ABSTRACT
O Complexo K. pneumoniae (C-Kp) é o principal grupo de bacilos Gram-negativos responsáveis por infecções nosocomiais graves em todo o mundo e o tratamento empírico dessas infecções usualmente inclui os carbapenêmicos. O principal mecanismo de resistência a essa classe de antimicrobianos é a expressão de carbapenemases, e no Brasil, mais frequentemente as do tipo KPC. Em março de 2019 a ceftazidima-avibactam foi disponibilizada para uso clínico no Brasil, sendo amplamente utilizada no tratamento infecções causadas por bacilos gram-negativos produtores de KPC. Diversos países já relataram a presença de K. pneumoniae produtores de KPC resistentes à ceftazidima-avibactam. No entanto, há poucos relatos dessa ocorrência no Brasil. O objetivo deste trabalho foi caracterizar genotipicamente e fenotipicamente isolados do C-Kp produtores de KPC, resistentes à ceftazidima-avibactam. No período de julho/2019 a julho/2021, 46 isolados do C-Kp, um por paciente, foram detectados em diferentes sítios de infecção ou culturas de vigilância de pacientes internados em hospitais privados de seis estados brasileiros. Os isolados tiveram seu genoma completo sequenciado nas plataformas MiSeq e MinION para determinação da variante alélica de blaKPC e avaliação do seu contexto genético. As taxas de resistência ao ertapenem e à ceftazidima-avibactam foram calculadas a partir de banco de dados. A clonalidade dos isolados foi avaliada por PFGE e MLST. A localização plasmidial do gene blaKPC foi confirmada por conjugação e/ou transformação. A concentração inibitória mínima (CIM) para betalactâmicos foi determinada por microdiluição em caldo segundo o BrCAST. Ensaios imunocromatográficos, NG-Test CARBA-5 e O.K.N.V.I. RESIST-5, foram avaliados quanto à sua performance na detecção de variantes KPC. A taxa de resistência ao ertapenem entre isolados do C-Kp aumentou 15,6% em 2019 para 27,3% em 2021. A taxa de resistência à ceftazidima-avibactam entre isolados do C-Kp resistentes ao ertapenem aumentou de 4,2% em 2019 para 17,2% em 2021. Onze isolados apresentaram novas variantes de KPC designadas KPC-103 a KPC-108 e KPC-139 a KPC-143. Os demais isolados apresentavam variantes de KPC já descritas, sendo a KPC-33 a variante mais frequente (36%). Quinze grupos clonais foram identificados, sendo que a maioria dos isolados pertencia ao ST11. O grupo clonal A foi o mais numeroso e pertencia ao ST258. A principal variante detectada nesse grupo foi a KPC-33. Diferentes grupos de incompatibilidade foram identificados em plasmídeos alberguando blaKPC, sendo os grupos IncN (n=12) e IncF, com replicons FII(K)-FIB (n=11), os grupos mais frequentes, seguido de InX3-IncU (n=9) e IncQ1 (n=1). Dos 46 isolados resistentes à ceftazidima-avibactam, 36 foram capazes de transferir o gene blaKPC para cepas receptoras. A maioria dos isolados foi sensível dose padrão ou sensível aumentabdo exposição ao meropenem e apresentaram redução significativa da CIM quando o avibactam foi adicionado ao aztreonam. O NG-Test CARBA-5 detectou 12 das 24 variantes testadas enquanto que o O.K.N.V.I. RESIST-5 detectou apenas nove variantes. A grande diversidade de variantes KPC, e a predominância do grupo clonal ST11, e da KPC- 33 retratam um cenário preocupante no Brasil
The K. pneumoniae Complex (C-Kp) is the main group of gram-negative bacilli responsible for serious nosocomial infections worldwide and the empirical treatment of these infections usually includes carbapenems. The main mechanism of resistance to this class of antimicrobials is the expression of carbapenemases, and in Brazil, more frequently the KPC type. In March 2019, ceftazidime-avibactam was available for clinical use in Brazil, being widely used to treat infections caused by KPC-producing gram-negative bacilli. Several countries have already reported the presence of KPC-producing K. pneumoniae resistant to ceftazidime-avibactam; however, there are few reports of this occurrence in Brazil. This work aimed to genotypically and phenotypically characterize KPC-producing C-Kp isolates resistant to ceftazidimeavibactam. From July 2019 to July 2021, 46 C-Kp isolates, one per patient, were detected in different sites of infection or surveillance cultures from patients admitted to private hospitals in six Brazilian states. The isolates had their complete genome sequenced on the MiSeq and MinION platforms to determine the allelic variant of blaKPC and evaluate its genetic context. Resistance rates to ertapenem and ceftazidime-avibactam were calculated from a database. The clonality of the isolates was evaluated by PFGE and MLST. The plasmid location of the blaKPC gene was confirmed by conjugation and/or transformation. The minimal inhibitory concentrations for beta-lactams were determined by broth microdilution according to BrCAST. Immunochromatographic assays, NG-Test CARBA-5 and O.K.N.V.I. RESIST-5, were evaluated for its performance in detecting KPC variants. The resistance rate to ertapenem among C-Kp isolates increased from 15.6% in 2019 to 27.3% in 2021. The resistance rate to ceftazidime-avibactam among ertapenem-resistant C-Kp isolates increased from 4.2% in 2019 to 17.2% in 2021. Eleven isolates showed new KPC variants designated KPC-103 to KPC-108 and KPC-139 to KPC-143. The remaining isolates presented previously described KPC variants, with KPC-33 being the most common variant (36%). Fifteen clonal groups were identified, with most isolates belonging to ST11. Different incompatibility groups were identified in plasmids harboring blaKPC, with the IncN (n=12) and IncF groups, with FII(K)-FIB(n=11) replicons, being the most frequent groups, followed by InX3-IncU (n= 9) and IncQ1 (n=1). All IncX3-IncU plasmids were approximately 46 kb in size and were identified among isolates belonging to the largest identified clonal group (A) and ST258. The main variant detected in this group was KPC-33. Of the 46 ceftazidime-avibactam-resistant isolates, 36 were able to transfer the blaKPC gene to recipient strains. Most isolates were susceptible standard dose or susceptible increased exposure to meropenem and showed a significant decrease in MIC when avibactam was added to aztreonam. The NG-Test CARBA-5 was able to detect 12 of the 24 variants evaluated while the O.K.N.V.I. RESIST-5 detected only nine variants. The great diversity of KPC variants, the predominance of the ST11 clonal group, and KPC-33 portray a worrying scenario in Brazil
Subject(s)
Aztreonam/agonists , Drug Resistance, Microbial , Ceftazidime/adverse effects , Klebsiella pneumoniae/classification , Anti-Infective Agents/analysis , Carbapenems/adverse effectsABSTRACT
Abstract The production of KPC (Klebsiella pneumoniae carbapenemase) is the major mechanism of resistance to carbapenem agents in enterobacterias. In this context, forty KPC-producing Enterobacter spp. clinical isolates were studied. It was evaluated the activity of antimicrobial agents: polymyxin B, tigecycline, ertapenem, imipenem and meropenem, and was performed a comparison of the methodologies used to determine the susceptibility: broth microdilution, Etest® (bioMérieux), Vitek 2® automated system (bioMérieux) and disc diffusion. It was calculated the minimum inhibitory concentration (MIC) for each antimicrobial and polymyxin B showed the lowest concentrations for broth microdilution. Errors also were calculated among the techniques, tigecycline and ertapenem were the antibiotics with the largest and the lower number of discrepancies, respectively. Moreover, Vitek 2® automated system was the method most similar compared to the broth microdilution. Therefore, is important to evaluate the performance of new methods in comparison to the reference method, broth microdilution.
Subject(s)
Humans , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , beta-Lactamases/metabolism , Enterobacteriaceae Infections/microbiology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests/methods , Bacterial Proteins/genetics , beta-Lactamases/genetics , beta-Lactams/pharmacology , Drug Resistance, Bacterial , Enterobacter/drug effects , Enterobacter/genetics , Enterobacter/isolation & purification , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Polymyxin B/pharmacologyABSTRACT
RESUMEN La emergencia de enterobacterias productoras de carbapenemasas de tipo Nueva Delhi Metalo beta-lactamasas (NDM), representan, hoy en día, un verdadero problema de salud pública mundial. La presencia de este mecanismo de resistencia limita o anula las opciones terapéuticas para combatir a estas bacterias. En Latinoamérica, las cifras son cada vez más elevadas, pues se reportan en Guatemala, Colombia, Chile, Argentina, entre otros. Perú no ha descrito, hasta la fecha, la presencia de este patrón de resistencia; sin embargo, desde hace varios años se presume de su existencia. Se describen nueve casos de Klebsiella pneumoniae NDM, como agentes infecciosos o colonizantes, en pacientes críticamente enfermos, en su mayoría con patología neuroquirúrgica, del Hospital Nacional Dos de Mayo, en Lima - Perú. Los pacientes de la serie descrita a continuación, representan los primeros reportes de Klebsiella pneumoniae NDM en el Perú.
ABSTRACT The emergence of Enterobacteria producing carbapenemases of type New Delhi Metalo beta-lactamases (NDM), >represent, today, a real problem of world public health. The presence of this resistance mechanism limits or nullifies the therapeutic options to combat these bacteria. In Latin America, the figures are getting higher, as they are reported in Guatemala, Colombia, Chile, Argentina, among others. Peru has not, to date, described the presence of this resistance pattern; however for several years it has been presumed to exist. Nine cases of Klebsiella pneumoniae NDM are described, as infectious or colonizing agents, in critically ill patients, mostly with neurosurgical pathology, of Hospital Nacional Dos de Mayo in Lima - Peru. The patients in the series described below represent the first reports of Klebsiella pneumoniae NDM in Peru.
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Bacterial Proteins/biosynthesis , beta-Lactamases/biosynthesis , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/enzymology , Peru , Microbial Sensitivity Tests , HospitalsABSTRACT
The emergence of β-lactamase-producing Enterobacteriaceae in the last few decades has become major challenge faced by hospitals. In this study, isolates of Klebsiella pneumoniae carbapenemase-2 (KPC-2)-producing K. pneumoniae from a tertiary hospital in Mato Grosso do Sul, Brazil, were characterized. Bacterial identification was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF; Bruker Daltonics, Germany) mass spectrometry. The minimum inhibitory concentrations of carbapenems were determined using the agar dilution method as recommended by the Clinical Laboratory Standards Institute guidelines. Carbapenemase production was detected using the modified Hodge test (MHT) and polymerase chain reaction (PCR), followed by DNA sequencing. Of 360 (12.2%) K. pneumoniae isolates obtained between May 2009 and May 2010, 44 (12.2%) were carbapenem nonsusceptible. Of these 44 isolates, thirty-six K. pneumoniae isolates that were positive by MHT and PCR carried the blaKPC-2 gene. Thus, KPC-2producing Klebsiella pneumoniae has been present in a Brazilian hospital located in the Midwest region since at least 2009.
Subject(s)
Humans , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/enzymology , beta-Lactamases , Brazil , DNA, Bacterial/genetics , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tertiary Care Centers , beta-Lactamases/geneticsABSTRACT
The present study proposed the isolation of arsenic resistant bacteria from wastewater. Only three bacterial isolates (MNZ1, MNZ4 and MNZ6) were able to grow in high concentrations of arsenic. The minimum inhibitory concentrations of arsenic against MNZ1, MNZ4 and MNZ6 were 300 mg/L, 300 mg/L and 370 mg/L respectively. The isolated strains showed maximum growth at 37 ºC and at 7.0 pH in control but in arsenite stress Luria Bertani broth the bacterial growth is lower than control. All strains were arsenite oxidizing. All strains were biochemically characterized and ribotyping (16S rRNA) was done for the purpose of identification which confirmed that MNZ1 was homologous to Enterobacter sp. while MNZ4 and MNZ6 showed their maximum homology with Klebsiella pneumoniae. The protein profiling of these strains showed in arsenic stressed and non stressed conditions, so no bands of induced proteins appeared in stressed conditions. The bacterial isolates can be exploited for bioremediation of arsenic containing wastes, since they seem to have the potential to oxidize the arsenite (more toxic) into arsenate (less toxic) form.
Subject(s)
Anti-Bacterial Agents/metabolism , Arsenic/metabolism , Drug Resistance, Bacterial , Enterobacter/drug effects , Klebsiella pneumoniae/drug effects , Wastewater/microbiology , Arsenites/metabolism , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Enterobacter/classification , Enterobacter/growth & development , Enterobacter/isolation & purification , Hydrogen-Ion Concentration , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/growth & development , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Oxidation-Reduction , Proteome/analysis , Ribotyping , /genetics , TemperatureSubject(s)
Female , Humans , Middle Aged , Community-Acquired Infections/microbiology , Klebsiella Infections/complications , Klebsiella pneumoniae/pathogenicity , Liver Abscess/microbiology , Serogroup , Acinetobacter baumannii/growth & development , Bacteremia , Brazil , Fatal Outcome , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/isolation & purification , Liver Abscess/blood , Microbial Sensitivity Tests , Species Specificity , Syndrome , VirulenceABSTRACT
O gênero Klebsiella compreende bactérias em forma de bastonete, gram-negativas e imóveis que pertencem à família Enterobacteriaceae. As espécies Klebsiella pneumoniae e Klebsiella oxytoca, patógenos oportunistas, são as mais importantes, sob o ponto de vista da clínica. [...] As beta-lactamases LEN são codificadas em cromossomos e são NSBLs. Recentemente a espécie K. variicola foi identificada. Esta espécie se caracteriza pela fixação de nitrogênio e nenhum gene constitutivo de beta-lactamase foi associado à esta espécie. Este estudo objetivou determinar o espectro de resistência de alelos de blaSHV identificados em K. pneumoniae e também identificar, em nível de espécie, isolados carreadores do gene blaLEN. Por PCR e sequenciamento, alelos de blaSHV e blaLEN foram definidos comparando suas sequências com bancos de dados usando as ferramentas blastN e blastP. Estes genes foram clonados e expressos em sistema heterólogo, e o espectro de resistência dos transformantes foi avaliado. A taxonomia genômica dos isolados deste gênero foi realizada com base em sequências de genes recuperados de genomas de espécies de Klebsiella e aplicou-se as abordagens MLSA, rMLST e cgMLST. Identificamos blaSHV-28 (n=2), blaSHV-110 (n=1) e alelos novos de blaSHV (n=3).
Os transformantes para estes genes apresentaram resistência à amoxicilina, ampicilina e piperacilina, o que corresponde a um espectro restrito de resistência. Quanto a blaLEN, foram identificados oito novos alelos em nove isolados. Dois deles eram idênticos aos encontrados no genoma de K. variicola AT-22 e todos estes alelos codificam NSBLs. A taxonomia genômica de Klebsiella definiu três grupos: K. variicola, K. pneumoniae e K.oxytoca. Curiosamente, alguns isolados de K. pneumoniae agruparam com o K. variicola AT-22, sugerindo pertencerem à esta espécie. Todos os isolados do grupo K. variicola albergavam o gene blaLEN indicando que esta seria a beta-lactamase constitutiva de K. variicola, e, por conseguinte, que o grupo KpIII é composto por isolados de K. variicola. Aqui nós determinamos que alelos de blaSHV cromossômicos, incluindo blaSHV-28 e blaSHV-110, previamente identificados como ESBLs, são NSBLs e concluímos que K. variicola pode estar sendo subestimada em infecções humanas.
The genus Klebsiella comprises non-motile, gram-negative, rod-shaped bacteria, presenting apolysaccharide-based capsule, belonging to the Enterobacteriaceae family. The two most clinicallyimportant species from the genus are the opportunistic pathogens Klebsiella pneumoniae andKlebsiella oxytoca. [...] LEN enzymes are chromosomal encoded with restrict activity against beta-lactams and confers resistance only to penicillins. More recently, K. variicola species were identifiedand this species has the ability of fixing nitrogen. There are few studies concerning this species andno constitutive beta-lactamase gene has been described in this species. This study aimed to determinethe resistance spectrum of blaSHV alleles identified in K. pneumoniae strains and also to identify, atspecies level, strains carrying blaLEN chromosomal class A beta-lactamase gene. PCR and sequencingwere performed and blaSHV, and blaLEN alleles were determined by comparing their sequences with thedatabase using blastN and blastP tools. These genes were cloned and expressed in a heterologoussystem, and the antibiotic resistance spectrum of the transformants was evaluated by susceptibilitytest.
The genomic taxonomy of strains from this genus were performed based on genes from draft andcomplete genome sequences from Klebsiella species using MLSA, rMLST and cgMLST approaches.Strains harboring blaSHV-28 (n=2), blaSHV-110 (n=1) and new blaSHV alleles (n=3) were identified. All thetransformants showed resistance to amoxicillin, ampicillin, piperacillin, ticarcillin, which correspond to arestricted spectrum of resistance. Concerning blaLEN alleles, here were identified eight new alleles innine strains. Two of them were identical to the one found in K. variicola AT-22 genome. The antibioticresistance profile presented by the transformants indicated that all these blaLEN alleles encode forNSBLs. The Klebsiella genomic taxonomy revealed three groups corresponding to K. variicola, K.pneumoniae and K. oxytoca species. Curiously, some K. pneumoniae grouped with the K. variicolaAT-22, suggesting that those strains, in fact, belong to K. variicola species and had been misidentifiedas K. pneumoniae. Moreover, it was observed that all strains from K. variicola cluster harbored theblaLEN gene. This finding indicated that the blaLEN gene is a constitutive K. variicola beta-lactamase, andtherefore, the KpIII group is composed by K. variicola strains. Here we determined that differentchromosomal blaSHV alleles, including blaSHV-28 e blaSHV-110, previously labeled as ESBLs, and the newones, are NSBLs. Also, we concluded that K. variicola can play an underestimated role in humaninfections.
Subject(s)
beta-Lactam Resistance , Genomics , Klebsiella pneumoniae/classification , Polymerase Chain ReactionABSTRACT
A produção de carbapenemases do tipo KPC tem se tornado um importante mecanismo de resistência aos carbapenemas na família Enterobacteriaceae. Embora seja descrita predominantemente em Klebsiella pneumoniae, a enzima KPC também tem sido encontrada em diferentes espécies de Enterobacteriaceae. Contudo, pouco se sabe sobre a epidemiologia da disseminação do gene blaKPC-2 nestas outras espécies. No Brasil, a enzima KPC foi relatada inicialmente em K. pneumoniae no Recife, em 2006, mas atualmente já se encontra disseminada pelo país, onde sua incidência tem aumentado significativamente. Portanto, o objetivo desse trabalho foi realizar a caracterização molecular de amostras brasileiras produtoras de KPC pertencentes a diferentes espécies de Enterobacteriaceae (excluindo K. pneumoniae), isoladas de diferentes estados brasileiros no período de 2009 a 2011. O perfil de resistência foi avaliado por difusão em ágar e E-test. A variante alélica de blaKPC, assim como a participação do transposon Tn4401 e a análise da presença de outros genes de beta-lactamases (TEM, SHV e CTX-M) foram realizadas por PCR e sequenciamento. Análise plasmidial e hibridação foram realizadas para determinar o ambiente genético do gene blaKPC. Para a tipagem molecular foi realizado PFGE e MLST (somente para Escherichia coli)Foram encaminhadas ao Laboratório de Pesquisa em Infecção Hospitalar da Fundação Oswaldo Cruz, 83 amostras produtoras de KPC-2 (correspondendo as espécies: Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Pantoea agglomerans, Providencia stuartii, Citrobacter freundii, Klebsiella oxytoca, Morganella morganii e Serratia marcescens) provenientes de 9 estados das regiões sudeste, nordeste e centro-oeste do país. Em amostras KPC positivas, foram encontrados altos percentuais de resistência à maioria dos antimicrobianos testados, inclusive tigeciclina (36,1 porcento não sensíveis) e polimixina B (16,5 porcento)...
The production of Klebsiella pneumoniae carbapenemase (KPC)-type enzymes has become an important mechanism of carbapenem resistance in the Family Enterobacteriaceae.Although it is predominantly described in Klebsiella pneumoniae, the KPC enzyme has also been found in different species of Enterobacteriaceae. Moreover, little is known about theepidemiology of the dissemination of blaKPC gene in other Enterobacteriaceae species. InBrazil, KPC was initially described in K. pneumoniae in Recife, state of Pernambuco, in 2006, but currently this enzyme is already disseminated throughout the country, where itsincidence has increased significantly. Thus, this study aimed to perform the molecular characterization of KPC-producing brazilian isolates belonging to different species of Enterobacteriaceae (non-K. pneumoniae) originated from different Brazilian states between2009 and 2011. The resistance profile was evaluated by disc-diffusion method and E-test. The allelic variant of the blaKPC gene, as well as the participation of Tn4401 and the presence of other beta-lactamase genes (TEM, SHV and CTX-M) were analyzed by PCR and genome sequencing. Plasmid analysis and hibridization were used to determine the genetic environment of the blaKPC gene. Molecular typing was done by PFGE and MLST (only forEscherichia coli). Eighty three unique clinical isolates of Enterobacteriaceae KPC-2-producers were referred to the Laboratório de Pesquisa em Infecção Hospitalar from Fundação Oswaldo Cruz, corresponding to 9 different species (Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Pantoea agglomerans, Providencia stuartii, Citrobacter freundii, Klebsiella oxytoca, Morganella morganii and Serratia marcescens) isolated from 9 states located in northeast, southeast and central regions of Brazil. Highresistance rates towards most of the antimicrobial agents tested, including tigecycline (36.1 percent nonsusceptible) and polymyxin B (16.5 percent) were detected...
Subject(s)
Humans , DNA Transposable Elements , Enterobacteriaceae , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/genetics , Molecular EpidemiologyABSTRACT
In this study, the discriminatory power of pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods for subtyping of 54 clinical isolates of Klebsiella pneumoniae were compared. All isolates were typeable by RAPD, while 3.6% of them were not typeable by PFGE. The repeatability of both typing methods were 100% with satisfying reproducibility (≥ 95%). Although the discriminatory power of PFGE was greater than RAPD, both methods showed sufficient discriminatory power (DI > 0.95) which reflects the heterogeneity among the K. pneumoniae isolates. An optimized RAPD protocol is less technically demanding and time consuming that makes it a reliable typing method and competitive with PFGE.
Subject(s)
Female , Humans , Male , Electrophoresis, Gel, Pulsed-Field , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/genetics , Molecular Typing/methods , Random Amplified Polymorphic DNA Technique , Genetic Variation , Genotype , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A new trend in cosmetic formulations is the use of biotechnological raw materials as the polysaccharides from Klebsiella pneumoniae, which are supposed to enhance cell renewal, improve skin hydration and micro-relief. Botanical extracts of Myrtus communis leaves contain different sugars, which may provide the same benefits. Thus, the objective of this study was to evaluate through objective and subjective analysis the immediate and long-term effects of cosmetic formulations containing polysaccharides biotechnologically-originated and / or the ones contained in Myrtus communis extracts. Three polysaccharide-based and placebo formulations were applied on the forearm skin of 40 volunteers. Skin hydration, transepidermal water loss (TEWL), viscoelasticity and skin micro-relief measurements were made before and 2 hours after a single application and after 15 and 30 day-periods of daily applications. Answers to a questionnaire about perceptions of formulation cosmetic features constituted the subjective analysis. All polysaccharide-based formulations enhanced skin hydration. Formulations with isolated or combined active substances improved skin barrier function as compared to placebo, in the short and long term studies. Formulations containing Myrtus communis extracts had the highest acceptance. Results suggest that daily use of formulations containing these substances is important for protection of the skin barrier function.
Uma nova tendência em formulações cosméticas é a utilização de matérias-primas biotecnológicas como os polissacarídeos de Klebsiella pneumoniae, que pode aumentar a renovação celular e melhor a hidratação e micro-relevo da pele. Por outro lado, o extrato vegetal de Myrtus communis contém diferentes polissacarídeos, que também podem proporcionar benefícios à pele. Assim, o objetivo do estudo foi a avaliação dos efeitos imediatos e em longo prazo, de formulações cosméticas contendo polissacarídeos obtidos por processo biotecnológico e/ou de extrato de M. communis por meio de análises objetivas e subjetivas. Três formulações contendo os polissacarídeos e um placebo foram aplicadas na pele dos antebraços de 40 voluntários. As medidas foram realizadas antes e após 2 horas da aplicação das formulações e após 15 e 30 dias de aplicações diárias em termos de hidratação da pele, perda transepidérmica de água (TEWL), viscoelasticidade e micro-relevo da pele. Para a análise subjetiva, os voluntários responderam um questionário a fim de obter-se informações sobre a percepção relativa à qualidade de cosméticos. Todas as formulações provocaram aumento da hidratação cutânea. As formulações que continham os polissacarídeos melhoraram a função barreira da pele, em curto e em longo prazo. A formulação contendo extrato de M. communis apresentou maior aceitação. Os resultados sugerem que o uso diário dos polissacarídeos avaliados é importante na proteção da função barreira da pele.
Subject(s)
Humans , Polysaccharides/analysis , Skin , Biotechnology/instrumentation , Myrtus communis/analysis , Additives in Cosmetics , Fluid Therapy/classification , Klebsiella pneumoniae/classificationABSTRACT
Objective: This study reports an outbreak of Klebsiella pneumoniae infections in 14 patients during a 2-month period (August-September, 2008) in the intensive care unit (ICU) of a teaching hospital in Kuwait. Materials and Methods: The clinical sources were blood (9), urine (3) and respiratory secretions (2) identified by the automated VITEK-2 ID System. Susceptibility testing was performed by the E-test method. Extended-spectrum β-lactamase (ESBL) production was assessed using the ESBL E-test and confirmed by PCR. Carriage of bla genes was determined by PCR and sequence analysis. The transferability of resistance phenotypes was demonstrated by conjugation experiments and clonal relatedness was determined by PFGE. Results: The isolates were susceptible to imipenem, meropenem, and tigecycline and produced ESBL. All isolates yielded an amplicon of 499 bp with universal consensus primers (MA primers). DNA sequence analysis showed that they all harboured blaCTX-M-15 and blaTEM-1 genes. The environmental isolate obtained from a suction machine was also CTX-M-15/TEM-1 producer. The resistance phenotypes were transferrable to the Escherichia coli J53 r strain. PFGE, revealed two clones, A and B, related with a Dice coefficient of >94.1%. A mortality rate of 21.4% was recorded. Conclusion: The outbreak was contained by robust and aggressive infection control measures. This study highlights the first outbreak of CTX-M-15-producing K. pneumoniae associated with high mortality in an adult medical ICU in Kuwait.
Subject(s)
Adult , Aged , Aged, 80 and over , Bacterial Typing Techniques , Bacteriological Techniques , Blood/microbiology , Conjugation, Genetic , Disease Outbreaks , Electrophoresis, Gel, Pulsed-Field , Female , Gene Transfer, Horizontal , Genotype , Hospitals, Teaching , Humans , Intensive Care Units , Klebsiella Infections/epidemiology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Kuwait/epidemiology , Male , Middle Aged , Molecular Typing , Polymerase Chain Reaction , Sequence Analysis, DNA , Sputum/microbiology , Urine/microbiology , beta-Lactamases/biosynthesis , beta-Lactamases/geneticsABSTRACT
Objective: Extended-spectrum beta-lactamases (ESBLs) are most prevalent in Klebsiella pneumoniae. This organism is frequently isolated from clinical specimens and can cause septicemia, pneumonia or urinary tract infection. There were occasionally suspicious outbreaks of ESBL-producing K. pneumoniae in patients' wards. The objective is to determine whether the randomly amplified polymorphic DNA (RAPD), which is a polymerase chain reaction (PCR)-based typing technique, can be used as a typing method for studying the molecular epidemiology of ESBL-producing K. pneumoniae. MATERIAL AND METHOD: The present study was carried out by using 30 ESBL-producing K. pneumoniae isolates obtained from different patients admitted to Siriraj Hospital between January and February 2004. RAPD was evaluated for three primers. All isolates were re-examined by using Southern blot hybridization. RESULTS: It was found that 29 DNA band patterns were generated individually by either AP4 or HLWL74 and R108 primers (30 patterns) for RAPD analysis and 30 patterns for Southern blot hybridization with class 1 integron (intI1) probe. Different patterns indicated that these 30 isolates could not be the cause of an outbreak in Siriraj Hospital. CONCLUSION: The RAPD typing is good and can be used as a screening, rapid and inexpensive'test for ESBL-producing K. pneumoniae during investigation of outbreaks.
Subject(s)
Bacterial Typing Techniques , Blotting, Southern , DNA/analysis , Gene Amplification , Genetic Testing , Genotype , Humans , Hybridization, Genetic , Klebsiella pneumoniae/classification , Polymorphism, Genetic , Random Amplified Polymorphic DNA Technique , Time Factors , beta-Lactamases/biosynthesisABSTRACT
PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microngram/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microngram/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Disease Outbreaks , Disease Susceptibility , Drug Resistance, Multiple, Bacterial , Genotype , Hospitals , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/classification , Korea , Phenotype , beta-Lactamases/classificationABSTRACT
In this study, we investigated the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates that were recovered from an outbreak in a Korean hospital. A new multilocus sequence typing (MLST) scheme for K. pneumoniae based on five housekeeping genes was developed and was evaluated for 43 ESBL-producing isolates from an outbreak as well as 38 surveillance isolates from Korea and also a reference strain. Overall, a total of 37 sequence types (STs) and six clonal complexes (CCs) were identified among the 82 K. pneumoniae isolates. The result of MLST analysis was concordant with that of pulsedfield gel electrophoresis. Most of the outbreak isolates belonged to a certain clone (ST2), and they produced SHV-1 and CTX-M14 enzymes, which was a different feature from that of the K. pneumoniae isolates from other Korean hospitals (ST20 and SHV-12). We also found a different distribution of CCs between ESBL-producing and -nonproducing K. pneumoniae isolates. The MLST method we developed in this study could provide unambiguous and well-resolved data for the epidemiologic study of K. pneumoniae. The outbreak isolates showed different molecular characteristics from the other K. pneumoniae isolates from other Korean hospitals.
Subject(s)
Humans , Electrophoresis, Gel, Pulsed-Field , Hospitals , Klebsiella pneumoniae/classification , Sequence Analysis, DNA , beta-Lactamases/biosynthesis , Nontuberculous Mycobacteria/drug effectsABSTRACT
PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90,6 percent of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.
Subject(s)
DNA, Bacterial/genetics , DNA, Intergenic/genetics , Klebsiella pneumoniae/genetics , Ribotyping/methods , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/isolation & purification , Polymerase Chain Reaction , Reproducibility of Results , /genetics , /geneticsABSTRACT
Klebsiella pneumoniae has long been a prominent cause of nosocomial infections and outbreaks have been observed in the intensive care units and in high risk groups. We present here a brief report on an outbreak of Klebsiella pneumoniae which occurred in a neonatal intensive care unit in our teaching hospital. As neonates are at highest risk for acquisition of Klebsiella pneumoniae producing extended spectrum beta-lactamase, infection control policies and procedures should be strictly followed to prevent such outbreaks.
Subject(s)
Anti-Bacterial Agents/pharmacology , Cefotaxime/pharmacology , Ceftazidime/pharmacology , Ceftriaxone/pharmacology , Cephalosporin Resistance , Cross Infection/epidemiology , Disease Outbreaks , Hospitals, Teaching , Humans , India/epidemiology , Infant, Low Birth Weight , Infant, Newborn , Intensive Care Units, Neonatal , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/classification , beta-Lactamases/biosynthesisABSTRACT
In the present study, isolation of anaerobic bacteria from 24 different eco-niches was carried out. A total number of 300 bacterial isolates, including 230 obligate and 70 facultative anaerobes were obtained using anaerobic techniques. All the isolates were initially screened for succinic acid production by Fluorescein test and TLC method. During screening, 10 isolates found to produce succinic acid were further examined by HPLC and then finally confirmed for succinic acid by LC-MS analysis. Amongst 10 isolates, isolate SAP, a facultative anaerobe isolated from buffalo rumen fluid, showed maximum yield of 2.1 g/l of succinic acid from 10 g of glucose in 24 hr under anaerobic condition. This isolate was identified as Klebsiella pneumoniae strain SAP by 16S rDNA sequence and signature sequence analysis. Mouse lethality test for the strain SAP showed LD50 value of 3.3 x 10(8) CFU/ml, which shows non-virulent nature of the strain. This strain may become a candidate strain for succinic acid production because of its osmotolerant nature and higher succinate:acetate ratio.
Subject(s)
Acetates/metabolism , Animals , Bacteria, Anaerobic/metabolism , Bacterial Proteins/metabolism , Bacterial Toxins/analysis , Base Sequence , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Fluorescein/diagnosis , Gas Chromatography-Mass Spectrometry , Humans , Klebsiella pneumoniae/classification , Mice , Osmosis/physiology , RNA, Ribosomal, 16S/genetics , Succinic Acid/metabolism , VirulenceABSTRACT
Respiratory isolates of Klebsiella pneumoniae in Korea during 2002-2003 were studied to determine the prevalence and types of extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC beta-lactamases (PABLs). ESBL-production was tested by double-disk synergy, and genotypes of beta-lactamases were determined by PCR and sequencing. ESBLs were detected in 28.4% of 373 isolates, and the most prevalent types were SHV-12 (63 isolates) and CTX-M-14 (9 isolates). Forty of 75 ESBL-producers (53.5%) also had PABLs: 21 isolates with CMY-2-like, 17 with DHA-1-like. Pulsed-field gel electrophoresis showed 19 types and 25 of 74 isolates had an identical pattern, indicating nosocomial spread. Dissemination of ESBL- and PABL-producing K. pneumoniae strains in Korea is a particular concern, as it limits the choice of antimicrobial agents for treatment of infections.
Subject(s)
Humans , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/biosynthesis , Base Sequence , Cross Infection/microbiology , DNA, Bacterial/genetics , Drug Resistance, Bacterial , Electrophoresis, Gel, Pulsed-Field , Genes, Bacterial , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/classification , Korea , Respiratory Tract Infections/drug therapy , beta-Lactamases/biosynthesisABSTRACT
Klebocin typing and antibiotic resistance have been studied for 518 strains of Klebsiella pneumoniae, [106 from intensive care unit (ICU) sites, 182 from ICU staff flora, 192 from patient flora and 38 from clinical specimens]. The overall typability was 71.62%. The most common mnemonic types among various sources were 111, 211, and 112. Of the total strains tested, 28.37% strains were found to be untypable. These strains are labelled as "444". When klebocin typing was used in association with antibiogram, in 86.84% cases of clinical infection probable source of infection could be detected. Thus a combination of two typing methods poses a significant contribution in epidemiological studies.