Alarming spread of vancomycin resistant enterococci in Sweden since 2007.

The total number of persons infected or colonised with vancomycin-resistant enterococci mandatorily reported to the Swedish Institute for Infectious Disease Control increased dramatically during 2007 and 2008. During a period of twenty months from 1 July 2007 to 28 February 2009, a total of 760 cases were reported compared with 194 cases reported during the entire period from 2000 to 2006. This rise was mainly attributed to a wide dissemination of vancomycin resistant enterococci which started in a number of hospitals in Stockholm in the autumn of 2007 and was followed by dissemination in various healthcare facilities (hospitals and homes for the elderly) in a further two Swedish counties in 2008. The majority of the cases (97%) were acquired in Sweden and among these, healthcare-acquired E. faecium vanB dominated (n=634). The majority of these isolates had identical or closely related pulsed-field gel electrophoresis patterns indicating clonal dissemination in the affected counties. The median minimum inhibitory concentration of vancomycin was 32 mg/L (ranging from 4 to >128 mg/L) and of teichoplanin 0.12 mg/L (ranging from 0.06 to 0.25 mg/L). Particular emphasis was placed on countermeasures such as screening, contact tracing, cleaning procedures, education in accurate use of infection control practices as well as increasing awareness of hygiene among patients and visitors. With these measures the dissemination rate decreased substantially, but new infections with the E. faecium vanB strain were still detected.

Genetic diversity of community-associated methicillin-resistant Staphylococcus aureus in southern Stockholm, 2000-2005.

This study investigated the molecular epidemiology of 104 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates from southern Stockholm during the period 2000-2005. The isolates were analysed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, staphylococcal chromosomal cassette (SCC) mec typing and detection of genes encoding Panton-Valentine leukocidin (PVL). Overall, 28 distinct PFGE patterns and 13 sequence types (STs) were identified. ST80, ST8, ST88 and ST150 were the major CA-MRSA clones in the area, and these accounted for 75% (78/104) of all CA-MRSA isolates. ST150 isolates, which have, to date, been found only in Sweden, were isolated exclusively from a group of homeless individuals. Eighty-six (83%) of the 104 isolates in the study possessed SCCmecIV, found in ten different STs, while 16 isolates possessed SCCmecV. The PVL genes were detected in 56% (58/104) of the isolates. Strain ST80-MRSA-IV carrying PVL genes predominated over the 6-year period and accounted for 38% of all isolates. However, a polyclonal tendency was observed among the CA-MRSA isolates recovered in recent years.

Emergence of VanD-type vancomycin-resistant Enterococcus faecium in Stockholm, Sweden.

A vancomycin-resistant Enterococcus faecium isolate from the urine of a liver transplant patient in Stockholm was found to contain a vanD gene. The sequence of the vanD PCR product shared 100% identity with the vanD5 allele. The isolate was resistant to a relatively high level of vancomycin (128 mg/L) and a low level of teicoplanin (4 mg/L). This is the first VanD-type vancomycin-resistant E. faecium isolate reported in Sweden. The emergence of this strain reinforces the necessity of infection control efforts to interrupt the spread of these organisms.

A case-cohort study to investigate concomitant waterborne outbreaks of Campylobacter and gastroenteritis in Söderhamn, Sweden, 2002-3.

Increased domestic, laboratory confirmed, Campylobacter notifications were reported in Siderhamn municipality, December 2002 and January 2003. Concurrently, during preliminary investigations a large outbreak of acute gastroenteritis was detected. Simultaneously, two studies were completed to identify risk factors for infection with Campylobacter and acute gastrointestinal infection (AGI): (1) a case-cohort study using Campylobacter cases (N = 101) with a large random sample from the municipal population as referents (N = 1000) and (2) a retrospective cohort study for the outcome AGI using the same sample. A postal questionnaire was used to collect demographic, clinical, water and food consumption data. Measures of association (risk ratio (RR), odds ratio (OR)) and 95% confidence intervals (CI) were calculated. Stool, environmental and water samples were tested by standard methods at Gävle Hospital and SMI laboratories respectively. In the case-cohort study, Camplylobacter cases were more likely than referents to consume communal water (OR = 12.6 (95% CI 1.7-92.3)). In the cohort study, risk of gastroenteritis was 2.3 times higher in those who consumed water (AR = 27.3%) than others (AR = 12%). Risk of illness was associated with the amount of water consumed in both studies. Campylobacter was detected in stools and Escherichia coli (E. coli) from routine communal water (CW) samples. Results suggest both Söderhamn outbreaks of Campylobacter and AGI were associated with consumption of CW. The method used strengthened epidemiological evidence and was efficient in the use of time and resources.

Comparison of genotypic and phenotypic methods for species identification of coagulase-negative staphylococcal isolates from blood cultures.

Coagulase-negative staphylococci are often isolated from blood cultures. Simple methods are needed for correct identification of those species most frequently found. In this study, PCR methods were developed for the identification of S. epidermidis and S. haemolyticus, based on DNA sequences in their 16S rDNA. The results obtained by these methods were compared with those obtained using a number of phenotypic methods, including two commercial kits API Staph and Staphzyme. Fifteen type collection strains and 133 blood culture isolates were tested. The sensitivity of the PCR for identification of S. epidermidis was 99% compared with API Staph, but the specificity was lower, 94%, because of positive results also for S. capitis. The results by the PCR for identification of S. haemolyticus correlated closely with the Staphzyme results, 13 isolates being identified by Staphzyme and 16 by the PCR. API Staph, however, identified only four clinical isolates as S. haemolyticus, probably too few. Among the individual phenotypic tests performed, a trehalose-mannitol agar method and a desferrioxamine disc diffusion test for the identification of S. epidermidis were found to be very accurate. Anaerobic growth after overnight incubation could be used to distinguish S. epidermidis from S. hominis. The conclusion is that a majority of all Gram-positive, catalase-positive and coagulase-negative blood culture isolates can be typed as regards species level using only a few genotypic and/or phenotypic tests.

Detecting methicillin-resistant Staphylococcus epidermidis--disc diffusion, broth breakpoint or polymerase chain reaction?

Growth conditions are important for the expression of resistance to methicillin among staphylococci. Consequently a phenotypic susceptibility test has to be chosen carefully to avoid false susceptible results. In this study we wanted to devise rapid and simple phenotypic tests whose results completely correlate with the presence of the methicillin resistance gene, mecA. A simplified polymerase chain reaction (PCR) method not needing separate DNA extraction from the tested bacteria was used to amplify a 449 bp region of the mecA gene. One hundred and ten strains of S. epidermidis were tested. The results were in complete agreement with those from a broth tube breakpoint test, known to identify more strains as resistant than does the method recommended by NCCLS. In disc diffusion test it was possible to clearly distinguish resistant from susceptible strains by using discs containing oxacillin, cephalexin and cephradine. A 5 micrograms cephradine disc was further analysed by testing another 441 consecutive clinical isolates of staphylococci. All resistant coagulase-negative staphylococci grew out to the edge of this disc, whereas susceptible strains showed an inhibition zone at least 10 mm in diameter. The 5 micrograms cephradine disc is recommended for routine work. The PCR method and broth tube breakpoint test are both reliable reference methods.

Urethritis associated with Chlamydia trachomatis: comparison of leukocyte esterase dipstick test of first-voided urine and methylene blue-stained urethral smear as predictors of chlamydial infection.

The use of nucleic acid amplification tests for the diagnosis of C. trachomatis has made it possible to send urine samples instead of urethral swab specimens to the laboratory. The sensitivity is very high, but not 100%, and we continue to perform a test for urethritis at our STD clinic. The aim of this study was to compare the performance of two alternative tests in the diagnosis of urethritis as predictors of C. trachomatis infection: the leukocyte esterase (LE) dipstick test of first-voided urine and polymorphonuclear leukocyte counts in a methylene blue-stained (MBS) urethral smear. Urine samples from 480 male patients attending an STD clinic were analysed using the LE test and LCR assay for C. trachomatis; urethral samples were analysed with MBS urethral smear and LCR. The majority (75.8%) of the 480 patients examined were asymptomatic. Chlamydial infection was detected in 50 patients. The sensitivity, specificity and positive predictive value of the LE test for predicting C. trachomatis infection were 46.0, 91.6 and 39.0%, respectively, among all patients examined and 25.9, 95.8 and 33.3%, respectively, among the asymptomatic patients. The corresponding values for the MBS urethral smear were 76.0, 82.1 and 33.0% among all patients and 63.0, 89.6 and 32.7% among the asymptomatic patients. At our STD clinic we chose to perform the examination of MBS urethral smears in the diagnosis of urethritis because of its higher sensitivity relative to the LE test for predicting C. trachomatis.

A comparison of methods to determine whether clinical isolates of Staphylococcus epidermidis from the same patient are related.

Staphylococcus epidermidis is a major cause of hospital-acquired infections but also part of the normal skin flora. A common clinical question is whether repeated isolation of S. epidermidis from one patient represents the same strain; because if different strains are isolated, they are often thought to be contaminants. In this study, different typing methods were compared to answer this question. Twenty isolates of S. epidermidis from five different patients were investigated. The isolates from each patient had identical or very similar antibiograms, and were recovered on different occasions. Typing was performed by antibiogram, biotype, slime production, plasmid profile, and pulsed-field gel electrophoresis (PFGE) banding pattern of SmaI digests of chromosomal DNA. In addition, the level of resistance to methicillin was determined by growth curves in broth containing methicillin for a series of different inocula for each isolate. The results showed that the isolates from each patient belonged to the same clone, but examples of instabilities in their antibiograms, plasmid profiles, as well as their PFGE banding patterns were seen. A change in the level of methicilli, resistance was observed in one strain; otherwise this characteristic was found to be strain-specific and stable in vivo. It was concluded that in combination with biotyping and antibiotic resistance testing the level of resistance to methicillin could be used as an aid to distinguish between two or more clinical isolates of S. epidermidis from the same patient.

Enhanced ability to colonize the skin: a possible explanation for the epidemic spread of certain strains of Staphylococcus epidermidis.

Experimental skin colonization was attempted on healthy volunteers using one epidemic and two non-epidemic strains of Staphylococcus epidermidis isolated from a bone marrow transplant unit. Although the three strains had similar biochemical reactions, they had different antibiograms and plasmid patterns, and the epidemic strain grew rather more slowly when in a mixture in broth. Two experiments involving sets of 5 volunteers were performed. The epidemic strain was mixed with one non-epidemic strain for experiment 1, and with the other for experiment 2. Each volunteer had an inoculum of a mixture of 10(7) cfu of each strain inoculated onto the antecubital fossae of both arms; one of the arms had had a prior treatment with chlorhexidine to see if this would prevent colonization. Quantitative skin cultures were continued until the test strains could no longer be isolated. Colonization occurred in all but one volunteer, and lasted from a few weeks to 17 months. Maximal counts of the epidemic strain were significantly higher than the maximal counts of the non-epidemic strains. Chlorhexidine had no effect in experiment 1, and caused a reduction in intensity and duration of colonization in experiment 2, although this did not achieve statistical significance. Plasmid patterns were unchanged throughout, but in two instances a variant of the epidemic strain that had lost resistance to methicillin and tobramycin was isolated together with the parent strain. The enhanced ability of the epidemic strain to colonize skin may be an important factor in allowing cross-infection.

Multiply antibiotic-resistant Staphylococcus epidermidis in patients, staff and environment--a one-week survey in a bone marrow transplant unit.

The distribution of Staphylococcus epidermidis resistant to ciprofloxacin and/or gentamicin was studied in an isolation unit for patients undergoing bone marrow transplantation. During 1 week all such strains colonizing patients or staff members or found on the clothes of staff or in the air were investigated. Antibiograms and plasmid profiles were used for epidemiological typing. Thirty-one different antibiograms were found. A few strains were widely spread and dominated quantitatively. Staff colonization was 23%, but contamination of their clothes was 82%. Two strains which colonized three of the patients were widely spread in the air. They were found in the corridor and in every patient room, even where the patient was not colonized. The main source of the environmental contamination seems to have been patients. Possible routes of infection were direct airborne transmission as well as passive transfer via staff.

Daily scrub with chlorhexidine reduces skin colonization by antibiotic-resistant Staphylococcus epidermidis.

The aim of this study was to establish whether long-term use of chlorhexidine would prevent skin colonization by antibiotic-resistant Staphylococcus epidermidis. Ten nurses, working on a ward for haematological disorders, volunteered to participate in the test. They washed one arm every morning for three weeks with chlorhexidine gluconate, ('Hibiscrub' ICI Pharmaceuticals). The other arm served as a negative control. Samples from the antecubital fossa of both arms were taken two to three times a week during the wash period and two weeks thereafter, giving a total of 216 samples. The appearance of resistant S. epidermidis with different antibiograms was analysed. During the wash period the total bacterial counts and the counts of the resistant S. epidermidis strains on the test arm were both about one-tenth of those on the control arm, a significant difference (P < 0.05). Moreover, there were significantly fewer resistant S. epidermidis on the test arm, 1.3 per sample, than on the control arm, 2.5 per sample (P < 0.01). Most of the resistant S. epidermidis were only found once or a few times on the same site, after which they disappeared, though a few persisted on the skin even during 'Hibiscrub' washing. In an agar dilution test, chlorhexidine minimum inhibitory concentrations (MICs) of persisting strains were the same as for strains disappearing from the skin following 'Hibiscrub' washing, 1.0 or 2.0 mg l-1, but somewhat higher than MICs of strains isolated from healthy carriers outside the hospital whose MICs were 0.5 mg l-1. The relative contribution to the skin counts by those S. epidermidis strains found only occasionally were compared with those found repeatedly but no difference in reduction was found between these categories during 'Hibiscrub' washing.

Reduction of bacterial surface contamination in the hospital environment by application of a new product with persistent effect.

The benefit of routine surface disinfection in hospitals has been discussed. In this study we have investigated a new product, Appeartex. After application on surfaces a remnant effect is achieved due to the positive charge of the active molecule. We studied the persistent effect of Appeartex one day after application in both an experimental study in the laboratory and a field study in a hospital ward. Surfaces of bedside tables were investigated. In the experimental study, large inocula of >or=10(6)cfu of S. aureus or enterococci were inoculated on to well-defined areas which had been treated/not treated with Appeartex. One hour later, samples were taken with a swab rinse technique. A reduction in the number of viable bacteria in the magnitude 10-10(3) cfu was seen due to the effect of Appeartex. In the field study the effect on naturally occurring low level contamination was studied. Defined surfaces on bedside tables used by patients were treated/not treated with Appeartex. One day later, samples were taken with contact agar plates and with a new swab method using two sequential nylon flocked swabs. Significantly fewer bacteria were found on Appeartex-treated surfaces compared with untreated surfaces. The median counts on Appeartex-treated surfaces were 10 cfu/50 cm(2), and on untreated surfaces 20 cfu/50 cm(2). There was no significant difference in the number of bacteria found by culture of samples taken with the contact agar method compared with samples taken using the nylon flocked swab method.

New technique to take samples from environmental surfaces using flocked nylon swabs.

Environmental surfaces near infected and/or colonised patients in hospitals are commonly contaminated with potentially pathogenic micro-organisms. At present, however, there is no standardised method for taking samples from surfaces in order to perform quantitative cultures. Usually contact plates or swabs are used, but these methods may give different results. The recovery rate of traditional swabbing, e.g. cotton or rayon, is poor. With a new type of swab utilising flocked nylon, the recovery may be enhanced up to three times compared with a rayon swab. In this study, we inoculated reference strains of Staphylococcus aureus and Enterococcus hirae onto a bedside table and took samples 1h later when inocula were dry. Sequential samples were taken from the same surface. A new sampling technique using two sequential nylon swabs for each sample was validated. The efficiency of the sampling, percentage recovery of the inoculum and the variation of culture results obtained from repeated experiments are described. Enhanced efficiency and higher recovery of inoculum were demonstrated using two sequential flocked nylon swabs for sampling.

Staphylococcus epidermidis--hospital epidemiology and the detection of methicillin resistance.

Infections in immunocompromised patients and in patients with indwelling prosthetic devices are often caused by hospital strains of Staphylococcus epidermidis resistant to methicillin. Tests for the detection of methicillin resistance, indicating resistance to all beta-lactam antibiotics, were evaluated in order to define a suitable screening test. A broth tube breakpoint test with a large inoculum, 10(7) colony forming units (cfu), gave the highest recovery of resistant strains. False resistance due to hyperproduction of beta-lactamase was excluded. The results correlated completely with the detection of the resistance gene, mecA, by the polymerase chain reaction. In 2/3 of the resistant strains tested the expression of the methicillin resistance was heterogeneous, only one cell in 10(2) to 10(4) expressed the resistance within 72 h in both. In broth screening tests an inoculum of at least 10(6) cfu therefore was required to detect all resistant strains within 24 h. Using agar dilution, 48 h incubation must be considered. In disc diffusion tests reliable results were obtained after only 16 h of incubation when discs containing cephradine 5 and 30 micrograms, oxacillin 1 microgram or cephalexin 30 micrograms were used, and the first disc is recommended for routine work. The epidemiology of S. epidermidis strains resistant to ciprofloxacin and/or gentamicin was studied in an isolation unit for patients undergoing bone marrow transplantation. Antibiograms and plasmids were used for typing and 31 such strains were found. Of 54 staff members 10 were colonized in the nares only, two in the nares and perineum and one in the nares and stool. In ambient air and on the clothes of staff a few of the strains predominated quantitatively. These strains colonized the skin of some of the patients who seemed to be the main dispersers. Possible routes of cross-infection were indirect contact transfer via the hands and clothes of staff (82% of the clothes were contaminated), and direct as well as indirect airborne transmission. To study the effects of chlorhexidine on skin bacteria, ten nurses washed one arm with chlorhexidine-detergent every morning for 3 weeks; the other arm served as control. The depression of the normal skin flora did not lead to a colonization with more antibiotic-resistant hospital strains. During the wash period the counts of antibiotic-resistant S. epidermidis on the treated arms were significantly reduced compared with the control arms, as also were the number of different strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Orthostatic reactions and blood volumes after moderate physical activation during acute febrile infections.

In 18 patients suffering from viral, mycoplasma or bacterial infections, orthostatic reaction and total haemoglobin were measured after termination of fever and 1 and 3 months thereafter. The results of the 3 months control were considered to represent the individual's normal values. The patients were randomized into two groups, one of which was subjected to a physical activity programme when febrile and the other, serving as control group, was confined to bed according to traditional clinical routine. The orthostatic reaction was measured as the mean heart rate during 10 min tilting on a tilt table. Plasma and red cell volumes were calculated from total haemoglobin, haemoglobin concentration and erythrocyte volume fraction. After fever heart rate during tilt was, in both groups, significantly higher than at the 3 months control but it was lower in the trained group (86 +/- 4 beats/min) than in the control group (100 +/- 3 beats/min) (P less than 0.05). Plasma volume and red cell volume being both numerically reduced in our patients after fever, showed a significant increase 3 months after illness only in the untrained control group. The results suggests that physical activity during acute febrile infections prevents the illness/bed rest-induced orthostatic deterioration and blood volume reduction.

Screening tests for the detection of methicillin resistance in Staphylococcus epidermidis.

Methods to detect resistance to methicillin in Staphylococcus epidermidis were studied in order to find a rapid screening test suitable for routine use. One hundred and forty-nine clinical isolates, 16 isolates from skin of healthy people and two reference strains were studied. Hypersecretion of beta-lactamase as a cause of methicillin resistance was eliminated in the strains studied. Tube and microtitre breakpoint, agar breakpoint and disc diffusion methods were compared. The breakpoint for methicillin resistance used was 16 mg/L in broth and 10 mg/L in agar. The discs used contained 1 and 5 micrograms oxacillin and 5 and 10 micrograms methicillin. Turbidimetric measurements in broth during incubation were carried out using the Bioscreen analysing system. The skin strains were founf to be susceptible in all tests. Using an inoculum of 10(7) cfu/mL 111/149 clinical isolates were classified as resistant after incubation for 24 h at 35 degrees C using the tube and microtitre breakpoint tests, incubation for 72 h did not increase this rate. When an inoculum of 10(5) cfu/mL was used 73% of these strains were identified within 24 h and all within 72 h with the tube breakpoint test. Using the microtitre breakpoint test, with an inoculum of 10(5) cfu/mL or lower, all resistant strains were not detected within 48 h. All agar breakpoint tests required 48 h incubation for reliable results. Only the 1 microgram oxacillin disc always separated strains found to be resistant or susceptible in the tube breakpoint test. The zone of inhibition was clearly readable after 16 h of incubation at 35 degrees C.

Muscle cell leakage of myoglobin after long-term exercise and relation to the individual performances.

Muscle protein release was studied during and after prolonged exercise by means of serum myoglobin determinations. Increased serum myoglobin levels were regularly found after performance of long-term exercise. The correlations (P less than 0.001) to muscle enzymes indicated nonselective release from muscle cells. Myoglobin measured after completed exercise was correlated to the finishing time in a ski race, i.e., the fast skiers showed lower myoglobin levels than slow skiers. Inexperienced skiers and also skiers older than 49 years of age were found to have higher myoglobin levels than the others although the mean finishing times were similar. Myoglobin measured during and after 5 h of bicycle exercise showed large differences even though the relative work load was identical. Myoglobin started to rise 1.2 h (median) after the commencement of the bicycle exercise. The time at which myoglobin started to rise correlated (P less than 0.01) with the myoglobin levels after completed exercise and was not related to the physical fitness of the individuals. The results indicated considerable individual differences with regard to muscle protein leakage during exercise. The leakage is not simply related to the individual's apparent physical fitness but probably also to constitutional factors, such as age and metabolic capacity of the muscle cells.

Influence of exercise intensity on ERK/MAP kinase signalling in human skeletal muscle.

The mitogen-activated protein (MAP) kinase pathways have been highlighted as a possible link between exercise and adaptive changes in skeletal muscle. In this study, the effect of exercise intensity on the activation of the ERK/MAP kinase pathway was investigated in human skeletal muscle. One-leg exercise at low (40% maximal oxygen consumption, VO2max for 30 min) and high (75% VO2max for 30 min) intensity resulted in 11.5+8. I-fold and 39.7+/-6.3-fold (mean +/-SEM) increases in ERK1/2 phosphorylation (P<0.001), respectively. The phosphorylation of MEK1/2, the upstream kinase of ERK1/2, increased with exercise intensity (P<0.05) to 2.5+/-0.9 and 4.8+/-1.1 times the basal level at the low and high intensity, respectively. The statistical analysis revealed a systematic difference between basal, low and high intensity exercise levels for both kinases. There was no change in the phosphorylation of either kinase in the non-exercised leg. The phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB), a possible downstream target of the ERK/MAP kinase signalling pathway, was unaffected by exercise. The phosphorylation of ERK1/2 was significantly higher in purified freeze-dried compared to crude wet muscle after exercise, whereas the opposite pattern was observed for CREB. In conclusion, phosphorylation of ERK1/2 and MEK1/2 increases in an exercise intensity-dependent manner in human skeletal muscle and this seems to originate in the muscle fibres themselves.

The role of a commercial latex agglutination test in the diagnosis of group B streptococcal infection in neonates.

An open prospective multicenter study was conducted in order to evaluate the Wellcogen Strep B latex agglutination test in the diagnosis of group B streptococcal (GBS) infections in neonates. Twenty-three (5.9%) of 391 urine specimens and 5 (1.2%) of 404 sera assayed were positive. The results of the urine tests corresponded to a sensitivity of 0.78 for bacteremic, 0.50 for non-bacteremic and 0.53 for all GBS associated (bacteremic, non-bacteremic and suspected) infections. After 20-25-fold concentration of urine specimens the sensitivity increased to 1.0 for bacteremic, 0.67 for non-bacteremic and 0.78 for all GBS associated infections. The specificity of the test was high (0.93 for concentrated urines), and the predictive value of a positive test (Pvpos) was 0.68. A positive latex test was highly predictive of positive surface cultures for GBS (Pv pos = 0.83 after concentration).