In vitro synergy screens of FDA-approved drugs reveal novel zidovudine- and azithromycin-based combinations with last-line antibiotics against Klebsiella pneumoniae.

Treatment of infections caused by multi-drug resistant (MDR) enterobacteria remains challenging due to the limited therapeutic options available. Drug repurposing could accelerate the development of new urgently needed successful interventions. This work aimed to identify and characterise novel drug combinations against Klebsiella pneumoniae based on the concepts of synergy and drug repurposing. We first performed a semi-qualitative high-throughput synergy screen (sHTSS) with tigecycline, colistin and fosfomycin (last-line antibiotics against MDR Enterobacteriaceae) against a FDA-library containing 1430 clinically approved drugs; a total of 109 compounds potentiated any of the last-line antibiotics. Selected hits were further validated by secondary checkerboard (CBA) and time-kill (TKA) assays, obtaining 15.09% and 65.85% confirmation rates, respectively. Accordingly, TKA were used for synergy classification based on determination of bactericidal activities at 8, 24 and 48 h, selecting 27 combinations against K. pneumoniae. Among them, zidovudine or azithromycin combinations with last-line antibiotics were further evaluated by TKA against a panel of 12 MDR/XDR K. pneumoniae strains, and their activities confronted with those clinical combinations currently used for MDR enterobacteria treatment; these combinations showed better bactericidal activities than usual treatments without added cytotoxicity. Our studies show that sHTSS paired to TKA are powerful tools for the identification and characterisation of novel synergistic drug combinations against K. pneumoniae. Further pre-clinical studies might support the translational potential of zidovudine- and azithromycin-based combinations for the treatment of these infections.

Intracranial atherosclerotic plaque enhancement and long-term risk of future strokes: A prospective, longitudinal study.

BACKGROUND AND PURPOSE: The prognostic significance of postcontrast enhancement of intracranial atheromatous plaque is uncertain. Prospective, long-term follow-up studies in Caucasians, using a multicenter design, are lacking. We aimed to evaluate whether this radiological sign predicts long-term new stroke in symptomatic and asymptomatic intracranial atherosclerotic disease (ICAD) patients. METHODS: This was a prospective, observational, longitudinal, multicenter study. We included a symptomatic and an asymptomatic cohort of ICAD patients that underwent 3T MRI including high-resolution sequences focused on the atheromatous plaque. We evaluated grade of stenosis, plaque characteristics, and gadolinium enhancement ratio (postcontrast plaque signal/postcontrast corpus callosum signal). The occurrence of new events was evaluated at 3, 6, 9, and 12 months and annually thereafter. The association between plaque characteristics and new stroke was studied using Cox multiple regression survival analysis and Kaplan-Meier curves. RESULTS: Forty-eight symptomatic and 13 asymptomatic patients were included. During 56.3 ± 16.9 months, 11 patients (18%) suffered a new event (seven ischemic, two hemorrhagic, and two transient ischemic attacks). A receiver operating characteristic curve identified an enhancement ratio of >1.77 to predict a new event. In a multivariable Cox regression, postcontrast enhancement ratio >1.77 (hazard ratio [HR]= 3.632; 95% confidence interval [CI], 1.082-12.101) and cerebral microbleeds (HR = 5.244; 95% CI, 1.476-18.629) were independent predictors of future strokes. Patients with a plaque enhancement ratio >1.77 had a lower survival free of events (p < .05). CONCLUSIONS: High intracranial postcontrast enhancement is a long-term predictor of new stroke in ICAD patients. Further studies are needed to elucidate whether postcontrast enhancement reflects inflammatory activity of intracranial atheromatous plaque.

Beyond CC398: Characterisation of Other Tetracycline and Methicillin-Resistant Staphylococcus aureus Genetic Lineages Circulating in Spanish Hospitals.

Tetracycline resistance (TetR) has been evidenced as a good phenotypic marker for detection of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of the clonal complex CC398. The aim of this study was to characterise a collection of 95 TetR-MRSA isolates, not belonging to the lineage CC398, that were obtained in a previous multicentre study, to detect other MRSA clonal complexes that could be associated with this phenotypic TetR marker. The TetR-MRSA isolates were recovered from 20 Spanish hospitals during 2016 and they were characterised to determine their antimicrobial resistance and virulence phenotypes/genotypes as well as the presence of the immune evasion cluster (IEC). A high proportion of isolates belonging to the CC1 lineage (46%) were observed, as well as to the CC5, CC8 and CC45 lineages (11% each one). Thirty-two different spa-types were identified, being predominantly CC1-t127 (40%) and CC45-t1081 (11%). The IEC system (with the gene scn as marker) was present in 73% of isolates and 16% produced the Panton Valentine leucocidin (PVL). A high proportion of MRSA-CC1 isolates were scn-negative (38.6%) and 52.9% of them were blaZ-negative. A multidrug resistance (MDR) phenotype was identified in 86% of MRSA isolates. The knowledge of other TetR-MRSA genetic lineages, in addition to CC398, is highly relevant, since most of them were MDR and some of them presented important virulence factors. Strains potentially associated with livestock (as the subpopulation CC1-t127-scn-negative) or with humans (as the CC45 lineage or the subpopulation CC1-scn-positive) have been found in this study. The use of tetracycline-resistance for detection, not only of CC398 but also of other LA-MRSA lineages should be tracked in the future.

Prevalence and Genetic Characteristics of Staphylococcus aureus CC398 Isolates From Invasive Infections in Spanish Hospitals, Focusing on the Livestock-Independent CC398-MSSA Clade.

BACKGROUND: Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported. OBJECTIVES: To determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates. METHODS: One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6-12 months in 17 Spanish hospitals (2018-2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing. RESULTS: Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin-inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p < 0.005). CONCLUSION: CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.

Use of MALDI-TOF MS (Bruker Daltonics) for identification of Mycobacterium species isolated directly from liquid medium.

OBJECTIVES: The aim of this study was to describe the evaluation of the use of MALDI-TOF MS for the identification of non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis directly from liquid MGIT cultures from January 2017 to December 2017. MATERIAL/METHODS: A total of 155 isolates (mainly respiratory) were analyzed by MALDI-TOF MS (Bruker Daltonics) directly from MGIT liquid medium with a previous extraction procedure. RESULTS: MALDI-TOF MS generated acceptable scores for 152 isolates (98.06%). Fifty isolates were identified as M. tuberculosis complex and the remaining 105 as NTM (M. abscessus subsp. abscessus, M. avium, M. celatum, M chelonae, M. chimaera, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. lentiflavum, M. mageritense, M. mucogenicum and M. xenopi). CONCLUSIONS: These results indicate that MALDI-TOF MS can be useful to identify mycobacteria directly from MGIT cultures and is an accurate, rapid and cost-effective system to be used as a routine method.

Characterization of AmpC ß-lactamase mutations of extensively drug-resistant Pseudomonas aeruginosa isolates that develop resistance to ceftolozane/tazobactam during therapy.

INTRODUCTION: We characterized AmpC ß-lactamase mutations that resulted in ceftolozane/tazobactam resistance in extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates recovered from patients treated with this agent from June 2016 to December 2018. METHODS: Five pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa XDR isolates were included among a total of 49 patients treated. Clonal relationship among isolates was first evaluated by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was further performed. AmpC mutations were investigated by PCR amplification of the blaPDC gene followed by sequencing. RESULTS: The ST175 high-risk clone was detected in four of the pairs of isolates and the ST1182 in the remaining one. All resistant isolates showed a mutation in AmpC: T96I in two of the isolates, and E247K, G183V, and a deletion of 19 amino acids (G229-E247) in the other three. The G183V mutation had not been described before. The five isolates resistant to ceftolozane/tazobactam showed cross-resistance to ceftazidime/avibactam and lower MICs of imipenem and piperacillin/tazobactam than the susceptible isolates. CONCLUSIONS: Ceftolozane/tazobactam resistance was associated in all of the cases with AmpC mutations, including a novel mutation (G183V) not previously described. There is a vital need for surveillance and characterization of emerging ceftolozane/tazobactam resistance, in order to preserve this valuable antipseudomonal agent.

Multicenter study of clinical non-ß-lactam-antibiotic susceptible MRSA strains: Genetic lineages and Panton-Valentine leukocidin (PVL) production.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is considered a major cause of healthcare-associated (HA) and community-acquired (CA) infections. Considering non-ß-lactam susceptibility as a potential marker for mecC-MRSA and CA-MRSA, the aim of this study was to determine the frequency and the associated genetic lineages of non-beta-lactam-antibiotic susceptible MRSA (NBLS-MRSA) strains in a multicenter study in Spain. METHODS: A collection of 45 NBLS-MRSA strains recovered in the period from January to June 2016 from 12 Spanish hospitals was analyzed. Molecular typing through spa-type characterization, agr group and multi-locus-sequence typing was performed. Methicillin-resistant genes (mecA and mecC) as well as immune evasion cluster (scn-chp-sak-sea-sep, considering scn gene as the marker of IEC system) and Panton-Valentine leukocidin (PVL) genes were determined with PCR/sequencing. RESULTS: The NBLS-MRSA phenotype was uncommon in the 12 hospitals analyzed (NBLS-MRSA/MRSA frequency: 0.3%-7.7%). All strains contained the mecA gene (and none contained mecC). Twenty-two different spa-types were detected among NBLS-MRSA strains, with spa-t008/agr-I the most prevalent (27%). The main clonal complexes were (CC/%): CC8/42.2%, CC5/33.3% and CC30/4.4%, with ST8 and ST5 as the main sequence types. The PVL toxin was present in 38% of strains (with spa-types t008, t024, t019, t044, t068, t318 and t3060). The IEC genes were detected in 78% of strains: IEC type-B (n=17), type-F (n=16), type-A (n=1) and type-E (n=1); 10 MRSA isolates were scn-negative. CONCLUSION: The NBLS-MRSA phenotype is uncommon in the analyzed hospitals; although no mecC-positive strains were detected, it could be a good marker for MRSA PVL-positive isolates (38%), frequently associated with CA-MRSA infections.

Detection of carbapenemases and other mechanisms of enzymatic resistance to ß-lactams in Enterobacteriaceae with diminished susceptibility to carbapenems in a tertiary care hospital.

INTRODUCTION: Our objective was to characterize the enzymatic ß-lactam resistance in clinical Enterobacteriaceae isolates with diminished susceptibility to carbapenems from 2013 to 2014 at Hospital Universitario Miguel Servet. MATERIAL/METHODS: A total of 63 clinical isolates were analyzed for the presence of carbapenemases (KPC, OXA-48 and MBL), ESBLs and AmpC enzymes by combined disk methods and PCR detection of carbapenemase-encoding and beta-lactamase-encoding genes. RESULTS: Fifteen isolates had a phenotypic test compatible with carbapenemase production; two of these were confirmed by PCR as OXA-48 producers. ESBL detection was positive in 27 isolates (43%); plasmid-mediated AmpC was detected in nine isolates (14.2%) and derepressed AmpC ß-lactamase was present in 18 isolates (28%). CONCLUSION: During the study period, the decreased susceptibility to carbapenems in Enterobacteriaceae in our area was not due to true carbapenemases but rather to ß-lactamase activity (82.5% were ESBL or AmpC producers), probably in combination with decreased permeability of the outer membrane.

Relative cerebral blood volume as a marker of durable tissue-at-risk viability in hyperacute ischemic stroke.

BACKGROUND AND PURPOSE: Selection of best responders to reperfusion therapies could be aided by predicting the duration of tissue-at-risk viability, which may be dependant on collateral circulation status. We aimed to identify the best predictor of good collateral circulation among perfusion computed tomography (PCT) parameters in middle cerebral artery (MCA) ischemic stroke and to analyze how early MCA response to intravenous thrombolysis and PCT-derived markers of good collaterals interact to determine stroke outcome. METHODS: We prospectively studied patients with acute MCA ischemic stroke treated with intravenous thrombolysis who underwent PCT before treatment showing a target mismatch profile. Collateral status was assessed using a PCT source image-based score. PCT maps were quantitatively analyzed. Cerebral blood volume (CBV), cerebral blood flow, and Tmax were calculated within the hypoperfused volume and in the equivalent region of unaffected hemisphere. Occluded MCAs were monitored by transcranial Duplex to assess early recanalization. Main outcome variables were brain hypodensity volume and modified Rankin scale score at day 90. RESULTS: One hundred patients with MCA ischemic stroke imaged by PCT received intravenous thrombolysis, and 68 met all inclusion criteria. A relative CBV (rCBV) >0.93 emerged as the only predictor of good collaterals (odds ratio, 12.6; 95% confidence interval, 2.9-55.9; P=0.001). Early MCA recanalization was associated with better long-term outcome and lower infarct volume in patients with rCBV<0.93, but not in patients with high rCBV. None of the patients with rCBV<0.93 achieved good outcome in absence of early recanalization. CONCLUSIONS: High rCBV was the strongest marker of good collaterals and may characterize durable tissue-at-risk viability in hyperacute MCA ischemic stroke.

Global study of IS6110 in a successful Mycobacterium tuberculosis strain: clues for deciphering its behavior and for its rapid detection.

The Mycobacterium tuberculosis insertion sequence IS6110, besides being a very useful tool in molecular epidemiology, seems to have an impact on the biology of bacilli. In the present work, we mapped the 12 points of insertion of IS6110 in the genome of a successful strain named M. tuberculosis Zaragoza (which has been referred to as the MTZ strain). This strain, belonging to principal genetic group 3, caused a large unsuspected tuberculosis outbreak involving 85 patients in Zaragoza, Spain, in 2001 to 2004. The mapping of the points of insertion of IS6110 in the genome of the Zaragoza strain offers clues for a better understanding of the adaptability and virulence of M. tuberculosis. Surprisingly, the presence of one copy of IS6110 was found in Rv2286c, as was recently described for a successful Beijing sublineage. As a result of this analysis, a rapid method for detecting this particular M. tuberculosis strain has been designed.

Unsuspected and extensive transmission of a drug-susceptible Mycobacterium tuberculosis strain.

BACKGROUND: A large and unsuspected tuberculosis outbreak involving 18.7% of the total of the tuberculosis cases studied, was detected in a population-based molecular epidemiological study performed in Zaragoza (Spain) from 2001 to 2004. METHODS: The Mycobacterium tuberculosis drug-susceptible strain, named MTZ strain, was genetically characterized by IS6110-RFLP, Spoligotyping and by MIRU-VNTR typing and the genetic patterns obtained were compared with those included in international databases. The characteristics of the affected patients, in an attempt to understand why the MTZ strain was so highly transmitted among the population were also analyzed. RESULTS: The genetic profile of the MTZ strain was rare and not widely distributed in our area or elsewhere. The patients affected did not show any notable risk factor for TB. CONCLUSION: The M. tuberculosis strain MTZ, might have particular transmissibility or virulence properties, and we believe that greater focus should be placed on stopping its widespread dissemination.

Efectividad de la relajacion fisica y mental en pacientes con hipertension inducida por el embarazo / Effectiveness of physical and mental relaxation in patients with pregnancy induced by hypertension

Estudio clinico experimental realizado en 15 mujeres con 22-42 semanas de embarazo y diagnostico hipertension inducida por el embarazo, sin historia de enfermedad cardiovascular o renal que no se encontraban en la fase aguda de la enfermedad, pero estaban hospitalizadas en el Hospital Regional Simon Bolivar de Bogota. Durante el estudio entre Febrero y Julio de 1989, como complemento del tratamiento medico-farmacologico, la enfermera implemento en el cuidado tecnicas de relajacion 2 veces durante un solo dia. Se evaluaron los niveles de ansiedad mediante el test de Hamilton, la presion arterial y las frecuencias cardiacas maternas y fetales como indicadores de la eficacia de este manejo en la reduccion de estres, considerado como factor predisponente o agravante de esta complicacion del embarazo. Las mujeres en quienes se realizo el estudio tenian entre 22 y 33 anos, a 10 de ellas se les habia hecho diagnostico de preeclampsia leve, a 4 de preeclampsia grave y a 1 de eclampsia. 9 eran primigestantes y 6 multigestantes. Antes de la relajacion la presion arterial sistolica se encontro entre 190 y 120 mm de Hg. y la diastolica entre 130-80 mm de Hg. comparativamente con las mediciones posteriores. Se evidencio disminucion mayor en la presion arterial diastolica continuado hasta 6 horas despues en el 13.3 por ciento . La ansiedad detectada en el 46.6 por ciento disminuyo o desaparecio inmediatamente despues del tratamiento. Las frecuencias cardiacas maternas y fetales no se aumentaron significativamente, por el contrario se mantuvo constante (mama) y en el 86.6 por ciento de los bebes. 6 ..