High-risk clones of carbapenem resistant Klebsiella pneumoniae recovered from pediatric patients in Southern Brazil.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) exhibit high mortality rates in pediatric patients and usually belong to international high-risk clones. This study aimed to investigate the molecular epidemiology and carbapenem resistance mechanisms of K. pneumoniae isolates recovered from pediatric patients, and correlate them with phenotypical data. Twenty-five CRKP isolates were identified, and antimicrobial susceptibility was assessed using broth microdilution. Carbapenemase production and ß-lactamase genes were detected by phenotypic and genotypic tests. Multilocus sequence typing was performed to differentiate the strains and whole-genome sequencing was assessed to characterize a new sequence type. Admission to the intensive care unit and the use of catheters were significantly positive correlates of CRKP infection, and the mortality rate was 36%. Almost all isolates showed multidrug-resistant phenotype, and most frequent resistant gene was blaKPC. We observed the dissemination of ST307 and clones belonging to CG258, which are considered high risk. In pediatric patients, these clones present with high genomic plasticity, favoring adaptation of the KPC and NDM enzymes to healthcare environments.

Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil.

OBJECTIVES: We aimed to investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) in paediatric patients from Hospital Pequeno Príncipe. The susceptibility profile was determined, and whole-genome sequencing (WGS) was used to analyse the genetic context of the strains. METHODS: Five VREfm isolates were recovered from sterile sites and surveillance cultures of two paediatric patients with acute lymphoblastic leukaemia. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the minimum inhibitory concentration (MIC) was assessed according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST). WGS was performed to analyse the genetic context of virulence and resistance genes, and in silico multilocus sequence typing was performed to identify the sequence typing of the strains. RESULTS: High-level vancomycin resistance was observed in all isolates (≥256 mg/L). WGS revealed the presence of mobile genetic elements, such as plasmids (rep2, rep11a, repUS15, rep17, and rep18a), insertion sequences, and phages. Multiple resistance genes (aac(6')-aph(2"), dfrG, ermB, and vanA) and virulence genes (acm and efaAfm) were identified. All the isolates were assigned to ST117 (ST1133 - via a novel MLST), an important epidemic lineage associated with nosocomial infections and outbreaks. CONCLUSION: Our results show that the ST117 (ST1133) VREfm isolates are circulating in paediatric patients, which raises a great concern. The development of new drugs as well as the implementation of an antimicrobial stewardship program are necessary for their correct management, limiting the spread of resistance in oncohematological patients.

Lower airway microbiota and decreasing lung function in young Brazilian cystic fibrosis patients with pulmonary Staphylococcus and Pseudomonas infection.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene that leads to respiratory complications and mortality. Studies have shown shifts in the respiratory microbiota during disease progression in individuals with CF. In addition, CF patients experience short cycles of acute intermittent aggravations of symptoms called pulmonary exacerbations, which may be characterized by a decrease in lung function and weight loss. The resident microbiota become imbalanced, promoting biofilm formation, and reducing the effectiveness of therapy. The aim of this study was to monitor patients aged 8-23 years with CF to evaluate their lower respiratory microbiota using 16S rRNA sequencing. The most predominant pathogens observed in microbiota, Staphylococcus (Staph) and Pseudomonas (Pseud) were correlated with clinical variables, and the in vitro capacity of biofilm formation for these pathogens was tested. A group of 34 patients was followed up for 84 days, and 306 sputum samples were collected and sequenced. Clustering of microbiota by predominant pathogen showed that children with more Staph had reduced forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) compared to children with Pseud. Furthermore, the patients' clinical condition was consistent with the results of pulmonary function. More patients with pulmonary exacerbation were observed in the Staph group than in the Pseud group, as confirmed by lower body mass index and pulmonary function. Additionally, prediction of bacterial functional profiles identified genes encoding key enzymes involved in virulence pathways in the Pseud group. Importantly, this study is the first Brazilian study to assess the lower respiratory microbiota in a significant group of young CF patients. In this sense, the data collected for this study on the microbiota of children in Brazil with CF provide a valuable contribution to the knowledge in the field.

First Genome Sequences of Two Multidrug-Resistant Candida haemulonii var. vulnera Isolates From Pediatric Patients With Candidemia.

Candida haemulonii is a complex formed by C. haemulonii sensu stricto, C. haemulonii var. vulnera, and C. duobushaemulonii. Members of this complex are opportunistic pathogens closely related to C. pseudohaemulonii, C. lusitaniae, and C. auris, all members of a multidrug-resistant clade. Complete genome sequences for all members of this group are available in the GenBank database, except for C. haemulonii var. vulnera. Here, we report the first draft genomes of two C. haemulonii var. vulnera (isolates K1 and K2) and comparative genome analysis of closely related fungal species. The isolates were biofilm producers and non-susceptible to amphotericin B and fluconazole. The draft genomes comprised 350 and 387 contigs and total genome sizes of 13.21 and 13.26 Mb, with 5,479 and 5,507 protein-coding genes, respectively, allowing the identification of virulence and resistance genes. Comparative analyses of orthologous genes within the multidrug-resistant clade showed a total of 4,015 core clusters, supporting the conservation of 24,654 proteins and 3,849 single-copy gene clusters. Candida haemulonii var. vulnera shared a larger number of clusters with C. haemulonii and C. auris; however, more singletons were identified in C. lusitaniae and C. auris. Additionally, a multiple sequence alignment of Erg11p proteins revealed variants likely involved in reduced susceptibility to azole and polyene antifungal agents. The data presented in this work will, therefore, be of utmost importance for researchers studying the biology of the C. haemulonii complex and related species.

Invasive candidiasis: Risk factor for mortality in a pediatric tertiary care hospital in south of Brazil.

BACKGROUND: Invasive candidiasis (IC) is a major cause of morbimortality in children. Previous studies described the clinical characteristics and risk factors for this infection; however, limited data are available on the predictors of mortality in these patients. In this context, we evaluated the risk factors associated with death due to IC in a pediatric tertiary care hospital in South of Brazil. METHODS: This is a retrospective, cross-sectional, observational, and analytical study of a series of pediatric patients with clinical and laboratory diagnosis of IC from March 2014 to September 2017. Univariate and multivariate analysis were performed to estimate the association between the characteristics of the patients and death. RESULTS: A total of 94 cases of IC were included. The incidence was 1.13 cases per 1000 patients/d, with a mortality rate of 14%. There was a predominance of non-albicans Candida (71.3%) in IC cases and, although there is no species difference in mortality rates, biofilm formation was associated with increased mortality. Clinical characteristics such as male sex, stay in the intensive care unit, and thrombocytopenia; comorbidities such as cardiological disease and renal insufficiency; and risks such as mechanical ventilation and dialysis were associated with increased mortality. CONCLUSION: Data from this study suggest that biofilm formation by Candida sp. is associated with increased mortality, and this is the first study to correlate the male sex and cardiological disease as risk factors for death in pediatric IC patients.

Matrix-assisted laser desorption ionization-time of flight: a promising alternative method of identifying the major coagulase-negative Staphylococci species.

INTRODUCTION.: This study aimed to evaluate different methods for differentiation of species of coagulase-negative staphylococci (CoNS) that caused infections in hospitalized immunocompromised patients. METHODS.: A total of 134 CoNS strains were characterized using four different methods. RESULTS.: The results of matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analysis were in complete agreement with those of tuf gene sequencing (kappa index = 1.00). The kappa index of Vitek 2® Compact analysis was 0.85 (very good) and that of the conventional method was 0.63 (moderate). CONCLUSIONS: . MALDI-TOF MS provided rapid and accurate results for the identification of CoNS (134; 100%).

Matrix-assisted laser desorption ionization-time of flight: a promising alternative method of identifying the major coagulase-negative Staphylococci species

Abstract INTRODUCTION. This study aimed to evaluate different methods for differentiation of species of coagulase-negative staphylococci (CoNS) that caused infections in hospitalized immunocompromised patients. METHODS. A total of 134 CoNS strains were characterized using four different methods. RESULTS. The results of matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analysis were in complete agreement with those of tuf gene sequencing (kappa index = 1.00). The kappa index of Vitek 2® Compact analysis was 0.85 (very good) and that of the conventional method was 0.63 (moderate). CONCLUSIONS . MALDI-TOF MS provided rapid and accurate results for the identification of CoNS (134; 100%).

Stenotrophomonas maltophilia: resistência emergente ao SMX-TMP em isolados brasileiros: uma realidade / Stenotrophomonas maltophilia: emerging resistance to TMP-SMX in Brazilian isolate: a reality

INTRODUÇÃO: Stenotrophomonas maltophilia é um importante patógeno hospitalar emergente, naturalmente resistente aos carbapenêmicos, que possui sulfametoxazol/trimetoprima (SMX-TMP) como opção terapêutica. Porém, relatos de resistência a essa droga começaram a aparecer. OBJETIVO: Avaliar o perfil de sensibilidade das cepas de S. maltophilia e comparar os resultados obtidos por diferentes metodologias. MÉTODOS: As cepas isoladas na rotina microbiológica do Hospital das Clínicas de São Paulo em 2007 foram avaliadas quanto ao perfil de suscetibilidade por disco-difusão e microdiluição. Respeitando a padronização do Clinical and Laboratory Standard Institute (CLSI), SMX-TMP e levofloxacino foram testados pelas duas técnicas, bem como a tigeciclina, interpretada segundo a agência norte-americana Food and Drug Administration (FDA). Ticarcilina/clavulanato, ceftazidima, imipenem, meropenem e moxifloxacino foram avaliados apenas por microdiluição. O percentual de correlação entre as metodologias foi analisado pelo programa WHONET®. RESULTADOS: Das 126 cepas, 1,6 por cento apresentaram resistência a SMX-TMP; 2,4 por cento, a levofloxacino; 23 por cento, a ticarcilina/clavulanato; 54 por cento, a ceftazidima. Todas apresentaram concentração inibitória mínima (CIM) < 2 µg/mL para tigeciclina e 96,8 por cento, CIM < 2 µg/mL para moxifloxacino. O índice de concordância categórica entre as metodologias foi de 100 por cento para SMX-TMP e de 89,6 por cento para levofloxacino. DISCUSSÃO E CONCLUSÃO: Embora SMX-TMP seja a droga de escolha para o tratamento de infecções por S. maltophilia, há possibilidade de resistência a esse antibiótico, dificultando a decisão terapêutica empírica, sendo fundamental a realização do teste de sensibilidade. A técnica de disco-difusão demonstrou boa correlação com a microdiluição. Entre as novas opções terapêuticas, tigeciclina e moxifloxacino apresentaram boa atividade in vitro.

INTRODUCTION: Stenotrophomonas maltophilia is an important emerging nosocomial pathogen naturally resistant to carbapenems. Although trimethoprim/sulfamethoxazole (TMP-SMX) is commonly used as a treatment option, resistance to this drug has been recently reported. OBJECTIVE: To evaluate the susceptibility profile of S. maltophilia strains and compare the results obtained by different methods. METHODS: As to susceptibility profile, all strains, which had been routinely isolated at Hospital das Clínicas, São Paulo, in 2007, were evaluated through disk diffusion and microdilution. In accordance with the Clinical and Laboratory Standard Institute (CLSI), TMP-SMX and levofloxacin were tested through both techniques, as well as tigecycline, which was interpreted in conformity with Food and Drug Administration (FDA) regulations. Ticarcillin/clavulanate, ceftazidime, imipenem, meropenem and moxifloxacin were evaluated only through microdilution. The correlation between these methods was assessed by WHONET®. RESULTS: 1.6 percent of 126 strains were resistant to TMP-SMX, 2.4 percent to levofloxacin, 23 percent to ticarcillin/clavulanate, and 54 percent to ceftazidime. All strains showed minimum inhibitory concentration (MIC) < 2 µg/mL for tigecycline and 96.8 percent MIC < 2 µg/mL for moxifloxacin. The correlation index between both methods was 100 percent for TMP-SMX and 89.6 percent for levofloxacin, respectively. DISCUSSION AND CONCLUSION: Although TMP-SMX is the standard treatment for S. maltophilia infections, there may be resistance to this antibiotic, which hinders the therapeutic approach, hence the significance of susceptibility tests. The disk diffusion technique showed a good correlation with microdilution. Among the new therapeutic options, both tigecycline and moxifloxacin presented significant activity in vitro.

New mutation in the CASR gene in a family with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT).

A loss of calcium-sensing receptor (CASR) function due to inactivating mutations can cause familial hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). NSHPT represents the most severe expression of FHH and courses as a life-threatening condition. The aim of this study was to identify and characterize a CASR mutation in a female infant brought to the health service due to dehydration, apathy, lack of breast feeding and severe hypercalcemia. Molecular analysis was performed on genomic DNA of the index case and her parents. A novel homozygous mutation (p.E519X) in CASR was identified in the proband; both mother and father had the same mutation in heterozygous state, confirming their FHH condition. The mutation results in a truncated and inactive protein due to the lack of transmembrane and intracellular domains. The identification of this novel CASR gene mutation established the basis of hypercalcemia in this family and allowed a genetic counseling.

New mutation in the CASR gene in a family with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) / Nova mutação no gene CASR em uma família com hipercalcemia hipocalciúrica familiar (HHF) e hiperparatireoidismo neonatal grave (HPTNG)

A loss of calcium-sensing receptor (CASR) function due to inactivating mutations can cause familial hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). NSHPT represents the most severe expression of FHH and courses as a life-threatening condition. The aim of this study was to identify and characterize a CASR mutation in a female infant brought to the health service due to dehydration, apathy, lack of breast feeding and severe hypercalcemia. Molecular analysis was performed on genomic DNA of the index case and her parents. A novel homozygous mutation (p.E519X) in CASR was identified in the proband; both mother and father had the same mutation in heterozygous state, confirming their FHH condition. The mutation results in a truncated and inactive protein due to the lack of transmembrane and intracellular domains. The identification of this novel CASR gene mutation established the basis of hypercalcemia in this family and allowed a genetic counseling.

Mutações inativadoras no gene do sensor do cálcio (CASR) podem causar hipercalcemia hipocalciúrica familiar (HHF) ou hiperparatireoidismo neonatal grave (HPTNSG). A HPTNS representa a forma mais grave da HHF cursando com risco de vida. O objetivo deste estudo foi identificar e caracterizar uma mutação no gene CASR de uma criança do sexo feminino levada ao hospital em decorrência de desidratação, apatia, dificuldade para mamar e hipercalcemia grave. A análise molecular foi realizada a partir do DNA genômico do caso índice e de seus pais. Uma nova mutação em homozigose (p.E519X) foi identificada no caso índice; ambos, mãe e pai, apresentaram a mesma mutação em heterozigose, o que os caracteriza como portadores de HHF. Essa alteração resulta em uma proteína truncada e inativa devido à falta dos domínios transmembrana e intracelular. A identificação dessa nova mutação estabeleceu a causa da hipercalcemia na família e permitiu o aconselhamento genético.