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.

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.

Bacterial biofilm formation on indwelling urethral catheters.

Urethral catheters are the most commonly deployed medical devices and used to manage a wide range of conditions in both hospital and community care settings. The use of long-term catheterization, where the catheter remains in place for a period >28 days remains common, and the care of these patients is often undermined by the acquisition of infections and formation of biofilms on catheter surfaces. Particular problems arise from colonization with urease-producing species such as Proteus mirabilis, which form unusual crystalline biofilms that encrust catheter surfaces and block urine flow. Encrustation and blockage often lead to a range of serious clinical complications and emergency hospital referrals in long-term catheterized patients. Here we review current understanding of bacterial biofilm formation on urethral catheters, with a focus on crystalline biofilm formation by P. mirabilis, as well as approaches that may be used to control biofilm formation on these devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Urinary catheters are the most commonly used medical devices in many healthcare systems, but their use predisposes to infection and provide ideal conditions for bacterial biofilm formation. Patients managed by long-term urethral catheterization are particularly vulnerable to biofilm-related infections, with crystalline biofilm formation by urease producing species frequently leading to catheter blockage and other serious clinical complications. This review considers current knowledge regarding biofilm formation on urethral catheters, and possible strategies for their control.

The phosphoenolpyruvate:sugar phosphotransferase system is involved in sensitivity to the glucosylated bacteriocin sublancin.

The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin.

In the absence of Lgt, lipoproteins are shed from Streptococcus uberis independently of Lsp.

The role of lipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) responsible for processing lipoproteins was investigated in Streptococcus uberis, a common cause of bovine mastitis. In the absence of Lgt, three lipoproteins [MtuA (SUB0473), Hap (SUB1625) and an extracellular solute-binding protein (SUB0365)] were detected in extracellular locations. All were shown by Edman degradation analysis to be cleaved on the carboxy side of the LXXC lipobox. Detection of MtuA, a lipoprotein shown previously to be essential for infectivity and virulence, was used as a surrogate lipoprotein marker to locate and assess processing of lipoproteins. The absence of Lgt did not prevent location of MtuA to the cell membrane, its location in the wild-type strain but, in contrast to the situation with wild-type, did result in a widespread location of this protein. In the absence of both Lgt and Lsp, MtuA was similarly released from the bacterial cell. In such strains, however, the cell-associated MtuA represented the full-length gene product, indicating that Lsp was able to cleave non-lipidated (lipo)proteins but was not responsible for their release from this bacterium.

Lipoprotein signal peptides are processed by Lsp and Eep of Streptococcus uberis.

Lipoprotein signal peptidase (lsp) is responsible for cleaving the signal peptide sequence of lipoproteins in gram-positive bacteria. Investigation of the role of Lsp in Streptococcus uberis, a common cause of bovine mastitis, was undertaken using the lipoprotein MtuA (a protein essential for virulence) as a marker. The S. uberis lsp mutant phenotype displayed novel lipoprotein processing. Not only was full-length (uncleaved) MtuA detected by Western blotting, but during late log phase, a lower-molecular-weight derivative of MtuA was evident. Similar analysis of an S. uberis double mutant containing insertions disrupting both lsp and eep (a homologue of the Enterococcus faecalis "enhanced expression of pheromone" gene) indicated a role for eep in cleavage of lipoproteins in the absence of Lsp. Such a function may indicate a role for eep in maintenance of secretion pathways during disruption of normal lipoprotein processing.