<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

Antibiotic synergy against biofilm-forming <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Eight antibiotics (aztreonam, ceftazidim, cefoperazon, cefepim, netilmicin, amikacin, ofloxacin and ciprofloxacin) exhibited antimicrobial activity individually and/or in combinations against 20 wild-type biofilm-forming strains of Pseudomonas aeruginosa. The strains were less susceptible in biofilm; in 10 strains antibiotic synergy was observed for the combination of aztreonam and ciprofloxacin. Synergy was also demonstrated in the case of β-lactams and aminoglycosides, β-lactams and fluoroquinolones, aminoglycosides and fluoroquinolones, and for monobactams and β-lactams although the strains were resistant to the individual antibiotics. Synergism or partial synergism was found with one or more antibiotic combinations against 32.4% of isolates.     

First Confirmation of Integron-Bearing Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis>

Six methicillin-resistant Staphylococcus aureus MRSA strains from two nosocomial infection cases described in a previous study [15], of which two occurred in March and the other four in May, 2005, were found to possess one copy of class 1 integron with aadA2 gene cassette located on chromosomes by Southern hybridization. Polymerase chain reaction (PCR) detection of mecA and pvl, SCCmec typing, multilocus sequence typing (MLST), spaA typing and coa typing were also performed. The results revealed 6 MRSA fell into the ST239-MRSA-III group (clonal complex 8), with the spaA type GKAOMQ and coa type HIJKL, whereas the pvl locus was not detected. DNA fingerprinting analysis by random amplified polymorphic DNA-PCR using three different assays were also performed, and all strains exhibited identical patterns, indicating that they were clonally related and might be mainly due to a specific clone in the hospital. This was the first time, to our knowledge, that class 1 integron-bearing MRSA (I-MRSA), simultaneously carrying two mobile genetic elements was confirmed: class 1 integron and SCCmec.     

<Emphasis Type="Italic">Escherichia coli,Pseudomonas aeruginosa</Emphasis>, and<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> Attachment Patterns on Glass Surfaces with Nanoscale Roughness

Attachment tendencies of Escherichia coli K12, Pseudomonas aeruginosa ATCC 9027, and Staphylococcus aureus CIP 68.5 onto glass surfaces of different degrees of nanometer-scale roughness have been studied. Contact-angle and surface-charge measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) were employed to characterize substrata and bacterial surfaces. Modification of the glass surface resulted in nanometer-scale changes in the surface topography, whereas the physicochemical characteristics of the surfaces remained almost constant. AFM analysis indicated that the overall surface roughness parameters were reduced by 60–70%. SEM, CLSM, and AFM analysis clearly demonstrates that although E. coli, P. aeruginosa and S. aureus present significantly different patterns of attachment, all of the species exhibited a greater propensity for adhesion to the “nano-smooth” surface. The bacteria responded to the surface modification with a remarkable change in cellular metabolic activity, as shown by the characteristic cell morphologies, production of extracellular polymeric substances, and an increase in the number of bacterial cells undergoing attachment.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type="Italic">in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> sigma B-dependent emergence of small-colony variants and biofilm production following exposure to <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> 4-hydroxy-2-heptylquinoline-<Emphasis Type="Italic">N-</Emphasis> oxide

Background  

Staphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.     

Desiccation tolerance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Staphylococcus aureus is a multidrug-resistant pathogen that not only causes a diverse array of human diseases, but also is able to survive in potentially dry and stressful environments, such as the human nose, on skin and on inanimate surfaces such as clothing and surfaces. This study investigated parameters governing desiccation tolerance of S. aureus and identified several components involved in the process. Initially, the role of environmental parameters such as temperature, growth phase, cell density, desiccation time and protectants in desiccation tolerance were determined. This established a robust model of desiccation tolerance in which S. aureus has the ability to survive on dry plastic surfaces for more than 1,097 days. Using a combination of a random screen and defined mutants, clpX, sigB and yjbH were identified as being required for desiccation tolerance. ClpX is a part of the ATP-dependent ClpXP protease, important for protein turnover, and YjbH has a proposed linked function. SigB is an accessory sigma factor with a role in generalized stress resistance. Understanding the molecular mechanisms that govern desiccation tolerance may determine the break points to be exploited to prevent the spread of this dangerous pathogen in hospitals and communities.     

Biofilm dispersion in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In recent decades, many researchers have written numerous articles about microbial biofilms. Biofilm is a complex community of microorganisms and an example of bacterial group behavior. Biofilm is usually considered a sessile mode of life derived from the attached growth of microbes to surfaces, and most biofilms are embedded in self-produced extracellular matrix composed of extracellular polymeric substances (EPSs), such as polysaccharides, extracellular DNAs (eDNA), and proteins. Dispersal, a mode of biofilm detachment indicates active mechanisms that cause individual cells to separate from the biofilm and return to planktonic life. Since biofilm cells are cemented and surrounded by EPSs, dispersal is not simple to do and many researchers are now paying more attention to this active detachment process. Unlike other modes of biofilm detachment such as erosion or sloughing, which are generally considered passive processes, dispersal occurs as a result of complex spatial differentiation and molecular events in biofilm cells in response to various environmental cues, and there are many biological reasons that force bacterial cells to disperse from the biofilms. In this review, we mainly focus on the spatial differentiation of biofilm that is a prerequisite for dispersal, as well as environmental cues and molecular events related to the biofilm dispersal. More specifically, we discuss the dispersal-related phenomena and mechanisms observed in Pseudomonas aeruginosa, an important opportunistic human pathogen and representative model organism for biofilm study.     

The antimicrobial potential of a new derivative of cathelicidin from <Emphasis Type="Italic">Bungarus fasciatus</Emphasis> against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32–128 μg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 μg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis> Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.     

Photodynamic antimicrobial activity of new porphyrin derivatives against methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Methicillin resistant Staphylococcus aureus (MRSA) with multiple drug resistance patterns is frequently isolated from skin and soft tissue infections that are involved in chronic wounds. Today, difficulties in the treatment of MRSA associated infections have led to the development of alternative approaches such as antimicrobial photodynamic therapy. This study aimed to investigate photoinactivation with cationic porphyrin derivative compounds against MRSA in in-vitro conditions. In the study, MRSA clinical isolates with different antibiotic resistance profiles were used. The newly synthesized cationic porphyrin derivatives (P_M, P_E, P_PN, and P_PL) were used as photosensitizer, and 655 nm diode laser was used as light source. Photoinactivation experiments were performed by optimizing energy doses and photosensitizer concentrations. In photoinactivation experiments with different energy densities and photosensitizer concentrations, more than 99% reduction was achieved in bacterial cell viability. No decrease in bacterial survival was observed in control groups. It was determined that there was an increase in photoinactivation efficiency by increasing the energy dose. At the energy dose of 150 J/cm² a survival reduction of over 6.33 log₁₀ was observed in each photosensitizer type. While 200 μM P_M concentration was required for this photoinactivation, 12.50 μM was sufficient for P_E, P_PN, and P_PL. In our study, antimicrobial photodynamic therapy performed with cationic porphyrin derivatives was found to have potent antimicrobial efficacy against multidrug resistant S. aureus which is frequently isolated from wound infections.     

Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Pre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen P. aeruginosa. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against P. aeruginosa.     

First Record of Larval Secretions of <Emphasis Type="Italic">Cochliomyia macellaria</Emphasis> (Fabricius, 1775) (Diptera: Calliphoridae) Inhibiting the Growth of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Maggot debridement therapy (MDT) consists on the intentional and controlled application of sterilized larvae of the order Diptera on necrotic skin lesions with the purpose of cleaning necrotic tissue and removing pathogenic bacteria. During MDT, a marked antimicrobial activity has been reported in literature specially associated with antibacterial substances from Lucilia sericata (Meigen); however, regarding Cochliomyia macellaria (Fabricius), little is known. This study aimed to evaluate in vitro inhibition of bacterial growth of Pseudomonas aeruginosa and Staphylococcus aureus in contact with excretions and secretions (ES) from C. macellaria larvae. Larval ES were extracted in sterile distilled water and divided in three groups: ES, containing 400 μL of autoclaved ES; ES+BAC, containing 400 μL of autoclaved ES+0.5-μL bacterial inoculum; and CONT-BAC, containing 400 μL of sterile distilled water +0.5 μL of bacterial inoculum. Aliquots of each experimental group were plated by spreading onto Petri dishes. Seedings were made at 0, 1, 2, 4, and 12 h after the extraction of ES. In ES+BAC groups, inhibition of S. aureus was verified between times 1 and 2 h and P. aeruginosa was inhibited between 0 and 4 h. There was no growth observed in any ES group. In the CONT-BAC groups, the number of colonies from time 4 h became countless for S. aureus and decreased for P. aeruginosa. As reported in the literature, we note here that ES have excellent bactericidal activity for both gram-positive and gram-negative bacteria, and this study shows for the first time the action of the bactericidal activity of exosecretions of C. macellaria against S. aureus and P. aeruginosa.     

Antibiofilm activity of <Emphasis Type="Italic">Andrographis paniculata</Emphasis> against cystic fibrosis clinical isolate <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Respiratory tract and device associated infections caused by biofilm forming Pseudomonas aeruginosa play a primary role in the pathogenesis and prognosis of cystic fibrosis (CF) diseases. The biofilm formed by these pathogens attributes to the antibiotic resistance and protection from host immune response. Once established, the pathogens respond poorly to therapeutic agents. Recently medicinal plants are largely explored as potential source of bioactive agents. In this context the present study reports the antibiofilm activity of the folkloric medicinal plant Andrographis paniculata against biofilm forming CF causative Pseudomonas aeruginosa isolated from CF sputum. P. aeruginosa was also assessed for their growth and development of the biofilm, phylogenetic relationship and antibiotic susceptibility. Antibiogram of the strains indicated that they were resistant to more than one antibiotic. Six extracts of A. paniculata showed significant antibiofilm activity. P. aeruginosa strains, KMS P03 and KMS P05, were found to be maximally inhibited by the methanol extract to an extent of 88.6 and 87.5% respectively. This is the first report on antibiofilm activity of A. paniculata extracts, and our results indicate scope for development of complementary medicine for biofilm associated infections.     

UDP-N-acetylglucosamine enolpyruvyl transferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K _m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.     

Transferable Carbenicillin Resistance in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

IN 1969, after carbenicillin had been in use for three years in this unit, highly resistant strains of Pseudomonas aeruginosa were isolated for the first time¹. Because these resistant strains included, from their first appearance, representatives of two unrelated types, it seemed likely that the resistance was transferable; this hypothesis was supported by experiments showing the transfer of carbenicillin resistance between Ps. aeruginosa and Escherichia coli K12 in vitro and in vivo^2–4;. The resistant Ps. aeruginosa produced a penicillinase (β lactamase) similar to that normally produced by some strains of Enterobacteria and different from that normally produced by Ps. aeruginosa^2,3, so it seemed likely that the Ps. aeruginosa had initially acquired resistance by the transfer of an R factor from a carbenicillin-resistant member of the Enterobacteriaceae colonizing the same burn. This hypothesis is now supported by a study on strains of Enterobacteria and Ps. aeruginosa isolated in a number of hospitals. We have also found evidence suggesting that Ps. aeruginosa which has acquired this R factor may not show resistance until it has been exposed repeatedly to carbenicillin.