Evidence of persistence of Prevotella spp. in the cystic fibrosis lung.

Purpose. Prevotella spp. represent a diverse genus of bacteria, frequently identified by both culture and molecular methods in the lungs of patients with chronic respiratory infection. However, their role in the pathogenesis of chronic lung infection is unclear; therefore, a more complete understanding of their molecular epidemiology is required.Methodology. Pulsed Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) assays were developed and used to determine the degree of similarity between sequential isolates (n=42) from cystic fibrosis (CF) patients during periods of clinical stability and exacerbation.Results. A wide diversity of PFGE and RAPD banding patterns were observed, demonstrating considerable within-genus heterogeneity. In 8/12 (66.7 %) cases, where the same species was identified at sequential time points, pre- and post-antibiotic treatment of an exacerbation, PFGE/RAPD profiles were highly similar or identical. Congruence was observed between PFGE and RAPD (adjusted Rand coefficient, 0.200; adjusted Wallace RAPD->PFGE 0.459, PFGE->RAPD 0.128). Furthermore, some isolates could not be adequately assigned a species name on the basis of 16S rRNA analysis: these isolates had identical PFGE/RAPD profiles to Prevotella histicola.Conclusion. The similarity in PFGE and RAPD banding patterns observed in sequential CF Prevotella isolates may be indicative of the persistence of this genus in the CF lung. Further work is required to determine the clinical significance of this finding, and to more accurately distinguish differences in pathogenicity between species.

Infection control and meticillin-resistant Staphylococcus aureus decolonization: the perspective of nursing home staff.

BACKGROUND: Infection control and meticillin-resistant Staphylococcus aureus (MRSA) in nursing homes have started to assume greater importance in practice and policy. AIM: To explore infection control and MRSA decolonization in nursing homes in Northern Ireland from the perspective of nursing home staff. METHODS: Semi-structured interviews with nursing home managers and focus group discussions with nursing home staff were conducted, transcribed verbatim and analysed via the framework method. FINDINGS: Six one-to-one interviews and six focus group discussions (N = 7, 6, 6, 5, 5 and 4 participants, respectively) were conducted. Three overarching themes with inter-related subthemes were identified as influencing infection control and MRSA decolonization in the nursing homes: organizational factors (e.g. time, financial resources, environment, management and culture), external factors [e.g. hospitals, regulation and general practitioners (GPs)], and residents and families. It was reported that when the workload was unmanageable, aspects of infection control were not adhered to and more financial resources were necessary. There was conflict in maintaining an environment that was both 'homely' and clinical, and it was difficult to achieve good infection control practices with confused residents, some families, GPs and members of staff who were resistant to change. Support for MRSA decolonization in nursing homes was tempered by the risk of recolonization, particularly from hospital admissions. CONCLUSIONS: Infection control and MRSA decolonization in the nursing home environment appear to be affected by many factors, some of which may be beyond the direct control of staff.

Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis.

BACKGROUND: Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF. METHODS: Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods. RESULTS: Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (κ=0.74) and B cepacia complex (κ=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3×10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×10(6) cfu/g, p=0.046). CONCLUSION: Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

Cluster randomised controlled trial of an infection control education and training intervention programme focusing on meticillin-resistant Staphylococcus aureus in nursing homes for older people.

The aim of this cluster randomised controlled trial was to test the impact of an infection control education and training programme on meticillin-resistant Staphylococcus aureus (MRSA) prevalence in nursing homes. Nursing homes were randomised to intervention (infection control education and training programme; N=16) or control (usual practice continued; N=16). Staff in intervention homes were educated and trained (0, 3 and 6 months) in the principles and implementation of good infection control practice with infection control audits conducted in all sites (0, 3, 6 and 12 months) to assess compliance with good practice. Audit scores were fed back to nursing home managers in intervention homes, together with a written report indicating where practice could be improved. Nasal swabs were taken from all consenting residents and staff at 0, 3, 6 and 12 months. The primary outcome was MRSA prevalence in residents and staff, and the secondary outcome was a change in infection control audit scores. In all, 793 residents and 338 staff were recruited at baseline. MRSA prevalence did not change during the study in residents or staff. The relative risk of a resident being colonised with MRSA in an intervention home compared with a control home at 12 months was 0.99 (95% confidence interval: 0.69, 1.42) after adjustment for clustering. Mean infection control audit scores were significantly higher in the intervention homes (82%) compared with the control homes (64%) at 12 months (P<0.0001). Consideration should be given to other approaches which may help to reduce MRSA in this setting.

Efficacy of a standard methicillin-resistant Staphylococcus aureus decolonisation protocol in routine clinical practice.

Decolonisation may reduce the risk of methicillin-resistant Staphylococcus aureus (MRSA) infection in individual carriers and prevent transmission to other patients. The aims of this prospective cohort study were to determine the long-term efficacy of a standardised decolonisation regimen and to identify factors associated with failure. Patients colonised with MRSA underwent decolonisation, which was considered to be successful if there was no growth in three consecutive sets of site-specific screening swabs obtained weekly post treatment. If patients were successfully decolonised, follow-up cultures were performed 6 and 12 months later. Of 137 patients enrolled, 79 (58%) were successfully decolonised. Of these 79, 53 (67%) and 44 (56%) remained decolonised at 6 and 12 months respectively. Therefore only 44/137 (32%) patients who completed decolonisation were MRSA negative 12 months later. Outcome was not associated with a particular strain of MRSA. Successful decolonisation was less likely in patients colonised with a mupirocin-resistant isolate (adjusted odds ratio: 0.08; 95% confidence interval: 0.02-0.30), in patients with throat colonisation (0.22; 0.07-0.68) and in patients aged >80 years (0.30; 0.10-0.93) compared with those aged 60-80 years. These findings suggest that although initially successful in some cases, the protocol used did not result in long-term clearance of MRSA carriage for most patients.

Treatment of a cochlear implant biofilm infection: a potential role for alternative antimicrobial agents.

OBJECTIVE: This study aimed to investigate antimicrobial treatment of an infected cochlear implant, undertaken in an attempt to salvage the infected device. METHODS: We used the broth microdilution method to assess the susceptibility of meticillin-sensitive Staphylococcus aureus isolate, cultured from an infected cochlear implant, to common antimicrobial agents as well as to novel agents such as tea tree oil. To better simulate in vivo conditions, where bacteria grow as microcolonies encased in glycocalyx, the bactericidal activity of selected antimicrobial agents against the isolate growing in biofilm were also compared. RESULTS: When grown planktonically, the S aureus isolate was susceptible to 17 of the 18 antimicrobials tested. However, when grown in biofilm, it was resistant to all conventional antimicrobials. In contrast, 5 per cent tea tree oil completely eradicated the biofilm following exposure for 1 hour. CONCLUSION: Treatment of infected cochlear implants with novel agents such as tea tree oil could significantly improve salvage outcome.

Can the use of a rapid polymerase chain screening method decrease the incidence of nosocomial meticillin-resistant Staphylococcus aureus?

Rapid detection of MRSA may be important for the control of MRSA spread in hospitals. The aim of this investigation was to compare the use of a rapid polymerase chain reaction (PCR) screening method with standard culture for the detection of meticillin-resistant Staphylococcus aureus (MRSA) colonisation and to determine its impact on the incidence of MRSA in two hospital wards. During the first phase of the investigation (four months), patients in a surgical ward were screened using the rapid PCR technique and patients in a medical/cardiology ward were screened with standard culture methods. During the second phase of the investigation (four months), MRSA screening methods were switched between the two wards. An audit of infection control practices on each ward was made at the end of each phase in order to check whether any changes had occurred that might influence the risks of MRSA transmission. Use of the rapid PCR method significantly reduced the median time between swabs being taken, to the results being telephoned to the wards (excluding weekends), from 47 to 21 h (P<0.001). However, comparison of MRSA incidence during use of PCR (20/1000 bed-days) and culture methods (22.1/1000 bed-days) revealed no significant difference in incidence on the surgical ward (P=0.69). Regarding the medical/cardiology ward, analysis of data was complicated by an increase in the detection of MRSA during the PCR phase (P<0.05). The study demonstrated that rapid PCR can significantly reduce the turnaround times but reducing the time between swabs being taken to results being telephoned to the ward is still not sufficient to limit the transmission of MRSA.

Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia).

AIMS: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci. METHODS AND RESULTS: Isolates of methicillin-resistant/-sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase-negative staphylococci (CONS) were habituated to sub-lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0.05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three-fifths of the antibiotics examined; no changes in TTO MIC were observed. CONCLUSIONS: TTO habituation 'stress-hardens' MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of TTO at sub-lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system.

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication.

Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation, immunohistochemistry, and the polymerase chain reaction.

Quantitative virus isolation, immunohistochemistry, polymerase chain reaction (PCR) assay, and a porcine circovirus 2 (PCV2)-specific antigen-capture enzyme-linked immunosorbent assay (ELISA) were used for differentiation between clinical and subclinical PCV2 infections of swine. Tissue samples from pigs experimentally infected with PCV2 and field cases of postweaning multisystemic wasting syndrome and PCV2-associated reproductive disorders were used in this evaluation. In initial studies on 6 PCV2 pools using 3 previously published PCR protocols for PCV2 detection, quantitative virus isolation, and antigen-capture ELISA, substantial differences in sensitivity were identified among these procedures. Examination of tissue samples from diseased and clinically normal pigs indicated that immunohistochemistry, quantitative virus isolation, and antigen-capture ELISA could be used to differentiate between clinical and subclinical PCV2 infections, but the PCR assay could not. Because subclinical infections of pigs with PCV2 are common, the use of nonquantitative PCR as a diagnostic tool for PCV2-related diseases should be discouraged and the PCV2-specific antigen-capture ELISA evaluated further.

Occurrence of Burkholderia cepacia in the hospital environment.

AIM: To determine the prevalence of Burkholderia cepacia from the environment in a regional adult cystic fibrosis (CF) care centre. METHODS: Two hundred and eighty-five environmental samples were taken from inpatient and outpatient environments within the CF centre. Following a non-selective enrichment step, broths were plated onto a selective medium and B. cepacia isolates were confirmed by phenotypic tests and also genotypically by species specific 16S rRNA PCR. RESULTS: B. cepacia was not detected from commonly shared items of equipment, staff hands, staff uniforms or toilets. In addition, the organism was not detected in toilet bowls, even in the B. cepacia unit. With regard to positive environments for B. cepacia, 4/10 (40%) of the outside surfaces and inner rims of patients' plastic disposable sputum collection containers and 4/17 (23.5%) of air from patients' rooms, following physiotherapy, were positive. CONCLUSIONS: All positive samples originated in the B. cepacia segregation area of the inpatient wards and B. cepacia was not detected in the non-cepacia area of the CF centre. Consequently, these two positive sites should therefore be treated as high risk, where organisms may be potentially transmitted from environment to patient. As there is now evidence to suggest differences in the virulence and hence clinical outcome of certain of the genomovars of this organism, this study may be important in highlighting the areas that patients who are B. cepacia positive should avoid, to minimise the risk of transmission and acquisition of this organism within the CF cepacia population. These results indicate that effective infection control procedures are required in patients' rooms following physiotherapy, for prevention of B. cepacia transmission and to avoid the transmission of different genomovar types within CF patients, who are already colonised with this pathogen.

Development of a new test for mechanical irritation: behind the knee as a test site.

BACKGROUND/AIMS: Certain consumer products, such as feminine hygiene products, and baby and adult diapers, are worn for prolonged periods of time in close proximity to the skin. A sensitive test method was needed to evaluate these products and their components for the potential to cause mechanical irritation during use. METHODS: We investigated several test sites on the body where normal, daily activities provided the opportunity for movement and, therefore, friction. These test sites were: the back of the knee, the axilla, and the wrist. Three exposure regimens were tested: 6-h daily for 4 days, 24-h daily for 3 days, and 24-h daily for 4 days. Controls consisted of satin fabric (a non-irritating material) or burlap (an irritating material). Test materials were secured to the test sites using an Ace elastic bandage (on the back of the knee), a snug fitting garment (on the axilla), or an athletic band (on the wrist). Test sites were scored for irritation on a visual scale prior to the initial application and 30-60 min after removal each day. Data were evaluated for the ability to reliably detect irritating materials, and for the ability to consistently differentiate between irritating and non-irritating materials. Quantitative measures of these abilities were used to rank the overall effectiveness of various test protocols as a model for mechanical irritation. RESULTS/CONCLUSION: The results of these studies indicated that a protocol using the back of the knee as a test site with an exposure regimen of 6-h daily for 4 days, best met the success criteria for an effective test system for evaluating mechanical irritation.

An anterior portal for wrist arthroscopy. Anatomical study and case reports.

We have found that the standard portals described limit arthroscopic access to and visualization of some areas of the radiocarpal joint. We describe a radial anterior portal, which we believe is useful in the arthroscopic treatment of wrist conditions and describe its use in clinical practice.

Detection of intrastrain antigenic variation of Bacteroides fragilis surface polysaccharides by monoclonal antibody labelling.

Bacteroides fragilis is a constituent of the normal resident microbiota of the human intestine and is the gram-negative obligately anaerobic bacterium most frequently isolated from clinical infection. Surface polysaccharides are implicated as potential virulence determinants. We present evidence of within strain immunochemical variation of surface polysaccharides in populations that are noncapsulate by light microscopy as determined by monoclonal antibody labelling. Expression of individual epitopes can be enriched from a population of an individual strain by use of immunomagnetic beads. Also, individual colonies in which either >94% or <7% of the bacteria carry an individual epitope retain this level of expression when subcultured into broth. In broth cultures where >94% of the bacteria carry a given epitope, there is no enrichment for other epitopes recognized by different polysaccharide-specific monoclonal antibodies. This intrastrain variation has important implications for the development of potential vaccines or immunodiagnostic tests.

Calculation of a new Meeh constant and experimental determination of burn size.

With advances in research techniques involving molecular biology, the need for precision in the determination of surface area is even greater than previously. This study sets out to determine the most accurate Meeh constant, k, for a commonly used weight range of experimental rats. The importance of a narrow weight range is appreciated and planometric data is presented from a series of adolescent rats that allow a new k value to be calculated. The figures presented here represent the largest number of animals, of a commonly used rat strain, in the most widely used weight range yet described. Pelts were removed from 16 anaesthetized male Sprague-Dawley rats after cervical dislocation. For each animal total body surface area (TBSA) was determined planometrically, along with premarked areas on the dorsal and ventral surfaces. Entering the planometrically measured data along with the animal's weight into the Meeh formula, a k value was obtained for each rat. Surface areas were then calculated for each animal using the mean k, 9.46, and compared against figures obtained by using historical values for the Meeh constant. Using the new k value a method is described for the accurate determination of total body surface area and premarked surface areas, in experimental rats of a specific weight range.

Regulation of the acute phase response genes alpha 1-acid glycoprotein and alpha 1-antitrypsin correlates with sensitivity to thermal injury.

BACKGROUND: The response to thermal injury is a complex physiologic process requiring communication between sites of injury and distal target organs. The liver, one of these target organs, synthesizes a family of secretory proteins, the acute phase reactants (APRs), that carries out specific protective functions. This study investigates the response of positively regulated (alpha 1-acid glycoprotein and alpha 1-antitrypsin) and negatively regulated (albumin) APR genes to severe thermal injury in three rat strains with differing abilities to survive thermal stress. METHODS: Age and weight matched male Buffalo, Sprague-Dawley, and Fischer 344, 12- to 16-week-old rats (275 to 325 gm) received a 40% total body surface area scald burn. Total RNA was isolated from livers at 0, 2, 5, 12, 24, and 48 hours. Northern blot hybridization was performed with 32P-labeled rat alpha 1-glycoprotein, rat albumin, and mouse alpha 1-antitrypsin cDNAs. Relative amounts of alpha 1-glycoprotein, alpha 1-antitrypsin, and albumin mRNAs were determined by means of densitometric analyses. RESULTS: All three strains elicit both a positive and negative acute phase (AP) response. Significant differences were observed in the degree and kinetics between strains. Those more sensitive to thermal injury exhibited a more intense positive AP response and possibly a delayed recovery. The AP response between these strains correlates with the variation in ability to survive severe trauma. CONCLUSIONS: The differences in the kinetics and intensity of induction of APR genes between Buffalo, Sprague-Dawley, and Fischer rat strains suggest that the least intense AP response and its timely recovery correlated with the ability to survive a severe thermal injury and that, conversely, the more intense and prolonged response correlated with sensitivity to severe thermal injury. We propose that this may be a basis for variation in survival to thermal injury.

Effect of thermal injury on the expression of transcription factors that regulate acute phase response genes: the response of C/EBP alpha, C/EBP beta, and C/EBP delta to thermal injury.

BACKGROUND: Eukaryotic organisms possess natural defense mechanisms that protect against stress stimuli. One such mechanism is the activation of families of stress response genes (e.g., the acute phase response). Transcription of many of these genes is regulated by the leucine zipper or bZIP proteins (CCAAT binding/enhancer binding proteins [C/EBPs]). The aim of this study was to show that the C/EBP transcription factor genes respond to thermal injury. METHODS: Age- and weight-matched male Buffalo, Sprague-Dawley, and Fischer 344 12- to 16-week-old rats (275 to 325 gm) received a 40% total body surface area scald burn. Total RNA was isolated from livers at 0, 2, 5, 12, 24, and 48 hours. Northern blot hybridization was performed with 32P-labeled C/EBP alpha, C/EBP beta, and C/EBP delta cDNAs. Relative amounts of each mRNA were determined by densitometric analyses. For Western analyses liver nuclear and cytoplasmic protein extracts were prepared from burned and control rats. Nuclear protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto a PVDF membrane, and detected by using an enhanced chemiluminescence detection kit. RESULTS: Expression of C/EBP genes is regulated in response to 40% total body surface area scald burn. A simultaneous decrease in C/EBP alpha and an increase in C/EBP beta and C/EBP delta mRNA levels occur in response to thermal injury. Western analyses detect changes in C/EBP alpha and C/EBP beta pool levels that suggest a differential regulation of these genes in response to thermal injury. CONCLUSIONS: The responses of C/EBP alpha, C/EBP beta, and C/EBP delta are similar in Buffalo, Sprague-Dawley, and Fischer rats. The induced level, however, of C/EBP beta mRNA was highest in the Sprague-Dawley strain and lowest in the Buffalo strain and correlates well with the mortality of these strains. Because C/EBP beta is associated with the transactivation of stress response genes, this may explain the intensity of the response in the susceptible strains. This agrees with our hypothesis that the higher degree of sensitivity of the Sprague-Dawley rat to stress relative to the Buffalo strain may be due to inherently higher levels of factors such as C/EBP whose functions are associated with activation of stress response genes.

Current treatment of severely burned patients.

OBJECTIVE: The authors provide an update on a multidisciplinary approach to the treatment of severely burned patients. A review of studies and clinical trials from the past to the present include fluid resuscitation, sepsis, immune function, hypermetabolism, early excision, wound healing, scar formation, and inhalation injury. SUMMARY BACKGROUND DATA: Advances in treating initial burn shock, infection control, early wound closure, and modulation of the hypermetabolic response have decreased morbidity and mortality in the last two decades. Specialized burn care centers, using a multidisciplinary approach, not only successfully treat large burns and their complications, but provide the necessary rehabilitation and psychological support required for readjustment back into society. CONCLUSIONS: Thermal injury results in a number of physiologic alterations that can be minimized by adequate fluid resuscitation to maintain tissue perfusion, early excision of burn wounds, and rapid wound coverage. These measures, in combination with antibiotic coverage and nutritional support in the form of early enteral tube feedings, will decrease the hypermetabolic response and the incidence of sepsis that can lead to hemodynamic instability and organ failure. Ongoing clinical trials using anabolic agents (e.g., recombinant human growth hormone) and pharmacologic agents that modulate inflammatory and endocrine mediators (e.g., ibuprofen and propranolol) show promise in the treatment of severe burn injuries.