Peter A. Lambert

Association between sciatica and Propionibacterium acnes

We hypothesised that the inflammation seen around the nerve root in patients with sciatica may be caused by microbial infection. We used a newly developed serological test to diagnose deep-seated infections caused by low virulent gram-positive microorganisms. 43 of 140 (31%) patients with sciatica tested positive. Intervertebral disc material from a further 36 patients with severe sciatica who had undergone microdiscectomy was cultured for the presence of microorganisms. 19 of these patients (53%) had positive cultures after long-term incubation. Propionibacterium acnes was isolated from 16 of the 19 (84%) positive samples. Low virulent microorganisms, in particular P acnes, might be causing a chronic low-grade infection in the lower intervertebral discs of patients with severe sciatica. These microorganisms could have gained access to the spinal disc after previous minor trauma.

Antimicrobial efficacy of eucalyptus oil and 1,8-cineole alone and in combination with chlorhexidine digluconate against microorganisms grown in planktonic and biofilm cultures

OBJECTIVES Effective disinfection and antisepsis is pivotal in preventing infections within the healthcare setting. Chlorhexidine digluconate (CHG) is a widely used disinfectant/antiseptic possessing broad-spectrum antimicrobial activity; however, its penetration into bacterial biofilms and human skin is poor. The aim of this study was to investigate the antimicrobial efficacy of crude eucalyptus oil (EO) and its main component 1,8-cineole (a recognized permeation enhancer), alone and in combination with CHG, against a panel of clinically relevant microorganisms grown in planktonic and biofilm cultures. METHODS MICs and minimum bactericidal/fungicidal concentrations were determined for each microorganism grown in suspension and biofilm using microbroth dilution and ATP bioluminescence, respectively. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between CHG and EO or 1,8-cineole. RESULTS Antimicrobial activity was demonstrated by CHG, EO and 1,8-cineole; however, CHG was significantly more active against microorganisms in both planktonic and biofilm modes of growth (P < 0.05). Crude EO was significantly more efficacious against microorganisms grown in suspension compared with 1,8-cineole (P < 0.05). Synergistic activity was demonstrated between CHG and both EO and 1,8-cineole against suspensions of Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli and Candida albicans, and biofilm cultures of MRSA and Pseudomonas aeruginosa. CONCLUSIONS In conclusion, CHG may be combined with either crude EO or its major component 1,8-cineole for enhanced, synergistic antimicrobial activity against a wide range of microorganisms in planktonic and biofilm modes of growth; however, the superior antimicrobial efficacy associated with crude EO alone, compared with 1,8-cineole, favours its combination with CHG.

Potassium fluxes, first indications of membrane damage in micro-organisms

A method is described whereby the leakage of potassium ions can be measured from microbial cells which have been treated with membrane active antimicrobial compounds. The method involves the use of specific ion electrodes and does not require the cells to be removed from suspension. The effect of two membrane-active antimicrobials; cetrimide upon E. coli and amphotericin upon C.albicans is related to the leakage of phosphate and 260 nm material.

Propionibacterium acnes:infection beyond the skin

Propionibacterium acnes is a Gram-positive bacterium that forms part of the normal flora of the skin, oral cavity, large intestine, the conjunctiva and the external ear canal. Although primarily recognized for its role in acne, P. acnes is an opportunistic pathogen, causing a range of postoperative and device-related infections. These include infections of the bones and joints, mouth, eye and brain. Device-related infections include those of joint prostheses, shunts and prosthetic heart valves. P. acnes may play a role in other conditions, including inflammation of the prostate leading to cancer, SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome, sarcoidosis and sciatica. If an active role in these conditions is established there are major implications for diagnosis, treatment and protection. Genome sequencing of the organism has provided an insight into the pathogenic potential and virulence of P. acnes.

Antimicrobial efficacy of chlorhexidine digluconate alone and in combination with eucalyptus oil, tea tree oil and thymol against planktonic and biofilm cultures of Staphylococcus epidermidis

OBJECTIVES Effective skin antisepsis and disinfection of medical devices are key factors in preventing many healthcare-acquired infections associated with skin microorganisms, particularly Staphylococcus epidermidis. The aim of this study was to investigate the antimicrobial efficacy of chlorhexidine digluconate (CHG), a widely used antiseptic in clinical practice, alone and in combination with tea tree oil (TTO), eucalyptus oil (EO) and thymol against planktonic and biofilm cultures of S. epidermidis. METHODS Antimicrobial susceptibility assays against S. epidermidis in a suspension and in a biofilm mode of growth were performed with broth microdilution and ATP bioluminescence methods, respectively. Synergy of antimicrobial agents was evaluated with the chequerboard method. RESULTS CHG exhibited antimicrobial activity against S. epidermidis in both suspension and biofilm (MIC 2-8 mg/L). Of the essential oils thymol exhibited the greatest antimicrobial efficacy (0.5-4 g/L) against S. epidermidis in suspension and biofilm followed by TTO (2-16 g/L) and EO (4-64 g/L). MICs of CHG and EO were reduced against S. epidermidis biofilm when in combination (MIC of 8 reduced to 0.25-1 mg/L and MIC of 32-64 reduced to 4 g/L for CHG and EO, respectively). Furthermore, the combination of EO with CHG demonstrated synergistic activity against S. epidermidis biofilm with a fractional inhibitory concentration index of <0.5. CONCLUSIONS The results from this study suggest that there may be a role for essential oils, in particular EO, for improved skin antisepsis when combined with CHG.

A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterization of type I cell surface-associated antigens.

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB(1)), representing 64 % of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

A new phylogenetic group of Propionibacterium acnes

Immunofluorescence microscopy-based identification of presumptive Propionibacterium acnes isolates, using the P. acnes-specific mAb QUBPa3, revealed five organisms with an atypical cellular morphology. Unlike the coryneform morphology seen with P. acnes types I and II, these isolates exhibited long slender filaments (which formed large tangled aggregates) not previously described in P. acnes. No reaction with mAbs that label P. acnes types IA (QUBPa1) and II (QUBPa2) was observed. Nucleotide sequencing of the 16S rRNA gene (1484 bp) revealed the isolates to have between 99.8 and 99.9 % identity to the 16S rRNA gene of the P. acnes type IA, IB and II strains NCTC 737, KPA171202 and NCTC 10390, respectively. Analysis of the recA housekeeping gene (1047 bp) did reveal, however, a greater number of conserved nucleotide polymorphisms between the sequences from these isolates and those from NCTC 737 (98.9 % identity), KPA171202 (98.9 % identity) and NCTC 10390 (99.1 % identity). Phylogenetic investigations demonstrated that the isolates belong to a novel recA cluster or lineage distinct from P. acnes types I and II. We now propose this new grouping as P. acnes type III. The prevalence and clinical importance of this novel recA lineage amongst isolates of P. acnes remains to be determined.

A randomized, prospective clinical trial to assess the potential infection risk associated with the PosiFlow needleless connector.

The microbial contamination rate of luers of central venous catheters (CVCs) with either PosiFlow needleless connectors or standard caps attached was investigated. The efficacy of 70% (v/v) isopropyl alcohol, 0.5% (w/v) chlorhexidine in gluconate 70% (v/v) isopropyl alcohol and 10% (w/v) aqueous povidone-iodine to disinfect the intravenous connections was also assessed. Seventy-seven patients undergoing cardiac surgery who required a CVC as part of their clinical management were randomly allocated either needleless connectors or standard caps. Patients were also designated to receive chlorhexidine/alcohol, isopropyl alcohol or povidone-iodine for pre-CVC insertion skin preparation and disinfection of the connections. After 72 h in situ the microbial contamination rate of 580 luers, 306 with standard caps and 274 with needleless connectors attached, was determined. The microbial contamination rate of the external compression seals of 274 needleless connectors was also assessed to compare the efficacy of the three disinfectants. The internal surfaces of 55 out of 306 (18%) luers with standard caps were contaminated with micro-organisms, whilst only 18 out of 274 (6.6%) luers with needleless connectors were contaminated (P<0.0001). Of those needleless connectors disinfected with isopropyl alcohol, 69.2% were externally contaminated with micro-organisms compared with 30.8% disinfected with chlorhexidine/alcohol (P<0.0001) and 41.6% with povidone-iodine (P<0.0001). These results suggest that the use of needleless connectors may reduce the microbial contamination rate of CVC luers compared with the standard cap. Furthermore, disinfection of needleless connectors with either chlorhexidine/alcohol or povidone-iodine significantly reduced external microbial contamination. Both these strategies may reduce the risk of catheter-related infections acquired via the intraluminal route.

Occurrence and function of membrane teichoic acids

Membrane teichoic acids, sometimes described as lipoteichoic acids, are important but not major components of nearly all Gram-positive bacteria. They appear on the outer surface of the cytoplasmic membrane and possess antigenic properties. Several functions have been ascribed to these glycerol phosphate polymers, including the binding of divalent cations required for optimal activity of membrane-bound enzymes, and the control of certain lytic enzymes. A substance that is identical or closely similar to membrane teichoic acid, lipoteichoic acid carrier, plays an important part in the biosynthesis of wall teichoic acid; it accepts polyol phosphate residues from CDP-glycerol or CDP-ribitol to form a polyol phosphate chain which is then transferred after the incorporation of a tri(glycerol phosphate) linkage unit, to the growing glycan chain of peptidoglycan.

Antimicrobial efficacy of copper surfaces against spores and vegetative cells of Clostridium difficile: the germination theory

OBJECTIVES Persistent contamination of surfaces by spores of Clostridium difficile is a major factor influencing the spread of C. difficile-associated diarrhoea (CDAD) in the clinical setting. In recent years, the antimicrobial efficacy of metal surfaces has been investigated against microorganisms including methicillin-resistant Staphylococcus aureus. This study compared the survival of C. difficile on stainless steel, a metal contact surface widely used in hospitals, and copper surfaces. METHODS Antimicrobial efficacy was assessed using a carrier test method against dormant spores, germinating spores and vegetative cells of C. difficile (NCTC 11204 and ribotype 027) over a 3 h period in the presence and absence of organic matter. RESULTS Copper metal eliminated all vegetative cells of C. difficile within 30 min, compared with stainless steel which demonstrated no antimicrobial activity (P < 0.05). Copper significantly reduced the viability of spores of C. difficile exposed to the germinant (sodium taurocholate) in aerobic conditions within 60 min (P < 0.05) while achieving a >or=2.5 log reduction (99.8% reduction) at 3 h. Organic material did not reduce the antimicrobial efficacy of the copper surface (P > 0.05). CONCLUSIONS The use of copper surfaces within the clinical environment and application of a germination solution in infection control procedures may offer a novel way forward in eliminating C. difficile from contaminated surfaces and reducing CDAD.