Microevolution, reinfection and highly complex genomic diversity in patients with sequential isolates of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic, extensively drug-resistant non-tuberculous mycobacterium. Few genomic studies consider its diversity in persistent infections. Our aim was to characterize microevolution/reinfection events in persistent infections. Fifty-three sequential isolates from 14 patients were sequenced to determine SNV-based distances, assign resistance mutations and characterize plasmids. Genomic analysis revealed 12 persistent cases (0-13 differential SNVs), one reinfection (15,956 SNVs) and one very complex case (23 sequential isolates over 192 months), in which a first period of persistence (58 months) involving the same genotype 1 was followed by identification of a genotype 2 (76 SNVs) in 6 additional alternating isolates; additionally, ten transient genotypes (88-243 SNVs) were found. A macrolide resistance mutation was identified from the second isolate. Despite high diversity, the genotypes shared a common phylogenetic ancestor and some coexisted in the same specimens. Genomic analysis is required to access the true intra-patient complexity behind persistent infections involving M. abscessus.

A whole-cell hypersensitive biosensor for beta-lactams based on the AmpR-AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20.

Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta-lactams are lacking. To address this gap, a biosensor based on the AmpC beta-lactamase regulation system (ampR-ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR-AmpC system is well-conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta-lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta-lactam hypersensitive strain, generating a whole-cell biosensor. The biosensor demonstrated dose-dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1-10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta-lactam antibiotics, specifically carbapenems, in different matrices.

Pyeloureteral Anastomosis as a Reconstructive Technique for Post-Renal Transplant Ureteral Stenosis.

OBJECTIVE: Ureteral stenosis in renal transplant patients is a frequent urological complication that involves significant morbidity and may compromise graft function. Despite the common use of minimally invasive techniques, surgery continues to be the definitive treatment for ureteral stenosis, and pyeloureteral anastomosis is an infrequent but effective technique in the management of this pathology and has been described as a safe treatment with a low percentage of complications. METHODS: This is a retrospective study of patients in whom surgical intervention via pyeloureteral anastomosis was carried out in our center in the last 12 years. A descriptive analysis of perioperative management, complications, and functional results is provided. A comparison of renal function at diagnosis and after surgery was made to evaluate the effectiveness of the procedure. RESULTS: Thirteen patients underwent surgery within the described time frame. Time to diagnosis of stenosis was 60 days [interquartile range (IQR) 31-368]. Creatinine at diagnosis was 2.2 mg/dL [IQR 1.9-3] with a glomerular filtration rate, estimated by the modification of diet in renal disease equation, of 29 mL/min/1.73 m2 [IQR 22.6-34.5]. Of these patients, 92.3% underwent percutaneous nephrostomy, and 38.5% also had a ureteral catheter. The mean duration of surgery was 265 minutes [IQR 240-300], and hospital stay was 9 days [IQR 7.5-16]. A double J was placed in all cases, which was maintained for 36 days [IQR 30-49]. Postoperative complications occurred in 15.4% of patients. Serum creatinine 1 year after surgery was 1.6 ± 0.4 mg/dL. Among the patients, 76.9% had no new pyelocalyceal dilatation on follow-up Doppler ultrasound scans at a mean follow-up time of 12 months. The restenosis rate was 23.1%, and all were successfully treated by endoscopic approach. There was an improvement in renal function figures at 1, 3, 6, and 12 months compared to renal function at diagnosis, both in terms of serum creatinine and glomerular filtration rate, with statistically significant results. CONCLUSION: Pyeloureteral anastomosis as a reconstructive technique of the urinary tract in renal transplant patients is an effective and reproducible technique with good long-term results.

Noncanonical Mismatch Repair Protein NucS Modulates the Emergence of Antibiotic Resistance in Mycobacterium abscessus.

NucS/EndoMS-dependent noncanonical mismatch repair (MMR) ensures the stability of genomic DNA in mycobacteria and acts as a guardian of the genome by preventing the accumulation of point mutations. In order to address whether the inactivation of noncanonical MMR could increase the acquisition of drug resistance by mutation, a ΔnucS strain was constructed and explored in the emerging pathogen Mycobacterium abscessus. Deletion of nucS resulted in a mutator phenotype with increased acquisition of resistance to macrolides and aminoglycosides, the two main groups of antimycobacterial agents for M. abscessus treatment, and also to second-line drugs such as fluoroquinolones. Inactivation of the noncanonical MMR in M. abscessus led to increases of 10- to 22-fold in the appearance of spontaneous mutants resistant to the macrolide clarithromycin and the aminoglycosides amikacin, gentamicin, and apramycin, compared with the wild-type strain. Furthermore, emergence of fluoroquinolone (ciprofloxacin) resistance was detected in a nucS-deficient strain but not in a wild-type M. abscessus strain. Acquired drug resistance to macrolides and aminoglycosides was analyzed through sequencing of the 23S rRNA gene rrl and the 16S rRNA gene rrs from independent drug-resistant colonies of both strains. When the acquisition of clarithromycin resistance was examined, a different mutational profile was detected in the M. abscessus ΔnucS strain compared with the wild-type one. To summarize, M. abscessus requires the NucS-dependent noncanonical MMR pathway to prevent the emergence of drug-resistant isolates by mutation. To our knowledge, this is the first report that reveals the role of NucS in a human pathogen, and these findings have potential implications for the treatment of M. abscessus infections. IMPORTANCE Chronic infections caused by M. abscessus are an emerging challenge in public health, posing a substantial health and economic burden, especially in patients with cystic fibrosis. Treatment of M. abscessus infections with antibiotics is particularly challenging, as its complex drug resistance mechanisms, including constitutive resistance through DNA mutation, lead to high rates of treatment failure. To decipher the evolution of antibiotic resistance in M. abscessus, we studied NucS-dependent noncanonical MMR, a unique DNA repair pathway involved in genomic maintenance. Inactivation of NucS is linked to the increase of DNA mutations (hypermutation), which can confer drug resistance. Our analysis detected increased acquisition of mutations conferring resistance to first-line and second-line antibiotics. We believe that this study will improve the knowledge of how this pathogen could evolve into an untreatable infectious agent, and it uncovers a role for hypermutators in chronic infectious diseases under antibiotic pressure.

Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis.

Rifampicin is a critical first-line antibiotic for treating mycobacterial infections such as tuberculosis, one of the most serious infectious diseases worldwide. Rifampicin resistance in mycobacteria is mainly caused by mutations in the rpoB gene; however, some rifampicin-resistant strains showed no rpoB mutations. Therefore, alternative mechanisms must explain this resistance in mycobacteria. In this work, a library of 11,000 Mycobacterium smegmatis mc2 155 insertion mutants was explored to search and characterize new rifampicin-resistance determinants. A transposon insertion in the MSMEG_1945 gene modified the growth rate, pH homeostasis and membrane potential in M. smegmatis, producing rifampicin resistance and collateral susceptibility to other antitubercular drugs such as isoniazid, ethionamide and aminoglycosides. Our data suggest that the M. smegmatis MSMEG_1945 protein is an ion channel, dubbed MchK, essential for maintaining the cellular ionic balance and membrane potential, modulating susceptibility to antimycobacterial agents. The functions of this new gene point once again to potassium homeostasis impairment as a proxy to resistance to rifampicin. This study increases the known repertoire of mycobacterial ion channels involved in drug susceptibility/resistance to antimycobacterial drugs and suggests novel intervention opportunities, highlighting ion channels as druggable pathways.

Cross Talk between α7 and α3ß4 Nicotinic Receptors Prevents Their Desensitization in Human Chromaffin Cells.

The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3ß4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3ß4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3ß4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3ß4 subtype is dependent on Ca2+ In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca2+ In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3ß4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.SIGNIFICANCE STATEMENT Desensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3ß4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca2+ to cooperate in the case of the α3ß4 current increase. Because choline is an α3ß4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.

Therapeutic concentrations of varenicline increases exocytotic release of catecholamines from human and rat adrenal chromaffin cells in the presence of nicotine.

Cardiovascular side effects of varenicline and a case report of a hypertensive crisis in a varenicline-prescribed patient with pheochromocytoma have been reported. The goal of the present study was to determine whether such side effects might derive, in part, from increased exocytosis of secretory vesicles and subsequent catecholamine release triggered by varenicline in human chromaffin cells of the adrenal gland. In this study, we performed electrophysiological plasma membrane capacitance and carbon fiber amperometry experiments to evaluate the effect of varenicline on exocytosis and catecholamine release, respectively, at concentrations reached during varenicline therapy (100 nM). Experiments were conducted in the absence or presence of nicotine, at plasma concentrations achieved right after smoking (250 nM) or steady-state concentrations (110 nM), in chromaffin cells of the adrenal gland obtained from human organ donors. Cells were stimulated with short pulses (10 ms) of acetylcholine (ACh; 300 µM) applied at 0.2 Hz, in order to closer mimic the physiological situation at the splanchnic nerve-chromaffin cell synapse. In addition, rat chromaffin cells were used to compare the effects obtained in cells from a more readily available species. Varenicline increased the exocytosis of secretory vesicles in human and rat chromaffin cells in the presence of nicotine, effects that were not due to an increase of plasma membrane capacitance or currents triggered by the nicotinic agonists alone. These results should be considered in nicotine addiction therapies when varenicline is used.

Control of Genome Stability by EndoMS/NucS-Mediated Non-Canonical Mismatch Repair.

The DNA repair endonuclease EndoMS/NucS is highly conserved in Archaea and Actinobacteria. This enzyme is able to recognize and cleave dsDNA carrying a mismatched base pair, and its activity is enhanced by the interaction with the sliding clamp of the replisome. Today, EndoMS/NucS has been established as the key protein of a non-canonical mismatch repair (MMR) pathway, acting specifically in the repair of transitions and being essential for maintaining genome stability. Despite having some particularities, such as its lower activity on transversions and the inability to correct indels, EndoMS/NucS meets the main hallmarks of a MMR. Its absence leads to a hypermutator phenotype, a transition-biased mutational spectrum and an increase in homeologous recombination. Interestingly, polymorphic EndoMS/NucS variants with a possible effect in mutation rate have been detected in clinical isolates of the relevant actinobacterial pathogen Mycobacterium tuberculosis. Considering that MMR defects are often associated with the emergence of resistant bacteria, the existence of EndoMS/NucS-defective mutators could have an important role in the acquisition of antibiotic resistance in M. tuberculosis. Therefore, a further understanding of the EndoMS/NucS-mediated non-canonical MMR pathway may reveal new strategies to predict and fight drug resistance. This review is focused on the recent progress in NucS, with special emphasis on its effect on genome stability and evolvability in Actinobacteria.

Detection of Low-Level Fosfomycin-Resistant Variants by Decreasing Glucose-6-Phosphate Concentration in Fosfomycin Susceptibility Determination.

Mutations that confer low-level fosfomycin resistance (LLFR) but not clinical resistance in Escherichia coli are increasingly reported. LLFR strains can become clinically resistant under urinary tract physiological conditions or may act as gateways for highly resistant subpopulations by the selection of additional LLFR mutations. Nevertheless, most LLFR strains are impossible to detect under routine fosfomycin susceptibility determinations. Here, we have explored the possibility of detecting LLFR variants by reducing glucose-6-phosphate (G6P) concentration in fosfomycin susceptibility testing for E. coli strains. As a proof of concept, fosfomycin minimal inhibitory concentrations (MICs) and disk diffusion susceptibility tests were performed for E. coli strain BW25113 and 10 isogenic derivatives carrying the most prevalent LLFR chromosomal mutations (∆uhpT, ∆glpT, ∆cyaA, and ∆ptsI) and their double combinations. Whereas standard G6P concentrations detected only ∆uhpT single and double variants, assays with reduced G6P detected all LLFR variants. In addition, G6P levels were determined to be ≤5 µg/mL in urine samples from 30 patients with urinary tract infection (UTI) caused by E. coli and 10 healthy volunteers, suggesting that most bacterial cells in uncomplicated UTIs are facing fosfomycin under low G6P concentration. Reducing G6P allows for the detection of LLFR variants, which may suppose a risk for future resistance development, especially in UTIs.

Anatomía patológica de la patología de tiroides y paratiroides. Sistema Bethesda del diagnóstico citológico de la patología de tiroides / Thyroid and parathyroid gland pathology. The Bethesda System for reporting thyroid cytopathology

INTRODUCCIÓN Y OBJETIVO: La PAAF es el mejor método para el manejo de pacientes con nódulos tiroideos, como método de cribado y para seleccionar a los pacientes que pueden ser sometidos a tratamiento quirúrgico. El objetivo de este artículo es hacer una breve descripción de las categorías diagnósticas del Sistema Bethesda en su segunda edición (2018). SÍNTESIS: El Sistema Bethesda establece 6 categorías diagnósticas: Insatisfactorio /No diagnóstico, Benigno, Atipia de significado Indeterminado/Lesión folicular de significado indeterminado, Neoplasia folicular/Sospechoso de neoplasia folicular, Sospechoso de Malignidad Maligno. Cada categoría lleva implícito el riesgo de malignidad y el manejo de estos pacientes, con lo cual el diagnóstico va a influir en la actitud a seguir. CONCLUSIONES: El Sistema Bethesda permite a los patólogos realizar informes sistematizados y al clínico establecer la actitud a seguir en función de cada categoría diagnóstica

INTRODUCTION AND OBJECTIVE: The FNA is the best method for the management of patients with thyroid nodules, as a screening method and for selecting patients who can undergo surgical treatment. The objective of this article is to make a brief description of the diagnostic categories of the Bethesda System in its second edition (2018). SYNTHESIS: The Bethesda System establishes 6 diagnostic categories: No diagnostic-Unsatisfactory, Benign, Atypia od Undetermined Significance/Follicular Lesion of Undetermined Significance, Follicular Neoplasm/Suspicious for a Follicular Neoplasm, Suspicious for Malignant and Malignant. CONCLUSIONS: The Bethesda System allows pathologists to make systematized reports and the clinician to establish the attitude to follow according to each diagnostic category

Contribution of hypermutation to fosfomycin heteroresistance in Escherichia coli.

OBJECTIVES: To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. MATERIALS AND METHODS: We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5-512 mg/L) and time-kill assays (64 and 307 mg/L). RESULTS: Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time-kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. CONCLUSIONS: The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.

4-Amino-1,2,4-triazole-3-thione as a Promising Scaffold for the Inhibition of Serine and Metallo-ß-Lactamases.

The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available ß-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of ß-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine ß-Lactamase (SBLs) KPC-2 and class B1 metallo ß-Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.

Effect of subinhibitory concentrations of ampicillin on Listeria monocytogenes / Efecto de las concentraciones subinhibitorias de ampicilina sobre Listeria monocytogenes

Listeria monocytogenes is an important cause of meningoencephalitis associated with high mortality. The treatment of choice for listeriosis is ampicillin alone or in combination with gentamicin or penicillin G. However, only low ampicillin concentrations are recorded in the central nervous system (CNS). In this study, we analysed the effect of subinhibitory concentrations of ampicillin on the morphology, growth and survival of L. monocytogenes. The non-inhibitory concentration (NIC), the minimum inhibitory concentration (MIC) and the MIC/NIC ratio were determined. Gram and Live/Dead staining showed aggregates of L. monocytogenes cells when grown at subinhibitory concentrations of ampicillin, with > 90% of viable cells. The L. monocytogenes strains tested showed an intermediate heteroresistance to ampicillin, characterised by a MIC/NIC ratio of 4. Our results seem to indicate that both intermediate heteroresistance and the formation of aggregates could play a role in the clinical failure of ampicillin in the treatment of CNS infections caused by L. monocytogenes. However, more studies are necessary to elucidate this question

Listeria monocytogenes es una importante causa de meningoencefalitis asociada con una elevada mortalidad. El tratamiento de elección para la listeriosis es ampicilina asociada o no a gentamicina o penicilina G. Sin embargo, en el sistema nervioso central (SNC) solo se alcanzan concentraciones bajas de ampicilina. En este estudio, analizamos el efecto de las concentraciones subinhibitorias de ampicilina sobre la morfología, el crecimiento y la supervivencia de L. monocytogenes. Para ello determinamos la concentración no inhibitoria (CNI), la concentración mínima inhibitoria (CMI) y el cociente CMI/CNI. Además, la tinción de Gram y la de vivos y muertos mostraron agregados de L. monocytogenes cuando crece a concentraciones subinhibitorias de ampicilina, con > 90% de las células viables. Las cepas de L. monocytogenes testadas mostraron heterorresistencia intermedia a la ampicilina, caracterizada por un cociente CMI/CNI de 4. Nuestros resultados parecen indicar que tanto la formación de agregados como la heterorresistencia intermedia podrían jugar un papel en el fracaso terapéutico observado en las infecciones del SNC causadas por L. monocytogenes. Sin embargo, se necesitan más estudios para aclarar esta cuestión

Effect of subinhibitory concentrations of ampicillin on Listeria monocytogenes.

Listeria monocytogenes is an important cause of meningoencephalitis associated with high mortality. The treatment of choice for listeriosis is ampicillin alone or in combination with gentamicin or penicillin G. However, only low ampicillin concentrations are recorded in the central nervous system (CNS). In this study, we analysed the effect of subinhibitory concentrations of ampicillin on the morphology, growth and survival of L. monocytogenes. The non-inhibitory concentration (NIC), the minimum inhibitory concentration (MIC) and the MIC/NIC ratio were determined. Gram and Live/Dead staining showed aggregates of L. monocytogenes cells when grown at subinhibitory concentrations of ampicillin, with >90% of viable cells. The L. monocytogenes strains tested showed an intermediate heteroresistance to ampicillin, characterised by a MIC/NIC ratio of 4. Our results seem to indicate that both intermediate heteroresistance and the formation of aggregates could play a role in the clinical failure of ampicillin in the treatment of CNS infections caused by L. monocytogenes. However, more studies are necessary to elucidate this question.

Phenylboronic Acids Probing Molecular Recognition against Class A and Class C ß-lactamases.

Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, ß-lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine ß-Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect ß-lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with ß-lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors.

Seawater salt-trapped Pseudomonas aeruginosa survives for years and gets primed for salinity tolerance.

BACKGROUND: In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood. RESULTS: In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions. CONCLUSIONS: The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.

[Laparoscopic renal cyst excision: Large simple renal cyst treatment with renal cell carcinoma cells in its wall. Histogenetic considerations and bibliographic review.] / Quistectomía laparoscópica: tratamiento de un quiste simple de gran tamaño con carcinoma de células renales en su pared. Consideraciones histogenéticas y revisión de la literatura.

OBJECTIVE: The incidence of simple renal cysts is very high, especially in elderly people. However, malignant transformation of a simple renal cyst is exceptional. Likewise, the treatment to be carried out, in these rare cases, is controversial, with respect to opting for radical renal surgery. METHODS: We present the case of a patient with a solid nodule in a large cyst. Complete removal of the cyst was performed by transperitoneal laparoscopic technique. The histopathological study of the surgical piece revealed the existence of a cyst with clear renal cell carcinoma with nucleolar grade 2. The clinical evolution has been satisfactory, performing a minimally invasive surgery (laparoscopic cyst excision). DISCUSSION: Although it is considered that surgical treatment of choice is radical surgery in these cases, we believe that nephron sparing surgery may represent a therapeutic option, taking into account the young age of our patient. A histogenetic hypothesis is discussed to explain the appearance of a clear cell tumor in a simple renal cyst. CONCLUSION: The development of a renal cell carcinoma in simple renal cysts is a very infrequent pathology.Laparoscopic total cystectomy is a minimally invasive therapeutic option for the treatment of renal cell carcinoma originating in a simple renal cyst, although it is of an important size. We establish the hypothesis of migration of the cells of the renal collecting tubes into the cyst wall to explain the malignant transformation of the renal simple cyst.

OBJETIVO: La incidencia de los quistes renales simples es muy frecuente, sobre todo en personas de edad avanzada. Sin embargo, la transformación maligna de un quiste renal simple es excepcional. Así mismo, el tratamiento a realizar, en estos casos raros, es un motivo de controversia, con respecto a optar por una cirugía radical renal.MÉTODOS: Presentamos el caso de un paciente con nódulo sólido en un quiste de gran tamaño. Se realiza extirpación completa del quiste mediante técnica de laparoscopia vía transperitoneal. El estudio histopatológico de la pieza quirúrgica revela la existencia un quiste con un carcinoma renal de células claras con grado nucleolar 2. La evolución clínica ha sido satisfactoria, realizando una cirugía de mínima invasión (quistectomía laparoscópica). DISCUSIÓN: Aunque se considera que el tratamiento quirúrgico es la cirugía radical en estos casos, nosotros consideramos que la cirugía preservadora de nefronas puede representar una opción terapéutica, teniendo en cuenta la edad de nuestro paciente. Se comenta una hipótesis histogenética para explicar la aparición de un tumor de células claras en un quiste renal simple. CONCLUSIONES: El desarrollo de un carcinoma de células renales en quistes renales simples es una patología muy infrecuente. La quistectomía total laparoscópica es una opción terapéutica mínimamente invasiva, para el tratamiento del carcinoma de células renales originado en un quiste renal simple, aunque éste sea de un tamaño importante. Proponemos la hipótesis de una migración de las células de los túbulos renales en la pared del quiste para explicar la transformación maligna del quiste simple renal.

N-acetylcysteine blocks SOS induction and mutagenesis produced by fluoroquinolones in Escherichia coli.

BACKGROUND: Fluoroquinolones such as ciprofloxacin induce the mutagenic SOS response and increase the levels of intracellular reactive oxygen species (ROS). Both the SOS response and ROS increase bacterial mutagenesis, fuelling the emergence of resistant mutants during antibiotic treatment. Recently, there has been growing interest in developing new drugs able to diminish the mutagenic effect of antibiotics by modulating ROS production and the SOS response. OBJECTIVES: To test whether physiological concentrations of N-acetylcysteine, a clinically safe antioxidant drug currently used in human therapy, is able to reduce ROS production, SOS induction and mutagenesis in ciprofloxacin-treated bacteria without affecting antibiotic activity. METHODS: The Escherichia coli strain IBDS1 and its isogenic mutant deprived of SOS mutagenesis (TLS-) were treated with different concentrations of ciprofloxacin, N-acetylcysteine or both drugs in combination. Relevant parameters such as MICs, growth rates, ROS production, SOS induction, filamentation and antibiotic-induced mutation rates were evaluated. RESULTS: Treatment with N-acetylcysteine reduced intracellular ROS levels (by ∼40%), as well as SOS induction (by up to 75%) and bacterial filamentation caused by subinhibitory concentrations of ciprofloxacin, without affecting ciprofloxacin antibacterial activity. Remarkably, N-acetylcysteine completely abolished SOS-mediated mutagenesis. CONCLUSIONS: Collectively, our data strongly support the notion that ROS are a key factor in antibiotic-induced SOS mutagenesis and open the possibility of using N-acetylcysteine in combination with antibiotic therapy to hinder the development of antibiotic resistance.

Quistectomía laparoscópica: tratamiento de un quiste simple de gran tamaño con carcinoma de células renales en su pared. Consideraciones histogenéticas y revisión de la literatura / Laparoscopic renal cyst excision: Large simple renal cyst treatment with renal cell carcinoma cells in its wall. Histogenetic considerations and bibliographic review

Objetivo: La incidencia de los quistes renales simples es muy frecuente, sobre todo en personas de edad avanzada. Sin embargo, la transformación maligna de un quiste renal simple es excepcional. Así mismo, el tratamiento a realizar, en estos casos raros, es un motivo de controversia, con respecto a optar por una cirugía radical renal. Métodos: Presentamos el caso de un paciente con nódulo sólido en un quiste de gran tamaño. Se realiza extirpación completa del quiste mediante técnica de laparoscopia vía transperitoneal. El estudio histopatológico de la pieza quirúrgica revela la existencia un quiste con un carcinoma renal de células claras con grado nucleolar 2. La evolución clínica ha sido satisfactoria, realizando una cirugía de mínima invasión (quistectomía laparoscópica). Discusión: Aunque se considera que el tratamiento quirúrgico es la cirugía radical en estos casos, nosotros consideramos que la cirugía preservadora de nefronas puede representar una opción terapéutica, teniendo en cuenta la edad de nuestro paciente. Se comenta una hipótesis histogenética para explicar la aparición de un tumor de células claras en un quiste renal simple. Conclusiones: El desarrollo de un carcinoma de células renales en quistes renales simples es una patología muy infrecuente. La quistectomía total laparoscópica es una opción terapéutica mínimamente invasiva, para el tratamiento del carcinoma de células renales originado en un quiste renal simple, aunque éste sea de un tamaño importante. Proponemos la hipótesis de una migración de las células de los túbulos renales en la pared del quiste para explicar la transformación maligna del quiste simple renal

Objective: The incidence of simple renal cysts is very high, especially in elderly people. However, malignant transformation of a simple renal cyst is exceptional. Likewise, the treatment to be carried out, in these rare cases, is controversial, with respect to opting for radical renal surgery. Methods: We present the case of a patient with a solid nodule in a large cyst. Complete removal of the cyst was performed by transperitoneal laparoscopic technique. The histopathological study of the surgical piece revealed the existence of a cyst with clear renal cell carcinoma with nucleolar grade 2. The clinical evolution has been satisfactory, performing a minimally invasive surgery (laparoscopic cyst excision). Discussion: Although it is considered that surgical treatment of choice is radical surgery in these cases, we believe that nephron sparing surgery may represent a therapeutic option, taking into account the young age of our patient. A histogenetic hypothesis is discussed to explain the appearance of a clear cell tumor in a simple renal cyst. Conclusion: The development of a renal cell carcinoma in simple renal cysts is a very infrequent pathology Laparoscopic total cystectomy is a minimally invasive therapeutic option for the treatment of renal cell carcinoma originating in a simple renal cyst, although it is of an important size. We establish the hypothesis of migration of the cells of the renal collecting tubes into the cyst wall to explain the malignant transformation of the renal simple cyst