Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo.

BACKGROUND: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments. RESULTS: In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsß genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39οC showed a decrease in cellular invasion and an increase in IL-8-but not in IL-6-production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39 °C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37 and 39 °C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39 °C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39 °C comparing with 37 °C. CONCLUSIONS: Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

Light Modulates Important Pathogenic Determinants and Virulence in ESKAPE Pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus.

Light sensing has been extensively characterized in the human pathogen Acinetobacter baumannii at environmental temperatures. However, the influence of light on the physiology and pathogenicity of human bacterial pathogens at temperatures found in warm-blooded hosts is still poorly understand. In this work, we show that Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa (ESKAPE) priority pathogens, which have been recognized by the WHO and the CDC as critical, can also sense and respond to light at temperatures found in human hosts. Most interestingly, in these pathogens, light modulates important pathogenicity determinants as well as virulence in an epithelial infection model, which could have implications in human infections. In fact, we found that alpha-toxin-dependent hemolysis, motility, and growth under iron-deprived conditions are modulated by light in S. aureus Light also regulates persistence, metabolism, and the ability to kill competitors in some of these microorganisms. Finally, light exerts a profound effect on the virulence of these pathogens in an epithelial infection model, although the response is not the same in the different species; virulence was enhanced by light in A. baumannii and S. aureus, while in A. nosocomialis and P. aeruginosa it was reduced. Neither the BlsA photoreceptor nor the type VI secretion system (T6SS) is involved in virulence modulation by light in A. baumannii Overall, this fundamental knowledge highlights the potential use of light to control pathogen virulence, either directly or by manipulating the light regulatory switch toward the lowest virulence/persistence configuration.IMPORTANCE Pathogenic bacteria are microorganisms capable of producing disease. Dangerous bacterial pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, are responsible for serious intrahospital and community infections in humans. Therapeutics is often complicated due to resistance to multiple antibiotics, rendering them ineffective. In this work, we show that these pathogens sense natural light and respond to it by modulating aspects related to their ability to cause disease; in the presence of light, some of them become more aggressive, while others show an opposite response. Overall, we provide new understanding on the behavior of these pathogens, which could contribute to the control of infections caused by them. Since the response is distributed in diverse pathogens, this notion could prove a general concept.

Widespread dispersion of the resistance element tet(B)::ISCR2 in XDR Acinetobacter baumannii isolates.

Acinetobacter baumannii is a significant nosocomial pathogen often associated with extreme drug resistance (XDR). In Argentina, isolates of A. baumannii resistant to tetracyclines have accounted for more than 40% of drug-resistant isolates in some hospitals. We have previously reported the dispersion of the tet(B) resistance element associated with the ISCR2 transposase in epidemiologically unrelated A. baumannii isolates recovered from 1983 to 2011. This study extends this surveillance to 77 recent (2009-2013) XDR A. baumannii isolates with different levels of minocycline susceptibility. Isolates were examined by a pan-PCR assay, which showed six different amplification patterns, and specific PCRs were used for the confirmation of the the ΔISCR2-tet(B)-tet(R)-ISCR2 element. The tet(B) gene was present in 66 isolates and the ISCR2 element in 68 isolates; the tet(B) gene was associated with ISCR2 in all tet(B)-positive isolates. We conclude that this element is widespread in XDR A. baumannii isolates from Argentina and could be responsible for the emergence of tetracycline resistance in recent years.

Preferential carriage of class 2 integrons in Acinetobacter baumannii CC113 and novel singletons.

Our understanding of the distribution of integrons associated with multidrug resistance in Acinetobacter baumannii isolates around the world remains incomplete. The association between the class 1 and 2 integron A. baumannii-positive isolates (n = 60), recovered since 1982 from 11 Argentinean hospitals, and the circulating lineages, was investigated. While class 2 integrons were highly significantly associated with clonal lineage CC113B/CC79P (P = 0·009) and novel singletons (P = 0·001), class 1 integrons were found not to be associated with CC109B/CC1P or other lineages. The study reveals a differential distribution of class 2 integrons in lineages, and suggests that the prevalence of intI2 in Argentina is related to the emergence of novel singletons in recent years and to the abundance of CC113B/CC79P, which has been the local dominant lineage for several decades.

Genetic Variability of AdeRS Two-Component System Associated with Tigecycline Resistance in XDR-Acinetobacter baumannii Isolates.

The emergence of tigecycline resistance has increased in the last years. Although tigecycline-resistant Acinetobacter baumannii isolates were described all over the world, few reports regarding the molecular basis of this resistance are available. It has been recognized that the overexpression of AdeABC efflux pump is related to the tigecycline-resistant phenotype. In 37 clinical A. baumannii isolates we first determined the tigecycline-resistant phenotype and then, within a selected group, we analyzed the sequence of the adeRS operon, which is involved in the expression of the AdeABC efflux pump. Nucleotide sequence analysis of adeR and adeS showed the presence of 5 and 16 alleles, respectively. These results expose a high genetic variability in both genes, the adeS gene being more susceptible to genetic variation. The presence of 2 AdeR and 2 AdeS new variants were reported. Two of the new AdeRS variants were present in the intermediate and the resistant tigecycline A. baumannii isolates, suggesting a putative role in the development of the observed phenotype. More studies need to be addressed to determine the role of the genetic variability observed in the adeRS operon.

First case of Streptococcus lutetiensis bacteremia involving a clindamycin-resistant isolate carrying the lnuB gene.

Here, we describe the first case of a Streptococcus lutetiensis isolate harboring the lnuB gene.

Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo.

Background: Staphylococcus aureus and Pseudomonas aeruginosa cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39°C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37°C, by using RNAseq and physiological assays including in-vivo experiments. Results: In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes on both species. Altered metabolic features at 39°C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39°C they upregulated ethanol oxidation related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα and pmsß genes at 39°C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphylxantin production and a decrease in biofilm formation at 39°C. On the side of PA monocultures, we observed increase in extracellular lipase and protease and biofilm formation at 39°C along with a decrease in motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in-vitro and in-vivo . S. aureus monocultured at 39°C showed a decrease in cellular invasion and an increase in IL-8 -but not in IL-6- production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39°C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37°C and 39°C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39°C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39°C comparing with 37°C. Conclusion: Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

Tn7::In2-8 dispersion in multidrug resistant isolates of Acinetobacter baumannii from Chile.

Acinetobacter baumannii is considered an important pathogen in our hospital environment having a well-known capacity to acquire different mechanisms of antibiotic resistance. Previous studies in our laboratory had exposed the high dispersion of class 2 integrons in this species. In the present study, we analyzed 7 multiresistant intI2 positive A. baumannii isolates, 6 of which were found to harbour the Tn7::In2-8 element. Our results demonstrate the unusually high distribution of Tn7::In2-8 among different A. baumannii clones from Chile, suggesting a particular behavior of these elements at geographical level.

Molecular characterization of KPC-2-positive Klebsiella pneumoniae isolates from a neurosurgical centre in Argentina.

Carbapenem-resistant Enterobacteriaceae is a growing concern worldwide. Klebsiella pneumoniae is an important nosocomial pathogen with a high capacity for nosocomial spread. We described the occurrence of plasmid-encoded KPC-2-harbouring K. pneumoniae isolates recovered from a neurosurgical centre in Argentina. The blaKPC-2 gene was surrounded by ISkpn6 and ISkpn7.

Whole-genome analysis and description of an outbreak due to carbapenem-resistant Ochrobactrum anthropi causing pseudo-bacteraemias.

Ochrobactrum anthropi, a rare human pathogen, has been isolated predominantly from patients with catheter-related bacteraemia and rarely from other infections. In 2016, six cases of pseudo-bacteraemia caused by carbapenem-resistant O. anthropi isolates were recovered from an Argentinian hospital. The resistant phenotype exposed by the isolates caught our attention and led to an extensive epidemiologic investigation. Here we describe the characterization of a carbapenem-resistant O. anthropi outbreak whose probable cause was by contaminated collection tubes. The genome analysis of one strain revealed the presence of various resistant determinants. Among them, a metal-dependent hydrolase of the ß-lactamase superfamily I, phnP, was found. Lately the recovery of unusual multidrug-resistant pathogens in the clinical setting has increased, thus emphasizing the need to implement standardized infection control practice and epidemiologic investigation to identify the real cause of hospital outbreaks.

First case of bacteraemia due to Acinetobacter schindleri harbouring blaNDM-1 in an immunocompromised patient.

Clinically significant NDM-1-producing Acinetobacter schindleri has not yet been described in the literature. We report the first case of bacteraemia due to an A. schindleri strain harbouring blaNDM-1 recovered from an immunocompromised patient. Our report reinforces the fact that NDM-1 can easily be acquired by Acinetobacter species.

[Influence of penicillin minimum inhibitory concentration in the synergy between penicillin and gentamicin in viridans-group streptococci]. / Influencia de la concentración inhibitoria mínima de penicilina en la acción sinérgica de su combinación con gentamicina frente a estreptococos del grupo viridans.

Penicillin resistance rates higher than 60% have been recorded in viridans group streptococci by some authors during the 90's and recently such resistance was associated with higher levels of mortality in bacteremia. The lowest minimum inhibitory concentration of penicillin for which synergy with aminoglycosides is not yet possible is still unknown. In order to try to dilucidate this puzzle, a study on the susceptibility to penicillin of 28 strains of viridans group streptococci isolated from significant samples in the Hospital de Pediatría "Prof. Dr. Juan P. Garrahan" was carried out. Seven mitis group isolates presenting different susceptibility patterns were selected for performing time-killing curves with penicillin, gentamicin, and penicillin plus gentamicin, using higher and lower penicillin concentrations than their minimal inhibitory concentrations. Synergy was not observed when the penicillin concentration was lower than the minimum inhibitory concentration, at least in these strains with minimum inhibitory concentrations of gentamicin > or = 16 microg/ml. When using penicillin in higher concentrations than the minimum inhibitory concentration, synergy was found in five of the seven strains. Aminoglycoside-modifying enzymes were found in the two other streptococci.

Unusual presentations of Comamonas kerstersii infection.

The association of Comamonas kerstersii with peritonitis resulting from the presence of perforated appendix has previously been described by our research team. In the present study, we describe the isolation of this microorganism from two forms of unusual presentations of C. kerstersii infection not previously described in the literature: localized intra-abdominal infection (psoas abscess) and pelvic peritonitis.

Relación entre automanejo y síntomas depresivos en personas con diabetes e hipertensión / Relationship between self-management and depressive symptoms in persons with diabetes and hypertension / Relação entre automanejo e sintomas depressivos em pessoas com diabetes e hipertensão

Resumen Introducción: Las personas con diabetes e hipertensión experimentan con mayor frecuencia sintomatología depresiva, lo cual contribuye a un inadecuado automanejo de la enfermedad, que implica tareas como: la toma de la medicación, asistir a las consultas médicas, el conocimiento de signos y síntomas, además de la toma de decisiones. Objetivo: Por lo anterior, el objetivo de este trabajo es identificar la relación entre automanejo en general y sus dimensiones con síntomas depresivos en personas con diabetes e hipertensión. Método: Estudio transversal-correlacional con n=205 pacientes con diagnóstico de diabetes (100) e hipertensión (105). Muestreo no probabilístico por conveniencia. Se utilizó la estadística descriptiva y r de Pearson. Se aplicaron los instrumentos Partners in Health (PIH) y The Patient Health Questionnaire (PHQ-8). Resultados: Se encontró correlación estadísticamente significativa entre la sintomatología depresiva y el automanejo (r=-0.308 ρ<0.001). Discusión y Conclusiones: Se concluye que la sintomatología depresiva se relaciona con el automanejo de la enfermedad de la siguiente manera: a mayor automanejo menor sintomatología de depresión, o a mayor sintomatología depresiva menor automanejo; dicha relación confirma que ambas variables se afectan mutuamente y/o mantienen una relación estrecha.

Abstract Introduction: Persons with diabetes and hypertension frequently suffer from depression as well, a situation which contributes to an inadequate management of the condition in terms of medication, medical consultations, signs, and decision making. Objective: To identify the relationship between general self-management and depression symptoms in persons with diabetes and hypertension. Method: This is a transversal and correlational study with a sample of 205 patients, 100 with a main diagnosis of diabetes, and 105 with a main diagnosis of hypertension. The sampling process was non-probabilistic and by convenience. Descriptive statistics, including Pearson's r were calculated. The Partners in Health (PIH) and Patient Health Questionnaire (PHQ-8) instruments were administered. Results: A statistically significant correlation between depression symptoms and diabetes and hypertension self-management was found (r=-0.308 ρ<0.001). Discussion and conclusions: Depression symptoms were related to how diabetes and hypertension are self-managed in a way that, the more self-management, the less depression symptoms, or the more depression symptoms, the less self-management.

Resumo Introdução: As pessoas com diabetes e hipertensão experimentam com maior frequência a sintomatologia depressiva, o qual contribui a um inadequado automanejo da doença, que envolve tarefas como: a toma da medicação, assistir às consultas médicas, o conhecimento de signos e sintomas, além da toma de decisões. Objetivo: Pelo anterior, o objetivo deste trabalho é identificar a relação entre automanejo em geral e suas dimensões com sintomas depressivos em pessoas com diabetes e hipertensão. Método: Estudo transversal-correlacional com n=205 pacientes com diagnóstico de diabetes (100) e hipertensão (105). Amostragem não probabilística por conveniência. Utilizou-se a estatística descritiva e r de Pearson. Aplicaram-se os instrumentos Partners in Health (PIH) e The Patient Health Questionnaire (PHQ-8). Resultados: Encontrou-se correlação estatisticamente significativa entre a sintomatologia depressiva e o automanejo (r=-0.308 ρ<0.001). Discussão e Conclusões: Conclui-se que a sintomatologia depressiva relaciona-se com o automanejo da doença da seguinte maneira: a maior automanejo, menor sintomatologia de depressão, ou a maior sintomatologia depressiva, menor automanejo; esta relação confirma que ambas variáveis afetam-se mutuamente e/ou mantem uma relação estreita.

Partial characterization of a basic protein from Crotalus molossus molossus (northern blacktail rattlesnake) venom and production of a monoclonal antibody.

The venom of Crotalus molossus molossus (blacktailed rattlesnake) is very basic compared to that of other Crotalinae venoms. Unlike other Crotalinae venoms that are separated by anion exchange chromatography, C. m. molossus venom must be fractionated by cation exchange chromatography. Electrophoretic titration (ET) was used to predict the isoelectric point (pI) and optimal conditions for isolation. The specific hemorrhagic activity for C. m. molossus venom was 7.5 mm/microg, making it one of the most hemorrhagic of Crotalinae venoms. Basic hemorrhagic and fibrinolytic proteins from the venom of C. m. molossus venom were further fractionated by cation exchange chromatography. A basic fibrinolytic/hemorrhagic protein (CMM4) was isolated. CMM4 has a molecular weight between 23 and 26 kDa and a pI of approximately 11.3. SDS electrophoresis revealed one band and ET curve revealed 3 bands with very similar surface charges at all pH. CMM4 did not activate plasminogen when tested with a Chrom Z-PLG assay. The proteins in CMM4 had similar N-terminal amino acid sequences to each other (D-Q-Q-N-L-P-Q-(S/A/R)-Y-(V/R/I)-E-L-V-V-V-A-D-H-R-L-F-M-K-Y-K-S-D-L- N-T). The differences in these proteins are in positions 8 and 10. CMM4 may contain isoforms that differ by minor sequence variations at their amino-termini. The amino acid sequences of CMM4 were very similar to other fibrinolytic and hemorrhagic metalloproteinases isolated from venoms of the genera Crotalus. The specific hemorrhagic activity of CMM4 decreased as the specific fibrinolytic activity increased. A monoclonal antibody (CMM1b) was produced against C. m. molossus venom that neutralized the hemorrhagic activity of some of its fractions. CMM1b also reacted with 11 of 29 venom samples tested via ELISA.

An internet database of crotaline venom found in the United States.

Many snake venoms have been shown to be complex mixtures of pharmacologically important molecules, some of which have potential therapeutic value in the treatment of clot-induced ischemia, cancer and other human disorders. The literature contains many references on how venom and/or venom components are being used in medicine. Within the United States, there are 44 subspecies of poisonous snakes. Despite this rather vast diversity, 90% of the venom-related biomedical research conducted on native snakes found in the United States has been done on a limited number of the more common species. Since the venoms from most of the native species are not available or characterized, their composition and potential usefulness in medicine and applied biomedical research has not been explored. The Natural Toxins Research Center (NTRC) at Texas A&M University-Kingsville has developed a serpentarium that presently houses a population of over 250 snakes composed of 11 species and 20 subspecies. These snakes are cataloged on the Internet database along with their geographical location data, proteolytic activities, high performance liquid chromatography (HPLC) and electrophoretic titration (ET) profiles. Many of these snake venoms have never been characterized and few locale-specific differences within a species have been examined. These venoms can be queried through an on-line search routine. The database will be a useful starting point for anyone interested in isolating fibrinolytic enzymes, specific toxins, hemorrhagins, or other pharmacologically active proteins from snake venoms.

A tender sinus does not always mean rhinosinusitis.

BACKGROUND: Sinus tenderness has not been quantitatively assessed. OBJECTIVE: We sought to compare sinus and systemic tenderness in rhinosinusitis, allergic rhinitis, and chronic fatigue syndrome (CFS), and healthy (non-CFS) groups. METHODS: Cutaneous pressures (kg/cm(2)) causing pain at 5 sinus and 18 systemic sites were measured in acute and chronic rhinosinusitis, active allergic rhinitis, healthy non-CFS/no rhinosinusitis, and CFS subjects. RESULTS: Sinus thresholds differed significantly (P

Screening for fibrinolytic activity in eight Viperid venoms.

Snake venoms contain direct-acting fibrinolytic metalloproteinases (MMP) that could have important applications in medicine. Fibrinolytic enzymes isolated from venom can induce in vitro clot lysis by directly acting on a fibrin clot. The most ideal fibrinolytic enzyme would have high affinity for clots, dissolve clots directly without causing hemorrhage, and would not be neutralized in vivo by endogenous metalloproteinase inhibitors. The purpose of this study was to compare DEAE/HPLC venom profiles from Viperid snakes and identify fractions that contain fibrinolytic activity with no hemorrhagic activity and are not neutralized by animal sera. The sera selected were from four (Virginia opossum, Gray woodrat, Mexican ground squirrel, and Hispid cottonrat) animals known to neutralize hemorrhagic activity in snake venoms. Nineteen fractions from the Viperid venoms had fibrinolytic activity. Agkistrodon venom fractions contained the highest specific fibrinolytic activities. A. piscivorus leucostoma fraction 4 contained a high specific fibrinolytic activity, no hemorrhagic activity, and the fibrinolytic activity was not neutralized by the proteinase inhibitors of the four animal sera. A. contortrix laticinctus fraction 1 also had a high specific fibrinolytic activity and no hemorrhagic activity. However, the fibrinolytic activity was neutralized by Didelphis virginiana (Virginia opossum) serum.