Estudo caso-controle dos aspectos clínicos, fatores de risco e mortalidade associados a infecções nosocomiais por Klebsiella pneumoniae produtoras de carbapenemases do tipo KPC

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2016.A resistência bacteriana aos antimicrobianos por Klebsiella pneumoniae Carbapenemase (KPC), principalmente em K. pneumoniae, representa um grave problema para o manuseio de infecções adquiridas no ambiente hospitalar. Carbapenêmicos, considerados a ?última linha de defesa? para diversos tipos de infecção por bactérias Gram-negativas, tornam-se ineficientes contra bactérias produtoras de carbapenemases, e poucos antibióticos estão disponíveis atualmente para o tratamento. O presente estudo caso-controle pareado, teve por objetivo identificar aspectos clínicos, fatores de risco e mortalidade associados a infecções causadas por K. pneumoniae produtora de carbapenemase do tipo KPC (Kp-KPC) em pacientes internados no Hospital Universitário da Universidade Federal de Santa Catarina (HU/UFSC) no período de Janeiro de 2012 a Dezembro de 2014. A identificação dos isolados e o antibiograma foram realizados por metodologia automatizada (Vitek 2®/Biomerieux). Foram utilizados dois testes fenotípicos, em diferentes momentos do estudo, para triagem dos isolados com resistência aos carbapenêmicos. Todos os isolados foram confirmados por metodologia molecular (Reação em Cadeia da Polimerase - PCR). Foram selecionados para o grupo caso 40 pacientes com Kp-KPC e para o grupo controle 40 pacientes com K. pneumoniae não produtora de carbapenemase do tipo KPC (Kp-não-KPC) isoladas de amostras de urina e hemocultura. Dados obtidos incluíram a origem do paciente no momento da internação, fatores de risco, como tempo de hospitalização antes da infecção, admissão em Unidade de Terapia Intensiva (UTI) antes do isolamento de K. pneumoniae, cirurgia, presença de dispositivos invasivos, uso prévio de antibióticos, tratamento empírico e definitivo, bem como comorbidades e desfecho clínico dos pacientes. Os dados foram coletados dos prontuários. A maioria dos pacientes transferidos para o HU/UFSC de outros hospitais pertencia ao grupo caso (P=0,010). Isso destaca a necessidade de realização de culturas de vigilância para pacientes transferidos para o HU/UFSC. Neste estudo, a admissão na UTI (Razão de Chance [RC], 3,115; Intervalo de Confiança [IC] 95%, 1,247-7,781; P=0,014), presença de cateter venoso (RC, 5,516; IC 95%, 1,109-27,429; P=0,023), presença de cateter urinário (RC, 3,484; IC 95%, 1,246-9,747; P=0,015) e uso prévio de antibióticos (RC, 3,444; IC 95%, 1,310-9,058; P=0,011) foram associados a infecções por Kp-KPC por análise univariada. Quando analisados os antibióticos ou as classes mais comumente utilizadas, foi encontrada diferença significativa para o grupo caso no uso de cefalosporinas deamplo espectro (P=0,039). A análise da terapia empírica mostrou que 71,4% dos pacientes que não a utilizaram foram a óbito. Dentre os pacientes que fizeram uso de terapia empírica, a mortalidade foi maior para os pacientes que receberam terapia apropriada (58,3% vs 37,5%). Após o resultado do antibiograma, a mortalidade foi maior para os pacientes que receberam terapia apropriada (55% vs 50%). Além disso, verificou-se que a mortalidade de quem fez uso de terapia definitiva combinada (70%) foi maior do que pacientes que utilizaram monoterapia (40%). A frequência da mortalidade total foi significantemente maior para o grupo caso (47,5% vs 25%, P=0,036). O número crescente de isolados, tanto em amostras clínicas quanto em culturas de vigilância, alerta para a necessidade de aprimoramento das medidas já adotadas a fim de controlar a disseminação do microrganismo no HU/UFSC. Cateteres venoso e urinário foram associados com infecções por Kp-KPC, por isso em internações mais longas, esses dispositivos devem ser revistos regularmente para verificar se ainda são necessários. Kp-KPC é um patógeno emergente associado à mortalidade no HU/UFSC. A frequência da mortalidade associada às limitadas opções terapêuticas ressalta a necessidade de detecção precoce, de medidas de prevenção de contato e o desenvolvimento de novos fármacos para o tratamento dessas infecções.Abstract : The bacterial resistance to antimicrobials by Klebsiella pneumoniae Carbapenemase (KPC), especially in Klebsiella pneumoniae, is a serious problem to handling healthcare associated infections. Carbapenems, considered the ?last-line agents" to treat several infections by Gram-negative bacteria become ineffective against carbapenem-producing bacteria, and few effective antibiotics are currently available to treatment. To identify clinical aspects, risk factors and mortality associated to infections caused by KPC-producing K. pneumoniae (Kp-KPC), this matched case-control study was performed at the University Hospital of the Federal University of Santa Catarina (HU/UFSC) from January 2012 through December 2014. The bacterial identification and antimicrobial susceptibility testing were performed by automatized methodology (Vitek 2®/Biomerieux). Two phenotypic tests were performed in different moment to screening the isolates with carbapenems resistance. All isolates were confirmed by molecular methodology (Polimerase Chain Reaction - PCR). Were selected isolates from urine and blood culture of forty patients with Kp-KPC (case) and forty patients with non-KPC-producing K. pneumoniae (Kp-non-KPC). Data obtained included origin of patient at the time of hospital admission, risk factors such as length of stay before infection, Intensive Care Unit (ICU) stay prior to K. pneumoniae isolation, surgery, use of invasive devices, prior antibiotic therapy, empiric therapy and definitive treatment, as well as comorbidities and outcomes. Data were collected from medical charts. Most of transferred patients to HU/UFSC from others hospitals belonged to case group (P=0.010). This highlight the need of surveillance cultures to patients transferred to HU/UFSC. In this study, stayed in ICU, (Odds Ratio [OR], 3.115; Confidence Intervals [CI] 95%, 1.247-7.781; P=0.014), use of venous catheter (OR, 5.516; CI 95%, 1.109-27.429; P=0.023), use of urinary catheter (OR, 3.484; CI 95%, 1.246-9.747; P=0.015) and prior antimicrobial use (OR, 3.444; CI 95%, 1.310-9.058; P=0.011) were associated with Kp-KPC infections by univariable analysis. The analysis of the antibiotics or class of antibiotics most commonly used, showed significant difference to case group for the use of extended-spectrum cephalosporins (P=0.039). The analysis of empirical therapy showed that 71.4% of patients who did not use empiric antibiotic died. Amongpatients who used empirical therapy, mortality was higher for patients who received appropriate therapy (58.3% vs 37.5%). After antimicrobial susceptibility testing, mortality was higher for patients who received appropriate therapy (55% vs 50%). Furthermore, it was found that mortality of patients who received definitive associated therapy (70%) was higher than patients who used monotherapy (40%) (P=0.370). The mortality frequency was significant higher for case group (47.5% vs 25%, P=0.036). The increase number of isolates, both in clinical samples and surveillance cultures, alert to the need to improve the measures already adopted in order to control the spread of the microorganisms in HU/UFSC. Venous and urinary catheters were associated with Kp-KPC infections, so in long-stay hospitalizations, these devices should be reviewed regularly to check whether they are still needed. Kp-KPC is an emerging pathogen associated with significant mortality in HU/UFSC. The mortality frequency associated with limited therapeutic options, highlight the need of early detection, contact prevention measures and development of new drugs for the treatment of these infections

In vitro selection of Neisseria gonorrhoeae unveils novel mutations associated with extended-spectrum cephalosporin resistance

The emergence of Neisseria gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESCs) is a worldwide concern because this class of antibiotics represents the last empirical treatment option for gonorrhea. The abusive use of antimicrobials may be an essential factor for the emergence of ESC resistance in N. gonorrhoeae. Cephalosporin resistance mechanisms have not been fully clarified. In this study, we mapped mutations in the genome of N. gonorrhoeae isolates after resistance induction with cefixime and explored related metabolic pathways. Six clinical isolates with different antimicrobial susceptibility profiles and genotypes and two gonococcal reference strains (WHO F and WHO Y) were induced with increasing concentrations of cefixime. Antimicrobial susceptibility testing was performed against six antimicrobial agents before and after induction. Clinical isolates were whole-genome sequenced before and after induction, whereas reference strains were sequenced after induction only. Cefixime resistance induction was completed after 138 subcultures. Several metabolic pathways were affected by resistance induction. Five isolates showed SNPs in PBP2. The isolates M111 and M128 (ST1407 with mosaic penA-34.001) acquired one and four novel missense mutations in PBP2, respectively. These isolates exhibited the highest minimum inhibitory concentration (MIC) for cefixime among all clinical isolates. Mutations in genes contributing to ESC resistance and in other genes were also observed. Interestingly, M107 and M110 (ST338) showed no mutations in key determinants of ESC resistance despite having a 127-fold increase in the MIC of cefixime. These findings point to the existence of different mechanisms of acquisition of ESC resistance induced by cefixime exposure. Furthermore, the results reinforce the importance of the gonococcal antimicrobial resistance surveillance program in Brazil, given the changes in treatment protocols made in 2017 and the nationwide prevalence of sequence types that can develop resistance to ESC

Avaliação de mutações após indução de resistência à cefixima em isolados clínicos de Neisseria gonorrhoeae e caracterização genômica de Neisseria elongata isolada de endocardite

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2021.Este estudo avaliou a presença de mutações no genoma de isolados de Neisseria gonorrhoeae após indução de resistência com cefixima e caracterizou o genoma de Neisseria elongata isolada de paciente com endocardite infecciosa (EI). Para o estudo de indução de resistência, foram selecionados seis isolados clínicos com diferentes genótipos da região da grande Florianópolis e duas cepas de referência (WHO F e WHO Y). Os isolados foram subcultivados com concentrações crescentes do antimicrobiano cefixima. Após a indução, foi determinada a concentração inibitória mínima (MIC) para seis antimicrobianos pelo método de ágar diluição. Os isolados clínicos foram sequenciados antes e após a indução e as cepas de referência somente após a indução utilizando a plataforma MiSeq Illumina. Os genomas dos isolados clínicos antes da indução de resistência foram montados utilizando SPAdes e anotados com Prokka. Para as cepas de referência, foram utilizados os genomas disponíveis no NCBI. Polimorfismos de único nucleotídeo (SNPs), inserções e deleções foram analisados por meio do mapeamento das leituras após a indução nos genomas montados, utilizando bwa. Samtools foi utilizado para manipulação do mapeamento. As variantes foram obtidas com o programa BCFtools e anotadas com o programa SnpEff. A indução foi finalizada após 138 subcultivos, com crescimento de todos os isolados em concentração do antimicrobiano acima do ponto de corte de resistência à cefixima de acordo com o BrCAST. Os isolados M111 e M128 (ST1407) apresentaram diversas mutações na proteína ligadora de penicilina (PBP2) e exibiram os maiores valores de MIC para cefixima dentre os isolados clínicos. M107 e M110 (ST338) não apresentaram mutações nos principais determinantes de resistência às cefalosporinas de espectro estendido (ESC), mas aumentaram em 127 vezes a MIC para cefixima. WHO Y, inicialmente resistente à cefixima, apresentou mutações no gene penB (porina) e nos genes mtrC e mtrD da bomba de efluxo MtrCDE. Para o estudo com N. elongata, foram avaliados os dados clínicos de um paciente com EI (M001) que foi a óbito durante a internação e realizado o sequenciamento do genoma completo do isolado. As leituras do sequenciamento foram utilizadas para recuperar 16 sequências desta espécie disponíveis no NCBI. Os genomas foram montados com SPAdes e Gfinisher e anotados utilizando Prokka. A análise de diversidade entre os isolados foi avaliada utilizando o programa PYANI. O core genoma foi obtido com o programa Roary e os SNPs foram extraídos com SNP-site. A filogenia do core genoma e de proteínas ortólogas foi realizada com o programa RAxML. A presença de genes de resistência e de virulência foi realizada com as bases de dados CARD e VFDB. Os isolados apresentaram uma grande diversidade genética. Dezessete diferentes genes de resistência foram encontrados em todos os isolados de N. elongata, com maior predominância de genes relacionados a sistemas de efluxo. Genes duplicados de pili foram encontrados somente no isolado M001, o que pode predizer uma maior capacidade de virulência. Os resultados obtidos no estudo apontam para aquisição de resistência às ESC por diferentes mecanismos e reforçam a importância do programa de vigilância antimicrobiana de N. gonorrhoeae no Brasil, devido a recente mudança de tratamento e prevalência de STs que podem adquirir resistência a esses antimicrobianos. O estudo genômico de N. elongata associado ao relato de caso, alerta a necessidade de rápida identificação microbiológica e intervenção médica para evitar desfechos desfavoráveis e fornece evidências que podem contribuir para outras investigações biológicas em espécies de Neisseria comensais.Abstract: This study evaluated the presence of mutations in the genome of Neisseria gonorrhoeae isolates after resistance induction to cefixime and characterized the genome of Neisseria elongata isolated from patient with infective endocarditis (EI). For the resistance induction study, six clinical isolates with different genotypes were selected from Florianopolis region and two reference strains (WHO F and WHO Y). The isolates were subcultured with increasing concentrations of the antimicrobial cefixime. After induction, the minimum inhibitory concentration (MIC) for six antimicrobials was determined by agar dilution method. Clinical isolates were sequenced before and after induction and reference strains only after induction using the Illumina MiSeq platform. The genomes of the clinical isolates before resistance induction were assembled using SPAdes and annotated with Prokka. For the reference strains, the genomes available at NCBI were used. Single nucleotide polymorphisms (SNPs), insertions, and deletions were analyzed by mapping reads after induction onto the assembled genomes using bwa. Samtools was used for mapping manipulation. Variants were obtained with the program BCFtools and annotated with the program SnpEff. Induction was completed after 138 subcultures, with the growth of all isolates at an antimicrobial concentration above the cutoff point for cefixime resistance according to BrCAST. Isolates M111 and M128 (ST1407) had several mutations in the penicillin-binding protein (PBP2) and exhibited the highest MIC values for cefixime among the clinical isolates. M107 and M110 (ST338) showed no mutations in key determinants of resistance to extended-spectrum cephalosporins (ESC) but increased the MIC for cefixime by 127-fold. WHO Y, initially resistant to cefixime, showed mutations in the penB (porin) gene and in the mtrC and mtrD genes of the MtrCDE efflux pump. For the study with N. elongata, the clinical data of one patient with EI (M001) who died during hospitalization were evaluated and the whole genome sequencing of the isolate was performed. The sequencing reads were used to retrieve 16 sequences of this species available at NCBI. Genomes were assembled with SPAdes and Gfinisher and annotated using Prokka. Diversity analysis among isolates was evaluated using the PYANI program. The core genome was obtained with the program Roary and SNPs were extracted with SNP-site. Phylogeny of the core genome and orthologous proteins was performed with the RAxML program. The presence of resistance and virulence genes was evaluated with the CARD and VFDB databases. The isolates showed a high genetic diversity. Seventeen different resistance genes were found in all N. elongata isolates, with a higher predominance of genes related to efflux systems. Duplicated pili genes were found only in isolate M001, which may predict a higher virulence capacity. The results achieved by the N. gonorrhoeae induced resistance study point to the acquisition of resistance to ESC by different mechanisms and reinforce the importance of N. gonorrhoea antimicrobial surveillance program in Brazil, due to the recent change of treatment and prevalence of STs that can acquire resistance to these antimicrobials. The genomic study of N. elongata associated with this case report highlights the need for rapid microbiological identification and medical intervention in order to avoid unfavorable outcomes and provides evidence that may contribute to further biological investigations of commensal Neisseria species

Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil.

Submitted by Nuzia Santos ([email protected]) on 2016-02-22T18:00:26Z No. of bitstreams: 1 Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil..pdf: 292609 bytes, checksum: 4bea0aaa468b44fe8ec1bba4d8017a9e (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-02-22T18:06:23Z (GMT) No. of bitstreams: 1 Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil..pdf: 292609 bytes, checksum: 4bea0aaa468b44fe8ec1bba4d8017a9e (MD5)Made available in DSpace on 2016-02-22T18:06:23Z (GMT). No. of bitstreams: 1 Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil..pdf: 292609 bytes, checksum: 4bea0aaa468b44fe8ec1bba4d8017a9e (MD5) Previous issue date: 2015Universidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilLaboratório Central de Saúde Pública de Santa Catarina. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular, Sorologia e Micobactérias. Florianópolis, SC, BrasilDrug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosis clinical isolates were analysed by spoligotyping and a partial region of the rpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoB in RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations within rpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil

Genomic Surveillance of SARS-CoV-2 in Healthcare Workers: A Critical Sentinel Group for Monitoring the SARS-CoV-2 Variant Shift

SARS-CoV-2 genome surveillance is important for monitoring risk groups and health workers as well as data on new cases and mortality rate due to COVID-19. We characterized the circulation of SARS-CoV-2 variants from May 2021 to April 2022 in the state of Santa Catarina, southern Brazil, and evaluated the similarity between variants present in the population and healthcare workers (HCW). A total of 5291 sequenced genomes demonstrated the circulation of 55 strains and four variants of concern (Alpha, Delta, Gamma and Omicron—sublineages BA.1 and BA.2). The number of cases was relatively low in May 2021, but the number of deaths was higher with the Gamma variant. There was a significant increase in both numbers between December 2021 and February 2022, peaking in mid-January 2022, when the Omicron variant dominated. After May 2021, two distinct variant groups (Delta and Omicron) were observed, equally distributed among the five Santa Catarina mesoregions. Moreover, from November 2021 to February 2022, similar variant profiles between HCW and the general population were observed, and a quicker shift from Delta to Omicron in HCW than in the general population. This demonstrates the importance of HCW as a sentinel group for monitoring disease trends in the general population

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems