Eluding antibiotic resistance: capitalizing on antimicrobial peptides interaction with the lipid bilayer

It is widely accepted that the emergence of drug-resistant pathogens is the result of the overuse and misuse of antibiotics. Infectious Disease Society of America, Center for Disease Control and World Health Organization continue to view, with concern, the lack of antibiotics in development, especially those against Gram-negative bacteria. Antimicrobial peptides (AMPs) have been proposed as an alternative to antibiotics due to their selective activity against microbes and minor ability to induce resistance. For example, the Food and Drug Administration approved Daptomycin (DAP) in 2003 for treatment of severe skin infections caused by susceptible Gram-positive organisms. Currently, there are 12 to 15 examples of modified natural and synthetic AMPs in clinical development. But most of these agents are against Gram-positive bacteria. Therefore, there is unmet medical need for antimicrobials used to treat infections caused by Gram-negative bacteria. In this study, we show that a pro-apoptotic peptide predominantly used in cancer therapy, (KLAKLAK)2, is an effective antimicrobial against Gram-negative laboratory strains and clinical isolates. Despite the therapeutic promise, AMPs development is hindered by their susceptibility to proteolysis. Here, we demonstrate that an all-D enantiomer of (KLAKLAK)2, resistant to proteolysis, retains its activity against Gram-negative pathogens. In addition, we have elucidated the specific site and mechanism of action of D(KLAKLAK)2 through a repertoire of whole-cell and membrane-model assays. Although it is considered that development of resistance does not represent an obstacle for AMPs clinical development, strains with decreased susceptibility to these compounds have been reported. Staphylococci resistance to DAP was observed soon after its approval for use and has been linked to alterations of the cell wall (CW) and cellular membrane (CM) properties. Immediately following staphylococcal resistance, Enterococci resistance to DAP was seen, yet the mechanism of resistance in enterococci remains unknown. Our findings demonstrate that, similar to S. aureus, development of DAP-resistance in a vancomycin-resistant E. faecalis isolate is associated with alterations of the CW and properties of the CM. However, the genes linked to these changes in enterococci appear to be different from those described in S. aureus.

Role of milk and meat products as vehicles for antibiotic-resistant bacteria

This subject is reviewed under the following headings: Microbial contamination of raw meat and raw milk; Antibiotic resistance of food-borne pathogens; Antibiotic resistance of commensal and potentially pathogenic bacteria as a new threat in food microbiology; Antibiotic-resistant staphylococci in fermented meat and [in] milk products; Antibiotic-resistant Enterococcus sp. in fermented meat and [in] milk products; Enterococci in farm animals and meat; Enterococci in fermented food; Molecular characterization of resistance of food-borne enterococci; and Further ecological and epidemiological considerations of resistant live bacteria in food. It is concluded that further research is needed, particularly into the possible transfer of the resistance of bacteria consumed in meat or milk products to the indigenous bacteria of the human consumer.

Antibiotic resistance spread in food

Nutritive and therapeutic treatment of farm animals with antibiotics, amounting to half of the world's antibiotic output, has selected for resistant bacteria that may contaminate the food produced. Antibiotic-resistant enterococci and staphylococci from animals are found in food when they survive the production processes, as in raw cured sausages and raw milk cheeses1. The broad host ranges of some plasmids and the action of transposons in many bacteria allow antibiotic-resistance genes to be communicated by conjugation between different species and genera2,3. A multi-antibiotic resistance plasmid from a lactococcus found in cheese provides a historical record of such events.

The treatment and outcome of peri-prosthetic infection of the ankle: a single cohort-centre experience of 34 cases

The treatment of peri-prosthetic joint infection (PJI) of the ankle is not standardised. It is not clear whether an algorithm developed for hip and knee PJI can be used in the management of PJI of the ankle. We evaluated the outcome, at two or more years post-operatively, in 34 patients with PJI of the ankle, identified from a cohort of 511 patients who had undergone total ankle replacement. Their median age was 62.1 years (53.3 to 68.2), and 20 patients were women. Infection was exogenous in 28 (82.4%) and haematogenous in six (17.6%); 19 (55.9%) were acute infections and 15 (44.1%) chronic. Staphylococci were the cause of 24 infections (70.6%). Surgery with retention of one or both components was undertaken in 21 patients (61.8%), both components were replaced in ten (29.4%), and arthrodesis was undertaken in three (8.8%). An infection-free outcome with satisfactory function of the ankle was obtained in 23 patients (67.6%). The best rate of cure followed the exchange of both components (9/10, 90%). In the 21 patients in whom one or both components were retained, four had a relapse of the same infecting organism and three had an infection with another organism. Hence the rate of cure was 66.7% (14 of 21). In these 21 patients, we compared the treatment given to an algorithm developed for the treatment of PJI of the knee and hip. In 17 (80.9%) patients, treatment was not according to the algorithm. Most (11 of 17) had only one criterion against retention of one or both components. In all, ten of 11 patients with severe soft-tissue compromise as a single criterion had a relapse-free survival. We propose that the treatment concept for PJI of the ankle requires adaptation of the grading of quality of the soft tissues. Cite this article: Bone Joint J 2014;96-B:772-7.

Strain typing and antimicrobial susceptibility of methicillin-resistant coagulase-positive staphylococcal species in dogs and people associated with dogs in Thailand

AIMS This study was to investigate and to characterize methicillin-resistant coagulase-positive staphylococci (MRCoPS) harboring in dogs and people associated with dogs in Thailand. METHODS AND RESULTS Staphylococci were collected from 100 dogs, 100 dog owners, 200 small animal veterinarians and 100 people without pet association. Species of MRCoPS were identified phenotypically and genotypically. Molecular characteristics were determined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and SCCmec typing, and antimicrobial susceptibility was assayed by broth microdilution and by microarray analysis for resistance genes. Methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSSc) and methicillin-resistant Staphylococcus aureus (MRSA) were isolated from dogs (45, 17 and 1%, respectively), veterinarians (8, 2 and 1·5%, respectively) and dog owners (3, 2 and 0%, respectively). Seventeen sequence types (STs) were identified among 83 MRSP isolates which specifically carried SCCmec V, II-III, ΨSCCmec57395 and three uncharacterized SCCmec types. MRSP ST 45, 68 and novel STs including 169, 178, 181 and 183 were shared among canine and human isolates. Most of MRSA ST398 and MRSSc carried SCCmec type V. The MRCoPS commonly displayed multiple resistances to tested antimicrobials and carried various resistance genes. CONCLUSION Variety of MRCoPS, especially new MRSP clones, distributed in dogs and people in Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY The existence of MRCoPS circulating between dogs and humans in Thailand provides indirect evidence of interspecies transmission and represents a potential public health hazard.

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment.

Microbiota at teeth and implants in partially edentulous patients. A 10-year retrospective study.

OBJECTIVE To determine the microbiota at implants and adjacent teeth 10 years after placement of implants with a sandblasted and acid-etched surface. MATERIAL AND METHODS Plaque samples obtained from the deepest sites of 504 implants and of 493 adjacent teeth were analyzed for certain bacterial species associated with periodontitis, for staphylococci, for aerobic gram-negative rods, and for yeasts using nucleic acid-based methods. RESULTS Species known to be associated with periodontitis were detectable at 6.2-78.4% of the implants. Significantly higher counts at implants in comparison with teeth were assessed for Tannerella forsythia, Parvimonas micra, Fusobacterium nucleatum/necrophorum, and Campylobacter rectus. Higher counts of periodontopathogenic species were detectable at implants of current smokers than at those of non-smokers. In addition, those species were found in higher quantities at implants of subjects with periodontitis. The prevalence of Prevotella intermedia, Treponema denticola, C. rectus, and moreover of Staphylococcus warneri might be associated with peri-implant inflammation. Selected staphylococcal species (not Staphylococcus aureus), aerobic gram-negative rods, and yeasts were frequently detected, but with the exception of S. warneri, they did not show any association with periodontal or peri-implant diseases. CONCLUSIONS Smoking and periodontal disease are risk factors for colonization of periodontopathic bacteria at implants. Those bacterial species may play a potential role in peri-implant inflammation. The role of S. warneri needs further validation.

Studies of the structure and function of a fibrinogen binding MSCRAMM(RTM) from Staphylococcus epidermidis

Coagulase-negative staphylococci (CNS) are recognized as important pathogens and are particularly associated with foreign body infections. S. epidermidis accounts for approximately 75% of the infections caused by CNS. Three genes, sdrF, sdrG, and sdrH, were identified by screening a S. epidermidis genomic library with a probe encompassing the serine-aspartate dipeptide repeat-encoding region (region R) of clfA from S. aureus. SdrG has significant amino acid identity to ClfA, ClfB and other surface proteins of S. aureus. SdrG is also similar to a protein (Fbe) recently described by Nilsson, et al. (Infection and Immunity, 1998, 66:2666–73) from S. epidermidis. The N-terminal domain (A region) of SdrG was expressed as a his-tag fusion protein in E. coli. In an ELISA, this protein, rSdrG(50-597) was shown to bind specifically to fibrinogen (Fg). Western ligand blot analysis showed that SdrG binds the Bβ chain of Fg. To further characterize the rSdrG(50-597)-Fg interaction, truncates of the Fg Bβ chain were made and expressed as recombinant proteins in E. coli. SdrG was shown to bind the full-length Bβ chain (1462), as well as the N-terminal three-quarters (1-341), the N-terminal one-half (1-220) and the N-terminal one-quarter (1-95) Bβ chain constructs. rSdrG(50-597) failed to bind to the recombinant truncates that lacked the N-terminal 25 amino acid residues of this polypeptide suggesting that SdrG recognizes a site within this region of the Bβ chain. Inhibition ELISAs have shown that peptide mimetics, including β1–25, and β6–20, encompassing this 25 residue region can inhibit binding of rSdrG(50-597) to Fg coated wells. Using fluorescence polarization we were able to determine an equilibrium constant (KD) for the interaction of rSdrG(50-597) with the Fg Bβ chain peptide β1–25. The labeled peptide was shown to bind to rSdrG(50-597) with a KD of 0.14 ± 0.01μM. Because rSdrG(50-597) recognizes a site in the Fg Bβ chain close to the thrombin cleavage site, we investigated the possibility of the rSdrG(50-597) site either overlapping or lying close to this cleavage site. An ELISA showed that rSdrG(50-597) binding to thrombin-treated Fg was significantly reduced. In a clot inhibition assay rSdrG(50-597) was able to inhibit fibrin clot formation in a concentration dependent manner. Furthermore, rSdrG(50-597) was able to inhibit clot formation by preventing the release of fibrinopeptide B as determined by HPLC. To further define the interaction between rSdrG(50-597) and peptide β6–20, we utilized an alanine amino acid replacement strategy. The residues in β6–20 that appear to be important in rSdrG(50-597) binding to Fg, were confirmed by the rSdrG(273-597)-β6–20 co-crystal structure that was recently solved by our collaborators at University of Alabama-Birmingham. Additionally, rSdrG(50-597) was not able to bind to Fg from different animal species, rather it bound specifically to human Fg in an ELISA. This suggests that the sequence variation between Fg Bβ chains of different species, specifically with in the N-terminal 25 residues, affects the ability of rSdrG(50-597) binding to Fg, and this may explain why S. epidermidis is primarily a human pathogen. ^

Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif

Surface proteins of Staphylococcus aureus are linked to the bacterial cell wall by sortase, an enzyme that cleaves polypeptides at the threonine of the LPXTG motif. Surface proteins can be released from staphylococci by treatment with hydroxylamine, resulting in the formation of threonine hydroxamate. Staphylococcal extracts, as well as purified sortase, catalyze the hydroxylaminolysis of peptides bearing an LPXTG motif, a reaction that can be inhibited with sulfhydryl-modifying reagents. Replacement of the single conserved cysteine at position 184 of sortase with alanine abolishes enzyme activity. Thus, sortase appears to catalyze surface-protein anchoring by means of a transpeptidation reaction that captures cleaved polypeptides as thioester enzyme intermediates.

The nasal microbiota in infants with cystic fibrosis in the first year of life: a prospective cohort study.

BACKGROUND Respiratory tract infections and subsequent airway inflammation occur early in the life of infants with cystic fibrosis. However, detailed information about the microbial composition of the respiratory tract in infants with this disorder is scarce. We aimed to undertake longitudinal in-depth characterisation of the upper respiratory tract microbiota in infants with cystic fibrosis during the first year of life. METHODS We did this prospective cohort study at seven cystic fibrosis centres in Switzerland. Between Feb 1, 2011, and May 31, 2014, we enrolled 30 infants with a diagnosis of cystic fibrosis. Microbiota characterisation was done with 16S rRNA gene pyrosequencing and oligotyping of nasal swabs collected every 2 weeks from the infants with cystic fibrosis. We compared these data with data for an age-matched cohort of 47 healthy infants. We additionally investigated the effect of antibiotic treatment on the microbiota of infants with cystic fibrosis. Statistical methods included regression analyses with a multivariable multilevel linear model with random effects to correct for clustering on the individual level. FINDINGS We analysed 461 nasal swabs taken from the infants with cystic fibrosis; the cohort of healthy infants comprised 872 samples. The microbiota of infants with cystic fibrosis differed compositionally from that of healthy infants (p=0·001). This difference was also found in exclusively antibiotic-naive samples (p=0·001). The disordering was mainly, but not solely, due to an overall increase in the mean relative abundance of Staphylococcaceae in infants with cystic fibrosis compared with healthy infants (multivariable linear regression model stratified by age and adjusted for season; second month: coefficient 16·2 [95% CI 0·6-31·9]; p=0·04; third month: 17·9 [3·3-32·5]; p=0·02; fourth month: 21·1 [7·8-34·3]; p=0·002). Oligotyping analysis enabled differentiation between Staphylococcus aureus and coagulase-negative Staphylococci. Whereas the analysis showed a decrease in S aureus at and after antibiotic treatment, coagulase-negative Staphylococci increased. INTERPRETATION Our study describes compositional differences in the microbiota of infants with cystic fibrosis compared with healthy controls, and disordering of the microbiota on antibiotic administration. Besides S aureus, coagulase-negative Staphylococci also contributed to the disordering identified in these infants. These findings are clinically important in view of the crucial role that bacterial pathogens have in the disease progression of cystic fibrosis in early life. Our findings could be used to inform future studies of the effect of antibiotic treatment on the microbiota in infants with cystic fibrosis, and could assist in the prevention of early disease progression in infants with this disorder. FUNDING Swiss National Science Foundation, Fondation Botnar, the Swiss Society for Cystic Fibrosis, and the Swiss Lung Association Bern.

The influence of the rearing period on intramammary infections in Swiss dairy heifers: A cross-sectional study

Healthy replacement heifers are one of the foundations of a healthy dairy herd. Farm management andrearing systems in Switzerland provide a wide variety of factors that could potentially be associated withintramammary infections (IMI) in early lactating dairy heifers. In this study, IMI with minor mastitispathogens such as coagulase-negative staphylococci (CNS), contagious pathogens, and environmentalmajor pathogens were identified. Fifty-four dairy farms were enrolled in the study. A questionnaire wasused to collect herd level data on housing, management and welfare of young stock during farm isitsand interviews with the farmers. Cow-level data such as breed, age at first calving, udder condition andswelling, and calving ease were also recorded. Data was also collected about young stock that spent aperiod of at least 3 months on an external rearing farm or on a seasonal alpine farm. At the quarterlevel, teat conditions such as teat lesions, teat dysfunction, presence of a papilloma and teat lengthwere recorded. Within 24 h after parturition, samples of colostral milk from 1564 quarters (391 heifers)were collected aseptically for bacterial culture. Positive bacteriological culture results were found in 49%of quarter samples. Potential risk factors for IMI were identified at the quarter, animal and herd levelusing multivariable and multilevel logistic regression analysis. At the herd level tie-stalls, and at cow-level the breed category “Brown cattle” were risk factors for IMI caused by contagious major pathogenssuch as Staphylococcus aureus (S. aureus). At the quarter-level, teat swelling and teat lesions were highlyassociated with IMI caused by environmental major pathogens. At the herd level heifer rearing at externalfarms was associated with less IMI caused by major environmental pathogens. Keeping pregnant heifersin a separate group was negatively associated with IMI caused by CNS. The odds of IMI with coagulase-negative staphylococci increased if weaning age was less than 4 months and if concentrates were fed tocalves younger than 2 weeks. This study identified herd, cow- and quarter-level risk factors that may beimportant for IMI prevention in the future.

The prevention and diagnosis of central venous catheter-related infection

The potential source of CVC colonisation was assessed. Isolates of coagulase-negative staphylococci (CoNS) recovered from the skin and CVC components of 3 cardiothoracic surgery patients were characterised by pulsed-field gel electrophoresis (PFGE). The genetic heterogeneity of CoNS isolated from the skin was demonstrated and specific genotypes implicated in catheter colonisation. In addition, phenotypic and genotypic typing techniques were assessed for their ability to characterise strains of CoNS recovered from 33 patients who developed catheter-related bloodstream infection (CR-BSI) on a bone marrow transplant (BMT) unit and Siaphylococcus aureus recovered from 6 cardiothoracic surgery patients with surgical site infection (SSI) following median sternotomy. This epidemiological investigation revealed that common strains of CoNS and 51 aureus where not associated with infection in patients with CR-BSI or sternal SSI during the study period. Furthermore, there was no correlation between phenotypic and genotypic characterisation results. The variable expression of phenotypic traits within strains of staphylococci was evident whilst PFGE and randomly amplified polymorphic DNA (RAPD) were highly discriminatory for the molecular characterisation of S. aureus and CoNS. This was highlighted in 8 stem cell transplant (SCT) patients whereby it was demonstrated that routine identification and characterisation of CoNS by phenotypic techniques may not be adequate for the diagnosis of CR-BSI by current guidelines. The potential of the lipid S ELISA to facilitate the diagnosis of CR-BSI in 38 haematology/SCT patients and sternal SSI in 57 cardiothoracic surgery patients was also assessed. The ELISA proved to be a sensitive test for the rapid serodiagnosis of infection due to staphylococci in immunocompetent patients. The acridine orange leucocyte cytospin test (AOLC) was also evaluated for the rapid diagnosis of CR-BSI in 16 haematology/SCT patients with Hickman CVC in situ. Although the sensitivity of the test was low, it may provide a useful adjunct to conventional methods for the in situ sampling of catheters to predict and diagnose CR-BSI, preventing the unnecessary removal of CVC.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.

Serodiagnosis of staphylococcal sepsis

The coagulase-negative staphylococci are the most frequent cause of sepsis associated with indwelling intravascular catheters. Current microbiological investigations to support the diagnosis of catheter-related sepsis (CRS) include the culture of blood and catheter tips, however positive results may reflect specimen contamination, or colonisation of the catheter rather than true sepsis. Previous serological approaches to assist in the diagnosis of CRS based on cellular staphylococcal antigens have been of limited value. In this current study, the serodiagnostic potential of an exocellular antigen produced by 7 strains of coagulase-negative staphylococci cultured in brain heart infusion broth was investigated. Antigenic material isolated by gel permeation from liquid culture was characterised by immunological techniques and chemical analysis. Characterisation of the exocellular antigen revealed a novel glycerophosphoglycolipid, termed lipid S. which shared antigenic determinants with lipoteichoic acid, but differed by comprising a glycerophosphate chain length of only 6 units. In addition, lipid S was immunologically distinct from diphosphatidyl glycerol, a constituent cell membrane phospho lipid. An indirect enzyme linked immunosorbent assay (ELISA) based on lipid S was subsequently developed and used to determine serum antibody levels (IgM and IgG) in 67 patients with CRS due to staphylococci, and 67 patients with a central venous catheter (CVC) in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the lipid S IgG ELISA was 75% and 90% respectively whilst the IgM assay had sensitivity and specificity of 52% and 85%. The addition of GullSORereagent to the EL1SA procedure to remove competing serum IgG and rheumatoid factor did not significantly improve the performance of the IgM assay. The serological response in serial serum samples of 13 patients with CRS due to staphylococci was investigated. Elevated levels of antibody were detected at an early stage of infection, prior to the isolation of microorganisms by standard culture methods, and before the clinical presentation of sepsis in 3 patients. The lipid S ELISA was later optimised and a rapid 4-hour assay developed for the serodiagnosis of CRS. Serum IgG levels were determined in 40 patients with CRS due to staphylococci and 40 patients with a CVC in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the rapid IgG assay was 70% and 100% respectively. Elevated serum antibody levels in patients with endocarditis, prosthetic joint infection and pyogenic spondylodiscitis due to Gram-positive cocci were also detected with the lipid S ELISA suggesting that the assay may facilitate the diagnosis of these infections. Unexpected increased levels of anti-lipid S IgG in 31% of control patients with sciatica suggested a possible microbial aetiology of this condition. Further investigation of some of these patients by culture of microdiscectomy tissue removed at operation, revealed the presence of low-virulent microorganisms in 37% of patients of which Propionibacterium aeries accounted for 85% of the positive culture isolates. The results suggested a previously unrecognised association between P. acnes and sciatica, which may have implications for the future management of the condition.

Antibacterial activity of low amperage and low voltage electric current (DC)

Infection is a major clinical problem associated with the use of intravenous catheters.The efficacy of a direct electric current (10µA, 9V) via electrode-conducting carbon impregnated catheters to prevent colonisation of catheters by micro-organisms was investigated. The range of organisms susceptible to 10µA was determined by a zone of inhibition test. The catheters acting as the anode and the cathode were inserted into a nutrient agar plate inoculated with a lawn of bacteria. There was no zone of inhibition observed around the anode. Organisms susceptible to 10µA at the cathode were Staphylococcus aureus (2 strains), Staphylococcus epidermidis (5 strains), Escherichia coli and Klebsiella pneumoniae (2 strains each), and one strain of the following micro-organisms: Staphylococcus hominis, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. The zones ranged from 6 to 16 mm in diameter according to the organisms under test. The zone size was proportional to the amperage (10 - 100 µA) and the number of organisms on the plate. Ten µA did not prevent adhesion of staphylococci to the cathode nor did it affect their growth in nutrient broth. However, it was bactericidal to adherent bacteria on the cathodal catheter and significantly reduced the number of bacteria on the catheter after 4 to 24 h application of electricity. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated.The mechanisms of the bactericidal activity associated with the cathode were investigated with S. epidermidis and S. aureus. The inhibition zone was greatly reduced in the presence of catalase. There was no zone around the cathode when the test was carried out under anaerobic conditions. Hydrogen peroxide was produced at the cathode surface under aerobic conditions, but not in the absence of oxygen. A salt-bridge apparatus was used to demonstrate further that hydrogen peroxide was produced at the cathode, and chlorine at the anode. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated. Antibacterial activity was reduced under anaerobic conditions, which is compatible with the role of hydrogen peroxide as a primary bactericidal agent of electricity associated with the cathode. A reduction in chloride ions did not significantly reduce the antibacterial activity suggesting chlorine plays only a minor role in the bactericidal activity against organisms attached to anodal electrode surfaces. The bactericidal activity of electric current associated with the cathode and H202 was greatly reduced in the presence of 50 μM to 0.5 mM magnesium ions in the test menstrum. Ten μA applied via the catheters did not prevent the initial biofilm growth by the adherent bacteria but reduced the number of bacteria in the biofilm by 2 log order aiter 24 h. The results suggested that 10 μA may prevent the colonisation of catheters by both the extra~ and intra-luminal routes. The localised production of hydrogen peroxide and chlorine and the intrinsic activity due to electric current may offer a useful method for the eradication of bacteria from catheter surfaces.