ß-lactam-induced OMV release promotes polymyxin tolerance in Salmonella enterica sv. Typhi.

The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a ß-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of ß-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of ß-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from ß-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that ß-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of ß-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.

Novel Megaplasmid Driving NDM-1-Mediated Carbapenem Resistance in Klebsiella pneumoniae ST1588 in South America.

Carbapenem-resistant Enterobacterales (CRE) is a critical public health problem in South America, where the prevalence of NDM metallo-betalactamases has increased substantially in recent years. In this study, we used whole genome sequencing to characterize a multidrug-resistant (MDR) Klebsiella pneumoniae (UCO-361 strain) clinical isolate from a teaching hospital in Chile. Using long-read (Nanopore) and short-read (Illumina) sequence data, we identified a novel un-typeable megaplasmid (314,976 kb, pNDM-1_UCO-361) carrying the blaNDM-1 carbapenem resistance gene within a Tn3000 transposon. Strikingly, conjugal transfer of pNDM-1_UCO-361 plasmid only occurs at low temperatures with a high frequency of 4.3 × 10-6 transconjugants/receptors at 27 °C. UCO-361 belonged to the ST1588 clone, previously identified in Latin America, and harbored aminoglycoside, extended-spectrum ß-lactamases (ESBLs), carbapenem, and quinolone-resistance determinants. These findings suggest that blaNDM-1-bearing megaplasmids can be adapted to carriage by some K. pneumoniae lineages, whereas its conjugation at low temperatures could contribute to rapid dissemination at the human-environmental interface.

[Evaluation of sensi-disk elution for colistin susceptibility determination in multidrug resistant gramnegative bacilli]. / Evaluación de la elución de sensidiscos para la determinación de susceptibilidad a colistín en bacilos gramnegativos multi-resistentes.

Using clinical strains of multidrug resistant (MDR) Gram negative bacilli, we compared MICs obtained from both broth microdilution, the reference method, and sensi-disk elution method. We found that, with A. baumannii exception, results were very similar. Sensi-disk elution method could be a good and reliable alternative for colistin resistance determination.

Draft genome sequence of a multidrug-resistant KPC-2 and SRT-2 co-producing Serratia marcescens strain isolated from a hospitalised patient in Chile.

OBJECTIVES: Serratia marcescens is a neglected opportunistic pathogen of public-health concern, especially due to its antimicrobial resistance features. Here we report the draft genome sequence of the first KPC-2 and SRT-2 co-producing S. marcescens strain (UCO-366) recovered from a catheter tip culture of a hospitalised patient in Santiago, Chile, in 2014. METHODS: Whole genomic DNA of strain UCO-366 was extracted and was sequenced using an Illumina NextSeq platform. De novo genome assembly was performed using Unicycler v.0.4.0 and the genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.4.8. Genomic features were analysed using bioinformatic tools available at the Center for Genomic Epidemiology, the Comprehensive Antibiotic Resistance Database (CARD) and Pathosystems Resource Integration Center (PATRIC). RESULTS: The genome size of strain UCO-366 was 5 267 357bp, with a G+C content of 59.7% and comprising 5299 coding sequences (CDS), 42 tRNAs and 115 pseudogenes. The genome of UCO-366 also included an IncL/M plasmid. The resistome comprised various antimicrobial resistance genes (ARGs) conferring resistance to carbapenems, cephalosporins, aminoglycosides, sulfonamides, chloramphenicol, rifampicin and fluoroquinolones. Importantly, S. marcescens UCO-366 harboured blaKPC-2 and blaSRT-2, representing the first description of these ß-lactamase genes in this species in Chile. CONCLUSION: Here we report the genome of the first KPC-positive multidrug-resistant S. marcescens strain identified in Chile, which co-harboured several ARGs. The genome sequence of S. marcescens UCO-366 provides an insight into the antimicrobial resistance characteristics of this species in this country and offers important data for further genomic studies on this critical priority pathogen.

Evaluación de la elución de sensidiscos para la determinación de susceptibilidad a colistín en bacilos gramnegativos multi-resistentes / Evaluation of sensi-disk elution for colistin susceptibility determination in multidrug resistant gramnegative bacilli

Resumen Utilizando cepas clínicas de bacilos gramnegativos multi-resistentes (MDR), comparamos las CIM obtenidas de la microdilución en caldo, el método de referencia y el método de elución de sensidiscos. Encontramos que, con la excepción de A. baumannii, los resultados fueron muy similares. El método de elución de sensidiscos podría ser una buena alternativa y confiable para la determinación de la resistencia a colistín.

Abstract Using clinical strains of multidrug resistant (MDR) Gram negative bacilli, we compared MICs obtained from both broth microdilution, the reference method, and sensi-disk elution method. We found that, with A. baumannii exception, results were very similar. Sensi-disk elution method could be a good and reliable alternative for colistin resistance determination.

Identification of Genes Involved in Biogenesis of Outer Membrane Vesicles (OMVs) in Salmonella enterica Serovar Typhi.

Outer membrane vesicles (OMVs) are nano-sized proteoliposomes discharged from the cell envelope of Gram-negative bacteria. OMVs normally contain toxins, enzymes and other factors, and are used as vehicles in a process that has been considered a generalized, evolutionarily conserved delivery system among bacteria. Furthermore, OMVs can be used in biotechnological applications that require delivery of biomolecules, such as vaccines, remarking the importance of their study. Although it is known that Salmonella enterica serovar Typhi (S. Typhi), the etiological agent of typhoid fever in humans, delivers toxins (e.g., HlyE) via OMVs, there are no reports identifying genetic determinants of the OMV biogenesis in this serovar. In the present work, and with the aim to identify genes participating in OMV biogenesis in S. Typhi, we screened 15,000 random insertion mutants for increased HlyE secretion. We found 9 S. Typhi genes (generically called zzz genes) determining an increased HlyE secretion that were also involved in OMV biogenesis. The genes corresponded to ompA, nlpI, and tolR (envelope stability), rfaE and waaC (LPS synthesis), yipP (envC), mrcB (synthesis and remodeling of peptidoglycan), degS (stress sensor serine endopeptidase) and hns (global transcriptional regulator). We found that S. Typhi Δzzz mutants were prone to secrete periplasmic, functional proteins with a relatively good envelope integrity. In addition, we showed that zzz genes participate in OMV biogenesis, modulating different properties such as OMV size distribution, OMV yield and OMV protein cargo.

Introduction of NDM-1 and OXA-370 from Brazil into Chile in strains of Klebsiella pneumoniae isolated from a single patient.

Carbapenemase-producing Enterobacteriaceae have rapidly disseminated worldwide and can colonize patients in healthcare centers. As in Chile the first isolations of NDM-1 and OXA-370 carbapenemases were related with a patient arriving from Brazil, the genetic relatedness of Klebsiella pneumoniae strains producers of these enzymes and isolated in both countries was assessed. PFGE analyses revealed that the isolates were clonally related, illustrating how travel contributes to the spread of multidrug-resistant microorganisms. In addition, the occurrence of three different carbapenemases in three different K. pneumoniae strains isolated from a single patient is described.

Incidencia de beta-lactamasas de espectro extendido en el Hospital Clínico de la Universidad de Chile / Incidence of extended-spectrum beta-lactamases in University of Chile Clinical Hospital

Las Betalactamasas de espectro extendido (BLEE) son enzimas de configuración plasmídica producidas por bacterias que hidrolizan los antibióticos betalactámicos. La aparición de cepas bacterianas productoras de BLEE, constituye un problema de salud pública que afecta a todo tipo de instituciones y a la comunidad en general. Este estudio tiene como objetivo determinar las incidencia de BLEE en pacientes hospitalizados del Hospital Clínico de la Universidad de Chile. Se estudió un total de 238 cepas en el periodo julio-agosto 2004; de las cuales 184 correspondieron a de E. coli, 39 a K. pneumoniae y 15 a K. oxytoca, utilizándose el test confirmatorio de BLEE según estándar NCCLS 2004. Se encontró una incidencia de BLEE para E. coli de un 10.3 por ciento, para K. pneumoniae de 28.2 por ciento y de K. oxytoca de 20 por ciento, respectivamente. La distribución de los aislamientos de las cepas BLEE (+) en los distintos servicios del Hospital fue diversa: E. coli se encontró principalmente, en los servicios de medicina y cirugía, y K. pneumoniae en los servicios críticos. Los resultados del estudio permitieron conocer la incidencia y distribución de BLEEs en el Hospital Clínico de la Universidad de Chile y justificaron la implementación de esta técnica de diagnóstico como parte de la rutina del laboratorio de microbiología.

Frecuencia de dislipidemia en pacientes con lupus eritematoso sistémico / Frequency of dislipidemia in patient with lupus eritematoso sistémico

El lupus eritematoso sistémico (LES) está asociado a una frecuencia significativamente mayor de enfermedad arteriosclerótica prematura y se conoce el rol de la dislipidemia como factor de riesgo para esta enfermedad. Objetivo: Determinar la frecuencia de dislipidemia en los pacientes con LES atendidos en el Servicio de Inmunorreumatología del Hospital Nacional Cayetano Heredia de Lima. Métodos: estudio descriptivo de corte transversal en el que se evaluó a pacientes con LES definido atendidos en la Clínica de LES del Servicio. Se procedió a evaluación clínica y determinación del pérfil lipídico. Se realizó una búsqueda de posibles factores relacionados con hipercolesterolemia: edad, sexo, edad de inicio de enfermedad, tiempo de tratamiento con corticoides, tratamiento con antimaláricos, hipertensión arterial, diabetes mellitus, estado postmenopáusico, compromiso neurológico, cardíaco o pulmonar por LES. Resultados: fueron evaluados 45 pacientes con LES definido, con edad promedio de 30 años (36 de 10,7 años), el 88,9 por ciento(40) fueron mujeres, el 97,8 por ciento recibió tratamiento con corticoides, el 24,4 por ciento, antimaláricos, y el 62,2 por ciento, ciclofosfamida. Se encontró que el 35.6 por ciento tenían compromiso cardíaco, el 40 por ciento, pulmonar, el 20 por ciento, neurológico, el 30,2 por ciento síndrome nefrótico y el 14 por ciento, insuficiencia renal crónica. presentaron dislipidemia 68,8 por ciento hipercolesterolemia (menor 200 mg/dl) 44,4 por ciento, hipertrigliceridemia 4,4 por ciento y mixta 20 por ciento. El índice LDL/HDL en el grupo sin dislipidemia fué 2,33 y en el grupo con dislipidemia 4,05. El grupo de pacientes que recibió antimaláricos presentó niveles menores de colesterol total (249,3 mgdl, DE 67,6 mg/dl vs 175,0 mg/dl, DE 30,9 mg/dl, p igual 0,04). Conclusión: en pacientes con LES se determinó una frecuencia elevada de hipercolesterolemia con niveles elevados de colesterol LDL e hipertrigliceridemia asociados a mayor riesgo de infarto de miocardio agudo en los próximos diez años. El uso de antimaláricos podría disminuir el riesgo de dislipidemia.