Unexpected synergistic and antagonistic antibiotic activity against Staphylococcus biofilms.

Objectives: To evaluate putative anti-staphylococcal biofilm antibiotic combinations used in the management of periprosthetic joint infections (PJIs). Methods: Using the dissolvable bead biofilm assay, the minimum biofilm eradication concentration (MBEC) was determined for the most commonly used antimicrobial agents and combination regimens against staphylococcal PJIs. The established fractional inhibitory concentration (FIC) index was modified to create the fractional biofilm eradication concentration (FBEC) index to evaluate synergism or antagonism between antibiotics. Results: Only gentamicin (MBEC 64 mg/L) and daptomycin (MBEC 64 mg/L) were observed to be effective antistaphylococcal agents at clinically achievable concentrations. Supplementation of gentamicin with daptomycin, vancomycin or ciprofloxacin resulted in a similar or lower MBEC than gentamicin alone (FBEC index 0.25-2). Conversely, when rifampicin, clindamycin or linezolid was added to gentamicin, there was an increase in the MBEC of gentamicin relative to its use as a monotherapy (FBEC index 8-32). Conclusions: This study found that gentamicin and daptomycin were the only effective single-agent antibiotics against established Staphylococcus biofilms. Interestingly the addition of a bacteriostatic antibiotic was found to antagonize the ability of gentamicin to eradicate Staphylococcus biofilms.

The biofilm eradication activity of acetic acid in the management of periprosthetic joint infection.

OBJECTIVES: Periprosthetic joint infection following joint arthroplasty surgery is one of the most feared complications. The key to successful revision surgery for periprosthetic joint infections, regardless of treatment strategy, is a thorough deep debridement. In an attempt to limit antimicrobial and disinfectant use, there has been increasing interest in the use of acetic acid as an adjunct to debridement in the management of periprosthetic joint infections. However, its effectiveness in the eradication of established biofilms following clinically relevant treatment times has not been established. Using an in vitro biofilm model, this study aimed to establish the minimum biofilm eradication concentration (MBEC) of acetic acid following a clinically relevant treatment time. MATERIALS AND METHODS: Using a methicillin-sensitive Staphylococcus aureus (MSSA) reference strain and the dissolvable bead assay, biofilms were challenged by 0% to 20% acetic acid (pH 4.7) for ten minutes, 20 minutes, 180 minutes, and 24 hours. RESULTS: The MBEC of acetic acid was found to be: 15%, 11%, 3.2%, and 0.8% following a ten-minute, 20-minute, 180-minute, and 24-hour treatment, respectively. CONCLUSION: This study found that the MBEC of acetic acid following a 10- or 20-minute treatment time exceeded its safety threshold, making these concentrations unsuitable as a topical debridement adjunct. However, a clinically acceptable concentration (5%) was still found to eliminate 96.1% of biofilm-associated MSSA following a 20-minute treatment time.Cite this article: S. T. J. Tsang, P. J. Gwynne, M. P. Gallagher, A. H. R. W. Simpson. The biofilm eradication activity of acetic acid in the management of periprosthetic joint infection. Bone Joint Res 2018;7:517-523. DOI: 10.1302/2046-3758.78.BJR-2018-0045.R1.

The dissolvable bead: A novel in vitro biofilm model for evaluating antimicrobial resistance.

In vitro biofilm assays are a vital first step in the assessment of therapeutic effectiveness. Current biofilm models have been found to be limited by throughput, reproducibility, and cost. We present a novel in vitro biofilm model, utilising a sodium alginate substratum for surface biofilm colony formation, which can be readily dissolved for accurate evaluation of viable organisms. The dissolving bead biofilm assay was evaluated using a range of clinically relevant strains. The reproducibility and responsiveness of the assay to an antimicrobial challenge was assessed using standardised methods. Cryo-scanning electron microscopy was used to image biofilm colonies. Biofilms were grown for 20h prior to testing. The model provides a reproducible and responsive assay to clinically-relevant antimicrobial challenges, as defined by established guidelines. Moreover cryo-scanning electron microscopy demonstrates that biofilm formation is localised exclusively to the alginate bead surface. Our results suggest that this simple model provides a robust and adaptable assay for the investigation of bacterial biofilms.

Molecular characterization of the oligopeptide permease of Salmonella typhimurium.

The oligopeptide permease (Opp) of Salmonella typhimurium is a periplasmic binding protein-dependent transport system and handles any peptides containing from two to five amino acid residues. Opp plays an important nutritional role and is also required for the recycling of cell wall peptides. We have determined the nucleotide sequence of the opp operon. In addition to the four opp genes identified previously by genetic means (oppABCD) a fifth gene, oppF, is shown to be cotranscribed as part of the opp operon. Using reverse genetics, we show that oppF also encodes an essential component of the Opp transport system. The five proteins, OppABCDF, are shown to be the only proteins required for Opp function. Regulation of opp expression and of the differential expression of genes within the operon is investigated. We have devised a simple means of constructing lacZ gene fusions to any S. typhimurium chromosomal gene in vivo, using derivatives of bacteriophage Mu. Using this procedure, opp-lacZ gene fusions were selected. The resultant Opp-LacZ hybrid proteins were used to show that OppB, OppC and OppD are membrane-associated proteins. A detailed comparison of the Opp components with those of other binding protein-dependent transport systems provides insight into the mechanisms and evolution of these transport systems.

TonB protein of Salmonella typhimurium. A model for signal transduction between membranes.

The tonB gene product is required for several outer membrane transport processes in bacteria. The tonB gene from Salmonella typhimurium was sequenced and found to be similar to that of Escherichia coli. The TonB protein is highly proline-rich and includes an unusual segment consisting of multiple X-Pro dipeptide repeats. A synthetic peptide corresponding to this segment has been used to raise anti-TonB antibodies. TonB was shown to be associated with the cytoplasmic membrane, apparently anchored via a single hydrophobic N-terminal segment. Protease accessibility studies, and the use of a series of TonB-beta-lactamase fusions, showed that the rest of the TonB protein is periplasmic. Unusually, export of TonB is not accompanied by cleavage of the N-terminal signal peptide. In the accompanying paper, we show that TonB interacts directly with the outer membrane FhuA (TonA) receptor. Thus, TonB must span the periplasm, providing a link between the cytoplasmic membrane and receptors in the outer membrane. On the basis of these data, and those published by other laboratories, we propose a model whereby TonB serves as a "mechanical" linkage that, by transmitting protein conformational changes from the cytoplasmic membrane across the periplasm, acts as a means of coupling energy to outer membrane transport processes. Such a mechanism has general implications for signal transduction within and between proteins.

Stage-specific serine and metallo-proteinase release by adult and larval Trichostrongylus vitrinus.

Proteinases were released in a stage-specific manner during in vitro culture by 4th larval stage and adult Trichostrongylus vitrinus. Substrate gel analyses and inhibitor studies revealed the presence of serine and metallo-proteinases, active over a broad pH range, which degraded proteins such as fibrinogen, plasminogen and fibronectin but not immunoglobulin. The adult proteinases were partially inhibited (43%) by immunoglobulin from immune lamb lymph compared to controls, indicating their relevance to parasite immunobiology in vivo.

Light emission from a Mudlux transcriptional fusion in Salmonella typhimurium is stimulated by hydrogen peroxide and by interaction with the mouse macrophage cell line J774.2.

Hydrogen peroxide is known to induce a multigenic response in Salmonella typhimurium cells. We have used a Mudlux transcriptional reporter system to identify and isolate fusions in the virulent strain SL1344 which respond to hydrogen peroxide in vitro by light production, and one of these fusions, MPG203, has been further characterized. Transient light production was observed from MPG203 at levels of hydrogen peroxide as low as 10 microM. However, high levels of this toxic oxidizing agent resulted in light suppression, particularly at low bacterial densities. This fusion was also shown to produce light following adhesion to cells of the mouse macrophage cell line J774.2. Furthermore, the response was greatly reduced in the presence of catalase, directly implicating hydrogen peroxide as the eliciting agent and suggesting the involvement of the hydrogen peroxide-induced bacterial stress response in the infection process. Chemiluminescence studies also indicated that inhibition of the respiratory burst may occur as the infection ratio is increased. In addition, the level of light produced from bacteria within individual macrophage cells was shown to vary.

Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent insertion element, IS 186.

Escherichia coli is known to contain more than one active transport system for nucleoside uptake. In the present study we report the sequence of a gene encoding a second nucleoside transport system, nupC (in addition to nupG). An open reading frame (ORF) of 1200 bp was identified that codes for a hydrophobic polypeptide of 43,560 Da and an NupC fusion protein was shown to be membrane associated. The native NupC protein is also identified, following over-expression. NupC exhibits short regions of homology to several membrane-associated proteins, including LacY and Cyd. Analysis of the nupC promoter region revealed the presence of at least two putative CRP-binding sites, centred at -40bp and -89bp, which probably flank a CytR-binding site. In addition, an adjacent IS 186 element was identified and found to reside within a putative terminator structure, downstream from the nupC ORF. This arrangement is shown to reflect the previously established gene order on the E. coli chromosome.

The Salmonella typhimurium AhpC polypeptide is not essential for virulence in BALB/c mice but is recognized as an antigen during infection.

The OxyR regulon is known to mediate protection against oxidizing agents in Salmonella typhimurium. We reported previously that ahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K. P. Francis, P. D. Taylor, C. J. Inchley, and M. P. Gallagher, J. Bacteriol. 179:4046-4048, 1997). We now report on the effects of disrupting ahp or oxyR on virulence in a BALB/c mouse model. Surprisingly, insertion of a Mudlux derivative within ahpC was found to result in attenuation, while irreversible inactivation of the locus through insertion of a cml cassette did not. An SL1344 derivative carrying an oxyR::kan disruption was also found to be as virulent as the parental strain. Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation. These results indicate that, although not essential for virulence, AhpC is expressed by S. typhimurium during infection of BALB/c mice and constitutes a target for the immune system.

Identification and localization of the membrane-associated, ATP-binding subunit of the oligopeptide permease of Salmonella typhimurium.

The OppF protein, a component of the oligopeptide permease of Salmonella typhimurium, is an ATP-binding protein and is believed to couple ATP hydrolysis to the transport process. This protein is an example of a large family of closely related proteins which couple ATP to a variety of different biological processes. The oppF gene has been cloned and sequenced. In order to identify and characterize its protein product we overproduced the protein from the cloned gene. Anti-OppF antibodies were raised against a synthetic peptide. Using these antibodies as a probe we identified OppF in wild-type and overproducing strains. Protease accessibility studies showed the protein to be a peripheral membrane protein located on the cytoplasmic side of the inner membrane. These findings have general implications for the organization and function of this class of prokaryotic and eukaryotic transport system.

Surface hygiene monitored using a reporter of fis in Escherichia coli.

AIMS: To examine the value of the fis promoter in monitoring regrowth of a surface-attached bacterial population following exposure to chemical stress using several candidate reporters, beta-galactosidase (lacZYA), bacterial luciferase (luxAB) and enhanced green fluorescent protein (EGFP). METHODS AND RESULTS: The pattern of expression for the reporters within Escherichia coli cells attached to surfaces was determined. Both the bacterial luciferase reporter and EGFP were readily detected, but EGFP was found to overcome problems associated with luciferase and beta-galactosidase. The effect of surface pretreatment, using polymer systems, on bacterial attachment and growth confirmed the usefulness of this approach. CONCLUSION: The fis promoter, combined with EGFP, can be used successfully to study adhesion, biocidal damage and recovery. The stability of the EGFP enabled the magnitude of the total recovery response to be monitored as cells remained fluorescent after the decline in fis expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The E. coli Pfis-egfp reporter system provides a new, versatile and sensitive tool to investigate bacterial adhesion both quantitatively and qualitatively.

The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation.

Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo-). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo- mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram-negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide-transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP-binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide-binding protein, which is periplasmically located in Gram-negative species, has a signal sequence characteristic of lipoproteins with an amino-terminal lipo-amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram-negative bacteria is the recycling of cell-wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signalling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.

Interactions between Salmonella typhimurium and Acanthamoeba polyphaga, and observation of a new mode of intracellular growth within contractile vacuoles.

Acanthamoeba polyphaga feeding on Salmonella typhimurium in a simple model biofilm were observed by light microscopy and a detailed record of interactions kept by digital image capture and image analysis. A strain of S. typhimurium SL1344 carrying a fis: gfp reporter construct (pPDT105) was used to assess intracellular growth in A. polyphaga on non-nutrient agar (NNA) plates. Invasion of the contractile vacuole (CV) was observed at a frequency of 1:100-1000 acanthamoebae at 35 degrees C. The salmonellae contained in CVs illustrated significant up-regulation of fis relative to extracellular bacteria, indicating that they were in the early stages of logarithmic growth, and reached numbers of 100-200 cells per vacuole after 4 days. This is the first report of this mode of intracellular growth. Up-regulation of fis was also observed in a proportion of S. typhimurium cells contained within food vacuoles. Filamentation of S. typhimurium and E. coli cells was frequently observed in coculture with A. polyphaga on NNA plates, with bacterial cells reaching lengths of up to 500 microm after 10 days' incubation at 35 degrees C. A. polyphaga was also seen to mediate bacterial translocation over the agar surface; egested salmonellae subsequently formed microcolonies along amoebal tracks. This illustrated intracellular survival of a fraction of the S. typhimurium population. These phenomena suggest that protozoa such as A. polyhaga may play an important role in the ecology of S. typhimurium in soil and aquatic environments.

Identification of the ahp operon of Salmonella typhimurium as a macrophage-induced locus.

Previously, we tagged a macrophage-induced Salmonella typhimurium locus with Mudlux (K. P. Francis and M. P. Gallagher, Infect. Immun. 61:640-649, 1993). The insertion lies within the OxyR-regulated ahpC locus and conveys alkyl peroxide sensitivity. Plasmid-encoded ahp reverses sensitivity but reduces luminescence. This suggests that OxyR is titrated by the multicopy ahp promoter.

Membrane topology of the integral membrane components, OppB and OppC, of the oligopeptide permease of Salmonella typhimurium.

The oligopeptide permease of Salmonella typhimurium is a periplasmic binding protein-dependent transport system. Five gene products, OppABCDF, are required for the functioning of this transporter, two of which (OppB and OppC) are highly hydrophobic, integral membrane proteins and are responsible for mediating passage of peptides across the cytoplasmic membrane. OppB and OppC are each predicted, from their sequences, to span the membrane many times. In this paper we describe experimental evidence confirming these predictions using a combination of biochemical, immunological and genetic procedures. Each of these two proteins is shown to span the membrane six times, with the N- and C-termini both being located at the cytoplasmic face of the membrane. Opp is apparently a typical member of the ABC (ATP-binding cassette) superfamily of transporters. These findings, therefore, have general implications for the organization and function of other ABC transporters, including the human multidrug resistance protein and the product of the cystic fibrosis gene.

Energy coupling to periplasmic binding protein-dependent transport systems: stoichiometry of ATP hydrolysis during transport in vivo.

Periplasmic binding protein-dependent transport systems mediate the accumulation of many diverse substrates in prokaryotic cells. Similar transport systems, including the P-glycoprotein responsible for multidrug resistance in human tumors, are also found in eukaryotes. The mechanism by which energy is coupled to the accumulation of substrate by these transport systems has been controversial. In this paper we demonstrate that ATP hydrolysis occurs in vivo concomitantly with transport. These data strongly suggest that ATP hydrolysis directly energizes substrate accumulation by these transport systems. The apparent stoichiometry is one to two molecules of ATP hydrolyzed per molecule of substrate transported.

Binding protein-dependent transport systems.

Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other. These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems. Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins. Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus. In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process.

Periplasmic binding protein-dependent transport systems: the membrane-associated components.

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.