Prevalence of antibiotic resistance and virulent factors in nosocomial clinical isolates of Pseudomonas aeruginosa from Panamá

ABSTRACT Background: Pseudomonas aeruginosa is an important causative agent of nosocomial infections. As pathogen, P. aeruginosa is of increasing clinical importance due to its ability to develop high-level multidrug resistance (MDR). Methods: The aim of the present study was to better understand the intrinsic virulence of circulating strains of Pseudomonas aeruginosa, by surveying and characterizing the antibiotic resistance profiles and prevalence of virulence factors in 51 clinical isolates of P. aeruginosa obtained from children admitted to Hospital del Niño-Panamá during the period of October 2016 until March 2017. Antimicrobial susceptibilities were assessed by determining the minimum inhibitory concentration for 12 antibiotics against P. aeruginosa clinical isolates using the VITEK system (https://www.biomerieux.com). Additionally, all isolates were examined by Polymerase Chain Reaction (PCR) for the presence of components of the MexAB-OprM efflux pump system (mexABR) and pyoverdine receptor genes and betalactamases resistance genes (ESBL) using gene-specific primers. Results: A total of 51 pyoverdine producing clinical isolates were analyzed, all of which expressed resistance genes such as genes of the MexAB-OprM efflux pump system (mexABR) and pyoverdine receptor genes (fpvA). Out of 51 MDR isolates, 22 were ESBL producers. The most common ESBL gene was blaTEM expressed by 43% of the isolates. The isolates tested in this study showed increased resistance to antibiotics in the following categories: (i) penicillins (ampicillin (69%), piperacillin (22%); (ii) pyrimethamines (trimethoprim, 65%); (iii) nitrofurans (nitrofurantoin, 63%), and (iv) third-generation cephalosporin cefotaxime (53%). These results underscore a high prevalence of MDR amongst clinical isolates from Panama. Conclusions: The present study indicates that prevalence of BlaTEM-carrying strains is increasing with subsequent multidrug resistance in Panamá and as well reported worldwide. The virulent factors identified in this study provide valuable information regarding the prevalence of resistance genes and their potential impact on treatments that exploit the unique physiology of the pathogen. To prevent further spread of MDR, the proportions of resistant strains of Pseudomonas aeruginosa should be constantly evaluated on healthcare institutions of Panamá. More importantly, this information can be used to better understand the evolution and dissemination of strains hoping to prevent the development of resistance in Pseudomonas aeruginosa. Future studies quantifying the expression of these virulent genes will emphasize on the acquisition of multidrug resistance.

Phylogeny of the Trichogramma endosymbiont Wolbachia, an alpha-proteobacteria (Rickettsiae) / Filogenia do endosimbionte Wolbachia em Trichogramma, an alpha-proteobacteria (Rickettsiae)

Abstract Wolbachia (Hertig) endosymbionts are extensively studied in a wide range of organisms and are known to be transmitted through the egg cytoplasm to the offsping. Wolbachia may cause several types of reproductive modifications in arthropods. In Trichogramma species, parthenogenesis-inducing Wolbachia bacteria allow females wasps to produce daughters from unfertilized eggs and these bacteria are present in at least 9% of all Trichogramma species. Phylogenetic studies have led to the subdivision of the Wolbachia clade in five supergroups (A, B, C, D and E) and Wolbachia from Trichogramma belong to supergroup B. Here, using the wsp gene, four groups of Wolbachia that infect Trichogramma species were distinguished and the addition of a new group "Ato" was suggested due to the addition of Wolbachia from Trichogramma atopovirilia (Oatman and Platner). Specific primers were designed and tested for the "Ato" group. Seventy-five percent of all evaluated Wolbachia strains from Trichogramma fell within "Sib" group.

Resumo Endosimbiontes do gênero Wolbachia (Hertig) são extensivamente estudados em uma ampla gama de organismos e são conhecidos por serem transmitidos via citoplasma do ovo hospedeiro para seu descendente. Wolbachia pode causar vários tipos de alterações reprodutivas nos artrópodes. Nas espécies de Trichogramma, a reprodução partenogenética induzida por Wolbachia, possibilita as fêmeas dos parasitoides a produção de fêmeas a partir de ovos não fertilizados e estas bactérias estão presentes em pelo menos 9% de todas as espécies de Trichogramma. Estudos filogenéticos têm levado a subdivisão do clado Wolbachia em cinco supergrupos (A, B, C, D and E). Wolbachia em Trichogramma pertence ao supergrupo B. Com o gene wsp foi possível se distinguir quatro grupos de Wolbachia que infectam Trichogramma e adicionar um novo grupo (Ato) devido a inclusão de Wolbachia detectada em Trichogramma atopovirilia (Oatman and Platner, 1983). Primers específicos foram construídos e testados para o grupo "Ato". Setenta e cinco por cento de todas as linhagens de Wolbachia que infectam Trichogramma se enquadraram dentro do grupo "Sib".

The Study of Otoacoustic Emissions and the Suppression of Otoacoustic Emissions in Subjects with Tinnitus and Normal Hearing: An Insight to Tinnitus Etiology

Introduction Analysis of the suppression effect is a simple method to evaluate cochlear status and central auditory mechanisms and, more specifically, the medial olivocochlear system. This structure may be involved in the generation of mechanisms that cause tinnitus and in the pathophysiology of tinnitus in patients with tinnitus and normal hearing. Objective To review the literature of the etiology of tinnitus on the lights of otoacoustic emissions in patients with normal hearing. Data Synthesis Individuals with tinnitus and normal hearing have a higher prevalence of alterations in transient-evoked otoacoustic emissions and distortion-product otoacoustic emissions than normal subjects. This fact suggests that dysfunctions of the outer hair cells (OHCs) might be important in the generation of the tinnitus; however, this feature is not always present in those who have the symptoms of tinnitus. Final Comments These findings suggest that OHC dysfunction is not necessary for tinnitus development—that is, there might be mechanisms other than OHC damage in the tinnitus development. On the other hand, OHC dysfunction alone is not sufficient to cause the symptom, because a great many individuals with OHC dysfunction did not complain about tinnitus. .

Autonomia do graduando em enfermagem na (re)construção do conhecimento mediado pela aprendizagem baseada em problemas / Autonomy of nursing undergraduate student in the (re)construction of knowledge mediated by problem-based learning / La autonomía del estudiante de enfermería en la (re)construcción del conocimiento mediada por el aprendizaje basado en problemas

Estudo qualitativo, método Bricolagem, que objetivou analisar como a Aprendizagem Baseada em Problemas (ABP) promove o desenvolvimento da autonomia do aluno no processo de aprender a aprender. Os sujeitos foram 16 alunos e dois tutores envolvidos na disciplina. A coleta dos dados combinou entrevista semiestruturada, observação participante, registro em portfólios, fichas de avaliação, e gravação em áudio das tutorias. A análise dos dados seguiu estratégias de interpretação definidas pelas autoras: leituras iniciais e aprofundada; construção e reunião de mapas de significados; elaboração, descrição e análise de categorias empíricas, à luz do referencial teórico. A ABP favorece a (re)construção de conhecimentos pela utilização de saberes e experiências prévias, que são compartilhados no pequeno grupo; pelo processo de teorização; e pela via do conhecimento pertinente - aquele passível de aplicação à prática. Concluímos que a ABP estimula o aprendizado contínuo, desenvolvendo no aluno autonomia no processo de aprender a aprender.

This is a qualitative study, using the ‘Do it yourself’ method, which aimed to analyze how Problem-Based Learning (PBL) promotes the development of learner’s autonomy in the process of learning to learn. The subjects were 16 students and two tutors involved in the discipline. Data collection techniques combined semi-structured interviews, participant observation, log in portfolios, evaluation forms, and audio recording of the tutorials. Data analysis followed interpretation strategies defined by the authors: initial and in depth readings; construction and assembly of meanings’ maps; development, description and analysis of empirical categories, in the light of the theoretical framework. The PBL favors the (re)construction of knowledge by the use of prior knowledge and experiences that are shared in small group; through the process of theorization; and by means of relevant knowledge - one that can be applied to practice. We conclude that PBL encourages continuous learning, developing in the student the autonomy in the process of learning to learn.

Estudio cualitativo, con método Bricolage, que tuvo como objetivo analizar cómo el Aprendizaje Basado en Problemas (ABP) promueve el desarrollo de la autonomía del alumno en el proceso de aprender a aprender. Los sujetos fueron 16 alumnos y dos tutores que participan en la disciplina. Para recolección de datos se combinaran entrevistas estructuradas, observación participante, registro en portfolios, formularios de evaluación y grabación de audio de los tutoriales. El análisis de los datos siguió las estrategias de interpretación definidos por los autores: lecturas iniciales y profundadas; construcción y montaje de mapas de significados; el desarrollo, descripción y análisis de categorías empíricas, a la luz de lo referencial teórico. El ABP favorece la construcción de conocimientos mediante el uso de saberes y experiencias que se comparten en grupos pequeños, a través del proceso de teorización, y por medio de los conocimientos pertinentes - uno que se puede aplicar a la práctica. Llegamos a la conclusión que el ABP promueve el aprendizaje continuo, el desarrollo de los estudiantes, la autonomía en el proceso de aprender a aprender.

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1.

Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.

Efflux as a mechanism of resistance to antimicrobials in Pseudomonas aeruginosa and related bacteria: unanswered questions

Pseudomonas aeruginosa is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents. This intrinsic multidrug resistance is caused by synergy between a low-permeability outer membrane and expression of a number of broadly-specific multidrug efflux (Mex) systems, including MexAB-OprM and MexXY-OprM. In addition to this intrinsic resistance, these and three additional systems, MexCD-OprJ, MexEF-OprN and MexJK-OprM promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes by mutational events. In addition to antibiotics, these pumps export biocides, dyes, detergents, metabolic inhibitors, organic solvents and molecules involved in bacterial cell-cell communication. Homologues of the resistance-nodulation-division systems of P. aeruginosa have been found in Burkholderia cepacia, B. pseudomallei, Stenotrophomonas maltophilia, and the nonpathogen P. putida, where they play roles in resistance to antimicrobials and/or organic solvents. Despite intensive studies of these multidrug efflux systems over the past several years, their precise molecular architectures, their modes of regulation of expression and their natural functions remain largely unknown

Anaerobiosis induced virulence of Salmonella typhi.

BACKGROUND & OBJECTIVES: Anaerobic conditions are frequently encountered by pathogens invading the gastrointestinal tract due to low/limiting oxygen conditions prevalent in the small intestine. This anaerobic stress has been suggested to enhance the virulence of gut pathogens. In the present study, we examined the effect of anaerobiosis on the virulence of Salmonella Typhi, a Gram negative bacteria which invades through the gut mucosa and is responsible for typhoid fever. METHODS: Salmonella Typhi (ty2) was cultured in aerobic and anaerobic conditions to compare its virulence by rabbit ileal loop assay, hydrophobicity assay, expression of outer membrane proteins (OMPs) and antioxidant enzymes assay. RESULTS: Anaerobically grown S. Typhi showed significantly higher cell surface hydrophobicity as compared to aerobic bacteria. In vivo toxin production by rabbit ileal loop assay also showed significantly higher fluid accumulation with anaerobic S. Typhi. Expression of OMPs in anaerobic S. Typhi showed a distinct induction of five outer membrane proteins. We observed that exposure of anaerobic S. Typhi to aerobic conditions induced significantly higher level of antioxidant enzymes like superoxide dismutase (SOD) and catalase. INTERPRETATION & CONCLUSION: Our results suggest that exposure of S. Typhi to anaerobic conditions enhances its virulence.

The mechanism of resistance of Pseudomonas aeruginosa to beta-lactam antibiotics and clinical significance / 华中科技大学学报（医学）（英德文版）

To study the resistant mechanism and clinical significance of pseudomonas aeruginosa to beta-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5 beta-lactam antibiotics was measured and their production of beta-lactamase and the beta-lactamase genes they carried detected. Furthermore, the relationship between the permeability, beta-lactamase and the clinical effects of beta-lactam antibiotics was observed. By using 14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPS) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P < 0.05). All strains harbored 1-4 beta-lactamase genes and produced beta-lactamase. Higher permeability rate and higher degree of stability to beta-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability and beta-lactamase. These results suggested that the permeability of outer membrane and beta-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa to beta-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.

Asp 280 residue is important in the activity of the Escherichia coli leader peptidase

Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.

Antibody response after immunization of Brazilian children with serogroup C meningococcal polysaccharide noncovalently complexed with outer membrane proteins

We have studied the antibody response of Brazilian vaccines to C meningococcal polysaccharide (C-PS) after one or two doses of a vaccine composed of C-PS, outer membrane proteins of B meningococci and aluminum hydroxide. Total IgG, IgG1 and IgG2 as well as bactericidal activity mediated by complement were measured in serum samples from children 3 to 83 months of age (post-vaccination IgG, IgG1 and IgG2 levels of 2.4 to 13.4 µg/ml; less than 18 to 67.8 U/ml and less than 8 to 106.8U/ml, respectively) and from individuals 10 to 14 years of age (post-vaccination IgG, IgG1 and IgG2 levels of 14.6 µg/ml, 23,7 U/ml and 112.0 U/ml, respectively). The antibody response, measured as IgG levels, was age-dependent. Although high antibody levels were demonstrableby enzyme-linked immunosorbent assay (ELISA), bactericidal activity was not demonstrable (less than 1:4) in serum from children aged less than 24 months. A significant bactericidal activity was detected in serum of children older than 49 months of age and in individuals 10 to 14 years of age. A predominance of IgG2 was observed in post-vaccination serum samples from children belonging to those two age groups. The antibody concentration sufficient to confer protection as well as the possible causes of the poor correlation observed between ELISA and bactericidal activity results are discussed

Immunological studies of Neisseria meningitidis andtigens: I. Interaction of protein P and its andtibodies evaluated by radioimmunoassay

A Neisseria menigitidis responsável pela meningite bacteriana de alta freqüência em nosso meio, tem como antígeno principais: o polissacarídeo capsular, proteína P e o carboidrato somático. A classificaçäo em grupos é baseada no políssacarídeo capsular ao passo que a proteína é tipo-específica. A aproximaçäo metodológica empregada para estudar as interaçöes da proteína de superfície com o anticorpo foi o radioimunoensaio. Foram testados os sístemas de coelho, carneiro e soro de convalescentes humanos. A proteína P antigênica foi marcada com "Iodo pelo método de cloramina-T. O título do soro é definido como a diluiçäo que precipita 50% de antígeno marcado em condiçöes experimentais padronizadas. A forma da curva de precipitaçäo dá uma indicaçäo da baixa afinidade entre antígeno-anticorpo no sistema de carneiro. No sistema humano, a linearidade da curva de precipitaçäo se manteve na diluiçäo de anticorpo por 64 vezes. Nos sistemas devem ser levados em consideraçäo fatores da ordem de baixa afinidade de antígeno-anticorpo, presença de pequeno número de grupos reativos ou baixo nível de determinantes antigênicos. É altamente desejável um melhor entidademento da proteína que poderia contribuir para o desenvolvimento de uma vacina capaz de prevenir contra meningite causada pelo sorotipos de maior incidência