To update or not to update the ESCMID guidelines for the diagnosis and treatment of biofilm infections - That is the question! The opinion of the ESGB board.

Background: The work on the ESGB guidelines for diagnosis and treatment of biofilm infections began in 2012 and the result was published in 2014. The guidelines have been and still are frequently cited in the literature proving its usefulness for people working with biofilm infections. At the ESGB Biofilm conference in Mallorca 2022 (Eurobiofilms2022) the board of the ESGB decided to evaluate the 2014-guidelines and relevant publications since 2014 based on a lecture given at the Eurobiofilms2022. Guideline methods: The Delphi method for working on production of guidelines and the current ESCMID rules for guidelines are presented. The criteria for evaluation of relevant literature are very strict and especially for treatment, most clinicians and regulatory authorities require convincing results from Level I (randomized controlled trials) publications to justify changes of treatments. The relevant new biofilm literature and the relevant biofilm presentations from the Eurobiofilms meetings and ECCMID conferences was used for evaluating the contemporary relevance of the ESGB 2014 guidelines. Diagnosis of biofilm infections: Several infectious diseases have been recognized as biofilm infections since 2014, but the diagnostic methods and therapeutic strategies are still the same as recommended in the 2014 ESGB guidelines which are summarized in this opinion paper. Treatment of biofilm infections: Some promising new in vitro and in vivo (animal experiments) observations and reports for therapy of biofilm infections are mentioned, but they still await clinical trials. Conclusion: The interim opinion at the present time (2022) is therefore, that the guidelines do not need revision now, but there is a need for survey articles discussing new methods of diagnosis and treatment of biofilm infections in order - hopefully - to give inspiration to conduct clinical trials which may lead to progress in diagnosis and treatment of patients with biofilm infections.

Transmitted drug resistance to antiretroviral drugs in Spain during the period 2019-2021.

To evaluate the prevalence of transmitted drug resistance (TDR) to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTI, NNRTI), protease inhibitors (PI), and integrase strand transfer inhibitors (INSTI) in Spain during the period 2019-2021, as well as to evaluate transmitted clinically relevant resistance (TCRR) to antiretroviral drugs. Reverse transcriptase (RT), protease (Pro), and Integrase (IN) sequences from 1824 PLWH (people living with HIV) were studied. To evaluate TDR we investigated the prevalence of surveillance drug resistance mutations (SDRM). To evaluate TCRR (any resistance level ≥ 3), and for HIV subtyping we used the Stanford v.9.4.1 HIVDB Algorithm and an in-depth phylogenetic analysis. The prevalence of NRTI SDRMs was 3.8% (95% CI, 2.8%-4.6%), 6.1% (95% CI, 5.0%-7.3%) for NNRTI, 0.9% (95% CI, 0.5%-1.4%) for PI, and 0.2% (95% CI, 0.0%-0.9%) for INSTI. The prevalence of TCRR to NRTI was 2.1% (95% CI, 1.5%-2.9%), 11.8% for NNRTI, (95% CI, 10.3%-13.5%), 0.2% (95% CI, 0.1%-0.6%) for PI, and 2.5% (95% CI, 1.5%-4.1%) for INSTI. Most of the patients were infected by subtype B (79.8%), while the majority of non-Bs were CRF02_AG (n = 109, 6%). The prevalence of INSTI and PI resistance in Spain during the period 2019-2021 is low, while NRTI resistance is moderate, and NNRTI resistance is the highest. Our results support the use of integrase inhibitors as first-line treatment in Spain. Our findings highlight the importance of ongoing surveillance of TDR to antiretroviral drugs in PLWH particularly with regard to first-line antiretroviral therapy.

Phenotypic and Genotypic Antimicrobial Susceptibility Testing of Chlamydia trachomatis Isolates from Patients with Persistent or Clinical Treatment Failure in Spain.

The aim of this multicentre project (seven hospitals across the Spanish National Health Service) was to study the phenotypic and genotypic susceptibility of C. trachomatis to the main antimicrobials used (macrolides, doxycycline, and quinolones) in isolates from patients with clinical treatment failure in whom reinfection had been ruled out. During 2018-2019, 73 clinical isolates were selected. Sixty-nine clinical specimens were inoculated onto confluent McCoy cell monolayers for phenotypic susceptibility testing. The minimum inhibitory concentration for azithromycin and doxycycline was defined as the lowest concentration associated with an at least 95% reduction in inclusion-forming units after one passage in the presence of the antibiotic compared to the initial inoculum for each strain (control). Sequencing analysis was performed for the genotypic detection of resistance to macrolides, analysing mutations in the 23S rRNA gene (at positions 2057, 2058, 2059, and 2611), and quinolones, analysing a fragment of the gyrA gene, and searching for the G248T mutation (Ser83->Ile). For tetracyclines, in-house RT-PCR was used to test for the tet(C) gene. The phenotypic susceptibility testing was successful for 10 isolates. All the isolates had minimum inhibitory concentrations for azithromycin ≤ 0.125 mg/L and for doxycycline ≤ 0.064 mg/L and were considered sensitive. Of the 73 strains studied, no mutations were found at positions T2611C or G248T of the gyrA gene. We successfully sequenced 66 isolates. No macrolide resistance-associated mutations were found at positions 2057, 2058, 2059, or T2611C. None of the isolates carried the tet(C) gene. We found no evidence for genomic resistance in this large, clinically relevant dataset.

Mechanisms of antibiotic resistance in Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa is a major cause of life-threatening acute infections and life-long lasting chronic infections. The characteristic biofilm mode of life in P. aeruginosa chronic infections severely limits the efficacy of antimicrobial therapies, as it leads to intrinsic tolerance, involving physical and physiological factors in addition to biofilm-specific genes that can confer a transient protection against antibiotics promoting the development of resistance. Indeed, a striking feature of this pathogen is the extraordinary capacity to develop resistance to nearly all available antibiotics through the selection of chromosomal mutations, evidenced by its outstanding and versatile mutational resistome. This threat is dramatically amplified in chronic infections, driven by the frequent emergence of mutator variants with enhanced spontaneous mutation rates. Thus, this mini review is focused on describing the complex interplay of antibiotic resistance mechanisms in P. aeruginosa biofilms, to provide potentially useful information for the design of effective therapeutic strategies.

Immunological profile of two canine breeds in an endemic region of Leishmania infantum.

Visceral leishmaniosis is the one of the most important protozoal zoonoses in Europe, and it is caused by Leishmania infantum, an intracellular protozoan parasite. The disease is endemic in dogs in the Mediterranean area. The main goal of this work is to correlate the levels of several cytokines linked to immune response against L. infantum infection in two canine breeds. Thirty-one Boxer and twenty-eight Ibizan Hound dogs living in the Valencian Community (East coast of Spain) were analyzed for the presence of anti-Leishmania antibodies in serum by IFAT test. Cytokines IFN-γ, TNF-α, IL-2, IL-6, IL-8, and IL-18 were determined by ELISA commercial tests. The levels of IFN-γ, IL-2, and IL-18 in our study, cytokines linked to a cellular immune response, were higher (p < 0.05) in the Ibizan Hound breed; IL-6 levels were higher, although not significant, and only levels of IL-8 were higher in Boxer than in Ibizan Hound. No expression of TNF-α was found. These results corroborate that Ibizan Hound can develop a protective response against canine leishmaniosis, while Boxer is a susceptible breed. The study of immunological aspects in the different canine breeds may represent a useful tool in the prediction of the disease.

Genetic Characterization of Non-Lymphogranuloma venereum Chlamydia trachomatis Indicates Distinct Infection Transmission Networks in Spain.

Chlamydia trachomatis infection is an important public health problem. Our objective was to assess the dynamics of the transmission of this infection, analysing the distribution of circulating ompA genotypes and multilocus sequence types of C. trachomatis in Spain as a function of clinical and epidemiological variables. During 2018 and 2019, we genetically characterized C. trachomatis in tertiary hospitals in six areas in Spain (Asturias, Barcelona, Gipuzkoa, Mallorca, Seville and Zaragoza), with a catchment population of 3.050 million people. Genotypes and sequence types were obtained using polymerase chain reaction techniques that amplify a fragment of the ompA gene, and five highly variable genes (hctB, CT058, CT144, CT172 and pbpB), respectively. Amplicons were sequenced and phylogenetic analysis was conducted. We obtained genotypes in 636/698 cases (91.1%). Overall and by area, genotype E was the most common (35%). Stratifying by sex, genotypes D and G were more common among men, and genotypes F and I among women (p < 0.05). Genotypes D, G and J were more common in men who have sex with men (MSM) than in men who have sex with women (MSW), in whom the most common genotypes were E and F. The diversity index was higher in sequence typing (0.981) than in genotyping (0.791), and the most common sequence types were ST52 and ST108 in MSM, and ST30, ST148, ST276 and ST327 in MSW. Differences in genotype distribution between geographical areas were attributable to differences in population characteristics. The transmission dynamics varied with sexual behaviour: the predominant genotypes and most frequent sequence types found in MSM were different to those detected in MSW and women.

Predictors of success/failure in the control of asthmatic smoking patients under conditions of clinical practice.

BACKGROUND: Tobacco smoking directly affects the airway, where it triggers a very strong local inflammatory response. OBJECTIVE: To determine the predictors of improvement or worsening of asthma control in asthmatic smokers. METHODS: Observational, prospective, multicenter, single cohort study, carried out in the outpatient pulmonology departments with a follow-up period of 6 months. The treatment was adjusted according to the indications of standard clinical practice. RESULTS: 196 patients were included, with a mean age of 54.64 years.39% of the patients were active smokers. Interpreting an Asthma Control Questionnaire (ACQ) score of ≤ 0.75 as asthma control, this was achieved in 30.2% of the cases. Patients with greater adherence were more likely to improve their asthma symptoms (p < 0.05), defined as a decrease in ACQ of 0.5 points or more at the final visit, while taking concomitant medication was a negative risk factor for improvement (p < 0.001). An eosinophil value >300 was a predictor for achieving control (p < 0.01). Patients treated with fluticasone propionate/formoterol versus those receiving budesonide/formoterol or beclomethasone/formoterol had a lower ACQ score (p < 0.01 and p < 0.01, respectively). CONCLUSION: Asthmatic patients with active tobacco exposure and a higher number of anti-asthma medications are more likely to have poorer control. Correct adherence to treatment is the main intervention to be performed to achieve the control. An eosinophil count greater than 300 was the main predictor for achieving control. Fluticasone propionate/formoterol FP/FORM was associated with a greater likelihood of improving ACQ score.

Prevalence of the fimbrial operon mrkABCD, mrkA expression, biofilm formation and effect of biocides on biofilm formation in carbapenemase-producing Klebsiella pneumoniae isolates belonging or not belonging to high-risk clones.

BACKGROUND: The role of mrkA adhesin expression, biofilm production, biofilm viability and biocides in the biofilm of carbapenemase-producing Klebsiella pneumoniae isolates was investigated. METHODS: Seventeen isolates representing different sequence types and carbapenemases were investigated. mrkA expression was determined by real-time reverse transcription polymerase chain reaction. Biofilm production (25°C and 37°C, with and without humidity) was determined by the crystal violet assay. The effect of isopropanol, povidone-iodine, sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, ethanol and triclosan on biofilm was determined. The effect of povidone-iodine on biofilm biomass and thickness was also determined by confocal laser scanning microscopy. RESULTS: mrkA expression ranged from 28.2 to 1.3 [high or intermediate level; 64% of high-risk (HR) clones] and from 21.5 to 1.3 (50% of non-HR clones). At 25°C, biofilm formation was observed in 41% of isolates (absence of humidity) and 35% of isolates (presence of humidity), whereas at 37°C, biofilm formation was observed in 76% of isolates with and without humidity. At 25°C, biofilm producers were more frequently observed in HR clones (45% with humidity and 55% without humidity) than non-HR clones (17% with and without humidity). Biofilm viability from day 21 was higher at 25°C than 37°C. The greatest decrease in biofilm formation was observed with povidone-iodine (29% decrease), which also decreased biofilm thickness. CONCLUSIONS: Biofilm formation in carbapenemase-producing K. pneumoniae is related to mrkA expression. Biofilm formation is affected by temperature (37°C>25°C), whereas humidity has little effect. Biofilm viability is affected by temperature (25°C>37°C). At 25°C, HR clones are more frequently biofilm producers than non-HR clones. Povidone-iodine can decrease biofilm production and biofilm thickness.

SARS-CoV-2 Vaccination Coverage and Factors Associated with Low Uptake in a Cohort of People Living with HIV.

People living with HIV (PLWH) are prioritised for SARS-CoV-2 vaccination due to their vulnerability to severe COVID-19. Therefore, the epidemiological surveillance of vaccination coverage and the timely identification of suboptimally vaccinated PLWH is vital. We assessed SARS-CoV-2 vaccination coverage and factors associated with under-vaccination among PLWH in Catalonia, Spain. As of 11.12.2021, 9945/14942 PLWH (66.6%) had received ≥1 dose of a SARS-CoV-2 vaccine. Non-Spanish origin (adjusted odds ratio (aOR) 0.64, 95% CI 0.59−0.70), CD4 count of 200−349 cells/µL (aOR 0.74, 95% CI 0.64−0.86) or 350−499 cells/µL (aOR 0.79, 95% CI 0.70−0.88), detectable plasma HIV-RNA (aOR 0.61 95% CI 0.53−0.70), and previous SARS-CoV-2 diagnosis (aOR 0.58 95% CI 0.51−0.65) were associated with under-vaccination. SARS-CoV-2 diagnosis (437 [9.5%] vs. 323 [3.5%], p < 0.001), associated hospitalisations (10 [2.3%] vs. 0 [0%], p < 0.001), intensive care unit admissions (6 [1.4%] vs. 0 [0%], p < 0.001), and deaths (10 [2.3%] vs. 0 [0%], p < 0.001) were higher among unvaccinated PLWH. Vaccination coverage was lower among PLWH with a CD4 count >200 cells/µL, detectable plasma HIV-RNA, previous SARS-CoV-2 diagnosis, and migrants. SARS-CoV-2 diagnosis, associated hospitalisations, and deaths among PLWH were lower among the vaccinated compared with the unvaccinated. SARS-CoV-2 vaccination prioritisation has not completely reached vulnerable PLWH with poorer prognosis. This information can be used to inform public health strategies.

Nivel de Actividad Física en supervivientes de cáncer de mama españolas. Aplicación del HUNT 1-Physical Activity Questionnaire / Nível de Atividade Física em sobreviventes espanholas de cancro de mama. Aplicação do HUNT 1-Physical Activity Questionnaire / Physical Activity Levels in Spanish Breast Cancer Survivors. Implementation of the HUNT1-Physical Activity Questionnaire

Objetivo: El objetivo de este estudio fue analizar los niveles de actividad física en supervivientes de cáncer de mama españolas a través de un cuestionario autoadministrado. Asimismo, se estudió la relación entre variables personales y clínicas, variables del entrenamiento y calidad de vida.Método: La presente investigación es de tipo no experimental, descriptiva y transversal. De una muestra significativa (n=386) de mujeres supervivientes de cáncer de mama, se registraron, mediante encuesta autoadministrada: datos antropométricos, sociodemográficos y clínicos; niveles de actividad física mediante cuestionario HUNT1-Physical Activity Questionnaire; y calidad de vida, mediante cuestionario específico para cáncer de mama Functional Assessment Cancer Theraphy-Breast (FACT-B).Resultados: El patrón de entrenamiento más habitual fue de 2-3 veces por semana (40.9%), a intensidad ligera (41.7%) en sesiones de 30-60 minutos (51%). La puntuación media en calidad de vida fue de 93±20 puntos (escala 0-148). El índice de masa corporal resultó influir tanto en nivel de actividad física, como en calidad de vida (p<0.001). Respecto a variables clínicas, se encontró relación entre administración de quimioterapia y frecuencia de entrenamiento (p<0.05); estadio de diagnóstico con puntuación total FACT-B (p<0.05); y existencia de comorbilidades sobre subescala física del test de calidad de vida (p<0.05).Conclusiones: Los resultados indicaron que la mayoría de supervivientes de cáncer de mama españolas no cumplen actualmente los niveles de actividad física recomendados. Del mismo modo sugieren que, aunque el estadio de diagnóstico no parece ser determinante en dicho hábito, sí afecta a su calidad de vida. (AU)

Objective: The objective of this study was to analyze the levels of physical activity in Spanish breast cancer survivors through a self-administered questionnaire, and to assess the relationship between personal and clinical variables, training variables and quality of life.Methods: The present research is non-experimental, descriptive and transversal. From a significant sample (n=386) of breast cancer survivors, the following were recorded by self-administered survey: anthropometric, sociodemographic and clinical data; physical activity levels by means of the HUNT1-Physical Activity Questionnaire; and quality of life, by means of the specific Functional Assessment Cancer Theraphy-Breast (FACT-B).Results: The most common training pattern was 2-3 times per week (40.9%), at light intensity (41.7%) in 30-60 minutes sessions (51%). The average quality of life score was 93±20 points (scale 0-148). The body mass index was found to influence both the level of physical activity and quality of life (p<0.001). Regarding clinical variables, we found a relationship between chemotherapy administration and training frequency (p<0.05); diagnostic stage with FACT-B total score (p<0.05); and existence of comorbidities on the physical subscale of the quality of life test (p<0.05).Conclusions: The results indicated that the majority of Spanish breast cancer survivors do not currently meet the recommended levels of physical activity. They also suggest that, although the stage of diagnosis does not seem to be a determining factor in this habit, it does affect their quality of life. (AU)

Objectivo: O objectivo deste estudo foi analisar os níveis de actividade física dos sobreviventes espanhóis do cancro da mama através de um questionário auto-administrado e avaliar a relação entre as variáveis pessoais e clínicas, as variáveis de treino e a qualidade de vida.Métodos: A presente investigação é não experimental, descritiva e transversal. A partir de uma amostra significativa (n=386) de sobreviventes de cancro da mama, foram registados, por inquérito auto-administrado: dados antropométricos, sociodemográficos e clínicos; níveis de actividade física, através do questionário HUNT1-Physical Activity Questionnaire; e qualidade de vida, através do questionário específico Functional Assessment Cancer Theraphy-Breast (FACT-B).Resultados: O padrão de treino mais comum foi 2-3 vezes por semana (40.9%), com intensidade luminosa (41.7%) em sessões de 30-60 minutos (51%). A pontuação média da qualidade de vida foi de 93±20 pontos (escala 0-148). O índice de massa corporal influenciou tanto o nível de actividade física como a qualidade de vida (p<0.001). Em relação às variáveis clínicas, encontramos uma relação entre a administração da quimioterapia e a frequência do treino (p<0.05); fase de diagnóstico com pontuação total FACT-B (p<0.05); e existência de comorbidades na subescala física do teste de qualidade de vida (p<0.05).Conclusões: Os resultados indicaram que a maioria dos sobreviventes espanhóis de cancro da mama não atinge actualmente os níveis recomendados de actividade física. Sugerem também que, embora a fase do diagnóstico não pareça ser um factor determinante neste hábito, ela afecta a sua qualidade de vida. (AU)

Rational Design of Simple Organocatalysts for the HSiCl3 Enantioselective Reduction of (E)-N-(1-Phenylethylidene)aniline.

Prolinamides are well-known organocatalysts for the HSiCl3 reduction of imines; however, custom design of catalysts is based on trial-and-error experiments. In this work, we have used a combination of computational calculations and experimental work, including kinetic analyses, to properly understand this process and to design optimized catalysts for the benchmark (E)-N-(1-phenylethylidene)aniline. The best results have been obtained with the amide derived from 4-methoxyaniline and the N-pivaloyl protected proline, for which the catalyzed process is almost 600 times faster than the uncatalyzed one. Mechanistic studies reveal that the formation of the component supramolecular complex catalyst-HSiCl3-substrate, involving hydrogen bonding breaking and costly conformational changes in the prolinamide, is an important step in the overall process.

Lumbar disc herniation surgery: Is it worth adding interspinous spacer or instrumented fusion with regard to disc excision alone?

BACKGROUND: Discectomy is sometimes associated with recurrence of disc herniation and pain after surgery. The evidence to use an interspinous dynamic stabilization system or instrumented fusion in association with disc excision to prevent pain and re-operation remains controversial. In this study, we analyzed if adding interspinous spacer or fusion, offers advantages in relation to microdiscetomy alone. METHODS: Patients with lumbar disc herniation were divided in 3 groups; microdiscectomy alone (MD), microdiscectomy plus interspinous spacer (IS) and open discectomy plus posterior lumbar interbody fusion (PLIF). The clinical efficacy was measured using the Owestry Disability Index (ODI). Other outcome parameters including visual analogue scale for pain (VAS) back and legs, length of stay, direct in-hospital cost, 90-day complication rate, and 1-year re-operation rate were also evaluated. RESULTS: A total of 103 patients whose mean age was 39.1 (±8.5) years were included. A significant improvement of the ODI and VAS back and legs pain baseline score was detected in the 3 groups. After 1 year, no significant differences in ODI, VAS back and legs pain were found between the 3 groups. There was an increase of 169% of the total direct in- hospital cost in IS group and 287% in PLIF group, in relation to MD (p < 0.001). Length of stay was 86% higher in the IS group and 384% longer in the PLIF group compared to MD (p < 0.001). The 1 year re-operation rates were 5.6%, 10% and 16.2% (p = 0.33). Discectomy seems to be the main responsible for the clinical improvement, without the interspinous spacer or fusion adding any benefit. The addition of interspinous spacer or fusion increased direct in-hospital cost, length of stay, and did not protect against re-operation.

Synergy between supported ionic liquid-like phases and immobilized palladium N-heterocyclic carbene-phosphine complexes for the Negishi reaction under flow conditions.

The combination of supported ionic liquids and immobilized NHC-Pd-RuPhos led to active and more stable systems for the Negishi reaction under continuous flow conditions than those solely based on NHC-Pd-RuPhos. The fine tuning of the NHC-Pd catalyst and the SILLPs is a key factor for the optimization of the release and catch mechanism leading to a catalytic system easily recoverable and reusable for a large number of catalytic cycles enhancing the long-term catalytic performance.

Treatment outcome in dually HIV-1 and HIV-2 coinfected patients living in Spain.

BACKGROUND: Whereas HIV-1 has spread globally, HIV-2 is mainly found in West Africa where dual HIV-1/HIV-2 coinfection is nowadays uncommon. Herein, we report the rate, main characteristics, and treatment outcomes of all dually infected patients living in Spain. METHODS: We identified retrospectively all persons coinfected with HIV-1 recorded at the Spanish HIV-2 registry. Dual infection had been confirmed using PCR in plasma and/or cells, and/or using discriminatory serological tests. RESULTS: From a total of 373 individuals with HIV-2 recorded at the Spanish registry, 34 (9.1%) were coinfected with HIV-1. Compared with HIV-2 monoinfected persons, dually infected patients were more often male (67.6%), presented with lower median CD4 cell counts (204 cells/µl), and had developed more frequently AIDS events (26.5%). Although 61.7% came from West Africa, 6 (17.6%) were native Spaniards. HIV-1 non-B subtypes were recognized in 75% of coinfected patients, being the most prevalent CRF02_AG. At baseline, 45% of dually infected patients had undetectable plasma HIV-2 RNA. After a median follow-up of 32 (13-48) months on antiretroviral therapy, dually infected patients achieved undetectable viremia in 85% for HIV-1, in 80% for HIV-2; and in 70% for both viruses. Median CD4 cell counts reached up to 418 cells/µl. CONCLUSION: Roughly 9% of individuals with HIV-2 infection living in Spain are coinfected with HIV-1. Overall, 70% of dually infected patients achieved viral suppression for both viruses under antiretroviral therapy. Given the relatively large population of West Africans living in Spain and the continuous migration flow from HIV-2 endemic areas, HIV-1/HIV-2 coinfection should always be excluded at first diagnosis in all HIV-seroreactive persons.

Whole-genome characterization and resistance-associated substitutions in a new HCV genotype 1 subtype.

Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using phylogenetic analysis and analysis of genetic distances in sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing subtypes. The whole genome was analyzed to confirm that the new isolate could be classified as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, which could limit the use of some inhibitors for treating this subtype. RAS studies of new subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are reported. In our case, the patient has not yet been treated, and the RAS report will be used to design the most effective treatment.

Social Behavior of Antibiotic Resistant Mutants Within Pseudomonas aeruginosa Biofilm Communities.

The complex spatial structure and the heterogeneity within biofilms lead to the emergence of specific social behaviors. However, the impact of resistant mutants within bacterial communities is still mostly unknown. Thus, we determined whether antibiotic resistant mutants display selfish or altruistic behaviors in mixed Pseudomonas aeruginosa biofilms exposed to antibiotics. ECFP-tagged P. aeruginosa strain PAO1 and its EYFP-tagged derivatives hyperproducing the ß-lactamase AmpC or the efflux pump MexAB-OprM were used to develop single or mixed biofilms. Mature biofilms were challenged with different concentrations of ß-lactams to monitor biofilm structural dynamics, using confocal laser scanning microscopy (CLSM), and population dynamics, through enumeration of viable cells. While exposure of single wild-type PAO1 biofilms to ß-lactams lead to a major reduction in bacterial load, it had little effect on biofilms formed by the resistant mutants. However, the most reveling finding was that bacterial load of wild-type PAO1 was significantly increased when growing in mixed biofilms compared to single biofilms. In agreement with CFU enumeration data, CLSM images revealed the amplification of the resistant mutants and their protection of susceptible populations. These findings show that mutants expressing diverse resistance mechanisms, including ß-lactamases, but also, as evidenced for the first time, efflux pumps, protect the whole biofilm community, preserving susceptible populations from the effect of antibiotics. Thus, these results are a step forward to understanding antibiotic resistance dynamics in biofilms, as well as the population biology of bacterial pathogens in chronic infections, where the coexistence of susceptible and resistant variants is a hallmark.

Ceftolozane/tazobactam for the treatment of multidrug resistant Pseudomonas aeruginosa: experience from the Balearic Islands.

A prospective, descriptive observational study of consecutive patients treated with ceftolozane/tazobactam in the reference hospital of the Balearic Islands (Spain), between May 2016 and September 2017, was performed. Demographic, clinical, and microbiological variables were recorded. The later included resistance profile, molecular typing, and whole genome sequencing of isolates showing resistance development. Fifty-eight patients were treated with ceftolozane/tazobactam. Thirty-five (60.3%) showed respiratory tract infections, 21 (36.2%) received monotherapy, and 37 (63.8%) combined therapy for ≥ 72 h, mainly with colistin (45.9%). In 46.6% of the patients, a dose of 1/0.5 g/8 h was used, whereas 2/1 g/8 h was used in 41.4%. In 56 of the cases (96.6%), the initial Pseudomonas aeruginosa isolates recovered showed a multidrug resistant (MDR) phenotype, and 50 of them (86.2%) additionally met the extensively drug resistant (XDR) criteria and were only susceptible colistin and/or aminoglycosides (mostly amikacin). The epidemic high-risk clone ST175 was detected in 50% of the patients. Clinical cure was documented in 37 patients (63.8%) and resistance development in 8 (13.8%). Clinical failure was associated with disease severity (SOFA), ventilator-dependent respiratory failure, XDR profile, high-risk clone ST175, negative control culture, and resistance development. In 6 of the 8 cases, resistance development was caused by structural mutations in AmpC, including some mutations described for the first time in vivo, whereas in the other 2, by mutations in OXA-10 leading to the extended spectrum OXA-14. Although further clinical experience is still needed, our results suggest that ceftolozane/tazobactam is an attractive option for the treatment of MDR/XDR P. aeruginosa infections.

Diagnóstico microbiológico de las infecciones relacionadas con la formación de biopelículas / Microbiological diagnosis of biofilm-related infections

Las infecciones asociadas a biopelículas suponen un grave problema sanitario ya que representan entre el 65 y el 80% de todas las infecciones. Estas son generalmente crónicas y están caracterizadas por la persistencia del microorganismo debido a su resistencia al sistema inmunitario y a los antimicrobianos. Las biopelículas se pueden localizar tanto en tejidos humanos como sobre dispositivos exógenos tales como catéteres, marcapasos, prótesis, implantes, sondas urinarias, etc. Tradicionalmente, los laboratorios de microbiología clínica realizan los estudios de sensibilidad sobre microorganismos en crecimiento planctónico. Sin embargo, de esta manera se pierden las características propias de la biopelícula con lo que la antibioterapia basada en estos estudios podría asociarse con fracaso terapéutico o recurrencias. El diagnóstico microbiológico y los estudios de sensibilidad en las infecciones relacionadas con biopelículas son complejos y, hoy por hoy, representan un reto que clínicos y microbiólogos han de abordar en equipo ya que no existe todavía un consenso global ni protocolos estandarizados

Biofilm-related infections represent a serious health problem, accounting for 65- 80% of all infections. The infections are generally chronic and characterized by the persistence of the microorganism, due to the increased resistance of biofilms to both the immune system and antimicrobials. Biofilms can be located to almost every human body tissue and on exogenous devices such as catheters, pacemakers, prosthetic material, implants, urinary catheters, etc. Traditional antimicrobial susceptibility studies in clinical microbiology laboratories have lied on the study of planktonic form of microorganisms. However, this approach might lead to miss the biofilm characteristics and to a treatment failure. Microbiological diagnosis and antimicrobial susceptibility studies of biofilm-related infections are complex and, nowadays, represent a challenge that clinicians and microbiologists have to address as a team in the absence of consensus or standardized protocols

Microbiological diagnosis of biofilm-related infections. / Diagnóstico microbiológico de las infecciones relacionadas con la formación de biopelículas.

Biofilm-related infections represent a serious health problem, accounting for 65- 80% of all infections. The infections are generally chronic and characterized by the persistence of the microorganism, due to the increased resistance of biofilms to both the immune system and antimicrobials. Biofilms can be located to almost every human body tissue and on exogenous devices such as catheters, pacemakers, prosthetic material, implants, urinary catheters, etc. Traditional antimicrobial susceptibility studies in clinical microbiology laboratories have lied on the study of planktonic form of microorganisms. However, this approach might lead to miss the biofilm characteristics and to a treatment failure. Microbiological diagnosis and antimicrobial susceptibility studies of biofilm-related infections are complex and, nowadays, represent a challenge that clinicians and microbiologists have to address as a team in the absence of consensus or standardized protocols.

Antibiotic treatment of biofilm infections.

Bacterial biofilms are associated with a wide range of infections, from those related to exogenous devices, such as catheters or prosthetic joints, to chronic tissue infections such as those occurring in the lungs of cystic fibrosis patients. Biofilms are recalcitrant to antibiotic treatment due to multiple tolerance mechanisms (phenotypic resistance). This causes persistence of biofilm infections in spite of antibiotic exposure which predisposes to antibiotic resistance development (genetic resistance). Understanding the interplay between phenotypic and genetic resistance mechanisms acting on biofilms, as well as appreciating the diversity of environmental conditions of biofilm infections which influence the effect of antibiotics are required in order to optimize the antibiotic treatment of biofilm infections. Here, we review the current knowledge on phenotypic and genetic resistance in biofilms and describe the potential strategies for the antibiotic treatment of biofilm infections. Of note is the optimization of PK/PD parameters in biofilms, high-dose topical treatments, combined and sequential/alternate therapies or the use antibiotic adjuvants.