Carbapenem-resistant hypervirulent ST23 Klebsiella pneumoniae with a highly transmissible dual-carbapenemase plasmid in Chile.

BACKGROUND: The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. RESULTS: Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM-1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. CONCLUSIONS: We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.

Inorganic Polyphosphate Affects Biofilm Assembly, Capsule Formation, and Virulence of Hypervirulent ST23 Klebsiella pneumoniae.

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to high mortality rates and propensity to cause severe community-acquired infections in healthy individuals. The ability to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Polyphosphate kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a proteomic analysis to gain further insights into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins involved in capsule synthesis (Wzi-Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB-LpxC-PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hvKP. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the ß-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

COVID-19 inactivated and non-replicating viral vector vaccines induce regulatory training phenotype in human monocytes under epigenetic control.

Background: Trained immunity is the enhanced innate immune response resulting from exposure to pathogens or vaccines against an unrelated pathogen stimulus. Certain vaccines induce a memory like response in monocytes and NK cells, leading to modulation in cytokine production, metabolic changes, and modifications in histone patterns. Here, we hypothesized that vaccination against SARS-CoV-2 could induce the training of monocytes in addition to stimulating the adaptive immune response. Methods: Therefore, we aimed to investigate the immunophenotyping, cytokine and metabolic profile of monocytes from individuals who were completely immunized with two doses of inactivated COVID-19 vaccine or non-replicating viral vector vaccine. Subsequently, we investigated the epigenetic mechanisms underlying monocyte immune training. As a model of inflammatorychallenge, to understand if the monocytes were trained by vaccination and how they were trained, cells were stimulated in vitro with the endotoxin LPS, an unrelated stimulus that would provoke the effects of training. Results: When challenged in vitro, monocytes from vaccinated individuals produced less TNF-α and those who received inactivated vaccine produced less IL-6, whereas vaccination with non-replicating viral vector vaccine induced more IL-10. Inactivated vaccine increased classical monocyte frequency, and both groups showed higher CD163 expression, a hallmark of trained immunity. We observed increased expression of genes involved in glycolysis and reduced IRG1 expression in vaccinated subjects, a gene associated with the tolerance phenotype in monocytes. We observed that both vaccines reduced the chromatin accessibility of genes associated with the inflammatory response, the inactivated COVID-19 vaccine trained monocytes to a regulatory phenotype mediated by histone modifications in the IL6 and IL10 genes, while the non-replicating viral vector COVID-19 vaccine trained monocytes to a regulatory phenotype, mediated by histone modifications in the IL6, IL10, TNF, and CCL2 genes. Conclusions: Our findings support the recognized importance of adopting vaccination against SARS CoV-2, which has been shown to be effective in enhancing the adaptive immune response against the virus and reducing mortality and morbidity rates. Here, we provide evidence that vaccination also modulates the innate immune response by controlling the detrimental inflammatory response to unrelated pathogen stimulation.

Antigenemia y reacción de polimerasa en cadena en tiempo real en el diagnóstico de enfermedad por citomegalovirus en adultos con virus de inmunodeficiencia adquirida / Antigenemia and real time polymerase chain reaction for diagnosis of cytomegal ovirus disease in HIV infected adults

Background: Cytomegalovirus (CMV) infection is frequent in HIV adults. It is unknown usefulness of quantitative methods for diagnosing the CMV disease in Chilean patients. Aim: To determine the performance of antigenemia and real time polymerase chain reaction (rtPCR) in the diagnosis of CMV disease in Chilean HIV adults. Method: Detection of CMV by viral isolation (AVR), antigenemia and quantitative rtPCR in HIV adults. Results: The 102 adults with suspected CMV disease had lower LTCD4 count and higher HIV viral load than 77 patients without suspicion (p < 0.05). Antigenemia and PCR were positive in 47 (46.1%) and 37 (36.3%) adults with clinical suspicion and in 2 (2.6%) and 4 (5.2%) of 77 without suspicion. The sensitivity, specificity, positive and negative predictive value of antigenemia and RPCtr were 92%, 80%, 72% and 95% and 72%, 95%, 92% and 80%, respectively. The cutoff values were ≥ lcell (+) and ≥ 5.5 log10 copies/2 x 10(6) cells. CMV was isolated in 6/179 patients (3.4%), all symptomatic. Conclusión: Positivity of antigenemia and rtPCR are similar for diagnosing CMV disease in Chilean HIV adults. AVR is inappropriate as a gold standard for its low performance.

Introducción: La infección por citomegalovirus (CMV) es frecuente en adultos con virus de inmunodeficiencia humana (VIH). No se ha establecido la utilidad de los métodos cuantitativos para diagnosticar enfermedad por CMV en pacientes chilenos. Objetivo: Determinar la positividad de antigenemia y reacción de polimerasa en cadena en tiempo real (RPC-TR) en el diagnóstico de enfermedad por CMV en adultos chilenos con infección por VIH. Metodología: Se detectó CMV mediante aislamiento viral rápido (AVR), antigenemia y reacción de polimerasa en cadena en tiempo real (RPC-TR) cuantitativa en adultos infectados por VIH, con y sin sospecha de enfermedad por CMV. Resultados: El recuento de LT CD4 fue menor y mayor la carga de VIH en 102 sintomáticos respecto a 77 asintomáticos (p < 0,05). La antigenemia y la RPC-TR fueron positivas en 46 y 36% de los enfermos y en 3 y 5% de los asintomáticos respectivamente. La sensibilidad, especificidad, valor predictor positivo y negativo de la antigenemia y la RPC-TR fueron 92%, 80%, 72% y 95% y 72%, 95%, 92% y 80%, respectivamente. Los valores de corte fueron ≥ 1 núcleo (+) y ≥ 5,5 log10 copias/2 x 10(6) céls. Se aisló CMV en 3,4%, todos los sintomáticos. Conclusión: La antigenemia y la RPC-TR tienen una positividad similar para diagnosticar enfermedad por CMV en adultos chilenos con infección por VIH. El AVR es inapropiado como referencia por su baja positividad.

[Antigenemia and real time polymerase chain reaction for diagnosis of cytomegalovirus disease in HIV infected adults]. / Antigenemia y reacción de polimerasa en cadena en tiempo real en el diagnóstico de enfermedad por citomegalovirus en adultos con virus de inmunodeficiencia adquirida.

BACKGROUND: Cytomegalovirus (CMV) infection is frequent in HIV adults. It is unknown usefulness of quantitative methods for diagnosing the CMV disease in Chilean patients. AIM: To determine the performance of antigenemia and real time polymerase chain reaction (rtPCR) in the diagnosis of CMV disease in Chilean HIV adults. METHOD: Detection of CMV by viral isolation (AVR), antigenemia and quantitative rtPCR in HIV adults. RESULTS: The 102 adults with suspected CMV disease had lower LTCD4 count and higher HIV viral load than 77 patients without suspicion (p < 0.05). Antigenemia and PCR were positive in 47 (46.1%) and 37 (36.3%) adults with clinical suspicion and in 2 (2.6%) and 4 (5.2%) of 77 without suspicion. The sensitivity, specificity, positive and negative predictive value of antigenemia and RPCtr were 92%, 80%, 72% and 95% and 72%, 95%, 92% and 80%, respectively. The cutoff values were ≥ lcell (+) and ≥ 5.5 log10 copies/2 x 10(6) cells. CMV was isolated in 6/179 patients (3.4%), all symptomatic. CONCLUSION: Positivity of antigenemia and rtPCR are similar for diagnosing CMV disease in Chilean HIV adults. AVR is inappropriate as a gold standard for its low performance.

5% lidocaine medicated plaster use in children with neuropathic pain from burn sequelae.

OBJECTIVE: Neuropathic pain is a challenge in children with burn sequelae. Although relatively infrequent, the intensity and chronicity of neuropathic pain negatively impact functionality and quality of life. The use of 5% lidocaine medicated plaster has not previously been reported in children. We explored the effectiveness and safety of 5% lidocaine medicated plaster to treat neuropathic pain in children with burn sequelae. DESIGN: Three-month prospective, uncontrolled study. SETTING: Corporation of Aid to Burned Children (COANIQUEM), a nonprofit pediatric burn rehabilitation center in Chile. SUBJECTS: Fourteen pediatric patients with burn sequelae neuropathic pain. OUTCOME MEASURES: Demographics, burn and pain evolution (type, intensity [using Wong-Baker FACES], and Douleur Neuropathique 4 [DN4]), and patient functionality. Plasma lidocaine levels were measured at 0, 12, 36, and 60 hours after treatment commencement. RESULTS: Fourteen patients were evaluable for plasma lidocaine levels. Twelve patients were available for clinical assessment (two patients lost to follow-up) [mean (standard deviation)]: age, 11 years 7 months (2 years 6 months); weight, 45 kg (11.9 kg); burn evolution, 5 years 6 months (4 years); time between burn and pain onset, 3 years 6 months (3 years 2 months); time between pain onset and treatment, 5.1 months (4.8 months); lidocaine, between < and ½ plaster; initial pain intensity (FACES), 6.8 (1.6); final pain intensity, 0 in 11/12 patients; DN4, initial-6, final-2.3. All patients reported improved functionality. Plasma lidocaine levels were ≤27.45 ng/mL (>180 times below critical levels). No adverse reactions occurred. CONCLUSIONS: These are the first published data suggesting that 5% lidocaine medicated plaster improves patient functionality, and is effective and safe for the treatment of neuropathic pain in pediatric patients with burn sequelae.