Barbro Isaksson

Pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs.

The pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were investigated by studying the postantibiotic effect (PAE) and initial killing. The influence of Ca2+ and albumin on these drugs was also evaluated. The PAE was studied by use of bioluminescence assay of bacterial ATP. Daptomycin at clinically achievable concentrations produced a dose-dependent PAE on E. faecalis (0.6 to 6.7 h) and S. aureus (1.0 to 6.3 h). The long PAE of daptomycin was seen simultaneously with a potent dose-dependent initial killing assayed by viable count determination. The initial change in bacterial ATP was not as extensive as the decrease in viability. Vancomycin at corresponding concentrations produced shorter PAEs on E. faecalis (0.5 to 1.0 h) and S. aureus (1.3 to 1.8 h). This coincides with a weak non-dose-dependent initial change in viability and intracellular ATP. The MICs of vancomycin were not influenced by different Ca2+ concentrations or by the addition of albumin to the broth. The MICs of daptomycin for both strains were lowered, and the PAEs were prolonged with increasing concentrations of Ca2+ in the broth. The PAE of daptomycin was Ca2+ dependent to the same extent as the MIC was. In the presence of physiological concentrations of albumin and free Ca2+, the PAEs of daptomycin on both strains were reduced and the MICs were increased in comparison with the results obtained in pure Mueller-Hinton broth with approximately the same free Ca2+ concentration. This decrease in daptomycin activity was considered to be due to the albumin binding of daptomycin. Despite the albumin binding of daptomycin, the PAE produced on E. faecalis and S. aureus in the presence of a physiological free Ca2+ concentration was still over 6 h at clinically achievable concentrations.

Division of the genus Enterococcus into species groups using PCR-based molecular typing methods.

Broad-range 16S rDNA PCR (BR-PCR) applied to DNA from 32 clinical enterococcal isolates and 12 other enterococci from a clinical reference collection followed by species-specific hybridization analysis identified 25 strains of Enterococcus faecalis and 19 Enterococcus species. Randomly amplified polymorphic DNA (RAPD) analysis using UPGMA clustering on the same material revealed four different clusters at a similarity level of 49%. Based on partial 16S rDNA sequence analysis of variable regions V4 and V9, it was possible to divide the 19 type strains specifying the genus Enterococcus into 12 different 16S rDNA species groups. The type strain distribution then served as a template for the analysis of the other 44 strains which were assigned to four different species groups (a-d) based on their 16S rDNA motifs. There was good agreement with the RAPD clusters. Species group a was an individual species line containing 25 strains that were identified as E. faecalis. Group b also represented an individual species line of 12 strains identified as E. faecium. The remaining seven strains that formed species groups c and d could not be fully identified to species by this analysis. It was concluded that BR-PCR of 16S rDNA followed by partial sequence analysis of the PCR products is a reliable technique for the identification and classification of enterococci. Further division of unresolved species groups should be achievable if regions other than V4 and V9 of 16S rDNA are also analysed.

A descriptive study of blood exposure incidents among healthcare workers in a university hospital in Sweden

In an attempt to document blood exposure incidents and compliance with recommended serological investigations, universal precautions and incident reporting routines, data was collected from occupational injury reports during a two-year period. In addition, a sample of healthcare workers (HCWs) answered a questionnaire about blood tests and work routines. In a third part of the study some HCWs were asked about the type and actual frequency of incidents, together with the number of reported incidents during the two-year study period. Of a total of 473 reported occupational blood exposures, the majority came from nurses and the minority from physicians. Most reported incidents occurred on hospital wards. The most common incidents were needlestick injuries, and 35% occurred when the needle was recapped. Medical laboratory technicians (MLT) reported significantly more mucocutaneous incidents than other professionals (P < 0.01). In 10% of the incidents, the patient had a known blood-borne infection. Serological investigations post-exposure varied among professional groups, and 35% were not tested. No seroconversion was shown in the HCWs tested. In the third part of the study, respondents recalled 1180 incidents, although only 9% of these had been reported. The majority occurred in operating theatres, and in connection with anaesthesia. There was a significant difference (P < 0.001) between the different professional groups with regard to the frequency of incident reporting. Physicians reported only 3% and MLTs 36% of the incidents. Eighty-one percent believed that the accident could have been avoided. Despite knowledge of universal precautions, professionals continue to behave in a risky manner, which can result in blood exposure incidents.

Antibiotic prescription practices, consumption and bacterial resistance in a cross section of Swedish intensive care units

Background: The purpose of this work was to study usage of antibiotics, its possible determinants, and patterns of bacterial resistance in Swedish intensive care units (ICUs).

Wound contamination in cardiac surgery

Objectives: To investigate the degree of bacterial contamination in the sternal wound during cardiac surgery and the sternal skin flora after operation in order to increase our understanding of the pathogenesis of sternal wound infections. Design: Prospective study where cultures were taken peri‐ and postoperatively from sternal wounds and skin. Setting: University Hospital. Patients: 201 cardiac surgery patients. Results: 89% of the patients grew bacteria from the subcutaneous sternal tissue. 98% of the patients showed bacterial growth on the surrounding skin at the end of the operation. We found both commensal and nosocomial bacteria in the sternal wound. These bacteria had different temporal distribution patterns. Coagulase‐negative staphylococci (CoNS) and Propionibacterium acnes (PA) were by far the most prevalent bacteria during and after the operation. Furthermore, 41% of patients had more than 10 000 CFU/pad CoNS on the skin. There was no correlation between length of operation and number of bacteria. Men displayed higher bacterial counts than women on the skin. Conclusion: Skin preparation with ethanol/chlorhexidine is unable to suppress the physiological skin flora for the duration of a heart operation. A decrease of CoNS and PA postoperatively can be caused by competitive recolonisation of commensal and nosocomial bacteria.

Detection and characterisation of SCCmec remnants in multiresistant methicillin-susceptible Staphylococcus aureus causing a clonal outbreak in a Swedish county

The purpose of this study was to investigate if multiresistant methicillin-susceptible Staphylococcus aureus (MR-MSSA) causing a clonal outbreak in Östergötland County, Sweden, were derived from methicillin-resistant S. aureus (MRSA) by carrying remnants of SCCmec, and, if so, to characterise this element. A total of 54 MSSA isolates with concomitant resistance to erythromycin, clindamycin and tobramycin from 49 patients (91% clonally related, spa type t002) were investigated with the BD GeneOhm MRSA assay and real-time polymerase chain reaction (PCR) targeting the SCCmec integration site/SCCmec right extremity junction. DNA sequencing of one isolate representing the MR-MSSA outbreak clone was performed by massive parallel 454 pyrosequencing. All isolates that were part of the clonal outbreak carried SCCmec remnants. The DNA sequencing revealed the carriage of a pseudo-SCC element 12 kb in size, with a genomic organisation identical to an SCCmec type ΙΙ element, except for a 41-kb gap. This study demonstrates the presence of a pseudo-SCC element resembling SCCmec type II among MR-MSSA, suggesting possible derivation from MRSA. The presence of SCCmec remnants should always be considered when SCCmec typing is used for MRSA detection, and may not be suitable in locations with a high prevalence of MR-MSSA, since this might give a high number of false-positive results.

Modified pulsed-field gel electrophoresis protocol for typing of enterococci

Controlling the spread of vancomycin‐resistant enterococci (VRE) is an important task in hospital epidemiology. Pulsed‐field gel electrophoresis (PFGE) has become the golden standard for molecular epidemiological characterisation of enterococcal isolates. For separation of DNA fragments by PFGE, different electrophoresis conditions have been recommended, but none of these protocols allows a satisfactory separation of both small and large DNA fragments of enterococci simultaneously. In this study we have speeded up the preparation of chromosomal DNA and defined new electrophoresis conditions that enhance separation of small and large DNA fragments for subtyping of enterococci with a 24 h PFGE.

Postantibiotic and bactericidal effect of imipenem against Pseudomonas aeruginosa.

The postantibiotic effect of imipenem onPseudomonas aeruginosa was studied at different inocula using one ATCC strain and four clinical isolates. The postantibiotic effect was measured using two different methods: viable counts and bioluminescence assay of intracellular bacterial ATP. The postantibiotic effect could be demonstrated with both methods (viable counts 1–2 h, ATP assay 3–5 h) for all strains at an inoculum of 106 CFU/ml. When the inoculum was raised to 108 CFU/ml, no postantibiotic effect could be observed with either method using routine growth conditions. This disappearance of the postantibiotic effect coincided with a loss of bactericidal effect of imipenem when high inocula were used. Improved oxygenation of the cultures restored the bactericidal and postantibiotic effects of imipenem at high inocula.

Surveillance of antibiotic resistance in ICUs in southeastern Sweden

Background: A study was designed to assess a computer‐based program for continuous registration of antibiotic resistance, statistics concerning severity of illness, and consumption of antibacterial drugs.

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

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