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.

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

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.

Insights into the behavior of six rationally designed peptides based on Escherichia coli's OmpA at the water-dodecane interface.

The Escherichia coli's membrane protein OmpA has been identified as a potential biosurfactant due to their amphiphilic nature, and their capacity to stabilize emulsions of dodecane in water. In this study, the influence of surfactant type, concentration, preservation time and droplet size on the crystallization of n-dodecane and water, in oil-in-water emulsions stabilized with six rationally designed Escherichia coli's OmpA-based peptides was investigated. A differential scanning calorimetry (DSC) protocol was established using emulsions stabilized with Tween 20® and Tween 80®. A relationship between the surfactant concentration and the crystallization temperatures of n-dodecane and water was observed, where the crystallization temperatures seem to be dependent on the preservation time. A deconvolution analysis shows that the peak morphology possibly depends on the interactions at the interface because the enthalpic contributions of each Gaussian peak remained similar in emulsions stabilized with the same peptide. Adsorption results show that the main driver for adsorption and thus stabilization of emulsions is polar interactions (e.g. H-bonding) through the hydrophilic parts of the peptides. Those peptides with a preponderance of polar interaction groups distribution (i.e. NH2, COOH, imidazole) showed the highest interfacial activity under favorable pH conditions. This suggests that custom-made peptides whose hydrophilic/hydrophobic regions can be fine-tuned depending on the application can be easily produced with the additional advantage of their biodegradable nature.

Immunogenicity of OmpA and OmpB antigens from Rickettsia rickettsii on mononuclear cells from Rickettsia positive Mexican patients.

Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects. Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production. Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids. Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.

Produção e purificação de imunoglobulinas Y policlonais anti-Leptospira spp / Production and purification of polyclonal anti-Leptospira immunoglobulin Y

Objetivou-se verificar se galinhas imunizadas com uma solução de Leptospira interrogans inativadas e proteínas de membrana externa do sorovar Hardjo, poderiam produzir anticorpos policlonais específicos anti-leptospiras, detectáveis em testes ELISA. Foram imunizados oito galinhas com 25 semanas de idade, da raça White Leghorn, sendo três imunizadas com uma suspensão de leptospiras inativadas, três com uma solução de proteínas de membrana externa extraída do sorovar Hardjo e duas controle. Coletas de sangue foram realizadas quinzenalmente e de ovos diariamente. A IgY foi purificada a partir da gema dos ovos utilizando para a delipidação o método de diluição em água ácida e a precipitação com sulfato de amônio. Nos testes ELISA realizados para verificar a especificidade da IgY, foi demonstrada a produção de anticorpos anti-Leptospira, tanto no soro quanto nas gemas purificadas. O pico de produção de anticorpos específicos ocorreu na 5º semana após a primeira imunização. Ficou demonstrada a possibilidade da indução da produção de anticorpos específicos em galinhas imunizadas com leptospiras do sorovar Hardjo inativadas, bem como, com proteínas de membrana externa (PME) extraidas desse sorovar. As galinhas imunizadas com uma suspensão de leptospiras inativadas ou com PME de Leptospira interrogans do sorovar Hardjo produziram anticorpos reativos a PME Hardjo detectáves por teste ELISA.

The aim was to determine whether hens immunized with an inactivated suspension of Leptospira and a solution of outer membrane proteins extracted from the serovar Hardjo, could produce specific polyclonal antibodies to Leptospira, detected in ELISA assay. Eight hens White Leghorn race with 25-weeks-old were immunized, three with an inactivated suspension of Leptospira, three with a solution of outer membrane proteins (OMP) extracted from the serovar Hardjo and two controls immunized with saline. Blood samples were collected fortnightly and eggs daily. The IgY was purified from the egg yolk using the method for the delipidation of dilution with water acidic and ammonium sulfate precipitation. The ELISA assay was performed to verify the specificity of the IgY, these was possible to observe the production of specific antibody to Leptospira both in serum and purified egg yolk. The specific antibody titers peaked in the fifth week post immunization. The production of polyclonal IgY was effective for producing high titers of specific antibodies.

Produção e purificação de imunoglobulinas Y policlonais anti-Leptospira spp / Production and purification of polyclonal anti-Leptospira immunoglobulin Y

Objetivou-se verificar se galinhas imunizadas com uma solução de Leptospira interrogans inativadas e proteínas de membrana externa do sorovar Hardjo, poderiam produzir anticorpos policlonais específicos anti-leptospiras, detectáveis em testes ELISA. Foram imunizados oito galinhas com 25 semanas de idade, da raça White Leghorn, sendo três imunizadas com uma suspensão de leptospiras inativadas, três com uma solução de proteínas de membrana externa extraída do sorovar Hardjo e duas controle. Coletas de sangue foram realizadas quinzenalmente e de ovos diariamente. A IgY foi purificada a partir da gema dos ovos utilizando para a delipidação o método de diluição em água ácida e a precipitação com sulfato de amônio. Nos testes ELISA realizados para verificar a especificidade da IgY, foi demonstrada a produção de anticorpos anti-Leptospira, tanto no soro quanto nas gemas purificadas. O pico de produção de anticorpos específicos ocorreu na 5º semana após a primeira imunização. Ficou demonstrada a possibilidade da indução da produção de anticorpos específicos em galinhas imunizadas com leptospiras do sorovar Hardjo inativadas, bem como, com proteínas de membrana externa (PME) extraidas desse sorovar. As galinhas imunizadas com uma suspensão de leptospiras inativadas ou com PME de Leptospira interrogans do sorovar Hardjo produziram anticorpos reativos a PME Hardjo detectáves por teste ELISA.(AU)

The aim was to determine whether hens immunized with an inactivated suspension of Leptospira and a solution of outer membrane proteins extracted from the serovar Hardjo, could produce specific polyclonal antibodies to Leptospira, detected in ELISA assay. Eight hens White Leghorn race with 25-weeks-old were immunized, three with an inactivated suspension of Leptospira, three with a solution of outer membrane proteins (OMP) extracted from the serovar Hardjo and two controls immunized with saline. Blood samples were collected fortnightly and eggs daily. The IgY was purified from the egg yolk using the method for the delipidation of dilution with water acidic and ammonium sulfate precipitation. The ELISA assay was performed to verify the specificity of the IgY, these was possible to observe the production of specific antibody to Leptospira both in serum and purified egg yolk. The specific antibody titers peaked in the fifth week post immunization. The production of polyclonal IgY was effective for producing high titers of specific antibodies.(AU)

Vaccination of pregnant cows with EspA, EspB, γ-intimin, and Shiga toxin 2 proteins from Escherichia coli O157:H7 induces high levels of specific colostral antibodies that are transferred to newborn calves.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of intestinal disease and hemolytic uremic syndrome, a serious systemic complication that particularly affects children. Cattle are primary reservoirs for EHEC O157:H7 and the main source of infection for humans. Vaccination of cattle with different combinations of bacterial virulence factors has shown efficacy in decreasing EHEC O157:H7 shedding. It is, therefore, important to demonstrate whether vaccination of pregnant cows with EHEC O157:H7 induces high titers of transferable antibodies to avoid early colonization of calves by the bacteria. In this study we evaluated the ability of EspA, EspB, the C-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C280) and inactivated Shiga toxin (Stx) 2 proteins to induce specific antibodies in colostrum and their passive transference to colostrum-fed calves. Friesian pregnant cows immunized by the intramuscular route mounted significantly high serum and colostrum IgG responses against EspB and γ-intimin C280 that were efficiently transferred to their calves. Antibodies to EspB and γ-intimin C280 were detected in milk samples of vaccinated cows at d 40 postparturition. Significant Stx2-neutralizing titers were also observed in colostrum from Stx2-vaccinated cows and sera from colostrum-fed calves. The results presented showed that bovine colostrum with increased levels of antibodies against EHEC O157:H7 may be obtained by systemic immunization of pregnant cows, and that these specific antibodies are efficiently transferred to newborn calves by feeding colostrum. Hyperimmune colostrum and milk may be an alternative to protect calves from early colonization by EHEC O157:H7 and a possible key source of antibodies to block colonization and toxic activity of this bacterium.

Macrophage-elicited osteoclastogenesis in response to Brucella abortus infection requires TLR2/MyD88-dependent TNF-α production.

Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. In this manuscript, we described an immune mechanism for inflammatory bone loss in response to infection by Brucella abortus. We established a requirement for MyD88 and TLR2 in TNF-α-elicited osteoclastogenesis in response to B. abortus infection. CS from macrophages infected with B. abortus induced BMM to undergo osteoclastogenesis. Although B. abortus-infected macrophages actively secreted IL-1ß, IL-6, and TNF-α, osteoclastogenesis depended on TNF-α, as CS from B. abortus-infected macrophages failed to induce osteoclastogenesis in BMM from TNFRp55⁻/⁻ mice. CS from B. abortus-stimulated MyD88⁻/⁻ and TLR2⁻/⁻ macrophages failed to express TNF-α, and these CS induced no osteoclast formation compared with that of the WT or TLR4⁻/⁻ macrophages. Omp19, a B. abortus lipoprotein model, recapitulated the cytokine production and subsequent osteoclastogenesis induced by the whole bacterium. All phenomena were corroborated using human monocytes, indicating that this mechanism could play a role in human osteoarticular brucellosis. Our results indicate that B. abortus, through its lipoproteins, may be involved in bone resorption through the pathological induction of osteoclastogenesis.

Brucella abortus induces the secretion of proinflammatory mediators from glial cells leading to astrocyte apoptosis.

Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage chemoattractant protein-1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55-/- mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-alpha signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis.

Role of the striatum in the humoral immune response to thymus-independent and thymus-dependent antigens in rats.

Since the role of striatal GABAergic medium-sized spiny (MSP) neurons in the modulation of the immune responses is largely unknown, we evaluated the humoral immune response in rats with bilateral lesion of the striatum caused by quinolinic acid, which destroys MSP neurons. Sham-operated rats and those with striatal lesions were immunized either with TNP-LPS, a T-independent antigen type 1, or one of several T-dependent antigens: ovoalbumin, bovine serum albumin, lysozyme, sheep red blood cells (SRBC) or outer membrane proteins (OMP) of Salmonella enterica serovar Typhimurium. The specific levels of serum IgM and IgG, as well as intestinal IgA antibodies were determined either by enzyme-linked immunosorbent assay (ELISA) or a haemagglutination assay 5 or 7 days after immunization. Our results show that the lesion of striatal MSP neurons attenuated the primary antibody response to the T-independent antigen type 1 (TNP-LPS), but increased the antibody response to T-dependent antigens (proteins, SRBC and OMP), indicating that the striatal neurons modulate the humoral immune response in rats. The mechanisms involved are probably related to a reduction in both the number of B cells and the expression of caveolin-1 in the spleen, as well as an increase in the number of CD4(+) T cells and in corticosterone levels of the serum.

The serotonin 5-HT7 receptor agonist LP-44 microinjected into the dorsal raphe nucleus suppresses REM sleep in the rat.

The effects of LP-44, a selective 5-HT7 receptor agonist, and of SB-269970, a selective 5-HT7 receptor antagonist, on spontaneous sleep were studied in adult rats implanted for chronic sleep recordings. The 5-HT7 receptor ligands were microinjected directly into the dorsal raphe nucleus (DRN) during the light period of the 12-h light/12-h dark cycle. Infusion of LP-44 (1.25-5.0 mM) into the DRN induced a significant reduction of rapid-eye-movement sleep (REMS) and of the number of REM periods. Similar effects were observed after the direct administration into the DRN of SB-269970 (0.5-1.0 mM). Pretreatment with a dose of SB-269970 (0.5 mM) that significantly affects sleep variables antagonized the LP-44 (2.5 mM)-induced suppression of REMS and of the number of REM periods. It is proposed that the suppression of REMS after microinjection of LP-44 into the DRN is related, at least in part, to the activation of GABAergic neurons in the DRN that contribute to long projections that reach, among others, the laterodorsal and pedunculopontine tegmental nuclei involved in the promotion of REMS.

Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni: potentiation of hemolytic activity by LipL32.

The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection.

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo.

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y. enterocolitica O:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y. enterocolitica O:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y. enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.