Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed. (C) 2004 Elsevier B.V. All rights reserved.

Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa

A colorimetric assay based on the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)} for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described. There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods. The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P. aeruginosa to bactericidal antibiotics.

The use of lytic bacteriophages in the prevention and eradication of biofilms of Proteus mirabilis and Escherichia coli.

Antibiotics have been the cornerstone of the clinical management of bacterial infections since their discovery in the early part of the last century. Eight decades later, their widespread, often indiscriminate use, has resulted in an overall reduction in their effectiveness, with reports of multidrug-resistant bacteria now commonplace. Increasing reliance on indwelling medical devices, which are inherently susceptible to biofilm-mediated infections, has contributed to unacceptably high rates of nosocomial infections, placing a strain on healthcare budgets. This study investigates the use of lytic bacteriophages in the treatment and prevention of biofilms of bacterial species commonly associated with infections of indwelling urological devices and catheter-associated urinary tract infections. The use of lytic bacteriophages against established biofilms of Proteus mirabilis and Escherichia coli is described, whereby biofilm populations have been reduced successfully by three to four log cycles (99.9-99.99% removal). The prevention of biofilm formation on Foley catheter biomaterials following impregnation of hydrogel-coated catheter sections with a lytic bacteriophage has also been investigated. This has revealed an approximate 90% reduction in both P. mirabilis and E. coli biofilm formation on bacteriophage-treated catheters when compared with untreated controls.

Bronchiolitis

INTRODUCTION: Bronchiolitis is the most common lower respiratory tract infection in infants, occurring in a seasonal pattern, with highest incidence in the winter in temperate climates and in the rainy season in warmer countries. Bronchiolitis is a common reason for attendance at and admission to hospital.

METHODS AND OUTCOMES: We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of prophylactic interventions for bronchiolitis in high-risk children? What are the effects of measures to prevent transmission of bronchiolitis in hospital? What are the effects of treatments for children with bronchiolitis? We searched: Medline, Embase, The Cochrane Library, and other important databases up to July 2010 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA).

RESULTS: We found 59 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions.

CONCLUSIONS: In this systematic review we present information relating to the effectiveness and safety of the following interventions: antibiotics, bronchodilators (oral, inhaled salbutamol, inhaled adrenaline [epinephrine], hypertonic saline), chest physiotherapy, continuous positive airway pressure, corticosteroids, fluid management, heliox, montelukast, nasal decongestants, nursing interventions (cohort segregation, hand washing, gowns, masks, gloves, and goggles), oxygen, respiratory syncytial virus immunoglobulins, pooled immunoglobulins, or palivizumab (monoclonal antibody), ribavirin, or surfactants.

Plasma cell polyp of the vocal fold

Plasma cell polyps of the vocal fold (plasma cell granulomas) are rare inflammatory polyps of the larynx. They should be included in the clinical and histological differential diagnosis of laryngeal polyps. Histologically they are polyclonal aggregates of plasma cells. It is essential to distinguish them from monoclonal, neoplastic plasma cell proliferations. The treatment of choice is surgical resection, although radiotherapy, laser ablation, antibiotics and steroids have been used successfully. We present a case of plasma cell granuloma presenting as a vocal fold polyp, treated surgically.

Incidence and risk factors for pulmonary exacerbation treatment failures in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa

Background: Pulmonary exacerbations (PEx) are responsible for much of the morbidity and mortality associated with cystic fibrosis (CF). However, there is a paucity of data on outcomes in CF PEx and factors influencing outcomes.

Methods: We reviewed all PEx in patients infected with Pseudomonas aeruginosa treated with parenteral antibiotics over 4 years at our center. Treatment failures were categorized a priori as those PEx requiring antibiotic regimen change, prolongation of therapy > 20 days because of failure to respond, an early recurrent event within < 45 days, or failure to recover lung function to > 90% of baseline FEV1.

Results: A total of 101 patients were followed for 452 PEx. Treatment failures were observed in 125 (28%) of PEx; antibiotic regimen change was observed in 27 (6%), prolongation of therapy in 29 (6%), early recurrent events in 63 (14%), and failure to recover lung function to > 90% of baseline FEV1 in 66 (15%). Demographic factors associated with one or more treatment failures per year included advanced airways disease, use of enteric feeds, CF-related diabetes, and CF liver disease but did not include female sex or F508del homozygosity. Increased treatment failure risk was associated with lower admission FEV1 and increased markers of inflammation. At therapeutic completion, increased inflammatory markers correlated with treatment failure. Failure rates decreased with increasing number of active antimicrobial agents used based on in vitro susceptibility (zero, 28/65 [43%]; one, 38/140 [27%]; two, 59/245 [24%]; three, 0/2 [0%]; P = .02).

Conclusions: One-fourth of PEx fail to respond adequately to initial management. Patient demographic and episode-specific clinical information can be used to identify individuals at increased risk of initial management failure.

Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial bio?lm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB,N-mercaptoacetyl-Phe-Tyr-amide (Ki 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in bio?lm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal bio?lms, and to eradicate bio?lm completely when used in combination with conventional antibiotics.

Prescribing Practices and Polypharmacy in Kitovu Hospital, Uganda.

This audit of prescribing practices explores recent trends at Kitovu Hospital, Uganda. The average number of drugs prescribed per patient was 2.89 ± 0.11, of which 1.79±0.09 were generics and 0.69±0.06 antibiotics. No injections were prescribed. Patient essential drug knowledge was 100% while the adequacy of labelling was 0%. The number of drugs prescribed correlated positively with patient age, was greater for female patients, similar for doctors and clinical officers but greater in medical (3.30±0.15, n=50) than surgical (2.48±0.13, n=50) outpatient clinics. The mean consultation time was 6.56 min and 10.25 min per patient in medical and surgical outpatient clinics respectively. The patient essential knowledge indicators were greatly improved but only modest reduction in polypharmacy was evident compared to the Ugandan Pharmaceutical Sector national survey of 2002. Antibiotic prescription was high and generic prescribing was found to be low. Policy changes are required to enhance rational drug use in the health sector in Uganda.

The Potential of Antimicrobial Peptides as Biocides

Antimicrobial peptides constitute a diverse class of naturally occurring antimicrobial molecules which have activity against a wide range of pathogenic microorganisms. Antimicrobial peptides are exciting leads in the development of novel biocidal agents at a time when classical antibiotics are under intense pressure from emerging resistance, and the global industry in antibiotic research and development stagnates. This review will examine the potential of antimicrobial peptides, both natural and synthetic, as novel biocidal agents in the battle against multi-drug resistant pathogen infections.

Population structure and characterization of viridans group streptococci (VGS) including Streptococcus pneumoniae isolated from adult patients with cystic fibrosis (CF)

A study was undertaken to examine the population structure of viridans group streptococci (VGS) in the sputum of adult patients with cystic fibrosis (CF). Freshly expectorated sputa (n=58) from 45 adult CF patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 190 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 12 species of VGS and included S. anginosus, S. australis, S. cristatus, S. gordonii, S. infantis, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pneumoniae, S. salivarius and S. sanguinis. The most frequently VGS organism isolated was S. salivarius (47/190; 24.7%), followed by S. mitts (36/190; 19%), S. sanguinis (25/190; 13.2%), S. oralis (20/190; 11.0%), S. pneumoniae (19/190; 10.0%), S. parasanguinis (16/190; 8.4%), S. infantis (11/190; 5.8%), S. gordonii (7/190; 3.7%), S. anginosus (4/190; 2.1%), S. cristatus (2/190; 1.1%), S. australis (1/190; 0.5%), S. mutans (1/190; 0.5%) and S. agalactiae (1/190; 0.5%). All, but four, patients harboured at least one VGS species, which ranged from one to five streptococcal species, with a mean of 2.85 species per patient. There was no clonality at the subspecies level employing ERIC RAPD PCR. Antibiotic susceptibility was determined by Minimum Inhibitory Concentration (MIC) testing against penicillin, erythromycin and ciprofloxacin. Overall, resistance to penicillin with all VGS was 73/190 (38.4%) and 167/190 (87.9%) for erythromycin. With regard to ciprofloxacin, 27/190 (14.2%) were fully resistant, whilst a further 21/190 (11.1%) showed intermediate resistance, which equated to approximately three quarters (74.7%) of isolates being fully sensitive to this agent. In addition, as a comparator control population, we examined antibiotic susceptibility, as above, in a non-CF population comprising 12 individuals (50 VGS isolates), who were not receiving chronic antibiotics. In comparison, 8% and 38% of VGS isolates from non-CF individuals were resistant by disk susceptibility testing to penicillin and erythromycin, respectively. None of the non-CF VGS organisms were resistant to ciprofloxacin, but 42% showed intermediate resistance. (C) 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Antibacterial activities of naturally occurring compounds against antibiotic-resistant Bacillus cereus vegetative cells and spores, Escherichia coli, and Staphylococcus aureus

After demonstrating the lack of effectiveness of standard antibiotics against the acquired antibiotic resistance of Bacillus cereus (NCTC 10989), Escherichia coli (NCTC 1186), and Staphylococcus aureus (ATCC 12715), we showed that the following natural substances were antibacterial against these resistant pathogens: cinnamon oil, oregano oil, thyme oil, carvacrol, (S)-perillaldehyde, 3,4-dihydroxybenzoic acid (beta-resorcylic acid), and 3,4-dihydroxyphenethylamine (dopamine). Exposure of the three pathogens to a dilution series of the test compounds showed that oregano oil was the most active substance. The oils and pure compounds exhibited exceptional activity against B. cereus vegetative cells, with oregano oil being active at nanogram, per milliliter levels. In contrast, activities against B. cereus spores were very low. Activities of the test compounds were in the following approximate order: oregano oil > thyme oil approximate to carvacrol > cinnamon oil > perillaldehyde > dopamine > beta-resorcylic acid. The order of susceptibilities of the pathogens to inactivation was as follows: B. cereus (vegetative) much greater than S. aureus approximate to E. coli much greater than B. cereus (spores). Some of the test substances may be effective against antibiotic-resistant bacteria in foods and feeds.

Detection of streptomycin residues in whole milk using an optical immunobiosensor

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples (similar to3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).

AUDIT OF ANTIBIOTIC USAGE IN A MEDIUM-SIZED GENERAL-HOSPITAL OVER AN 11-YEAR PERIOD - THE IMPACT OF ANTIBIOTIC POLICIES

The objective of the present study was to evaluate trends in antibiotic expenditure over an 11-year period (1982-1992) in a 370-bed district general hospital in Northern Ireland and to examine the impact of two separate antibiotic policies on antibiotic usage. A further objective was to examine the attitudes of prescribers to the second policy. Drug utilization review was used to collect information on antibiotic expenditure and usage before and after introduction of separate antibiotic policies in 1985 (not intensively monitored) and 1989 (intensively monitored). A mail questionnaire was used to determine the attitudes of prescribers. The first policy (1985) showed no benefits with regard to the number of antibiotic entities stocked (45 before, 45 after), number of dosage units issued (9.35 increase) or expenditure (33.35 increase). The 1989 policy led to significant reductions in the number of antibiotic entities stocked (28.9%), number of antibiotics issued (11.9%) and expenditure (6.1%). Expenditure began to spiral upwards when active monitoring of the second policy was suspended. The majority of prescribers (87.2%) who responded to the questionnaire (56.5% response rate) felt that the 1989 policy made a positive contribution to antibiotic usage in the hospital.

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro.

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1) ) and high (10(7) PFU mL(-1) ) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 µg mL(-1) , respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.

Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis

Cystic fibrosis (CF) is the most common inherited lethal disease in Caucasians which results in multiorgan dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR Delta F508 (Delta F508) mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1 beta. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type but not in Delta F508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-Delta F508 macrophages than in WT macrophages. An autophagosome is a compartment that engulfs nonfunctional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and Delta F508 macrophages. However, autophagy dysfunction is more pronounced in Delta F508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.