Disseminação de Klebsiella pneumoniae multirresistente: produção de biofilme, avaliação da virulência e do fitness bacteriano em amostras clínicas produtoras de KPC

Klebsiella pneumoniae is often involved with healthcare-associated infections (HAI) in Brazil, and has a great ability to develop or acquire antimicrobial resistance. This study investigated the clonal dissemination and prevalence of sequence types (STs) in clinical strains of K. pneumoniae carrying blaCTX-M e blaKPC genes, as well as the presence of genes encoding virulence factors and the ability of these strains to produce biofilms. In addition, the impact of carbapenem and polymyxin-resistance on bacterial fitness of carbapenemase-producing K. pneumoniae (KPC) strains was also examined. We randomly selected non-duplicated K. pneumoniae isolates from a collection recovered from inpatients at the Clinical Hospital of the Federal University of Uberlândia (HC-UFU) from June 2009 to July 2015. The study included broad-spectrum cephalosporin and/or carbapenems-resistant strains. For investigation of the blaCTX-M and blaKPC resistance genes and its association with virulence genes (fimH, fimA, wabG, iucC, rmpA, ecpA, mrkD e khe), the strains were evaluated by PCR. DNA sequencing was performed to confirm the presence of blaKPC-2 gene. The pulsotypes, STs and clonal complexes (CCs) were determined by PFGE and MLST, respectively. Initial adhesion and biofilm formation were examined by quantitative assays and the results confirmed by scanning electron microscopy. For fitness evaluation, in vitro pairwise competition experiments were carried out using three strains of K. pneumoniae: one resistant to carbapenem, harboring 5 of the 8 evaluated virulence genes, another less virulent, but resistant to carbapenem and polymyxin, and ATCC 10031 multisensitive strain. For epidemiological evaluations, sixty K. pneumoniae strains were randomly selected, of which 30 carbapenem-sensitive (KpSC) strains isolated from infections; 30 carbapenem-resistant K. pneumoniae (KpRC) strains, 20 isolated from infections and 10 from colonizations. Significant differences were found when patients infected by KpSC and KpRC were compared, especially the high previous use of β-lactams (53%), carbapenems (73.3%) and polymyxin B (43.3%). Inadequate therapy, although prevalent in 56.7% of the patients, was much higher (70%) among those with KpRC infections. In total, 75% of the strains were characterized as multiresistant. Furthermore, we observed high consumption of cefepime, ceftriaxone and carbapenems by defined daily doses analysis, with an upward trend between the beginning and the end of the period of study. Regarding the presence of resistance genes, 80% of strains carried blaCTX-M gene and 100% of carbapenem-resistant isolates the blaKPC-2 gene. Virulence genes were detected with high frequencies in both groups, unrelated to carbapenem resistance. However, among the KpRC strains, the fimH, fimA and wabG genes were predominant (83%). It was observed polyclonal dissemination of strains with predominance of MLST STs 11 and 340, belonging to the clonal complex 258. All K. pneumoniae strains evaluated could adhere to an unmodified polystyrene surface. However, the statistical analysis showed that three strains had remarkably higher adhesion rates than the others (p<0.001). Compared to control, all strains had the ability to produce biofilm, without significant differences. However, the evaluation of biomass by crystal violet showed that 60% of the isolates were weak biofilm producers, results confirmed by scanning electron microscopy. According to the fitness results, the polymyxin-resistant strain was found to have more significant growth rates (p=0.030) and, in competition experiments between the two multiresistant clinical strains, the carbapenem-resistant strain showed a significantly lower fitness cost when compared to the carbapenem and polymyxin-resistant strain. The knowledge of the virulence factors and the pathogenic potential of ESBL and carbapenemases-producing K. pneumoniae contributes to a better understanding of colonization, pathogenicity and persistence of these microorganisms in the hospital environment and can provide tools to improve the treatment of serious infections as well as subsidies to improve infection control and prevention measures.FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas GeraisTese (Doutorado)Klebsiella pneumoniae é um agente etiológico frequente em infecções relacionadas à assistência à saúde no Brasil e possui uma grande capacidade de desenvolver ou adquirir resistência aos antimicrobianos. Este estudo investigou a disseminação clonal e os tipos de sequência (STs) predominantes em amostras clínicas de K. pneumoniae apresentando genes blaCTX-M e blaKPC, bem como a presença de genes para fatores de virulência e a capacidade de produção de biofilmes. Adicionalmente, também foi examinado o impacto das resistências aos carbapenêmicos e polimixina no fitness bacteriano de K. pneumoniae produtoras de carbapenemase (KPC). Para as avaliações epidemiológicas foram selecionadas randomicamente 60 amostras não-duplicadas de K. pneumoniae, sendo 30 amostras de infecção K. pneumoniae sensível aos carbapenêmicos (KpSC), 20 infecções e 10 colonizações por K. pneumoniae resistente aos carbapenêmicos (KpRC). As amostras foram selecionadas a partir de uma coleção recuperada de pacientes internados no Hospital de Clínicas da Universidade Federal de Uberlândia (HC-UFU) no período de junho de 2009 a julho de 2015. Para investigação dos genes de resistência blaCTX-M e blaKPC e genes de virulência fimH, fimA, wabG, iucC, rmpA, ecpA, mrkD e khe, as amostras foram avaliadas por reação em cadeia da polimerase (PCR). A confirmação de blaKPC-2 foi realizada por sequenciamento genético. Os pulsotipos, STs e complexos clonais foram determinados por Pulsed Field Gel Electrophoresis (PFGE) e Multilocus Sequence Typing (MLST). A adesão inicial e a formação de biofilme foram examinadas por ensaios quantitativos, e os resultados confirmados por microscopia eletrônica de varredura. Para avaliação do fitness foram utilizados experimentos de competição em pares, in vitro, utilizando três amostras de K. pneumoniae: uma resistente aos carbapenêmicos, contendo 5 dos 8 genes de virulência avaliados, uma resistente aos carbapenêmicos e polimixina, menos virulenta, e a amostra ATCC 10031 multissensível. Diferenças importantes foram encontradas na caracterização dos grupos KpSC e KpRC, destacando-se o elevado uso prévio de β-lactâmicos (53%), carbapenêmicos (73,3%) e polimixina B (43,3%) naqueles infectados por amostras resistentes. A terapia inadequada embora tenha sido prevalente em 56,7% dos casos, foi bem maior (70%) entre pacientes com infecções por KpRC. No total, 75% das amostras foram caracterizadas como multirresistentes. Além disso, foi observado alto consumo em DDD de cefepime, ceftriaxona e carbapenêmicos, com tendência ascendente entre o início e o final do período estudado. Em relação à presença de genes de resistência, 80% das amostras apresentaram o gene blaCTX-M e 100% das amostras com resistência aos carbapenêmicos possuíam o gene blaKPC. Os genes de virulência foram detectados com frequências elevadas em ambos os grupos, sem relação com a resistência aos carbapenêmicos. Entretanto, entre as amostras de KpRC, os genes fimH, fimA e wabG foram predominantes (83%). Observou-se disseminação policlonal das amostras com predominância dos STs 11 e 340, pertencentes ao complexo clonal 258. Todas as amostras de K. pneumoniae avaliadas foram capazes de aderir a uma superfície de poliestireno não modificada. No entanto, a análise estatística evidenciou que três amostras apresentaram taxas de adesão notavelmente mais altas do que as demais (p<0.001). Comparados ao controle, todas as amostras apresentaram a habilidade de produzir biofilme, sem diferenças significativas. No entanto, a avaliação da biomassa por cristal violeta evidenciou que 60% das amostras eram produtoras fracas de biofilme, resultado confirmado pela microscopia eletrônica de varredura. De acordo com os resultados do fitness, observou-se que a amostra resistente à polimixina apresentou taxas de crescimento mais significativas (p=0,030) e na competição entre as duas amostras clínicas multirresistentes, a amostra resistente ao carbapenêmico mostrou custo de fitness significativamente menor quando comparado à amostra resistente aos carbapenêmicos e à polimixina. O conhecimento dos fatores de virulência e o potencial patogênico de K. pneumoniae produtoras de ESBL e carbapenemases contribui para melhor compreensão da colonização, patogenicidade e persistência desses micro-organismos no ambiente hospitalar e pode fornecer ferramentas para melhorar o tratamento de infecções graves, assim como subsídios para aprimorar as medidas de controle e prevenção de infecções

Uncontrolled headache induced by oxcarbazepine

Headache induced by acute exposure to a specific drug constitutes an idiosyncratic side effect. Metabolic imbalance appears as the leading aetiology, among several other hypotheses. Either primary headaches show a higher susceptibility to this idiosyncratic reaction or a drug-induced primary headache evolves in intensity and duration, becoming uncontrolled until the complete discontinuation of the drug in consideration. The goal of this study is to describe three patients diagnosed with migraine and epilepsy (both under control) who evolved into status migrainosus after the introduction of oxcarbazepine (OXC), as part of a switch off from carbamazepine (CBZ). Twenty-four to seventy-two hours following the switch, all patients developed intractable headache, despite the use of different symptomatic drugs. Complete recovery of the headache symptoms occurred only after OXC was discontinued. We discuss the potential mechanisms associated to OXC and status migrainosus, drug-induced headaches and uncontrolled headaches

Exploring the staphylococcus aureus in patients infected of the tertiary-care university hospital: results of the retrospective cohort study

Objective: To establish a baseline of knowledge regarding about inappropriate therapy, virulence and resistance in a cohort of patients infected with S. aureus. Methods: Retrospective cohort study in tertiary-care university hospital was employed to evaluate the risk factors and the impact of inappropriate therapy among patients with Staphylococcus aureus infections, resistance and virulence. To assess the presence of the genes was performed PCR. Results: Patients with MRSA were older and hospitalized 17 days longer than those with MSSA infection, which were in ICU with a bloodstream infection. 50.0% received inadequate antibiotic therapy and we found virulence factors associated with MRSA (mecA, LukS, fnbB and clfA genes). Conclusion: These data show that surveillance studies related to Staphylococcus aureus infections remain essential to identify resistance and inform policy on resistance

Biofilm formation of Brazilian meticillin-resistant Staphylococcus aureus strains: prevalence of biofilm determinants and clonal profiles

Biofilms plays an important role in medical-device-related infections. This study aimed to determine the factors that influence adherence and biofilm production, as well as the relationship between strong biofilm production and genetic determinants in clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). Fifteen strains carrying different chromosomal cassettes recovered from hospitalized patients were selected; five SCCmecII, five SCCmecIII and five SCCmecIV. The SCCmec type, agr group and the presence of the virulence genes (bbp, clfA, icaA, icaD, fnbB, bap, sasC and IS256) were assessed by PCR. PFGE and multilocus sequence typing (MLST) techniques were also performed. The initial adhesion and biofilm formation were examined by quantitative assays. The surface tension and hydrophobicity of the strains were measured by the contact angle technique to evaluate the association between these parameters and adhesion ability. SCCmecIII and IV strains were less hydrophilic, with a high value for the electron acceptor parameter and higher adhesion in comparison with SCCmecII strains. Only SCCmecIII strains could be characterized as strong biofilm producers. The PFGE showed five major pulsotypes (AE); however, biofilm production was related to the dissemination of one specific PFGE clone (C) belonging to MLST ST239 (Brazilian epidemic clonal complex). The genes agrI, fnbB and IS256 in SCCmecIII strains were considered as genetic determinants associated with strong biofilm-formation by an ica-independent biofilm pathway. This study contributes to the understanding of biofilm production as an aggravating factor potentially involved in the persistence and severity of infections caused by multidrug-resistant MRSA belonging to this genotype.We thank FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais, proceeding APQ 01398-11) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, PDSE proceeding 8952/11-6) for the financial support and scholarships. We also thank Dr Teruyo Ito, Juntendo University, Japan, and Dr Elsa Masae Mamizuka, Universidade de São Paulo, Brazil, for kindly providing the control strains used in this study.info:eu-repo/semantics/publishedVersio

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Perfil epidemiológico de infecção por enterococos resistentes à vancomicina em hospital universitário com alta prevalência de pacientes colonizados

Globally the vancomycin-resistant enterococci (VRE) remains an important cause of infection related to health care. The study aimed to characterize samples of VRE isolated from patients hospitalized during the epidemic and endemic periods, determining the risk of colonized patients developing infection, its relationship with consumption antibiotics and the role of colonization pressure. Additionally, we investigated the presence of virulence determinants in samples recovered from colonization and infection. We conducted longitudinal cohort study of patients colonized and infected by Enterococcus faecium (VREfm) and E. faecalis (VREfc) resistant to vancomycin, by active search for the Microbiology Laboratory of the Clinical Hospital of the Federal University of Uberlândia, during the period of January 2010 to June 2012. The identification and antimicrobial resistance were determined by automated system Vitek®2. A record following the model of NHSN (\"National Healthcare Safety Network\") was completed for each patient, considering clinical, demographic and epidemiological factors. The risk factors were evaluated by univariate analysis and multivariate logistic regression, and Pearson and Spearman tests were used to correlate two variables, the antimicrobial DDD per 1000 patient-days and the number of VRE per 1000 patient-days. Virulence genes asa1, gelE, esp and hyl, and vanA resistance were detected by polymerase chain reaction. Among the 171 patients evaluated, VRE was the most frequent micro-organism (92%). Twenty-two patients (12.9%) developed infection by VRE on average 14 days after colonization with the prevalence of urinary tract infections (72%). Among patients infected most used urinary catheter as the most frequent device (86%). The acquisition rate of VRE was 1.92/1000 patient-days in early August 2010 and the end of January 2011 there were no cases of VREfm, when it was observed the end of the outbreak and the begin of VREfm endemicity in the hospital (0.555 VRE/1000 patient-days). There was a relationship between the temporal and spatial infected patients with evidence of cross-transmission mainly in the Intensive Care Unit for Adults. Only the use of aminoglycosides has been previously considered an independent risk factor for VRE infection (P=0.0013), however, the use of glycopeptides was correlated with the presence of that micro-organism in a hospital (r=0.717, P=0.03). Although the colonization pressure with VRE was high with variations from 0.004 to 1.32% during the 30-month study was not statistically associated with the development of VRE infection. An association was observed between samples with high-level resistance to streptomycin and penicillin and ampicillin resistance in samples VREfm, however, for samples of VREfc, the high level of resistance to gentamicin was more frequently (77%) associated only with penicillin resistance. All samples VREfm carried the vanA gene and were resistant to teicoplanin, expressing high levels of resistance to vancomycin (MIC &#8805; 256 &#956;g/ml). The esp gene was the most frequent detected in 82.4% of samples colonization and 76.5% of the clinical samples. We showed high prevalence of VREfm in a tertiary hospital, independently associated with the previous use of aminoglycosides and glycopeptides consumption.Fundação de Amparo a Pesquisa do Estado de Minas GeraisMestre em Imunologia e Parasitologia AplicadasGlobalmente o Enterococo Resistente à Vancomicina (VRE) continua sendo uma causa importante de infecção relacionada à assistência à saúde. O trabalho teve como objetivo caracterizar amostras de VRE isoladas de pacientes hospitalizados durante os períodos epidêmico e endêmico, determinando o risco de pacientes colonizados desenvolver infecção, sua relação com o consumo de antibióticos e o papel da pressão de colonização. Adicionalmente, foi investigada a presença de determinantes de virulência em amostras recuperadas de colonização e infecção. Foi realizado estudo longitudinal de coorte de pacientes colonizados e infectados por Enterococcus faecium (VREfm) e E. faecalis (VREfc) resistentes à vancomicina, por busca ativa no Laboratório de Microbiologia do Hospital de Clínicas da Universidade Federal de Uberlândia, durante o período de janeiro de 2010 a junho de 2012. A identificação e a resistência aos antimicrobianos foram determinadas pelo sistema automatizado Vitek®2. Uma ficha seguindo o modelo do NHSN ( National Healthcare Safety Network ) foi preenchida para cada paciente, considerando fatores demográficos, clínicos e epidemiológicos.Os fatores de risco foram avaliados por análise univariada e regressão logística multivariada, e os testes de Pearson e Spearman foram utilizados para correlacionar duas variáveis, DDD do antimicrobiano por 1000 pacientes-dia e o número de VRE por 1000 pacientes-dia. Os genes de virulência asa1, gelE, esp e hyl, e resistência vanA foram detectados por reação em cadeia da polimerase. Entre os 171 pacientes avaliados, VREfm foi o micro-organismo mais frequente (92%). Vinte e dois pacientes (12,9%) desenvolveram infecção pelo VRE, em média 14 dias após a colonização com a predominância das infecções urinárias (72%). Entre os pacientes infectados a maioria usou sonda vesical como procedimento invasivo mais frequente (86%). A taxa de aquisição do VREfm foi 1,92/1000 pacientes-dia no início de agosto de 2010 e no final de janeiro de 2011 não houve o isolamento de VREfm, quando foi observado o término do surto de VREfm e início da endemicidade no hospital (0,555 VRE/1000 pacientes-dia). Houve relação temporal e espacial entre os pacientes infectados com evidência de transmissão cruzada principalmente na Unidade de Terapia Intensiva de Adultos. Somente o uso prévio de aminoglicosídeos foi considerado fator de risco independente para infecção pelo VRE (P=0,0013); no entanto, o consumo de glicopeptídeos foi correlacionado com a presença desse micro-organismo no hospital (rs= 0,717, P=0,03). Apesar da pressão de colonização por VRE ter sido elevada com variações de 0,004 a 1,32% durante os 30 meses de estudo, não foi relacionada estatisticamente com o desenvolvimento de infecção pelo VRE. Foi observada associação entre as amostras com alto nível de resistência à estreptomicina e resistência à penicilina e ampicilina nas amostras de VREfm, entretanto, para as amostras de VREfc, o elevado nível de resistência à gentamicina foi mais frequente (77%) associado somente com a resistência à penicilina. Todas as amostras de VREfm carreavam o gene vanA e foram resistentes a teicoplanina, expressando elevados níveis de resistência à vancomicina (MIC &#8805; 256 &#956;g/ml). O gene esp foi o mais frequente, detectado em 82,4% das amostras de colonização e em 76,5% das amostras clínicas. Mostramos alta prevalência de VREfm em um hospital terciário, independentemente associado com o uso prévio de aminoglicosídeos e o consumo de glicopeptídeos

The nares as a CA-MRSA reservoir in the healthy elderly

Abstract:INTRODUCTION:The frequency of methicillin-resistant Staphylococcus aureus (MRSA) has increased in the community. This study evaluated the prevalence of MRSA and community-acquired (CA)-MRSA in 120 healthy elderly.METHODS:The MRSA were evaluated for the presence of the IS256, mecA, agr, icaA, icaD, fnbB , and pvl genes with PCR. Results: Frequency of S. aureus and MRSA colonization was 17.8% and 19%, respectively. CA-MRSA isolate showed SCC mec IV, fnbB+ , and icaD+ .CONCLUSIONS:CA-MRSA was detected, with genotype determined as SCC mec type IV/IS256/ fnbB+ / icaA / icaD+ / bbp-/agr2 / bap / pvl, characterizing this population as a possible reservoir of this organism in the community

Spread of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clones in patients with ventilator-associated pneumonia in an adult intensive care unit at a university hospital

Background:In Brazil, ventilator-associated pneumonia (VAP) caused by carbapenem resis- tant Acinetobacter baumanniiand Pseudomonas aeruginosaisolates are associated with signi&#64257;cant mortality, morbidity and costs. Studies on the clonal relatedness of these isolates could lay the foundation for effective infection prevention and control programs.Objectives: We sought to study the epidemiological and molecular characteristics of A. baumannii vs. P. aeruginosaVAP in an adult intensive care unit (ICU).Methods: It was conducted a cohort study of patients with VAP caused by carbapenem resistant A. baumanniiand P'. aeruginosaduring 14 months in an adult ICU. Genomic studies were used to investigate the clonal relatedness of carbapenem resistant OXA-23-producing A. baumanniiand P. aeruginosaclinical isolates. The risk factors for acquisition of VAP were also evaluated. Clinical isolates were collected for analysis as were samples from the environment and were typed using pulsed &#64257;eld gel electrophoresis.Results: Multivariate logistic regression analysis identi&#64257;ed trauma diagnosed at admission and inappropriate antimicrobial therapy as independent variables associated with the development of A. baumanniiVAP and hemodialysis as independent variable associated with P. aeruginosaVAP. All carbapenem resistant clinical and environmental isolates of A. baumanniiwere OXA-23 producers. No MBL-producer P. aeruginosawas detected. Molecular typing revealed a polyclonal pattern; however, clone A (clinical) and H (surface) were the most frequent among isolates of A. baumanniitested, with a greater pattern of resistance than other isolates. In P. aeruginosathe most frequent clone I was multi-sensitive.Conclusion: These &#64257;ndings suggest the requirement of constant monitoring of these microor- ganisms in order to control the spread of these clones in the hospital environment