Immunological variants of the aerobactin-cloacin DF13 outer membrane protein receptor IutA among enteric bacteria

Mouse monoclonal antibodies (MAbs) were generated against a 76-kDa IutA receptor of pathogenic avian Escherichia coli 15972. Six of the eight IutA-specific MAbs isolated (AB1 to AB6) were shown to be directed toward membrane-exposed conformational epitopes, although they did not interfere with the uptake of ferric aerobactin and cloacin DF13 as assessed by competition experiments with purified ligands. The two remaining IutA MAbs (AB9 and AB10) recognized linear epitopes buried in the IutA molecule. The panel of IutA MAbs was used to characterize IutA variants occurring in strains of E. coli, Klebsiella pneumoniae, Enterobacter spp., and Shigella spp., resulting in the identification of four immunological groups of IutAs. MAb AB9 defined an epitope conserved in all IutA variants. In addition, the panel of IutA MAbs served to identify the presence of IutA in wild-type bacteria grown in the presence of diphenylamine to reduce the expression of O-specific polysaccharide.

Capsule polysaccharide is a bacterial decoy for antimicrobial peptides

Antimicrobial peptides (APs) are important host weapons against infections. Nearly all APs are cationic and their microbicidal action is initiated through interactions with the anionic bacterial surface. It is known that pathogens have developed countermeasures to resist these agents by reducing the negative charge of membranes, by active efflux and by proteolytic degradation. Here we uncover a new strategy of resistance based on the neutralization of the bactericidal activity of APs by anionic bacterial capsule polysaccharide (CPS). Purified CPSs from Klebsiella pneumoniae K2, Streptococcus pneumoniae serotype 3 and Pseudomonas aeruginosa increased the resistance to polymyxin B of an unencapsulated K. pneumoniae mutant. Furthermore, these CPSs increased the MICs of polymyxin B and human neutrophil alpha-defensin 1 (HNP-1) for unencapsulated K. pneumoniae, Escherichia coli and P. aeruginosa PAO1. Polymyxin B or HNP-1 released CPS from capsulated K. pneumoniae, S. pneumoniae serotype 3 and P. aeruginosa overexpressing CPS. Moreover, this material also reduced the bactericidal activity of APs. We postulate that APs may trigger in vivo the release of CPS, which in turn will protect bacteria against APs. We found that anionic CPSs, but not cationic or uncharged ones, blocked the bactericidal activity of APs by binding them, thereby reducing the amount of peptides reaching the bacterial surface. Supporting this, polycations inhibited such interaction and the bactericidal activity was restored. We postulate that trapping of APs by anionic CPSs is an additional selective virulence trait of these molecules, which could be considered as bacterial decoys for APs.

Quinolones sensitize gram-negative bacteria to antimicrobial peptides

The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. Antimicrobial peptides (APs) make up the front line of defense in those areas exposed to microorganisms, and there is intensive research to explore their use as new antibacterial agents. On the other hand, it is known that subinhibitory concentrations of antibiotics affect the expression of numerous bacterial traits. In this work we evaluated whether treatment of bacteria with subinhibitory concentrations of quinolones may alter the sensitivity to APs. A 1-h treatment of Klebsiella pneumoniae with 0.25 x the MIC of ciprofloxacin rendered bacteria more sensitive to polymyxins B and E, human neutrophil defensin 1, and beta-defensin 1. Levofloxacin and nalidixic acid at 0.25 x the MICs also increased the sensitivity of K. pneumoniae to polymyxin B, whereas gentamicin and ceftazidime at 0.25 x the MICs did not have such an effect. Ciprofloxacin also increased the sensitivities of K. pneumoniae ciprofloxacin-resistant strains to polymyxin B. Two other pathogens, Pseudomonas aeruginosa and Haemophilus influenzae, also became more sensitive to polymyxins B and E after treatment with 0.25 x the MIC of ciprofloxacin. Incubation with ciprofloxacin did not alter the expression of the K. pneumoniae loci involved in resistance to APs. A 1-N-phenyl-naphthylamine assay showed that ciprofloxacin and levofloxacin increased the permeabilities of the K. pneumoniae and P. aeruginosa outer membranes, while divalent cations antagonized this action. Finally, we demonstrated that ciprofloxacin and levofloxacin increased the binding of APs to the outer membrane by using dansylated polymyxin B.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

Clinical relevance of the ESKAPE pathogens.

In recent years, the Infectious Diseases Society of America has highlighted a faction of antibiotic-resistant bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) - acronymically dubbed 'the ESKAPE pathogens' - capable of 'escaping' the biocidal action of antibiotics and mutually representing new paradigms in pathogenesis, transmission and resistance. This review aims to consolidate clinically relevant background information on the ESKAPE pathogens and provide a contemporary summary of bacterial resistance, alongside pertinent microbiological considerations necessary to face the mounting threat of antimicrobial resistance.

Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens

The emergence of multidrug-resistant pathogens within the clinical environment is presenting a mounting problem in hospitals worldwide. The 'ESKAPE' pathogens (Enterococcusfaecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have been highlighted as a group of causative organisms in a majority of nosocomial infections, presenting a serious health risk due to widespread antimicrobial resistance. The stagnating pipeline of new antibiotics requires alternative approaches to the control and treatment of nosocomial infections. Atmospheric pressure nonthermal plasma (APNTP) is attracting growing interest as an alternative infection control approach within the clinical setting. This study presents a comprehensive bactericidal assessment of an in-house-designed APNTP jet both against biofilms and planktonic bacteria of the ESKAPE pathogens. Standard plate counts and the XTT metabolic assay were used to evaluate the antibacterial effect of APNTP, with both methods demonstrating comparable eradication times. APNTP exhibited rapid antimicrobial activity against all of the ESKAPE pathogens in the planktonic mode of growth and provided efficient and complete eradication of ESKAPE pathogens in the biofilm mode of growth within 360 s, with the exception of A. baumannii where a >4log reduction in biofilm viability was observed. This demonstrates its effectiveness as a bactericidal treatment against these pathogens and further highlights its potential application in the clinical environment for the control of highly antimicrobial-resistant pathogens.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

Rapid-bTB: a novel lateral flow test able to differentiate Mycobacterium bovis from Mycobacterium tuberculosis in sputum

Background: A novel lateral flow, immunochromatographic assay (LFD) specific for Mycobacterium bovis, the cause of bovine tuberculosis and zoonotic TB, was recently developed at Queen’s University Belfast. The LFD detects whole M. bovis cells, in contrast to other commercially available LFD tests (BD MGITTM TBc ID, SD Bioline TB Ag MPT 64, Capilia TB-Neo kit) which detect MPT64 antigen secreted during growth. The new LFD test has been evaluated in the veterinary context, and its specificity for M. bovis in the broadest sense (i.e. subsp. bovis, subsp. caprae and BCG) and sensitivity to detect M. bovis in positive MGIT™ liquid cultures was demonstrated comprehensively.
Methods: Preliminary work was carried out by researchers at Queen’s University Belfast to optimise sputum sample preparation, estimate the limit of detection (LOD) of the LFD with M. bovis-spiked sputum samples, and check LFD specificity by testing a broad range of non-tuberculous Mycobacterium spp. (NTM) and other bacterial genera commonly encountered in sputum samples (Haemophilus, Klebsiella, Pseudomonas, Staphylococcus). In the Cameroon laboratory direct detection of M. bovis in human sputa was attempted, and 50 positive sputum MGIT™ cultures and 33 cultures of various Mycobacterium spp. originally isolated from human sputa were tested.
Results: Sputum sample preparation consisted of digestion with 1% NALC for 30 min, centrifugation at 3000g for 20 min, PBS wash, centrifugation again, and pellet resuspended in KPL blocking buffer before 100 µl was applied to the LFD. The LOD of the LFD applied to M. bovis-spiked sputum was estimated to be 104 CFU/ml. A small number of confirmed Ziehl-Neelsen ‘3+’ M. bovis positive sputum samples were tested directly but no positive LFD results were obtained. All of the sputum MGIT™ cultures and mycobacterial cultures (including M. tuberculosis, M. africanum, M. bovis, M. intracellulare, M. scrofulaceum, M. fortuitum, M. peregrinum, M. interjectum) tested LFD negative when read after 15 min except for the M. bovis cultures, thereby confirming specificity of LFD for M. bovis in the clinical microbiology context.
Conclusions: Results indicate that the ‘Rapid-bTB’ LFD is a very specific test, able to differentiate M. bovis from M. tuberculosis, M. africanum, and a range of NTM isolated from human sputa in MGITTM liquid cultures. However, the LFD lacks sufficient sensitivity to be applied earlier in the diagnostic process to directly test human sputa.

ISCR and their association with antibiotic resistance genes

A prevalência de bactérias resistentes a antibióticos em ambiente hospitalar tem vindo a tornar-se dramática e preocupante a nível mundial. Contudo, com a utilização inadequada de antibióticos em áreas tão diversas como a veterinária, a aquacultura e a agricultura, esta deixou de estar confinada ao ambiente hospitalar, sendo o ambiente um reservatório natural de microganismos resistentes a estes compostos. O conhecimento detalhado dos determinantes de resistência a antibióticos presentes nestes ambientes, sejam estes genes de resistência ou estruturas envolvidas na sua mobilização, é fundamental, não só do ponto de vista do conhecimento como para a eventual implementação de medidas de contenção da sua disseminação. Neste contexto, são necessários estudos que permitam conhecer o panorama mais realista da distribuição destes determinantes de resistência a antibióticos, quer no meio ambiente quer no ambiente clínico. Assim, constituiu objectivo principal deste trabalho contribuir para o conhecimento do panorama actual da prevalência e distribuição dos elementos ISCR, bem como de outros determinantes de resistência a antibióticos em espécies de Gram-negativo clinicamente relevantes (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii e Citrobacter freundii) recolhidas a partir de amostras de pacientes do Hospital Infante D. Pedro, EPE, Aveiro, Portugal, entre 2006 e 2008. Adicionalmente, foram também recolhidas bactérias de Gram-negativo do meio ambiente, a partir de amostras de águas e de vísceras de peixes, nas quais foram igualmente pesquisados os elementos acima referidos. À excepção dos isolados de E. coli e de Gramnegativo ambientais, todas as estirpes estudadas foram seleccionadas com base no seu perfil de multirresistência a antibióticos. Os resultados mostraram que em todas as espécies recolhidas no ambiente hospitalar foi detectada a presença de elementos ISCR, do tipo ISCR1 ou ISCR2. O elemento ISCR1, foi encontrado em isolados de E. coli, K. pneumoniae e C. freundii e o elemento ISCR2 em isolados de A. baumannii. Não foi detectada a presença de ISCRs nos isolados de Gram-negativo ambientais, o que sugere que a ocorrência dos mesmos é fortemente influenciada pela pressão selectiva exercida pelo ambiente em que os microrganismos se encontram. Genes qnr e integrões de classe 1 foram os determinantes de resistência mais frequentemente encontrados associados aos elementos ISCR1. Os vários determinantes de resistência foram encontrados em diferentes contextos genéticos e localizados em estruturas móveis, nomeadamente em plasmídeos. O elemento ISCR2 presente em isolados de A. baumannii encontra-se associado ao gene sul2 em todos os isolados, dentro de um mesmo contexto genético e com uma localização cromossomal. Contudo, o contexto genético encontrado nestes isolados é novo não tendo sido descrito até à data em outros microrganismos. O presente estudo constitui a primeira descrição de elementos ISCR intrinsecamente ligados a genes de resistência a antibióticos, em Portugal. Uma vez que estes elementos parecem ser responsáveis pela mobilização de um grande número de genes de resistência a antibióticos, a sua elevada incidência entre estirpes resistentes e multirresistentes, bem como a sua associação com genes de resistência é preocupante e requer vigilância.

Compósitos de polímeros naturais e nanopartículas metálicas

Esta tese descreve diversas estratégias de preparação assim como a caracterização de nanocompósitos com base em distintos biopolímeros. Em particular foi estudada a incorporação de nanopartículas (NPs) metálicas, nomeadamente de Ag, Cu e Au. Estes nanomateriais apresentam um potencial prático enorme em diversas áreas, no entanto foi investigada especificamente a sua aplicação como materiais antimicrobianos. No primeiro capítulo apresenta-se uma revisão bibliográfica, onde são realçados os principais tópicos discutidos ao longo da tese. Inicialmente apresenta-se uma contextualização deste trabalho sendo seguidamente apresentadas algumas considerações sobre nanocompósitos e o seu impacto tecnológico atual. Em seguida, descrevem-se as vantagens do uso de NPs como cargas nos materiais compósitos especificamente no caso de bionanocompósitos. Foi focado o uso da celulose como matriz uma vez que foi o composto “base” usado neste trabalho. Fez-se a descrição exaustiva das metodologias existentes na literatura para a preparação dos nanocompósitos celulósicos com diferentes NPs metálicas assim como das respetivas aplicações. Dentro das aplicações, foi dado especial destaque às propriedades antimicrobianas dos materiais preparados seja a nível da sua atividade antibacteriana ou antifúngica. Esta introdução privilegia o trabalho relacionado diretamente com os sistemas descritos nos capítulos subsequentes. No segundo capítulo apresentam-se os resultados obtidos para nanocompósitos de prata em matriz celulósica. Através do uso de metodologias, tais como a síntese in situ e a pós-deposição, foram preparados diversos materiais usando dois substratos celulósicos distintos nomeadamente a celulose vegetal e bacteriana. Estes nanocompósitos foram caracterizados em termos da sua morfologia e composição química, verificando-se a importância destas características na sua atividade antibacteriana. Foi verificado que nanocompósitos com teores de Ag de 5 x 10-4 (% m/m) são suficientes para obter atividade antibacteriana. A libertação de Ag(I) foi estudada em alguns destes materiais de modo a tentar perceber o mecanismo subjacente a este tipo de nanocompósitos. No terceiro capítulo é apresentado o estudo de NPs coloidais de Ag e Au como cargas para a preparação de nanocompósitos à base de quitosano nãomodificado e modificado quimicamente (derivado solúvel em água e derivado anfifílico). Foram preparados filmes finos de espessura de 9-14 μm, caracterizando-se as suas propriedades óticas e antibacterianas. As propriedades óticas foram ajustadas, quer pela variação do teor de NPs de Ag (0,3-3,9% m/m) ou pela utilização de amostras de NPs com distribuição de tamanho de partícula distinta. Foi investigada a atividade antibacteriana tanto para bactérias Gram-negativas (Klebsiella pneumoniae e Escherichia coli) como para Gram-positivas (Staphylococcus aureus). Para nanocompósitos preparados com o quitosano não modificado verificou-se uma dependência em função do teor de Ag. No caso do uso de derivados modificados, os materiais preparados mostraram uma eficiência superior, mesmo sem NPs de Ag. No quarto capítulo é apresentada a síntese e caracterização de nanocompósitos de pululano e NPs de Ag. Neste estudo é avaliada a atividade antifúngica dos filmes compósitos preparados contra o Aspergillus niger usando protocolos padrão. Estes materiais foram preparados na forma de filmes (66-74 μm de espessura) por evaporação de solvente da mistura de pululano e coloides de Ag. Foi observado o aumento da inibição do fungo na presença dos nanocompósitos, tendo sido pela primeira vez mostrado o efeito disruptivo destes materiais sobre os esporos do A. niger através da análise das imagens de SEM. Este efeito ocorre na presença dos filmes devido à presença das cargas de NPs de Ag dispersas no pululano. O desenvolvimento de materiais de papel com NPs de Cu é um desafio devido à propensão destas espécies em oxidar sob condições ambiente. No quinto capítulo é descrita pela primeira vez o estudo comparativo do crescimento e estabilidade de NPs de Cu em celulose vegetal e bacteriana. Para além disso foi avaliado o uso de nanoestruturas com diferentes dimensionalidades como cargas, nomeadamente nanoesferas e nanofios. Foi observado que o uso de nanofios aumenta a resistência à oxidação destes nanocompósitos para tempos de exposição ao ar mais prolongados. As matrizes celulósicas apresentam comportamento distinto no crescimento e/ou adsorção das NPs de Cu. A celulose bacteriana foi o substrato mais eficiente para retardar a oxidação das NPs. A atividade antibacteriana destes nanocompósitos foi avaliada. Ao longo desta dissertação são apresentados métodos distintos para a obtenção de nanocompósitos com base em biopolímeros e NPs metálicas. Estes estudos permitiram não só a preparação de novos nanocompósitos mas também compreender e otimizar os mecanismos subjacentes à sua preparação. Ao mesmo tempo, este trabalho contribuiu para a transferência de tecnologia e conhecimento entre a área da Nanotecnologia e a área dos materiais derivados de fontes renováveis. As propriedades apresentadas por estes nanomateriais mostraram a sua possível aplicação como novos materiais antimicrobianos, no entanto é possível antecipar futuras aplicações em outras áreas tecnológicas.

Potential health benefits of Artemisia gorgonum extracts

Relevância etnofarmacológica: Artemisia gorgonum (Asteraceae), conhecida como “losna ou lorna”, é usada em Cabo Verde na medicina tradicional para o tratamento de inflamações, febre e gastroenterites. Estudos recentes sugerem que artimetina, isolada a partir de Artemisa gorgonum, poderia ser usada para o tratamento da malária devido à sua atividade antiplasmodial. Objetivo do estudo: Avaliação in vitro da atividade anti-microbiana e sinergética dos extratos hidroetanol (70%) e metanol de A. gorgonum (EHAG e EMAG) em bactérias do trato urinário e uma espécie de fungo. A atividade antioxidante dos extratos de hidroetanol (70%), metanol, clorofórmio e clorofórmio-metanol (1:2), e o efeito protetor de EHAG contra lesões hepáticas em ratos induzidos com CCl4 também foram analisados. Material e métodos: A atividade antimicrobiana dos extratos de A. gorgonum foi testada in vitro contra sete estirpes de microrganismos, incluindo bactérias Gram-positivas, Gram-negativas e uma espécie de fungo. O método DAA (Decimal assay for additivity) foi determinado para atividade antibacteriana do EHAG contra Pseudomonas aeruginosa. O efeito antioxidante in vitro de vários extratos de A. gorgonum foi analisado pelo método DPPH. A lesão hepática foi induzida por injeção intrapeitoral do CCl4. Seguidamente, os ratos foram administrados oralmente com EHAG, diariamente, por um período de 7 dias. Resultados e Discussão: Foi observada atividade antibacteriana dos extratos de EHAG e EMAG contra todos os microrganismos usados neste estudo. O crescimento das estirpes de Escherichia coli e Pseudomonas aeruginosa foi o mais inibido por ambos os extratos, apresentando valores significativos, enquanto o crescimento das estirpes S. aureus e Klebsiella spp. foi o menos afetado. Candida albicans foi inibida fortemente pelo EMAG. As combinações de extrato hidroetanólico com antibióticos demonstraram atividade antibacteriana sinergética contra todos os patogénicos testados. Em contrapartida, a combinação de extrato metanólico com antibióticos permitiu observar efeitos antagónicos contra todas as bactérias, exceto Klebsiella spp. que apresentou atividade sinérgica. O EHAG e EMAG mostraram efeito significativo na eliminação do radical DPPH. A atividade hepatoprotetora foi observada em ratos previamente administrados com CCl4. Estes estudos evidenciam os potenciais benefícios de A. gorgonum.

Cinnamaldehyde increases the susceptibility of quorum-sensing-mediated biofilms to conventional antibiotics

The role of bacterial communication, also known as quorum sensing is an important mechanism in biofilm formation which is fundamental to the development of anti-biofilm strategies. In this current study, the synergy between a quorum sensing inhibitor (cinnamaldehyde) and two antibiotics (ceftazidime and levofloxacin) was evaluated in an attempt to develop a strategy for biofilm disruption using the high-throughput minimum biofilm eliminating concentration (MBEC) assay. Klebsiella pneumoniae and Proteus mirabilis biofilms of initial broth suspensions of 108 colony forming units (CFU) per mL, cultivated on the pegs of the MBEC device were challenged with 5120 µg/ml of ceftazidime and levofloxacin in a double dilution assay in the presence of 500 µM cinnamaldehyde. The minimum inhibitory concentrations (MIC) in the presence of cinnamaldehyde for ceftazidime and levofloxacin were 0.125% (640 µg/mL) and 0.0625% (320 µg/mL) respectively with no significant bacterial growth on LB agar. The MBECs for ceftazidime and levofloxacin were above 5120 and 2560 µg/mL respectively which yielded over 70% reduction in both Klebsiella pneumoniae and Proteus mirabilis biofilms. The above results indicate the possibility that the synergy between antimicrobial agents may lead to biofilm eradication.

The in vitro assessment of the synergistic effects of antibiotics and wound dressings on biofilms from diabetic foot pathogens

The impact of biofilm in the effective control of wound microbiome is an ongoing dilemma which has seen the use of different treatment strategies. The effects of wound dressings and antibiotics on both planktonic bacteria and biofilms have been separately evaluated in previous studies. In this current study, the combined antimicrobial effects of some selected wound dressings (silver-impregnated: Acticoat and Silvercel; and honey-impregnated: Medihoney™ Apinate) and antibiotics (ceftazdime and levofloxacin) on Klebsiella pneumoniae and Proteus mirabilis in their quasi-biofilm state were assessed using zone of inhibition (ZOI) test. Before the addition of the wound dressings, bacterial suspension of 108 colony forming units per mL and different concentrations of ceftazidime and levofloxacin (256, 512, 1024 and 5120µg/mL) of a final volume of 1mL were inoculated on Mueller Hinton agar and allowed to dry. Wound dressings cut into circular shapes (2cm diameter) were aseptically placed on the agar plates and incubated at 35 – 37°C for 24 hours. ZOIs associated with Acticoat, Silvercel and Medihoney™ Apinate dressings were compared with that of Atrauman (non-medicated control) dressing. All three dressings showed significant (p < 0.05) biofilm-inhibiting activity against both bacteria at antibiotic concentrations of 1024 and 5120µg/mL with ZOI between 17.5 and 35mm.

In Vitro Assessment of the Synergy between Polymyxin B (PMB) and Polymyxin B Nonapeptide (PMBN) and Antibiotics on Biofilms from Diabetic Foot Infections

Background: The increasing resistance of Gram-negative bacteria isolated from nosocomial infections and chronic wounds, such as diabetic foot ulcers has renewed research interests in the use of polymyxins in the treatment of multidrug resistant infections. The added resistance conferred by biofilm development in such infections and the absence of novel antibiotics presuppose that polymyxins are the likely drugs of choice in spite of their nephrotoxicity. The effects of PMB and PMBN have been previously assessed on planktonic bacteria isolated from various infections. Methods: This current study assessed the synergy between a PMB/PMBN and two antibiotics (ceftazidime and levofloxacin) in an attempt to develop a strategy for biofilm disruption using the Minimum Biofilm Eradication Concentration Physiology and Genetic assay (MBEC™ P & G, Innovotech Inc, Edmonton, Alberta, Canada) according to manufacturer’s instructions. Klebsiella pneumoniae (K. pneumoniae) and Proteus mirabilis (P. mirabilis) biofilms of initial broth suspensions of 108 colony forming units per mL, cultivated on the pegs of the MBEC device were challenged with 5120 µg/mL of both ceftazidime and levofloxacin in a ten-fold dilution assay and in the presence of 100 and 500 µg/mL PMB and PMBN. Results: From table of results (Table 1), it can be deduced that both ceftazidime and levofloxacin are very effective in inhibiting biofilm development (as shown by percentage inhibition (PI)) when augmented with PMB and PMBN. This is about 100-fold increase in efficacy when compared to the antibiotics used on their own. The percentage reduction (PR) in biofilm was also increased considerably when PMB and PMBN concentrations were increased to 500 µg/mL. PMB was more effective than its less antibacterial derivative PMBN. Levofloxacin was also found to be more effective than ceftazidime when combined with both PMB and PMBN due to its enhanced cell-membrane permeability and as an anti-DNA replication uncoupling agent. Conclusion: The above results indicate that the synergy between antibiotics and cell membrane permeabilising agents may provide alternate strategies towards biofilm eradication

Antibiotic resistance in enterobacteriaceae isolated from Portuguese deli meats

This study aimed to identify the presence of β-lactam-resistant bacteria in different types of Portuguese deli meats. The numbers of ampicillin resistant bacteria varied from negative in 25 g to 1.0 × 108colony-forming units/g. Within 78 randomly selected β-lactam-resistant bacteria, 24 different resistant phenotypes were found and 35.9% were multidrug resistant (MDR). The majority (87.2%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the blaTEM gene was detected in 23 out of 67 isolates (34.3%) and 16 of them presented MDR phenotypes. Four Klebsiella oxytoca isolates (6%) harbored a gene for the CTX-M/OXY-type enzyme. The direct sequencing of their purified amplicons confirmed the presence of three types of blaOXYgenes (blaOXY-1, blaOXY-2 and blaOXY-5). These results suggest that without good hygienic practices, deli meats may act as a vehicle of transfer of β-lactam-resistant bacteria to the gastrointestinal tract of consumers.