Antimicrobial Heteroresistance: an Emerging Field in Need of Clarity

SUMMARY: "Heteroresistance" describes a phenomenon where subpopulations of seemingly isogenic bacteria exhibit a range of susceptibilities to a particular antibiotic. Unfortunately, a lack of standard methods to determine heteroresistance has led to inappropriate use of this term. Heteroresistance has been recognized since at least 1947 and occurs in Gram-positive and Gram-negative bacteria. Its clinical relevance may be considerable, since more resistant subpopulations may be selected during antimicrobial therapy. However, the use of nonstandard methods to define heteroresistance, which are costly and involve considerable labor and resources, precludes evaluating the clinical magnitude and severity of this phenomenon. We review the available literature on antibiotic heteroresistance and propose recommendations for definitions and determination criteria for heteroresistant bacteria. This will help in assessing the global clinical impact of heteroresistance and developing uniform guidelines for improved therapeutic outcomes.

Interfacial self-assembly of a bacterial hydrophobin

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded β-sheet. This form then self-assembles into a well-ordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Predatory Bdellovibrio bacteria use gliding motility to scout for prey on surfaces

Bdellovibrio bacteriovorus is a famously fast, flagellate predatory bacterium, preying upon Gram-negative bacteria in liquids; how it interacts with prey on surfaces such as in medical biofilms is unknown. Here we report that Bdellovibrio bacteria "scout" for prey bacteria on solid surfaces, using slow gliding motility that is present in flagellum-negative and pilus-negative strains.

Bdellovibrio predation in the presence of decoys:Three-way bacterial interactions revealed by mathematical and experimental analyses

Bdellovibrio bacteriovorus is a small, gram-negative, motile bacterium that preys upon other gram-negative bacteria, including several known human pathogens. Its predation efficiency is usually studied in pure cultures containing solely B. bacteriovorus and a suitable prey. However, in natural environments, as well as in any possible biomedical uses as an antimicrobial, Bdellovibrio is predatory in the presence of diverse decoys, including live nonsusceptible bacteria, eukaryotic cells, and cell debris. Here we gathered and mathematically modeled data from three-member cultures containing predator, prey, and nonsusceptible bacterial decoys. Specifically, we studied the rate of predation of planktonic late-log-phase Escherichia coli S17-1 prey by B. bacteriovorus HD100, both in the presence and in the absence of Bacillus subtilis nonsporulating strain 671, which acted as a live bacterial decoy. Interestingly, we found that although addition of the live Bacillus decoy did decrease the rate of Bdellovibrio predation in liquid cultures, this addition also resulted in a partially compensatory enhancement of the availability of prey for predation. This effect resulted in a higher final yield of Bdellovibrio than would be predicted for a simple inert decoy. Our mathematical model accounts for both negative and positive effects of predator-prey-decoy interactions in the closed batch environment. In addition, it informs considerations for predator dosing in any future therapeutic applications and sheds some light on considerations for modeling the massively complex interactions of real mixed bacterial populations in nature.

Lactobacilli possess inhibitory activity against dipeptidyl peptidase-4 (DPP-4)

Dipeptidyl peptidase 4 (DPP-4) enzymatically inactivates incretin hormones, and DPP-4 inhibitor drugs are clinically approved therapies for type 2 diabetes. The primary substrates of DPP-4 are produced in the intestinal lining and we therefore investigated whether lactobacilli colonizing the gut can inhibit this enzyme. Fifteen Lactobacillus strains (Lb 1-15) from human infant faecal samples were isolated, identified, extracted and screened for inhibitory activity against DPP-4. Activity was compared against Lactobacillus reference strains (Ref 1-7), a Gram positive control (Ctrl 1) and two Gram negative controls (Ctrl 2-3). A range of DPP-4 inhibitory activity was observed (10-32%; P<0.05-0.001). Strains of L. fabifermentans (25%), L. plantarum (12-24%) and L. fermentum (14%) had significant inhibitory activity. However, we also noted that E. coli (Ctrl 2) and S. Typhimurium (Ctrl 3) had the greatest inhibitory activity (30-32%). Contrastingly, some isolates (Lb 12-15) and reference cultures (Ref 1-4) instead of inhibiting DPP-4 actually enhanced it, perhaps indicating the presence of X-prolyl-dipeptidyl-amino-peptidase (PepX). This provides a future rationale for using probiotic bacteria or their components for management of type 2 diabetes via DPP-4 inhibition.

Functional domains of the human epididymal protease inhibitor, eppin

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin’s domains are involved in the protein’s antibacterial activity, only the Kunitz domain is required for selective protease inhibition.

Drug delivery strategies for photodynamic antimicrobial chemotherapy: From benchtop to clinical practice

Light and photosensitizer-mediated killing of many pathogens, termed photodynamic antimicrobial chemotherapy (PACT), has been extensively investigated in vitro. A wide range of organisms from the Gram-positive Staphylococcus aureus to the Gram-negative Pseudomonas aeruginosa have been proven to be susceptible to PACT. Multidrug-resistant strains are just as susceptible to this treatment as their naive counterparts. Both enveloped and non-enveloped viruses have demonstrated susceptibility in vitro, in addition to fungi and protozoa. Significantly, however, no clinical treatments based on PACT are currently licensed. This paper provides a comprehensive review of work carried out to date on delivery of photosensitizers for use in PACT, including topical, intranasal and oral/buccal delivery, as well as targeted delivery. We have also reviewed photo-antimicrobial surfaces. It is hoped that, through a rational approach to formulation design and subsequent success in small-scale clinical trials, more widespread use will be made of PACT in the clinic, to the benefit of patients worldwide. (C) 2009 Elsevier B.V. All rights reserved.

“Shotgun” cloning of skin peptide precursors from skin secretion-derived cDNA libraries of the Fujian large-headed frog, Limnonectes fujianensis

Amphibian skin secretions are rich sources of biologically-active peptides and several studies involving molecular cloning of their biosynthetic precursors have revealed that many exhibit highly-conserved domain architectures with an associated high degree of primary structural conservation of the signal peptides. This conservation of primary structure is reflected at the level of nucleotide sequence — a finding that has permitted our group to design primers to these sites facilitating “shotgun” cloning using cDNA libraries from uninvestigated species. Here we describe the results of such an approach using a skin secretion-derived cDNA library from the Fujian large-headed frog, Limnonectes fujianensis, a completely unstudied species. In over 50 clones studied by this approach, 12 were found to encode peptides of different primary structure. Representatives of 5 different families of antimicrobial peptides derived from the skins of ranid frogs were found and these were brevinin-1 (n = 3), the ranatuerin-2 (n = 3), esculentin-2 (n = 1), temporin (n = 1) and chensinin (n = 1). Three clones encoded peptides that were novel with no homologues present in contemporary on-line databases. These included two related 16-mer peptides, named peptides SC-16a and b, and an unrelated 24-mer, named peptide AG-24. Preliminary biological characterisation of SC-16a has demonstrated an antimicrobial activity against Gram-negative bacteria with a minimal inhibitory concentration of 35 µM with no observable haemolysis up to 200 µM. This finding may suggest that this peptide represents a novel class of antimicrobial with little effect on eukaryotic membranes.

A potent novel antimicrobial peptide related to caerin 1.12 from the skin secretion of the Australian frog, Litoria aurea

Skin secretions from Australian frogs of the genus Litoria have been extensively studied for many years and are known to contain a large array of antimicrobial peptides that often bear their specific names — caerins (L. caerulea), aureins (L. aurea), citropins (L. citropa) and maculatins (L. genimaculata) — and each group displays distinct primary structural attributes. During a systematic transcriptome cloning study using a cDNA library derived from skin secretion of L. aurea, a series of identical clones were identified that encoded a novel 25-mer antimicrobial peptide that displayed 92% structural identity with caerin 1.12 from L. caerulea, differing in amino acid sequence at only two positions — Arg for Gly at position 7 and Leu amide for Ser amide at the C-terminus. The novel peptide had conserved Pro residues at positions 15 and 19 that flank a flexible hinge region which previous studies have suggested are important for effective orientation of the two alpha-helices within the bacterial membrane resulting in lysis of cells. As the two substitutions in the novel peptide serve to increase both positive charge and hydrophobicity, we synthesised a replicate and determined its minimal inhibitory concentration (MIC) against Gram positive Staphylococcus aureus and Gram negative Escherichia coli. The MICs for these organisms were 3 µM and 4 µM, respectively, indicating a high potency and haemolysis was

A novel and potent trypsin inhibitor peptide, AC-17, from the skin secretion of the edible frog, Rana esculenta

Protease inhibitors are found in many venoms and evidence suggests that they occur widely in amphibian skin secretions. Kunitz inhibitors have been found in the skin secretions of bombinid toads and ranid frogs, Kazal inhibitors in phyllomedusine frogs and Bowman–Birk inhibitors in ranid frogs. Selective protease inhibitors could have important applications as therapeutics in the treatment of diseases in which discrete proteases play an aetiologcal role. Here we have examined the skin secretion of the edible frog, Rana esculenta, for protease inhibitors using trypsin as a model. HPLC fractions of secretions were screened for inhibitory activity using a chromogenic substrate as reporter. Three major peptides were resolved with trypsin inhibitory activity in HPLC fractions — one was a Kunitz-type inhibitor, a second was a Bowman–Birk inhibitor but the third represented a novel class of trypsin inhibitor in European frog skin. Analysis of the peptide established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17. Peptide AC-17 resembled a typical “Rana box” antimicrobial peptide but while it was active against Escherichia coli (MIC 30 µM) it was devoid of activity against Staphylococcus aureus and of haemolytic activity. In contrast, the peptide was a potent inhibitor of trypsin with a Ki of 5.56 µM. AC-17 represents the prototype of a novel trypsin inhibitor from the skin secretion of a European ranid frog that may target a trypsin-like protease present on the surface of Gram-negative bacteria.

Complete Genome Sequence of Propionibacterium acnes Type IB Strain 6609

Propionibacterium acnes is an anaerobic Gram-positive bacterium that forms part of the normal human cutaneous microbiota and is thought to play a central role in acne vulgaris, a chronic inflammatory disease of the pilosebaceous unit (I. Kurokawa et al., Exp. Dermatol. 18:821-832, 2009). Here we present the whole genome sequence of P. acnes type IB strain 6609, which was recovered from a skin sample from a woman with no recorded acne history and is thus considered a nonpathogenic strain (I. Nagy, Microbes Infect. 8:2195-2205, 2006).

Draft Genome Sequence of an Antibiotic-Resistant Propionibacterium acnes Strain, PRP-38, from the Novel Type IC Cluster

Propionibacterium acnes, a non-spore-forming, anaerobic Gram-positive bacterium, is most notably recognized for its association with acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821–832, 2009). We now present the draft genome sequence of an antibiotic-resistantP. acnesstrain, PRP-38, isolated from an acne patient in the United Kingdom and belonging to the novel type IC cluster. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Degradation of 1,3-dichloropropene by Pseudomonas cichorii 170

The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source, Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible, The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13063, Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.

Application of atmospheric pressure nonthermal plasma for the in vitro eradication of bacterial biofilms

The use of atmospheric pressure nonthermal plasma represents an interesting and novel approach for the decontamination of surfaces colonized with microbial biofilms that exhibit enhanced tolerance to antimicrobial challenge. In this study, the influence of an atmospheric pressure nonthermal plasma jet, operated in a helium and oxygen gas mixture under ambient pressure, was evaluated against biofilms of Bacillus cereus,Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa. Within <4 min of plasma exposure, complete eradication of the two Gram-positive bacterial biofilms was achieved. Although Gram-negative biofilms required longer treatment time, their complete eradication was still possible with 10 min of exposure. Whilst this study provides useful proof of concept data on the use of atmospheric pressure plasmas for the eradication of bacterial biofilms in vitro, it also demonstrates the critical need for improved understanding of the mechanisms and kinetics related to such a potentially significant approach. © 2012 Federation of European Microbiological Societies.