Identification of mechanisms modulating chlorhexidine and octenidine susceptibility in Proteus mirabilis.

AIMS: We aimed to identify mechanisms underlying the tolerance of P. mirabilis-a common cause of catheter associated urinary tract infection-to the clinically used biocides chlorhexidine (CHD) and octenidine (OCT). METHODS AND RESULTS: We adapted three clinical isolates to grow at concentrations of 512 µg ml-1 CHD and 128 µg ml-1 OCT. Genetic characterisation and complementation studies revealed mutations inactivating the smvR repressor and increasing smvA efflux expression were associated with adaptation to both biocides. Mutations in mipA (encoding the MltA interacting protein) were less prevalent than smvR mutations and only identified in CHD adapted populations. Mutations in the rppA response regulator were exclusive to one adapted isolate and were linked with reduced polymyxin B susceptibility and a predicted gain of function after biocide adaptation. Biocide adaptation had no impact on crystalline biofilm formation. CONCLUSIONS: SmvR inactivation is a key mechanism in both CHD and OCT tolerance. MipA inactivation alone confers moderate protection against CHD, and rppA showed no direct role in either CHD or OCT susceptibility.

Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

De-repression of the smvA efflux system arises in clinical isolates of Proteus mirabilis and reduces susceptibility to chlorhexidine and other biocides.

Proteus mirabilis is a common pathogen of the catheterised urinary tract and often described as intrinsically resistant to the biocide chlorhexidine (CHD). Here we demonstrate that de-repression of the smvA efflux system has occurred in clinical isolates of P. mirabilis and reduces susceptibility to CHD and other cationic biocides. Compared to other isolates examined, P. mirabilis RS47 exhibited a significantly higher CHD MIC (≥512 µg/ml) and significantly greater expression of smvA. Comparison of the RS47 smvA and cognate smvR repressor with sequences from other isolates, indicated that RS47 encodes an inactivated smvR. Complementation of RS47 with a functional smvR from isolate RS50a (which exhibited the lowest smvA expression and lowest CHD MIC) reduced smvA expression by ∼59-fold, and markedly lowered the MIC of CHD and other cationic biocides. Although complementation of RS47 did not reduce MICs to concentrations observed in isolate RS50a, the significantly lower polymyxin B MIC of RS50a indicated that differences in LPS structure are also a factor in P. mirabilis CHD susceptibility. To determine if exposure to CHD can select for mutations in smvR, clinical isolates with the lowest CHD MICs were adapted to grow at increasing concentrations of CHD up to 512 µg/ml. Analysis of the smvR in adapted populations indicated that mutations predicted to inactivate smvR occurred following CHD exposure in some isolates. Collectively, our data show that smvA de-repression contributes to reduced biocide susceptibility in P. mirabilis, but differences in LPS structure between strains are also likely to be an important factor.

Pseudomonas aeruginosa adapts to octenidine in the laboratory and a simulated clinical setting, leading to increased tolerance to chlorhexidine and other biocides.

BACKGROUND: Octenidine is frequently used for infection prevention in neonatal and burn intensive care units, where Pseudomonas aeruginosa has caused nosocomial outbreaks. AIM: To investigate the efficacy and impact of using octenidine against P. aeruginosa. METHODS: Seven clinical isolates of P. aeruginosa were exposed to increasing concentrations of octenidine over several days. Fitness, minimum bactericidal concentrations after 1 min, 5 min and 24 h, and minimum inhibitory concentrations (MICs) of a variety of antimicrobials were measured for the parental and octenidine-adapted P. aeruginosa strains. Octenidine and chlorhexidine MICs of a population of P. aeruginosa isolated from a hospital drain trap, exposed to a diluted octenidine formulation four times daily for three months, were also tested. FINDINGS: Some planktonic cultures of P. aeruginosa survived >50% of the working concentration of an in-use octenidine formulation at the recommended exposure time. Seven strains of P. aeruginosa stably adapted following continuous exposure to increasing concentrations of octenidine. Adaptation increased tolerance to octenidine formulations and chlorhexidine up to 32-fold. In one strain, it also led to increased MICs of antipseudomonal drugs. Subsequent to continuous octenidine exposure of a multi-species community in a simulated clinical setting, up to eight-fold increased tolerance to octenidine and chlorhexidine of P. aeruginosa was also found, which was lost upon removal of octenidine. CONCLUSION: Incorrect use of octenidine formulations may lead to inadequate decontamination, and even increased tolerance of P. aeruginosa to octenidine, with resulting cross-resistance to other biocides.

Growth media and assay plate material can impact on the effectiveness of cationic biocides and antibiotics against different bacterial species.

The effectiveness of several cationic disinfectants as well as colistin and polymyxin B were assessed under different growth conditions against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains. These conditions included different media (MH1, MH2, TSB and LB) and plate material (polypropylene and polystyrene). Results showed that Minimum inhibitory and bactericidal concentrations (MIC/MBC) values of colistin and polymyxin B were significantly lower on polypropylene plates when compared to polystyrene plates regardless of media used. There were also differences in MIC/MBC values to certain biocides e.g. chlorhexidine and octenidine particularly for S. aureus and E. coli strains, with polypropylene again showing lower values. Other biocides appear to be mostly unaffected by plate type. Whether biocide efficacy was altered by media composition was organism dependent with S. aureus and E. coli more affected than P. aeruginosa. Lower MIC values were more commonly associated with MH2 media and higher MIC values with TSB media for both polypropylene and polystyrene plates, although there were exceptions. Results obtained for standard strains were, in general, indicative for other S. aureus, E. coli and P. aeruginosa strains tested. This study demonstrates the importance of media composition and plate material on biocide effectiveness and highlights the need for optimized disinfectant testing methods. SIGNIFICANCE AND IMPACT OF THE STUDY: There are an increasing number of reports of bacterial strains that are multi-drug resistant. The use of biocides as part of infection control is crucial in helping to combat the spread of these particular strains. Unlike for antibiotics, there are few standardized measuring techniques to understand if an isolate has become more resistant to biocides. This study demonstrates the importance of media composition and plate material on variation and reporting of susceptibility of several bacterial species to specific cationic biocides. It is a useful comparison study to highlight the need to standardize biocide susceptibility testing.

Varying activity of chlorhexidine-based disinfectants against Klebsiella pneumoniae clinical isolates and adapted strains.

BACKGROUND: Control of multi-drug-resistant (MDR) organisms relies increasingly on the use of biocides, including chlorhexidine, to limit the risk of infection. The concentration and formulation of chlorhexidine can vary hugely between products. AIM: To establish the activity of chlorhexidine and in-use chlorhexidine formulations against 14 clinical Klebsiella pneumoniae strains isolated before and since the use of chlorhexidine became routine, and strains that have adapted following sublethal chlorhexidine exposure. METHODS: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of five chlorhexidine-containing formulations were measured at 5min, 15min, 30min and 24h for the panel of K. pneumoniae strains. FINDINGS: After 5min, MBCs of five formulations varied from 0.006 to >50% working concentration (WC) or from 78 to 2500µg/mL chlorhexidine. For one formulation, MBCs were >50% WC for five of the 14 strains, and for another formulation, four of the 14 strains could resist 25% WC. NCTC 13368 was consistently most tolerant to chlorhexidine, whereas the strains isolated before the use of chlorhexidine became routine were more sensitive. One pre-chlorhexidine era and five modern strains increased MICs up to 16-fold following exposure to sublethal concentrations of chlorhexidine. A hand disinfectant with MBCs of 0.39% WC for all six of the wild-type strains, had MBCs of 50% WC for the chlorhexidine-adapted strains. CONCLUSION: Not all chlorhexidine formulations kill MDR K. pneumoniae after the recommended exposure time. Activity, especially against chlorhexidine-adapted strains, depends on additional ingredients. Careful formulation of chlorhexidine products is therefore important to maintain and enhance the activity of chlorhexidine products, and avoid potential breakdown in infection control.

Establishment of a multi-species biofilm model to evaluate chlorhexidine efficacy.

BACKGROUND: Chronic infections, for example, diabetic foot ulcers, have a large impact in terms of patient morbidity and mortality. These wounds are characterized by complex polymicrobial communities of bacteria, which may include a number of difficult-to-eradicate multidrug-resistant pathogens. AIM: To establish a multi-species biofilm model to test the efficacy of chlorhexidine and chlorhexidine-containing formulas in eradication of polymicrobial biofilms. METHODS: A Centers for Disease Control and Prevention bioreactor was used to establish a multi-species biofilm incorporating Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis with equal numbers of each pathogen. This model was used to test the effectiveness of chlorhexidine at controlling the pre-formed biofilm. FINDINGS: Chlorhexidine digluconate (CHD) was added to the bioreactor at a range of concentrations. K. pneumoniae and P. aeruginosa survived within multi-species biofilms, up to and including 4% CHD, whereas S. aureus was reduced to below the level of detection at 1%. Wiping the biofilm-containing coupons from the bioreactor with chlorhexidine-containing medical wipes resulted in >3 to <4log10 reduction after 24h, for all species. When the coupons were embedded in a simulated wound bed, formed in an agar plate, CHD-containing medical dressings completely eliminated S. aureus (>8log10 reduction), but had minimal effect (<3log10) against the other species tested. CONCLUSION: The study demonstrates that the effectiveness of chlorhexidine may be limited in settings where it is required to act on multi-species biofilms. This may compromise the ability of chlorhexidine to control the infection and spread of these pathogens.

Evaluation of the effectiveness of hydrogen-peroxide-based disinfectants on biofilms formed by Gram-negative pathogens.

BACKGROUND: Hydrogen peroxide (H2O2)-based disinfectants are widely used in a number of different healthcare settings to control bacterial colonization and contamination, and reduce the risk of cross-infection. Efficacy tests of these formulations are performed on planktonic cultures, although it is well known that biofilms are the dominant form of bacterial contamination and more difficult to eradicate. AIM: To determine if the biofilms of three different Gram-negative pathogens associated with multi-drug-resistant phenotypes can be eradicated effectively using different H2O2-based disinfectants. METHODS: Planktonic cultures and single-species 24-h biofilms of seven strains of Acinetobacter spp., seven strains of Klebsiella pneumoniae and seven strains of Pseudomonas aeruginosa, including clinical isolates, were exposed to working concentrations of H2O2 and H2O2-based formulations for 1 min to 24h. Survival was monitored. FINDINGS: The levels of susceptibility of planktonic cultures to unformulated and formulated H2O2 were similar in all organisms and strains tested, with minimum inhibitory concentrations ranging from 0.5 to 20mM H2O2. However, biofilms showed up to 266-fold less sensitivity to H2O2 and its formulations. The level of reduced susceptibility correlated with the strain's propensity to form biofilm, and differed between species. The two formulations with additional acidic active ingredients performed better at short exposure times, whereas ethanol-containing products required longer exposure times to be effective. CONCLUSION: Biofilms of a significant number of clinical isolates of multi-drug-resistant nosocomial pathogens are not susceptible to working concentrations of several H2O2-based disinfectants. This may compromise the ability to control these pathogens with such products.