Assessment of elimination efficacy on foodborne pathogenic microbes and foulant precipitates using phytic acid and sulfamic acid.

This research investigated the comparative efficacy of sulfamic acid (SA) and phytic acid (PA), both individually and in combination, for treating potential foodborne pathogens and pre-formed foulants. Pathogens studied included Listeria monocytogenes, E. coli DH5α, Salmonella typhimurium, Staphylococcus aureus, and vegetative Bacillus cereus, in suspended aqueous solutions, as well as Pseudomonas aeruginosa biofilm on quartz glass surfaces. Inactivation kinetics for Listeria monocytogenes revealed concentration-dependent rate constants (k) of 6.6(±0.2) × 10-6 M and 2.8(±0.1) × 10-8 M for single treatments of SA and PA, respectively, and ranged from 6.9(±0.3) to 50.7(±2.3) × 10-6 M for combined treatments with PA pre-treatment concentrations of 75-758 µM. Observable cellular abnormalities in Listeria monocytogenes, such as membrane vesiculation, chelation, cellular disruption, biomolecule leakage, and lipid peroxidation, were identified after exposure to PA or SA, either individually or in combination. The optimized combined treatment of PA and SA achieved significant removal (i.e., >3-log; 99.9%) of potential foodborne pathogens under simulated food-washing process conditions. Additionally, over 90% descaling efficacy was observed for pre-formed foulants such as CaCO3 precipitates and Pseudomonas aeruginosa biofilm on quartz glass surfaces with the combined treatment. These findings provide novel insights into the versatile utility of PA and SA for optimizing combinational water disinfection systems and addressing (in)organic foulant scaling on surfaces in the food processing industry.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response.

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

[Criterium pencil mines as an alternative to platinum rods (Inoclic) in achievement of the antibiogram in agar medium for countries with limited resources?] / Les mines de crayon pour critérium comme alternative aux tiges de platine (Inoclic) dans la réalisation de l'antibiogramme en milieu gélosé pour les pays à ressources limitées ?

The realization of the antibiotic susceptibility test in agar is the routine bacteriological examination for the determination and monitoring of bacterial susceptibility to antibiotics. In this study, we report the comparative results between pencil leads for criterium, as an alternative to platinum rods in the realization of the antibiotic susceptibility test. METHODOLOGY: Experimental study evaluating the comparability of the results between Criterium and Inoclic mines (by counting bacterial cells on agar after 5 successive dilutions of reason 10 from a bacterial suspension obtained after piercing through a colony; by measuring the inhibition diameters of 4 ATCC reference bacterial strains on an antibiogram in an agar medium) and evaluating the sterility of the criterium mines by culturing them on enriched broth (heart - brain type). RESULTS: 42 bacterial strains were used for bacterial cell counting. The results were of the same order of magnitude (107 CFU/mL) between Inoclic and criterium mines, for all strains and at all dilutions. The antibiotic susceptibility tests performed for the 4 reference strains by the Inoclics and criterium mines all complied (100%) with the expected limits for determining their sensitivity profile to the antibiotics tested. Compared to the bacterial growth inhibition diameters on antibiotic susceptibility tests, no intra-operator variability was observed, while significant inter-operator variability (both with Inoclic and 0.5 mm criterium mines) was observed with some strains and for inhibition diameters greater than 10 mm. The enriched broth cultures (BCC) and their subculture carried out on 10 criterium mines from 5 different batches were negative. CONCLUSION: Criterium mines seem to be a serious and less expensive alternative to Inoclic for the realization of antibiotic susceptibility testing in our resource-limited countries.

A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa.

The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

Fight Against Antimicrobial Resistance: We Always Need New Antibacterials but for Right Bacteria.

Antimicrobial resistance in bacteria is frightening, especially resistance in Gram-negative Bacteria (GNB). In 2017, the World Health Organization (WHO) published a list of 12 bacteria that represent a threat to human health, and among these, a majority of GNB. Antibiotic resistance is a complex and relatively old phenomenon that is the consequence of several factors. The first factor is the vertiginous drop in research and development of new antibacterials. In fact, many companies simply stop this R&D activity. The finding is simple: there are enough antibiotics to treat the different types of infection that clinicians face. The second factor is the appearance and spread of resistant or even multidrug-resistant bacteria. For a long time, this situation remained rather confidential, almost anecdotal. It was not until the end of the 1980s that awareness emerged. It was the time of Vancomycin-Resistance Enterococci (VRE), and the threat of Vancomycin-Resistant MRSA (Methicillin-Resistant Staphylococcus aureus). After this, there has been renewed interest but only in anti-Gram positive antibacterials. Today, the threat is GNB, and we have no new molecules with innovative mechanism of action to fight effectively against these bugs. However, the war against antimicrobial resistance is not lost. We must continue the fight, which requires a better knowledge of the mechanisms of action of anti-infectious agents and concomitantly the mechanisms of resistance of infectious agents.

Boron Doped Diamond as a Low Biofouling Material in Aquatic Environments: Assessment of Pseudomonas aeruginosa Biofilm Formation.

Boron doped diamond (BDD), given the robustness of the material, is becoming an electrode of choice for applications which require long-term electrochemical monitoring of analytes in aqueous environments. However, despite the extensive work in this area, there are no studies which directly assess the biofilm formation (biofouling) capabilities of the material, which is an essential consideration because biofouling often causes deterioration in the sensor performance. Pseudomonas aeruginosa is one of the most prevalent bacterial pathogens linked to water-related diseases, with a strong capacity for forming biofilms on surfaces that are exposed to aquatic environments. In this study, we comparatively evaluate the biofouling capabilities of oxygen-terminated (O-)BDD against materials commonly employed as either the packaging or sensing element in water quality sensors, with an aim to identify factors which control biofilm formation on BDD. We assess the monospecies biofilm formation of P. aeruginosa in two different growth media, Luria-Bertani, a high nutrient source and drinking water, a low nutrient source, at two different temperatures (20 and 37 °C). Multispecies biofilm formation is also investigated. The performance of O-BDD, when tested against all other materials, promotes the lowest extent of P. aeruginosa monospecies biofilm formation, even with corrections made for total surface area (roughness). Importantly, O-BDD shows the lowest water contact angle of all materials tested, that is, greatest hydrophilicity, strongly suggesting that for these bacterial species, the factors controlling the hydrophilicity of the surface are important in reducing bacterial adhesion. This was further proven by keeping the surface topography fixed and changing surface termination to hydrogen (H-), to produce a strongly hydrophobic surface. A noticeable increase in biofilm formation was found. Doping with boron also results in changes in hydrophobicity/hydrophilicity compared to the undoped counterpart, which in turn affects the bacterial growth. For practical electrochemical sensing applications in aquatic environments, this study highlights the extremely beneficial effects of employing smooth, O-terminated (hydrophilic) BDD electrodes.

Garlic clove extract assisted silver nanoparticle - Antibacterial, antibiofilm, antihelminthic, anti-inflammatory, anticancer and ecotoxicity assessment.

Facile and low cost garlic clove extract based silver nanoparticles was synthesized and its broad spectrum of therapeutic activity including antibiofilm, antiparasitic and anti-breast cancer activity was evaluated. The synthesized garlic­silver nanoparticles (G-AgNPs) were characterized by various physico-chemical techniques. G-AgNPs showed good optical property, highly crystalline nature, spherical shape and uniformly dispersed with size measuring between 10 and 50 nm. G-AgNPs have shown greater anti-bacterial and antibiofilm activity on clinically important pathogens methicillin-resistant S. aureus and P. aerigunosa at 100 µg ml-1. The efficacy of G-AgNPs against earthworm evidenced its effectiveness as anti-helminthic agent in treating intestinal parasites. The significant inhibition of BSA protein denaturation proves its anti-inflammatory property. In addition, G-AgNPs have shown remarkable anticancer effect and significantly inhibited the human breast cancer cell (MCF-7) viability at 100 µg ml-1 after 24 h. A noticeable change in the morphology of MCF-7 cells was also noticed. G-AgNPs were non-toxic to human HEK293 embryonic cells. Also, the non-toxic nature of G-AgNPs to C. cornuta and no morphological, physiological changes proved its safety to the environment. It is concluded that G-AgNPs have a broad range of biological applications and it can be used as an eco-friendly material without having negative effects in the environment.

Assessment of biofilm formation by pseudomonas aeruginosa and hydrodynamic evaluation of microtiter plate assay.

OBJECTIVE: To assess the biofilm formation in clinical and environmental isolates of Pseudomonas aeruginosa and to evaluate the hydrodynamics in microtiter plate assay and compare it with conventional assays for biofilm formation. METHODS: The cross-sectional study was conducted at the Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan, in 2013-14, while the computational work was done at the National University of Science and Technology, Islamabad. The study comprised environmental and clinical isolates of pseudomonas aeruginosa. Pseudomonas citramide agar was used as a selective media, and further confirmation was done by biochemical tests. Biofilm formation was assessed by Congo red assay, air liquid interfaceassay and microtiter plate assay. Computational Fluid Dynamics (CFD) simulations were also used to improve the microtiter plate assay for biofilm formation assessment. Polymerase chain reaction was used for screening of pelA and pelG genes. RESULTS: Of the 50 isolates, 25(50%) each were environmental and clinical. The number of biofilm producers observed in Congo red assay, air liquid interface assay and microtiter plate assay were 7(14%), 15(30%) and 30(60%) respectively. Biofilm former gene pelA was observed in 22(44%) isolates while 36(72%) isolates showed the presence of pelG gene. CONCLUSIONS: Microtiter plate assay was found to be a reliable method to detect biofilm forming pseudomonas aeruginosa isolates which further provides a base for development of methods to detect biofilms readily and accurately.

Regulação da expressão de pioverdina dependente de contato em pseudomonas aeruginosa / Regulation of surface-dependent pyoverdine expression in Pseudomonas aeruginosa

A gama-proteobactéria Pseudomonas aeruginosa é um patógeno oportunista humano frequentemente associado a pacientes com queimadura grave e aos portadores de fibrose cística. O estabelecimento de infecção depende de uma série de fatores que contribuem para a virulência deste patógeno, dentre eles a produção de sideróforos e outros sistemas de captação de ferro. Pioverdina é o principal sideróforo sintetizado por bactérias do gênero Pseudomonas e linhagens deficientes na sua produção são incapazes de estabelecer infecção em modelos animais. A regulação da biossíntese deste sideróforo envolve a agregação entre as células, indicando a dependência de contato para completa indução da sua produção. O contato com uma superfície altera o comportamento das células e diversos fenótipos são dependentes deste sinal mecânico. PrlC é uma oligopeptidase A putativamente envolvida na degradação de peptídeo-sinais e PA14_00800, uma pequena proteína com domínio de função desconhecida, codificada por um gene imediatamente à jusante de prlC. Existem poucos trabalhos na literatura sobre PrlC e seus homólogos e nenhuma informação sobre PA14_00800. Este trabalho teve como objetivo elucidar o envolvimento de PrlC e PA14_00800 na regulação da produção de pioverdina por células em contato com uma superfície. Para estabelecer uma correlação na expressão destes genes, um estudo da organização gênica foi realizado por RT-PCR, confirmando que eles fazem parte do mesmo operon e, portanto, que a expressão destes genes é regulada pelos mesmos fatores. Ensaios classicamente modulados pelo segundo mensageiro c-di-GMP, como formação de biofilme e motilidade, não apresentaram variações nas linhagens mutantes ΔprlC, ΔPA14_00800 ou Δoperon, indicando que a deleção destes genes não altera significativamente os níveis de c-di-GMP nas células. A motilidade do tipo swarming é, no entanto, severamente afetada na linhagem ΔPA14_00800 quando o meio de cultura não contém cloreto de cálcio e glicose, indicando um defeito na sinalização celular ou requerimento energértico desta linhagem nestas condições. PA14_00800 regula a fluorescência de P. aeruginosa em meio sólido e semissólido, mas não em meio líquido. Esta fluorescência depende tanto de pioverdina quanto de PQS, umamolécula de comunicação celular fluorescente, e a possibilidade de outros fatores estarem envolvidos neste fenótipo ainda está sob investigação. Análise do transcritoma por RNASeq com a linhagem ΔPA14_00800 comparada à linhagem parental foi realizada a partir de colônias destas linhagens crescidas em M9 modificado. Genes envolvidos no sistema de secreção do tipo III e do tipo VI e na biossíntese de PQS apareceram dentre os genes diferencialmente expressos, bem como genes para o catabolismo de glicose. Este trabalho foi o primeiro a investigar o papel de PA14_00800 na fisiologia de P. aeruginosa, e os conhecimentos adquiridos aqui podem ser transpostos, com cautela, para compreensão da função dos homólogos de PA14_00800 em outras bactérias

The gamma-proteobacterium Pseudomonas aeruginosa is a human opportunistic pathogen frequently associated with patients with severe burns and those with cystic fibrosis. The establishment of infection depends on several factors that contribute to the virulence of this pathogen, among them siderophore production and other iron uptake systems. Pyoverdine is the main siderophore synthesized by the bacteria of the genus Pseudômonas and pyoverdinedeficient strains are unable to establish infection in animal models. The regulation of biosynthesis of this siderophore involves cell aggregation, indicating contact dependency for complete induction of pyoverdine production. Surface contact alters cell behavior and several phenotypes are dependent on this mechanical cue. PrlC is an oligopeptidase A putatively involved in peptide-signals degradation and PA14_00800, a small protein with a domain of unknown function, encoded by a gene immediately downstream of prlC. There are few papers in the literature on PrlC and its homologues and no information on PA14_00800. This work aimed to elucidate the role of PrlC and PA14_00800 in surface-dependent regulation of pyoverdine production. To establish a correlation in the expression of these genes, a study of the gene organization was performed by RT-PCR, confirming that they are part of an operon and therefore the expression of these genes is regulated by the same factors. Traits classically modulated by the second messenger c-di-GMP, such as biofilm formation and motility, did not show variations in the ΔprlC, ΔPA14_00800 or Δoperon, indicating that the deletion of these genes does not significantly alter the levels of c-di-GMP within the cells. Swarming motility is, however, severely affected in the strain ΔPA14_00800 when the culture medium does not contain calcium chloride and glucose, indicating a cell signaling defect or energetic requirement under these conditions. PA14_00800 regulates surface-dependent fluorescence of P. aeruginosa, in solid and semi-solid medium. This fluorescence depends on both pyoverdine and PQS, a fluorescent cell-to-cell communication molecule, and the investigation of other putative factors involved in this phenotype is still under study. Transcriptomic analysis by RNASeq with the strain ΔPA14_00800 compared to PA14 was performed from colonies ofthese strains grown in modified M9 1% agar. Genes involved in the type III and type VI secretion systems, in PQS biosynthesis and glucose catabolism were differentially expressed. This work was the first to investigate the role of PA14_00800 in the physiology of P. aeruginosa, and the knowledge obtained here can be cautiously transposed to understanding the role of PA14_00800 homologues in other bactéria

Critical Assessment of Methods to Quantify Biofilm Growth and Evaluate Antibiofilm Activity of Host Defence Peptides.

Biofilms are multicellular communities of bacteria that can adhere to virtually any surface. Bacterial biofilms are clinically relevant, as they are responsible for up to two-thirds of hospital acquired infections and contribute to chronic infections. Troublingly, the bacteria within a biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotic to kill cells within a biofilm when compared to planktonic bacterial cells. Identifying and optimizing compounds that specifically target bacteria growing in biofilms is required to address this growing concern and the reported antibiofilm activity of natural and synthetic host defence peptides has garnered significant interest. However, a standardized assay to assess the activity of antibiofilm agents has not been established. In the present work, we describe two simple assays that can assess the inhibitory and eradication capacities of peptides towards biofilms that are formed by both Gram-positive and negative bacteria. These assays are suitable for high-throughput workflows in 96-well microplates and they use crystal violet staining to quantify adhered biofilm biomass as well as tetrazolium chloride dye to evaluate the metabolic activity of the biofilms. The effect of media composition on the readouts of these biofilm detection methods was assessed against two strains of Pseudomonas aeruginosa (PAO1 and PA14), as well as a methicillin resistant strain of Staphylococcus aureus. Our results demonstrate that media composition dramatically alters the staining patterns that were obtained with these dye-based methods, highlighting the importance of establishing appropriate biofilm growth conditions for each bacterial species to be evaluated. Confocal microscopy imaging of P. aeruginosa biofilms grown in flow cells revealed that this is likely due to altered biofilm architecture under specific growth conditions. The antibiofilm activity of several antibiotics and synthetic peptides were then evaluated under both inhibition and eradication conditions to illustrate the type of data that can be obtained using this experimental setup.

Simplifying Piperacillin/Tazobactam Dosing: Pharmacodynamics of Utilizing Only 4.5 or 3.375 g Doses for Patients With Normal and Impaired Renal Function.

OBJECTIVES: To evaluate the pharmacodynamic exposure of piperacillin/tazobactam across the renal function range using 4.5 or 3.375 g dosing regimens. METHODS: A 5000-patient Monte Carlo simulation was conducted to determine the probability of achieving 50% free time above the minimum inhibitory concentration ( fT > MIC) for piperacillin. Proposed regimens, using solely 4.5 or 3.375 g strengths, were compared with regimens listed in piperacillin/tazobactam prescribing information over creatinine clearance (CrCl) ranges of 120 mL/min to hemodialysis. The probability of target attainment (PTA) at MICs ≤ 16 µg/mL was compared between proposed and standard regimens. RESULTS: At CrCl 41 to 120 mL/min, prolonged infusions of 4.5 g (3 hours) and 3.375 g (4 hours) every 6 hours resulted in ≥95% PTA versus ≥76% for standard regimens (0.5 hour). At CrCl 20 to 40 mL/min, 4.5 and 3.375 g every 8 hours as prolonged infusions achieved slightly higher PTA (≥98%) versus standard regimens (≥93%). Similarly, PTA achieved with prolonged infusions of 4.5 and 3.375 g every 12 hours (≥93%) was comparable with those of standard regimens (≥91%) at CrCl 1 to 19 mL/min. In hemodialysis, 100% PTA was achieved with prolonged infusion regimens. CONCLUSION: Piperacillin/tazobactam regimens designed around the 4.5 or 3.375 g dose and prolonged infusions provided similar or better PTA at MICs ≤ 16 µg/mL compared with standard regimens. These observations may support the stocking and use of a single piperacillin/tazobactam strength to simplify dosing.

Emergence of complex behavior in pili-based motility in early stages of P. aeruginosa surface adaptation.

Pseudomonas aeruginosa move across surfaces by using multiple Type IV Pili (TFP), motorized appendages capable of force generation via linear extension/retraction cycles, to generate surface motions collectively known as twitching motility. Pseudomonas cells arrive at a surface with low levels of piliation and TFP activity, which both progressively increase as the cells sense the presence of a surface. At present, it is not clear how twitching motility emerges from these initial minimal conditions. Here, we build a simple model for TFP-driven surface motility without complications from viscous and solid friction on surfaces. We discover the unanticipated structural requirement that TFP motors need to have a minimal amount of effective angular rigidity in order for cells to perform the various classes of experimentally-observed motions. Moreover, a surprisingly small number of TFP are needed to recapitulate movement signatures associated with twitching: Two TFP can already produce movements reminiscent of recently observed slingshot type motion. Interestingly, jerky slingshot motions characteristic of twitching motility comprise the transition region between different types of observed crawling behavior in the dynamical phase diagram, such as self-trapped localized motion, 2-D diffusive exploration, and super-diffusive persistent motion.

Assessment of biofilm removal capacity of a broad host range bacteriophage JHP against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an efficient biofilm-dwelling microbial pathogen, associated with nosocomial infections. These biofilm-associated infections are resistant to antibiotics and immune defenses, therefore pose major problem against their treatment. This scenario demands alternative therapeutic regimens, and bacteriophage therapy is one among potential strategies for clinical management of multiple drug resistance. In this investigation, the efficacy of a bacteriophage, JHP, is evaluated to eradicate P. aeruginosa biofilms. Growth kinetics of P. aeruginosa biofilm revealed that the highest cell density biofilm (1.5 × 1016 CFU/mL) was established within the polystyrene microtiter plate at 72 h post inoculation. Pseudomonas aeruginosa biofilms of different ages, treated with JHP (0.6 MOI) for different post-infection durations, reduced biomass from 2 to 4.5 logs (60-90%). JHP treatment before biofilm development reduced the bacterial load up to 9 logs (>95% bacterial load reduction) as compared with untreated control, which highlights its potential to prevent biofilm formation in indwelling medical devices. Combinations of JHP with other phages or antibiotics could be an efficient alternative for P. aeruginosa biofilm removal in clinical and industrial settings.

Assessment of early combination effects of colistin and meropenem against Pseudomonas aeruginosa and Acinetobacter baumannii in dynamic time-kill experiments.

BACKGROUND: In view of the paucity of clinical evidence, in vitro studies are needed to find antibiotic combinations effective against multidrug-resistant Gram-negative bacteria. Interpretation of in vitro effects is usually based on bacterial growth after 24 h in time-kill and checkerboard experiments. However, the clinical relevance of the effects observed in vitro is not established. In this study we explored alternative output parameters to assess the activities of colistin and meropenem against Pseudomonas aeruginosa and Acinetobacter baumannii. METHODS: Four strains each of P. aeruginosa and A. baumannii were exposed to colistin and meropenem, alone and in combination, in 8 h dynamic time-kill experiments. Initial (1 h), maximum and 8 h bacterial reductions and the area under the bacterial time-kill curve were evaluated. Checkerboards, interpreted based on fractional inhibitory concentration indices after 24 h, were performed for comparison. RESULTS: In the time-kill experiments, the combination resulted in enhanced 1 h, maximum and 8 h bacterial reductions against 2, 3 and 5 of 8 strains, respectively, as compared to the single drugs. A statistically significant reduction in the area under the time-kill curve was observed for three strains. In contrast, the checkerboards did not identify synergy for any of the strains. CONCLUSIONS: Combination effects were frequently found with colistin and meropenem against P. aeruginosa and A. baumannii in time-kill experiments but were not detected with the checkerboard method. We propose that the early dynamics of bacterial killing and growth, which may be of great clinical importance, should be considered in future in vitro combination studies.

Assessment of antivirulence activity of several d-amino acids against Acinetobacter baumannii and Pseudomonas aeruginosa.

OBJECTIVES: Biofilm formation and bacterial adherence are important requirements for persistence, multidrug resistance and infection. The d-amino acids play a role as modulators of bacterial growth and persistence, though their ability to inhibit biofilms is much debated. In this study, we analysed the effects of 18 different d-amino acids on the pathogens Acinetobacter baumannii and Pseudomonas aeruginosa. METHODS: In vitro assays were carried out to analyse the effect of d-amino acids on bacterial growth, biofilm formation/disassembly, capacity to attach to eukaryotic cells and cellular death. In addition, in vivo assays were performed in mice, using experimental models of sepsis and pneumonia. RESULTS: Biofilm formation was inhibited in A. baumannii by d-His, d-Cys and d-Trp (35%-86%) at 2 mM and in P. aeruginosa by d-Cys, d-Trp and d-Tyr (10%-30%) at 4 mM. Attachment to the A549 human alveolar cells was reduced in A. baumannii by d-Cys, d-His, d-Met, d-Val and d-Ser, and in P. aeruginosa by d-Arg and d-Trp. Growth was inhibited in A. baumannii by d-Cys and d-Trp, and in P. aeruginosa by d-Trp. In virulence assays, incubation of alveolar cells infected with P. aeruginosa with d-Cys, d-Trp and d-Arg reduced cell death (56%-45%). However, no significant effect of d-amino acids was observed in vivo. CONCLUSIONS: Some d-amino acids can inhibit bacterial growth, biofilm formation and adherence to eukaryotic cells in A. baumannii and P. aeruginosa, and showed a protective effect against infection of alveolar cells with P. aeruginosa. Despite the fact that some considerable protection was observed in mice, survival differences between treated and control groups were not statistically significant.

Optimizing intravenous fosfomycin dosing in combination with carbapenems for treatment of Pseudomonas aeruginosa infections in critically ill patients based on pharmacokinetic/pharmacodynamic (PK/PD) simulation.

OBJECTIVE: The purpose of the study was to determine the optimal dosing regimen of intravenous fosfomycin for the treatment of Pseudomonas aeruginosa (PA) based on PK/PD targets. METHOD: A total of 120 PA isolates were recovered from various clinical specimens at university hospital in Thailand. Minimum Inhibitory Concentrations (MICs) of all the isolates were determined by the E-test method. PK parameters were obtained from a published study. Monte Carlo simulation was performed to calculate the percentage of target attainment (PTA) and cumulative fraction of response (CFR). RESULTS: MIC90 of fosfomycin alone, fosfomycin in combination with carbapenem, carbapenems alone and carbapenems in combination with fosfomycin were >1,024, 1,024, >32 and 32µg/ml, for multidrug resistant (MDR)-PA and 512, 128, 8 and 3µg/ml respectively, for non-MDR PA. Approximately 40% of the non-MDR PA were carbapenem-resistant strains. For non-MDR PA with CRPA, fosfomycin 16g continuous infusion in combination with carbapenems provided %PTA of approximately 80 and %CFR of > 88. While, %PTA and %CFR > 90 were achieved with fosfomycin 24g/day prolonged infusion in combination with carbapenem. CONCLUSIONS: Prolonged infusion of fosfomycin 16 - 24g combined with extended carbapenem infusion could be used in non-MDR PA treatment with CRPA.

Assessment of the contribution of chemoreceptor-based signalling to biofilm formation.

Although it is well established that one- and two-component regulatory systems participate in regulating biofilm formation, there also exists evidence suggesting that chemosensory pathways are also involved. However, little information exists about which chemoreceptors and signals modulate this process. Here we report the generation of the complete set of chemoreceptor mutants of Pseudomonas putida KT2440 and the identification of four mutants with significantly altered biofilm phenotypes. These receptors are a WspA homologue of Pseudomonas aeruginosa, previously identified to control biofilm formation by regulating c-di-GMP levels, and three uncharacterized chemoreceptors. One of these receptors, named McpU, was found to mediate chemotaxis towards different polyamines. The functional annotation of McpU was initiated by high-throughput thermal shift assays of the receptor ligand binding domain (LBD). Isothermal titration calorimetry showed that McpU-LBD specifically binds putrescine, cadaverine and spermidine, indicating that McpU represents a novel chemoreceptor type. Another uncharacterized receptor, named McpA, specifically binds 12 different proteinogenic amino acids and mediates chemotaxis towards these compounds. We also show that mutants in McpU and WspA-Pp have a significantly reduced ability to colonize plant roots. Data agree with other reports showing that polyamines are signal molecules involved in the regulation of bacteria-plant communication and biofilm formation.

Fitness cost of antibiotic susceptibility during bacterial infection.

Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contribute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on how to best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance. Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strains during antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result in concomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of this accepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genes across many functional categories and operons that contributed to maximal in vivo fitness during lung infections in animal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivo fitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteria Acinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our results show that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitness advantages that enhance virulence in drug-resistant microbes.

A comparative study on edible Agaricus mushrooms as functional foods.

Agaricus bisporus is a cultivated mushroom; A. bitorquis, A. campestris and A. macrosporus are edible mushrooms growing wild in nature. A chemical characterization was carried out with samples that originated in Serbia. Antioxidant, antimicrobial and anti-quorum sensing properties of their methanolic and ethanolic extracts were assessed. A. campestris had the lowest caloric value and total sugar content and showed the highest concentration in organic and phenolic acids, as also in tocopherols (mainly γ-tocopherol). In general, the methanolic extracts showed higher antioxidant, but lower antibacterial and antifungal potential than ethanolic ones. Sub-inhibitory concentrations of the ethanolic extracts demonstrated reduction of virulence factors, AQ inhibition zones, twitching and swimming motility. The biofilm forming capability of P. aeruginosa PAO1 was also reduced in a concentration-dependent manner at sub-MIC values. The extracts of the tested Agaricus species are a promising source of antioxidant, antimicrobial and antiquorum sensing compounds.