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.

Processing methods for photoacoustic Doppler flowmetry with a clinical ultrasound scanner.

Photoacoustic flowmetry (PAF) based on time-domain cross correlation of photoacoustic signals is a promising technique for deep tissue measurement of blood flow velocity. Signal processing has previously been developed for single element transducers. Here, the processing methods for acoustic resolution PAF using a clinical ultrasound transducer array are developed and validated using a 64-element transducer array with a -6 dB detection band of 11 to 17 MHz. Measurements were performed on a flow phantom consisting of a tube (580 µm inner diameter) perfused with human blood flowing at physiological speeds ranging from 3 to 25 mm / s. The processing pipeline comprised: image reconstruction, filtering, displacement detection, and masking. High-pass filtering and background subtraction were found to be key preprocessing steps to enable accurate flow velocity estimates, which were calculated using a cross-correlation based method. In addition, the regions of interest in the calculated velocity maps were defined using a masking approach based on the amplitude of the cross-correlation functions. These developments enabled blood flow measurements using a transducer array, bringing PAF one step closer to clinical applicability.

From medical imaging data to 3D printed anatomical models.

Anatomical models are important training and teaching tools in the clinical environment and are routinely used in medical imaging research. Advances in segmentation algorithms and increased availability of three-dimensional (3D) printers have made it possible to create cost-efficient patient-specific models without expert knowledge. We introduce a general workflow that can be used to convert volumetric medical imaging data (as generated by Computer Tomography (CT)) to 3D printed physical models. This process is broken up into three steps: image segmentation, mesh refinement and 3D printing. To lower the barrier to entry and provide the best options when aiming to 3D print an anatomical model from medical images, we provide an overview of relevant free and open-source image segmentation tools as well as 3D printing technologies. We demonstrate the utility of this streamlined workflow by creating models of ribs, liver, and lung using a Fused Deposition Modelling 3D printer.

Further bacteriological evaluation of the TOUL mobile system delivering ultra-clean air over surgical patients and instruments.

Two mobile TOUL-400 units (types 1 and 2) that produce an exponential ultra-clean air flow (EUA) via a mobile screen were evaluated (maximum height from floor to centre of screen: type 1, 1.4m; type 2, 1.6m). Bacterial deposition rates were lowered by >60% (P=0.001) over a table area of 1.7 m (length)x1.0m (width) with the TOUL-400 type 1 unit, and the mean air count at 1.0m from the screen was reduced from 23 to 1.6 colony-forming units (CFU)/m3 in experiments in a room with six air changes/h (ACH). The corresponding reductions were two- to three-fold greater in an operating room (OR) with 16 ACH due to higher bacterial contamination levels in the control experiments. The dramatic but localized reduction of the deposition rate recorded on one 14-cm settle plate (>2376-fold at 0.8m from the screen in the OR) apparently reflected the focus of the EUA. The impact of the TOUL-400 unit was underestimated by almost 100-fold by the air counts of bacteria recorded in parallel at the same sampling point (26.5-fold reduction). During sham coronary angiography and sham hip arthroplasty performed in a room with six ACH, ultra-clean air (<10 CFU/m3) was obtained over the incision area with the TOUL-400 type 2 unit when the EUA was undisturbed (maximum screen-wound distance 1.7 m). In actual coronary angiography (room with six ACH, screen-wound distance 2.0-2.3m) and various surgical procedures in the OR (screen-wound distance 1.4-1.8m), ultra-clean air was obtained at the wound in three of 18 instances, characterized by undisturbed air flow and a maximum distance of 1.8 m. The newly developed TOUL-300 surgical instrument table (1.3-1.7 x 0.6m), equipped at one end with the same EUA unit as the TOUL-400 unit, was evaluated for a room with six ACH and an OR with 16 ACH. It yielded ultra-clean air at 0.8m (1.9 CFU/m3, 96% reduction, P=0.01) and reduced the deposition rate by >60% over most of the table surface. Simplified positioning of the screen or a longer reach, plus a mechanism for precise focusing of the air flow on to the wound area would increase the clinical utility of the TOUL EUA system.

Evaluation of a disk approximation test of inducible beta-lactamases in Enterobacteriacae and Pseudomonas aeruginosa.

A modified disk approximation test of inducible beta-lactamases in Enterobacteriacae and Pseudomonas aeruginosa was evaluated. The amount of inducer was adapted to produce the smallest possible zone of inhibition and the distance between the centre of the disks was standardized. Among several beta-lactam antibiotic disks tested, imipenem (0.06 microgram per disk) or cefoxitin (10 micrograms per disk) placed at a distance of 14-16 mm from a cefotaxime disk (30 micrograms) most efficiently revealed inducible beta-lactamases. A positive induction test (primarily expected with Enterobacter spp, Citrobacter freundii, Serratia spp, Pseudomonas aeruginosa and indole positive Proteus spp) may serve as a warning of the risk of selecting mutants with beta-lactamase mediated cross-resistance during systemic treatment with ureidopenicillins, later generations of cephalosporins or monobactams. Such resistance was exemplified by characterization of some laboratory mutants with hyperproduction of beta-lactamases. However, evaluation of the specificity and sensitivity of the disk approximation test (and other indirect induction tests) still remains to be done.

Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity.

To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.

beta-Lactamase substrate profiles of coagulase-negative skin staphylococci from orthopaedic inpatients and staff members.

Three different beta-lactamase substrate profiles were identified in 95 isolates of coagulase-negative staphylococci (CNS), of 57 different phenotypes, from 16 orthopaedic inpatients and staff members in one ward, by applying a bacterial whole-cell assay based on the hydrolysis of cefazolin, cephaloridine and nitrocefin. The typability of the assay was 93%, and 91% of the CNS isolates could be classified. To assess the discrimination between the beta-lactamase profiles obtained in the whole-cell assay, beta-lactamase extracts from 19 of the CNS isolates were used for estimation of their relative beta-lactamase substrate affinity index (RSAI). The RSAI assay was able to type previously unclassifiable or nontypable isolates. Two of the profiles obtained with the whole-cell assay were similar to those of the Staphylococcus aureus controls producing A or D and B or C beta-lactamases respectively. The distribution of beta-lactamase substrate profiles among the CNS isolates indicated an efficient spread of these drug resistance genes.

Single versus triple dose antimicrobial prophylaxis in elective abdominal surgery and the impact on bacterial ecology.

The results of 28 months of surveillance of postoperative wound infection rates in elective abdominal surgery using triple or single dose antimicrobial prophylaxis are reported. No significant differences in infection rates were seen in 217 patients undergoing gastric, biliary and pancreatic surgery who received either triple or single dose cefuroxime. Also, no significant differences in infection rates were seen in 198 patients undergoing jejunal, ileal and colorectal surgery who received either triple or single dose cefuroxime plus metronidazole. Our findings are consistent with those of other studies and support the conclusion that single dose prophylaxis is as effective as triple dose in elective abdominal surgery. During the study period there was no change in the pattern of bacteria isolated or in cefuroxime resistance rates from infected sites of patients from the Department of Surgery. No increased rate of cefuroxime resistance was seen in urinary isolates from patients elsewhere in the hospital.

Tympanostomy tubes for prevention of purulent otitis media.

A recently developed animal model was used to study the effect of tympanostomy tubes (TTs) on the spontaneous development of purulent otitis media. In 35 rats with soft-palate clefts a TT was inserted into the right tympanic membrane. The left ear was left intact. Serous effusion occurred in the attic space within 2 days after surgery, whether or not the middle ear cavity (MEC) was artificially ventilated. Between days 7 and 21 the intact-ear MEC was gradually filled with effusion material that turned purulent. Effusion material did not develop in the mesotympanum and hypotympanum of the intubated ears. Microbiologic examination of the effusion material showed a microflora similar to that in the nasopharynx. Ventilation through a TT reduced the number of colonized MECs (4 vs. 10) on day 21. In the individual culture-positive MEC with a TT there were fewer colonies than in the corresponding ear without a TT. These results support the contention that a TT may prevent the development of purulent otitis media.

Treatment of early recurrences of acute purulent otitis media; value of myringotomy.

The present investigation was carried out in order to study the efficacy of oral antibiotics (A) versus oral antibiotics plus acute myringotomy (B) in the treatment of early recurrences of acute purulent otitis media. Seventy-nine children with early recurrences (arisen within 4 weeks of a primary episode) were randomly allocated to one of the two treatment groups (A or B). Eleven of 41 (26.8%) children in group A, and 12/38 (31.6%) in group B had healed at 4 weeks (p greater than 0.1). Seventeen children of both groups had secretory otitis media at 4 weeks and new relapses had occurred in 13 children in group A and 9 in group B. No difference between the groups was noted regarding the number of otitis episodes during the next 5 months. Thus, acute myringotomy could not be proven to affect the clinical course of an early recurrence of acute purulent otitis media as compared with that after treatment with oral antibiotics alone.

Efficacy of Metronidazole in experimental Bacteroides fragilis otitis media.

The efficacy of metronidazole in otitis media due to Bacteroides fragilis was evaluated in a guinea pig model. Fifty-nine animals received an injection of 10(8) live B. fragilis bacteria through the tympanic membrane into the right middle ear cavity and metronidazole therapy was started 7 days later. On day 14 after challenge the animals were sacrificed and their middle ears analysed. Intraperitoneal injection of 6 mg metronidazole (about 20 mg per kg) once daily, 6 mg twice daily and 15 mg (50 mg per kg) once daily reduced the incidence of culture positive ears from 13/17 among untreated controls to 7/17 (p = 0.08), 3/10 (p = 0.05) and 2/15 (p = 0.001), respectively. The therapeutic efficacy of metronidazole in experimental otitis media was less than that expected from the concentrations of drug recorded in serum and the drug levels presumed to be achieved in the middle ear effusion.

Tympanostomy tubes prevent the development of experimentally induced purulent otitis media.

Cleavage of the soft palate will cause purulent otitis media bilaterally in 100% of experimental animals (the rat) within 2 weeks. In this model a tympanostomy tube was inserted unilaterally. At 2, 7, and 21 days after the surgery the middle ear status was mapped in the otomicroscope and microbiological samples collected from the middle ear cavities and the nasopharynx. All intact ears became infected and developed purulent otitis media. Most of the tubulated ears exhibited normal middle ear cavities and remained culture negative. The few ventilated ears which were infected, however, showed a lower bacterial count than the corresponding intact ear. Thus in this model the insertion of a tympanostomy tube prevents the purulent otitis media from occurring.

Experimental otitis media in guinea pigs. Serum antibody response to Bacteroides fragilis, Propionibacterium acnes and Peptostreptococcus anaerobius upon challenge.

The production of antibodies in serum was studied after inoculation of Bacteroides fragilis, Propionibacterium acnes or Peptostreptococcus anaerobius into the middle ear of guinea pigs. Inoculation of B. fragilis resulted in IgG and IgM serum antibodies at an early stage of the infection, while IgA antibodies were seldom detected and at a later stage. In contrast, inoculation of P. acnes or P. anaerobius induced no serum antibody response. Of these three species B. fragilis induced the most intense otitis, whereas P. anaerobius failed to induce otitis.

Relapse of acute purulent otitis media: antibiotic sensitivities of nasopharyngeal pathogens.

The present investigation was conducted to find out if a relapse of acute purulent otitis media is associated with a decreased sensitivity of nasopharyngeal pathogens to commonly used antimicrobial agents. All but one of 63 children with relapse included in this study yielded one or more of the classical middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Branhamella catarrhalis, S. pyogenes) in their nasopharynx (NPH) secretions. S. pneumoniae was the predominating isolate from NPH (71% of the patients) as well as from middle ear effusion (53%). At a control visit 4 weeks after the start of antibiotic therapy, 91% were NPH carriers of potential pathogens and S. pneumoniae was still the most common isolate (53%). Beta-lactamase was produced by 55% of B. catarrhalis isolates from the NPH specimens on the first visit, but only by 33% of B. catarrhalis isolates on the control visit. Two NPH isolates of H. influenzae produced beta-lactamase. One isolate of S. pneumoniae (serotype 18) was intermediately sensitive to phenoxymethylpenicillin. Generally low MICs were found for erythromycin and cefaclor. H. influenzae isolates were generally sensitive to ampicillin in vitro, but only 1 isolate was fully sensitive to erythromycin. B. catarrhalis isolates were uniformly sensitive to doxycycline and trimethoprim/sulphamethoxazole. No tolerance to penicillin was demonstrated in S. pneumoniae and H. influenzae. The present data indicate that the relapse of acute otitis media is not associated with development of tolerance or resistance to therapeutic antimicrobials commonly used.

Microbiological evaluation of a commercial transport system for urine samples.

The aim of this study was to evaluate a commercial tube prepared with boric acid, sodium formate and sorbitol (Hemogard Vacutainer tubes, Becton-Dickinson, HG tubes). Fourteen bacterial strains were incubated in urine in HG tubes and conventional tubes. During a 24-h period, most of the microorganisms grew readily in conventional tubes at room temperature, whereas the bacterial counts were comparatively unchanged when chilled or kept in HG tubes. The bacterial counts of Alcaligenes faecalis and lactobacilli decreased by two 10 logarithms in the HG tubes, at room temperature. Experiments were also performed with the addition of various antibiotics. Ciprofloxacin caused a decrease in E. coli counts regardless of type of tube used, while the HG tubes and the conventional tubes kept chilled conserved bacterial counts upon challenge with fosfomycin, trimethoprim or mecillinam. Bedside cultures from 154 outpatients were sampled and divided into three tubes. One conventional tube was sent to our laboratory by ordinary chilled transport. Another conventional tube and one HG tube were transported to the laboratory without chilling. Cultures were performed upon arrival at the laboratory and then 24, 48 and 72 h after primary sampling. Within 24 h of sampling, no significant differences in bacterial counts were observed between chilled conventional tubes and the HG tubes at room temperature. However, in the HG tubes a significant change in enterococcal counts was noted within 48 h.

Oxygen and serum complement in phagocytosis and killing of Propionibacterium acnes.

Phagocytosis and killing of Propionibacterium acnes by human peripheral blood leukocytes were promoted by normal human serum and by serum depleted of immunoglobulin G and M. Serum that had been heat-inactivated, depleted of complement component C3, or depleted of C4 and factor B, did not promote phagocytosis or killing. Depletion of complement-component C4 or factor B from normal human serum did not impair its capacity to promote phagocytosis and killing of P. acnes. These results indicate that phagocytosis of P. acnes is mediated by complement component C3, activated either by the classical or by the alternative pathway. P. acnes was phagocytosed as efficiently under anaerobic as under aerobic conditions. The bacteria were, however, killed at a considerably slower rate by anaerobically incubated leukocytes than by leukocytes incubated in air. Thus, oxygen dependent mechanisms were essential for killing of P. acnes.

Penicillin tolerance in Arcanobacterium haemolyticum.

It was previously found that Arcanobacterium haemolyticum, which can cause tonsillitis with exanthema, is not eradicated from the pharynx by administration of phenoxymethylpenicillin, despite minimum inhibitory concentration values of 0.015-1.0 micrograms/ml. Therefore, recent clinical isolates were studied for penicillin tolerance by using a disk diffusion screening test and a pour plate assay. Macrobroth dilution minimum inhibitory and bactericidal concentrations and antibiotic kill kinetics were determined for 4 isolates. Tolerance was present in 38 of 40 clinical isolates with the disk diffusion assay. With the pour plate assay all 40 isolates were tolerant, 34 of them highly tolerant. The presence of the tolerant phenotype was confirmed by macrobroth dilution assays. It is concluded that A. haemolyticum is often penicillin-tolerant, suggesting that phenoxymethylpenicillin administration would be ineffective in eradicating A. haemolyticum from the pharynx.

Drug-resistant coagulase-negative skin staphylococci. Evaluation of four marker systems and epidemiology in an orthopaedic ward.

Drug-resistant coagulase-negative staphylococci (DRCNS) in orthopaedic patients and ward staff were studied. A significant increase in the DRCNS carriage rate was observed among the 16 patients studied after 14 days of hospitalization with levels approaching that of the staff. Patients receiving dicloxacillin prophylaxis (n = 9) were more likely to be colonized with methicillin-resistant CNS, while patients receiving no antibiotics (n = 7) became to a larger extent colonized with multiple DRCNS. The combined data from species determination, biochemical, plasmid, and antibiogram typing revealed a considerable diversity among DRCNS; 64 types were distinguished among 112 DRCNS isolates selected for study after exclusion of apparently duplicate isolates. Plasmid plus antibiogram typing yielded almost as many types (61); whereas species determination plus antibiogram distinguished only 33 types. Although a novel computerized 96-reaction biotyping method alone enabled differentiation of 17 biotypes, most DRCNS isolates belonged to one of three major biotypes limiting the usefulness of this method. Ten of the 64 (16%) DRCNS types identified comprised 50 of the 112 (45%) isolates. These were isolated from staff and from patients on day 14, suggesting a nosocomial origin.