglutination assay, eight of the 11 strains bound Cn type I and seven bound Fg, whereas no strain bound immobilised IgG. Binding to immobilised Cn-I, Fg, Lm and Vn was abolished when the bacterial cells were treated with proteases or heat, indicating cell-surface receptors with protein characteristics. Cell-surface extracts of S. lugdunensis 2342 were able to totally inhibit binding of the homologous strain and S. aureus Cowan 1 to latex-immobilised proteins Cn-I, Lm, Vn, Fn and Fg. The binding of 125I-labelled Cn IV by S. lugdunensis 2342, was heat sensitive, whereas the binding to S. aureus Cowan 1 was heat resistant. The strains gave negative results in tests for the presence of protein A with a S. aureus protein A gene probe and with sensitised red blood cells. No production of heat-stable nuclease (TNase) could be detected by monoclonal antibodies against TNase or by the polymerase chain reaction with an oligonucleotide sequence from S. aureus TNase as primer.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effect on group A streptococci of loracarbef versus cefadroxil, cefaclor and penicillin V.

The in vitro activity of loracarbef, penicillin V, cefaclor and cefadroxil against log and stationary phase cultures of group A streptococci was compared. MICs and MBCs were determined with the broth dilution method and by a modified agar plate dilution technique where the beta-lactams were inactivated after the MICs were determined allowing inhibited but not killed organisms to grow on further incubation. The MICs of loracarbef and the two cephalosporins were 16-32 times higher than those of penicillin V. In plate dilution the MBC/MIC ratios of all agents were < or = 2 for log phase cultures. With stationary phase cultures, especially in the broth dilution test, the MBC/MIC ratios of loracarbef and the two cephalosporins were > or = 32 for a large number of strains. The phenotype response of stationary phase cultures to beta-lactam antibiotics may not only be related to the physiological status of the streptococci, to the culture conditions and to the beta-lactam under test. The present investigation indicated that the phenotypic response was also an intrinsic property of certain strains.

Evaluation of four qualitative methods for detection of beta-lactamase production in Staphylococcus and Micrococcus species.

Four qualitative methods for the detection of beta-lactamase production in Staphylococcus and Micrococcus species were evaluated and compared with a quantitative macroiodometric reference method. The disc diffusion test with penicillin G and the cloverleaf method could not separate beta-lactamase-positive from beta-lactamase-negative strains. Two applications of the chromogenic cephalosporin test, using uninduced strains and strains grown on blood agar plates, gave a large number of false negative and false positive results. False negative reactions were most common among uninduced strains, while the false positive reactions were most often recorded for Staphylococcus saprophyticus. A high degree of efficiency was recorded for the nitrocefin spot test, using induced strains grown on antibiotic susceptibility agar, and for the starch-iodine plate method. The starch-iodine plate with methicillin as inducer gave the most reliable results.

Determination of interpretive breakpoints for ceftazidime disc-diffusion susceptibility testing using single-strain regression analysis.

Interpretive breakpoints for ceftazidime disc-diffusion susceptibility testing were determined using single-strain regression analysis (SRA). Regression lines were determined for a total of 58 strains representing 15 species, from inhibition zone diameters obtained for discs containing six different ceftazidime concentrations. Statistical analysis for excluding non-linearity of test-results was performed. A minimum of five tests on consecutive days was required for maximal precision of regression analysis according to the SRA-method. Calculated regression lines showed similarities within individual and groups of bacterial species. A minimum of five strains could be used to represent these groups. Interpretive breakpoints according to recommended MIC-limits were determined for each species taking into consideration confidence limits for zone correlates of MIC-values. Single-strain regression analysis for the determination of interpretive breakpoints for ceftazidime disc-diffusion susceptibility testing in individual laboratories.

Determination of species- and laboratory-related interpretive breakpoints for doxycycline susceptibility testing using single-strain regression analysis.

Single-strain Regression Analysis (SRA) was performed for doxycycline on a total of 68 bacterial strains representing 16 different species. Species- and laboratory-related zone diameter breakpoints were determined and compared with histograms of zone diameter values obtained from 942 routine susceptibility tests. Calculated breakpoints were similar within individual species. When considering the homogeneity of susceptibility groups within bacterial species, the calculated breakpoints gave rise to relatively few interpretive errors. In contrast, general breakpoints for doxycycline as recommended by the Swedish Reference Group (SRG) (R less than = 20 mm and S greater than = 26 mm) would give rise to a high proportion of false interpretations in the present laboratory. Fifty-three per cent of H.influenzae strains would have been assigned to the wrong susceptibility group. For E.coli and K.pneumoniae, 22 and 35 per cent, respectively, would have been erroneously categorized using SRG breakpoints. E.cloacae and E.aerogenes would have been assigned another category in 39 and 50 per cent, respectively. The procedure for setting species-specific and laboratory-related interpretive breakpoints is described. Determination of species- and laboratory-related interpretive breakpoints using SRA provides a new approach towards improved accuracy of disc-diffusion susceptibility testing.

Single-strain regression analysis for quality control of cephalothin-susceptibility testing and determination of interpretive breakpoints.

Histogram analysis of inhibition zone diameters around the 30 micrograms cephalothin disk for E. coli, P. mirabilis, and K. pneumoniae in samples from 1975 to 1982 showed a marked reproducibility of the disk-diffusion antibiotic-susceptibility test in the routine laboratory. A comparison of interpretive breakpoints with histograms for E. coli, P. mirabilis, K. pneumoniae, S. aureus, coagulase-negative staphylococci, and S. faecalis showed a higher proportion of possible misinterpretations using the breakpoints of the Swedish Reference Group, SRG, as compared to international (NCCLS) breakpoints. Further analysis using single-strain regression analysis revealed two major causes of interpretive errors. Firstly, the laboratory-related regression line for a bacterial species can be different from the general regression line of the reference laboratory. This difference has to be corrected by using species-related breakpoints. For E. coli, a species-specific breakpoint was determined to R = greater than 13 mm. Secondly, MIC limits recommended for the susceptibility categories of cephalothin by SRG are lower than the international limits and close to the true MIC values of many bacterial isolates, leading to misinterpretations due to the methodological variation. These studies suggest an adoption of international MIC limits for the susceptibility categories of cephalothin in Scandinavia. The "I" category should denote an indeterminate zone. A multi-laboratory quality control assessment using histogram analysis is recommended with optional single-strain regression analysis to determine breakpoints for problem combinations of bacterial species and antibiotics.