Identification and antimicrobial susceptibility profile of bacteria isolated from primary endodontic infections.

This study aimed to identify and characterize the antimicrobial susceptibility profile of bacteria found in primary endodontic infections in the teeth of patients treated at the Dental Clinic of the University of Ribeirão Preto, São Paulo, Brazil. From September to December 2019, samples were obtained from 21 patients with primary endodontic infections. The collections were carried out in triplicate using paper cones placed close to the total length of the root canal. Bacterial isolation was performed in Brain Heart Infusion agar, Blood agar, and other selective culture media cultured at 37°C for up to 48 h under aerobiosis and microaerophilic conditions. The bacterial species were identified using the Vitek 2 automated system. The disk diffusion method on agar Müeller-Hinton was used to assess antimicrobial susceptibility with the recommended antimicrobials for each identified bacterial species. A total of 49 antibiotics were evaluated. Fifteen of the 21 samples collected showed bacterial growth, and 17 bacterial isolates were found. There were 10 different bacterial species identified: Enterococcus faecalis (four isolates), Streptococcus mitis/oralis (three isolates), Streptococcus anginosus (three isolates) being the most common, followed by Staphylococcus epidermidis, Enterococcus faecium, Streptococcus constellatus, Streptococcus alactolyticus, Enterobacter cloacae, Klebsiella variicola, and Providencia rettgeri (one isolate of each species). The analysis demonstrated significant susceptibility to most of the tested antibiotics. However, some Enterococcus isolates resisted the antibiotic's erythromycin, ciprofloxacin, and tetracycline. A Staphylococcus epidermidis isolate was characterized as multidrug-resistant. Five Streptococcus isolates were non-susceptible to all antibiotics tested.

Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

High Prevalence of Panton-valentine Leukocidin-encoding Genes in Methicillin-resistant Staphylococcus aureus Isolated from Inpatients with Invasive Infections at a University Hospital in Southern Brazil.

BACKGROUND: Staphylococcus aureus is a major cause of a wide diversity of infections in humans, and the expression of Panton-Valentine Leukocidin (PVL) has been associated with severe clinical syndromes. OBJECTIVES: The present study aimed to investigate the prevalence of PVL-encoding genes in S. aureus isolated from clinical samples of inpatients with invasive infections in a teaching hospital in Southern Brazil. Furthermore, phenotypic and genotypic characteristics of bacterial isolates were analyzed. METHODS: A total of 98 S. aureus isolates recovered from different body sites were characterized according to their antimicrobial susceptibility profile, methicillin-resistance and SCCmec typing, genetic relatedness and occurrence of virulence-encoding genes, such as icaA, lukS-PV/lukF-PV, and tst. RESULTS: Sixty-eight (69.4%) isolates were classified as methicillin-resistant, and among them, four (5.9%) did not harbor the mecA gene. The mecA-harboring methicillin-resistant S. aureus (MRSA) isolates were grouped into SCCmec types I (6.3%), II (64.1%), III (6.3%), IV (15.6%), V (4.7%), and VI (1.6%). One isolate (1.6%) was classified as non-typeable (NT). Seventy isolates (71.4%) were classified as multidrug-resistant. The overall prevalence of virulence-encoding genes was as follows: icaA, 99.0%; tst, 27.5%; and lukS-PV/lukF-PV, 50.0%. The presence of tst gene was significantly higher (p < 0.001) in methicillin-susceptible S. aureus (MSSA) compared to MRSA isolates. CONCLUSION: The present study reports a high prevalence of multidrug-resistant S. aureus harboring lukS-PV/lukF-PV and tst genes in invasive infections. The continuous monitoring of the antimicrobial susceptibility profile and virulence of S. aureus is an important measure for the control of infections caused by this bacterium.

High mortality from carbapenem-resistant Klebsiella pneumoniae bloodstream infection.

In this study, it was evaluated clinical data of 107 patients with bloodstream infection (BSI) by Klebsiella pneumoniae and performed phenotypic and molecular analyzes in 50.5% (54/107) of the samples, those that showed a resistance profile to carbapenemics. The blaKPC gene was present in 90.4% (49/54) of the samples, blaNDM gene in one sample and, in 7.4% (4/54) of the samples, no carbapenemase gene was found. In the similarity analysis, it was found 4 main clones and 11 samples were not genetically related. The median age of the patients was 58 (40-70) years old and 60.7% (65/107) were male. When comparing two groups of patients with BSI due to K. pneumoniae with and without resistance to carbapenems, the variables ICU permanence, renal failure (IR), previous use of antimicrobials, Charlson's comorbidity index (ICCi), some invasive procedures and death showed a statistically significant difference (p < 0.05). And when relating death as a dependent variable, IR, liver failure and patients with BSI XDR or PDR, were predictors of increased mortality. Our study showed a higher mortality rate in patients with BSI due to carbapenem-resistant pneumonia with additional resistance or not to polymyxins.

Colonization by multidrug-resistant microorganisms of hospitalized newborns and their mothers in the neonatal unit context.

INTRODUCTION: The mother plays a fundamental role in the constitution and regulation of her child's healthy microbiota, however, preterm newborns are separated from their mothers soon after birth and transferred to Neonatal Intensive Care Units, being exposed the constant risk for the development of multidrug-resistant microorganisms' infections. The aim of this study was to explore the multidrug-resistant microorganism colonization of hospitalized babies and their mothers in the neonatal unit context. METHODOLOGY: A prospective case study conducted with hospitalized babies and their mothers in the Neonatal Unit at a university hospital. The sample was composed of 433 binomials (mother-child). Colonization culture samples were taken at the moment of the baby's discharge, via two swabs in the oral, nasal, axillary, inguinal, and rectal regions. RESULTS: The colonization incidence among the binomials, 30 (6.9%) were both colonized by multi-resistant microorganisms. Mothers of colonized babies (24.4%) demonstrated a higher chance of colonization in comparison to mothers of non-colonized babies (11.9%) (p = 0.002). Relationships were drawn between baby colonization and prematurity, extremely low birth weight, and non-exclusive maternal breastfeeding (p<0.05). ESBL-producing Gram-negative microorganisms were more frequent in the cultures of the binomials, with 35.9% of the babies colonized with Klebsiella spp. ESBL and 42.0% of the mothers with Escherichia coli ESBL. Furthermore, 50% of the binomials were colonized with E. coli ESBL. CONCLUSION: The prematurity, extremely low birth weight, and non-exclusive breastfeeding at hospital discharge were associated with baby colonization by multidrug-resistant microorganism. Furthermore, mothers of colonized children presented higher chances of colonization.

Colonization profile and duration by multi-resistant organisms in a prospective cohort of newborns after hospital discharge.

The aim of this study was to determine the spontaneous decolonization period and characteristics in a prospective cohort of newborns colonized by multidrug-resistant organisms, after their discharge from the neonatal intensive care unit. Multidrug resistance is defined as bacterial non-susceptibility to ≥ 1 agent of ≥ 3 antimicrobial categories. In total, 618 newborns were included in the study, of which 173 (28.0%) presented a positive culture for multidrug-resistant microorganisms, and of these, 52 (30.1%) were followed up in this study. The most frequent intrinsic factors were be born by cesarean section (86.5%), prematurity (84.6%), and very low birth weight (76.9%). The extrinsic factors were having remained hospitalized for an average of 27 days, during which 67.3% were submitted to invasive procedures and 88.5% received antimicrobials. The intrinsic and extrinsic factors of newborns were not associated to a decolonization period longer or shorter than 3 months, which was the average period of decolonization found in the present study. From the totality of colonization cultures sampled at hospital discharge, the Gram-negative Extended Spectrum ß-lactamase producing bacteria were the most common, with 28.9% of babies colonized by Klebsiella spp. The median period of decolonization by multidrug-resistant microorganisms in the newborns population after hospital discharge was 3 months, but was highly dependent on the microbial species, and this period was not associated to any intrinsic and extrinsic factors of the newborn.

Escherichia coli Bloodstream Infections in Patients at a University Hospital: Virulence Factors and Clinical Characteristics.

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates are responsible for many bloodstream infections. The aim of this study was to characterize E. coli isolated from the bloodstreams of patients (n = 48) at the University Hospital in Brazil. Epidemiological data were obtained through the analysis of medical records and laboratory tests. By PCR analysis, we investigated the presence of virulence factors (VFs), pathogenicity islands (PAIs), extended-spectrum ß-lactamase (ESBL), phylogenetic classifications (A, B1, B2, C, D, E, and F) and molecular genotype by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The mortality analysis showed that 33.3% of the deaths were associated with bacteraemia due to E. coli infections; in addition, an age between 60 and 75 years (p < 0.001; OR = 6.3[2.1-18.9]) and bacteraemia with an abdominal origin (p = 0.02; OR = 5[1.2-20.5]) were risk factors for the severity of the infection. Additionally, the presence of the afa gene was associated with mortality due to E. coli bacteraemia (p = 0.027; OR = 11.4[1.5-85.7]). Immunosuppression (27.1%), intestinal diseases (25.0%) and diabetes (18.8%), were prevalent among patients, and most of the bacteraemia cases were secondary to urinary tract infections (50.0%). The serum resistance gene traT was present in 77.1% of isolates, group capsular 2 (kpsMT II) was present in 45.8% and the K5 capsule was present in 20.8% of isolates. The isolates also showed a high prevalence for the siderophore yersiniabactina (fyuA) (70.8%) and PAI IV536 (77.1%). Phylogenetic analysis showed that group B2 (45.8%) was the most prevalent, and was the phylogroup that had a higher prevalence of VFs and PAIs. However, in this study, a considerable number of isolated bacteria were classified as group B1 (18.8%) and as group E (14.6%). Eight (16.7%) isolates were resistant to third and fourth generation cephalosporin and group CTX-M-1 (CTX-M-15) was the most prevalent ESBL type. The molecular genotyping showed two clonal lineages and several isolates that were not related to each other. This study provides additional information on the epidemiological and molecular characteristics of E. coli bloodstream infections in Brazil.

Prevalence and antimicrobial susceptibility profile of Streptococcus agalactiae in pregnant women seen at the University Hospital of Londrina, Paraná, Brazil / Prevalência e sensibilidade aos antimicrobianos de Streptococcus agalactiae em gestantes atendidas no Hospital Universitário de Londrina, Paraná, Brazil

A retrospective study of pregnant women seen at the University Hospital of Londrina, Paraná, Brazil was performed to determine the prevalence of Group B Streptococcus (GBS) vaginal-rectal colonization, and the GBS susceptibility for antimicrobials used in intrapartum antibiotic prophylaxis. A vaginal-rectal swab was collected from 2,901 women between 35 and 37 weeks of gestation. Of these, 527 (18.2%) had a positive culture for GBS, and 0.4%, 10.2% and 10% of the isolates were resistant to penicillin, erythromycin and clindamycin, respectively. These results highlight the importance of continuous surveillance of GBS colonization in pregnant women for preventing GBS infections in neonates.

Um estudo retrospectivo foi realizado com gestantes atendidas no Hospital Universitário de Londrina, Paraná, Brasil para determinar a prevalência de colonização vaginal-retal por estreptococos do Grupo B (EGB) e o perfil de sensibilidade de EGB aos antimicrobianos utilizados para a antibioticoterapia profilática intraparto. Swabs vaginais-retais foram coletados de 2.901 mulheres entre a 35ª e 37ª semana de gestação. Destes, 527 (18,2%) apresentaram cultura positiva para EGB, e 0,4%, 10,2% e 10% dos isolados foram resistentes à penicilina, eritromicina e clindamicina, respectivamente. Estes resultados destacam a importância de vigilância contínua da colonização por EGB em gestantes para a prevenção de infecções em neonatos por EGB.

Bacteremia causada por Staphylococcus aureus: Uma análise de quinze anos da sensibilidade a antimicrobianos em um hospital terciário do Brasil / Bacteremia caused by Staphylococcus aureus: a fifteen-year analysis of antimicrobial susceptibility in a tertiary hospital in Brazil / Bacteremia causada por Staphylococcus aureus: Un análisis de quince años de la sensibilidad a los antimicrobianos en un hospital terciario de Brasil

Justificativa e Objetivos: Infecções da corrente sanguínea por Staphylococcus aureus constituem uma das principais causas de morbidade e mortalidade em todo mundo. O tratamento de infecções por S. aureus é complexo, em parte, devido à elevada prevalência de resistência aos antimicrobianos. Compreender a epidemiologia e os padrões de resistência deste microrganismo é um ponto crítico para a prescrição empírica adequada de antimicrobianos. Assim, este estudo teve por objetivo avaliar a evolução de resistência antimicrobiana de S. aureus em um período de quinze anos. Métodos: Foram analisados os perfis de sensibilidade para os antimicrobianos ciprofloxacina (5µg); clindamicina (2µg); eritromicina (15µg); gentamicina (10µg); oxacilina (30µg); penicilina (10U); rifampicina (5µg); sulfametoxazol-trimetoprima (23.75/1.25µg) e tetraciclina (30µg) em 720 isolados de S. aureus provenientes de hemoculturas em um hospital terciário do sul do Brasil. Os valores de sensibilidade adotados foram aqueles contidos no CLSI, 2017. Os dados foram obtidos do Sistema de Informação AGTA Healthcare, módulo LABHOS®. Resultados: A frequência média de S. aureus resistente a meticilina foi de 43,74%. Com exceção de penicilina, ocorreu variação significativa da resistência para todos os antimicrobianos no período avaliado (ρ<0,001). Ciprofloxacina (51,14%), eritromicina (44,99%) e clindamicina (39,85%) apresentaram os maiores índices de resistência com tendência de aumento. Surpreendentemente, gentamicina (4%) e sulfametoxazol-trimetoprima (4%) apresentaram queda significativa nos percentuais de resistência. Para vancomicina, do ano 2010 a 2015, observou-se um aumento das concentrações inibitórias mínimas. Conclusão: Embora o índice de resistência tenha aumentado nos quinze anos para a maioria dos antimicrobianos, para sulfametoxazol-trimetoprima e gentamicina ocorreu redução significativa. Este estudo evidenciou, ainda, a emergência do fenótipo S. aureus com resistência intermediária a vancomicina.(AU)

Background and Objectives: Bloodstream infections caused by Staphylococcus aureus are a major cause of morbidity and mortality worldwide. Treatment of S. aureus infections is complex, in part, due to the high prevalence of antimicrobial resistance. Understanding the epidemiology and resistance patterns of this microorganism is a critical point for the proper empirical prescription of antimicrobials. Thus, this study aimed to evaluate the evolution of antimicrobial resistance of S. aureus in a period of fifteen years. Methods: Antimicrobial susceptibility profiles was determined for cefoxitin (30µg), penicillin (10 U), erythromycin (15 µg), clindamycin (2 µg), gentamycin (10 µg),ciprofloxacin (5 µg), sulfamethoxazole-trimethoprim (23.75/1.25 µg), rifampicin (5 µg), and tetracycline (30µg) in 720 S. aureus isolated from blood cultures in a tertiary hospital in southern Brazil were analyzed. Sensiblity values was determined according to Clinical Laboratory Standards Institute (CLSI, 2017).The data were obtained from the AGTA Healthcare Information System, LABHOS® module. Results: The mean frequency of methicillin-resistant S. aureus was 43.74%. Except for penicillin, there was a significant variation of resistance for all antimicrobials in the period evaluated (ρ<0.001). Ciprofloxacin (51.14%), erythromycin (44.99%) and clindamycin (39.85%) had the highest rates of resistance, with tendency to increase. Surprisingly, gentamicina (4%) and sulfamethoxazole-trimethoprim (4%) showed a significant percentage decrease in resistance. For vancomycin, from 2010 to 2015, it was observed an increase in minimum inhibitory concentrations. Conclusion: Although the resistance rate increased in the fifteen years for most antimicrobials, for sulfamethoxazole-trimethoprim and gentamicin a significant reduction occurred, indicating a possible clonal change. This study also evidenced the emergence of S. aureus with intermediate resistance to vancomycin phenotype.(AU)

Justificación y objetivos: Infecciones del flujo sanguíneo por Staphylococcus aureus constituyen una de las principales causas de morbilidad y mortalidad en todo el mundo. El tratamiento de las infecciones por S. aureus es complejo, en parte debido a la elevada prevalencia de resistencia a los antimicrobianos. Comprender la epidemiología y los patrones de resistencia de este microorganismo es un punto crítico para la prescripción empírica adecuada de antimicrobianos. Así, este estudio tuvo por objetivo evaluar la evolución de resistencia antimicrobiana de S. aureus en un período de quince años. Métodos: Se analizaron los perfiles de sensibilidad a los antimicrobianos ciprofloxacino (5µg); clindamicina (2µg); eritromicina (15 µg); gentamicina (10µg); oxacilina (30µg); penicilina (10U); rifampicina (5µg); sulfametoxazol-trimetoprima (23.75 / 1.25 µg) y tetraciclina (30µg) de 720 S. aureus aislados de hemocultivos de un hospital terciario del sur de Brasil. Los valores de sensibilidad adoptados fueron aquellos contenidos en el Clinical Laboratory Standards Institute (CLSI, 2017). Los datos fueron obtenidos del Sistema de Información AGTA Healthcare, módulo LABHOS®. Resultados: La frecuencia media de S. aureus resistente a meticilina fue de 43,74%. Con excepción de la penicilina, hubo variación significativa de la resistencia para todos los antimicrobianos en el período evaluado (ρ<0,001). Ciprofloxacino (51,14%), eritromicina (44,99%) y clindamicina (39,85%) presentaron los mayores índices de resistencia con tendencia de aumento. Sorprendentemente, gentamicina (4%) y sulfametoxazol-trimetoprim (4%) presentaron una caída significativa en los porcentajes de resistencia. Para vancomicina del año 2010 a 2015 se puede evidenciar un aumento de las concentraciones inhibitorias mínimas. Conclusiones: Aunque la resistencia a antimicrobianos aumentó en los quince años para la mayoría de los antimicrobianos, para sulfametoxazol-trimetoprim y gentamicina se produjo una reducción significativa, indicando un posible cambio clonal. Este estudio evidenció, además, la emergencia del fenotipo S. aureus con resistencia intermedia a vancomicina.(AU)

Spread of multidrug-resistant high-risk Klebsiella pneumoniae clones in a tertiary hospital from southern Brazil.

Klebsiella pneumoniae is among the most important pathogens found in hospitals. The emergence of multiple antibiotic resistant K. pneumoniae associated with its virulence factors is a worldwide concern and its early identification is crucial, especially for controlling the spread of emerging clones. This article reports a high prevalence of multiresistant K. pneumoniae in a university hospital in southern Brazil, harboring several virulence and ß-lactamase encoding genes, including pandrug-resistant high-risk international clones belonging to the clonal group 258 (ST11, ST15, ST101, ST258, ST340 and ST874).

Antimicrobial activity evaluation and comparison of methods of susceptibility for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter spp. isolates

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.

Antimicrobial activity evaluation and comparison of methods of susceptibility for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter spp. isolates.

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.

Molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolates from a university hospital in Brazil.

INTRODUCTION: The emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kpn) isolates is attracting significant attention in nosocomial infection settings. K. pneumoniae is the main pathogen that harbours blaKPC genes. METHODOLOGY: This study evaluated 54 K. pneumoniae carbapenem-resistant isolates from patients hospitalized at the University Hospital of Londrina, between July 2009 and July 2010. The isolates were phenotypically screened for carbapenemase production and submitted for genotypic confirmation by polymerase chain reaction (PCR) for KPC, metallo-ß-lactamases, OXA-48, and extended-spectrum beta-lactamase genes. The absence of outer membrane proteins (OMP) was investigated by SDS-PAGE. The susceptibility profile was determined by broth microdilution, according to Clinical and Laboratory Standards Institute protocol. RESULTS: All isolates were phenotypically positive for class A carbapenemase production, but negative for metallo-ß-lactamase activity. PCR analysis demonstrated that all isolates carried blaKPC genes and sequencing showed that all strains belonged to KPC-2 subtype. Four strains did not show porin expression, and all isolates were resistant to ertapenem, meropenem, and imipenem. Susceptibility rates reached 35.2% for gentamicin, 85.2% for polymixyn B, 87% for colistin, and 98.1% for both tigecycline and fosfomycin. Pulsed-field gel electrophoresis showed six clones, and three of them predominated among the isolates. CONCLUSIONS: KPC-2-producing K. pneumoniae is becoming predominant among carbapenem-resistant K. pneumoniae isolates at the hospital. The association of the enzyme KPC with other resistance determinants, such as loss of porins, may increase the severity of the situation of nosocomial infections. There is an urgent need to develop strategies for infection control and prevention.

Effect of a Metalloantibiotic Produced by Pseudomonas aeruginosa on Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae.

Multidrug-resistant organisms (MDRO) are a great problem in hospitals, where thousands of people are infected daily, with the occurrence of high mortality rates, especially in infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-producing Kpn). The challenge is to find new compounds that can control KPC producing-Kpn infections. The aim of this study was to evaluate the antibiotic activity of the F3d fraction produced by the Pseudomonas aeruginosa LV strain against clinical isolates of KPC-producing Kpn. The results showed that the minimum inhibitory concentration of F3d (62.5 µg mL(-1)), containing an organic metallic compound, killed planktonic cells of KPC-producing Kpn strains after 30 min of incubation. At the same concentration, this fraction also showed an inhibitory effect against biofilm of these bacteria after 24 h of incubation. Treatment with the F3d fraction caused pronounced morphological alterations in both planktonic and biofilm cells of the bacteria. The inhibitory effect of the F3d fraction seems to be more selective for the bacteria than the host cells, indicating its potential in the development of new drugs for the treatment of infections caused by KPC-producing Kpn and other MDRO.

Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.

Comparação do sistema de identificação automatizado Vitek 2 e PCR-ITS para caracterização das espécies dos isolados clínicos de Candida spp / Comparison of Vitek-2 automated identification system and PCR-ITS for species characterization of clinical isolates of Candida spp

As infecções graves causadas pelo gênero Candida têm se tornado um desafio na questão diagnóstica, no intuito de se detectar e identificar o agente etiológico de forma ágil, precisa e padronizada nos laboratórios clínicos. A predição da susceptibilidade aos antifúngicos, bem como a necessidade da geração de dados epidemiológicos reforçam a importância da identificação rotineira adequada das espécies de leveduras envolvidas em infecções. Dentre as 200 espécies de Candida já descritas, C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. guilliermondii, C. krusei e C. lusitaniae são mais frequentemente relacionadas a infecções em humanos. Todos os métodos fenotípicos de identificação de Candida apresentam limitações,em especial na caracterização de espécies não C. albicans, porém, a aplicação de métodos moleculares pode refletir no aumento de custo e tempo despendido para a obtenção de resultados laboratoriais. A fim de avaliara aplicação do sistema automatizado Vitek 2-YST ID (bio Merieux) aliado ao uso de agar cromogênico na identificação rotineira de espécies de Candida, foram testados 44 isolados de infecção invasiva por inoculação em agar cromogênico e no painel automatizado e realização de amplificação do DNA relativo às regiões do espaçador interno transcrito 1 e 2 do rRNA (PCR-ITS). Oligo nucleotídeos espécie específicos foram utilizados e o tamanho do produto amplificado foi correlacionado aos demais resultados. O sistema automatizado identificou 95,4% dos isolados quando em associação com as características coloniais observadas no meio cromogênico, porém, o uso de PCR-ITS ou metodologias mais sensíveis seria necessário para solucionar os demais resultados, ambíguos e errôneos.

Serious infections caused by genus Candida have become a challenge in the diagnostic question, in order todetect and identify the etiologic agent of agile, precise and standardized form in manner clinical laboratories. The prediction of susceptibility to antifungal agents, and the need to generate epidemiological data highlight the importance of routine identification of yeast species involved in infections. Among the 200 Candida species already described, C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. guilliermondii, C. krusei and C. lusitaniae are most often related with infections in humans. All of the phenotypic methods of identification of Candida have limitations, especially the characterization of the species not C. albicans, however, the application of molecular methods may reflect the increased cost and spent time for obtain results on laboratory. In order to evaluate the implementation of the automated system Vitek 2 ID - YST(bioMerieux) combined with the use of chromogenic agar in the routine identification of Candida species were tested 44 isolates from invasive infection by inoculation of chromogenic agar and automated panel and realization DNA amplification for the internal transcribed spacer regions of rRNA 1 and 2 (ITS - PCR). Oligonucleotides specific species were used and the size of the amplified product was correlated to other results. The automated system identified 95.4 % of the isolates when in association with colonial features observed in chromogenic medium, however, the use of PCR -ITS or more sensitive methodologies would be needed to solve the other results, ambiguous and erroneous.

Impacto de um bundle nas taxas de pneumonia associada à ventilação mecânica (PAV) em uma unidade de terapia intensiva pediátrica em Londrina-PR / Impact of the ventilator bundle on ventilator-associated pneumonia (VAP) rates in a pediatric intensive care unit in Londrina-PR

Infecções associadas aos cuidados de saúde (IRAS) constituem importante preocupação à segurança do paciente ao redor do mundo. Pneumonia associada à ventilação mecânica (VAP) é a principal causa de morte entre IRAS, com mortalidade de 15 a 70%, dependendo da população de pacientes. O Centro de Controle e Prevenção de Doenças (CDC) recomendou que todas as UTIs implementassem um bundle depara reduzir a taxa de PAV. Os objetivos do presente estudo foram analisar o efeito do pacote de intervenções de controle da infecção, a educação, as taxas de PAV na Unidade Pediátrica de Terapia Intensiva (UTIP)do Hospital Universitário de Londrina-PR. Este estudo foi realizado entre janeiro e dezembro de 2013 e consistiu em três períodos: pré-intervenção, intervenção e pós-intervenção. A intervenção educativa foi dada a 86 trabalhadores da saúde (PS), e um questionário também foi realizado no pré e pós-intervenção. No geral,foram avaliadas 135 oportunidades de atendimento ao paciente. A higiene das mãos diminuiu do período pré para o pós-intervenção, entretanto a intubação gástrica por via oral, a manutenção da cabeceira da cama entre 30-45º, a pressão do cuff endotraqueal e remoção de condensação do circuito respirador aumentaram significativamente do período pré para o pós-intervenção. A taxa de PAV foi 49,6% durante o período de pré-intervenção e 17,5% durante o período pós-intervenção demonstrando uma redução de 64,8%. Nossos resultados mostram que a implementação do pacote de intervenções de controlo da infecção foi associada com uma redução significativa na taxa de VAP.

Healthcare-associated infections (HAI) are an important patient safety concern around the globe. Ventilator associated pneumonia (VAP) is the leading cause of death among HAI, with attributable mortality ranging from 15 to 70% depending on the patient population. The Center for Disease Control and Prevention (CDC) has recommended that all ICUs implement a ventilator bundle to reduce the VAP rate. The purposes of the present study were to examine the effect of the bundle of infection control interventions, education, VAP rates in the Pediatric Intensive Care Unit (PICU) of the Hospital Universitário of Londrina-PR. This study was conducted between January and December 2013 and consisted of three periods: pre-intervention, intervention and post-intervention. An educational intervention was given to 86 health care workers (HCWs) about bundles to prevent VAP, and a questionnaire was also performed pre and post-intervention. Overall, 135 opportunities of patient care were evaluated. The compliance with hand hygiene and the use of gloves and gowns did not improve, but orotracheal intubation, maintenance of the patients in a 30-45º head of bed elevation, endotracheal cuff pressure and removal of condensate from ventilator circuts increased significantly when comparing pre-and post-intervention. The VAP rate was 49.6% during the pre-intervention period and 17.5% during the post-intervention period demonstrating a 64.8% reduction in VAP rate. Our results show that implementation of the bundle of infection control interventions was associated with a significant reduction in VAP rate.

Evolução da resistência de Klebsiella pneumoniae no Hospital Universitário de Londrina no período de 2000 a 2011 / Evolution of resistance of Klebsiella pneumoniae in Londrina University Hospital from 2000 to 2011

O presente estudo teve como objetivo avaliar a evolução da resistência a antimicrobianos em isolados clínicos de Klebsiella pneumoniae, no período de 2000 a 2011, no Hospital Universitário da Universidade Estadual de Londrina (HU-UEL). Foi realizada uma análise retrospectiva de 2.318 testes de sensibilidade aos antimicrobianos de K. pneumoniae, a partir de um banco de dados do setor de Microbiologia do Laboratório Clínico do HU. No período de 2000 a 2009, o principal mecanismo de resistência aos antimicrobianos β-lactâmicos observado foi a produção de β-lactamases do tipo ESBL (β-lactamase de espectro ampliado),que pode ser verificado pelo aumento da resistência de K. pneumoniae às cefalosporinas de 3a. e 4a. geração. No entanto, a partir de 2009 apareceram as primeiras cepas de K. pneumoniae produtoras de carbapenemase, comprometendo a eficácia dos carbapenêmicos. Os índices de resistência ao ertapenem variaram de 16%,em 2005, para 40%, em 2011. Outra classe de antimicrobianos comprometida foi a das fluoroquinolonas; para ciprofloxacina, os índices de resistência variaram de 13% a 62%, em 2001 e 2011, respectivamente.Os aminoglicosídeos tiveram oscilações de resistência durante o período estudado, chegando, em 2011,a valores de 56% e 30% para gentamicina e amicacina, respectivamente. Enquanto isso, sulfametoxazol/trimetoprim e piperacilina/tazobactam alcançaram índices de resistência de 60%, nesse mesmo período. O aumento de resistência em K. pneumoniae neste hospital evidencia a necessidade de adequação do tratamento de infecções por este agente e de adoção o de medidas apropriadas que visem ao controle de infecções, bem como ao uso adequado dessas drogas.

The present study aimed to evaluate the evolution of antibiotic resistance in clinical isolates of Klebsiella pneumoniae in the period of 2000 to 2011, at the University Hospital of Londrina (HU-UEL). A retrospective analysis of 2,318 antimicrobial susceptibility tests of Klebsiella pneumoniae was performed from a database of the Clinical Laboratory of Microbiology of the University Hospital. In the period of 2000 to 2009, the main mechanism of resistance observed to β-lactam antimicrobials was due to the production of ESBL β-lactamase type (β-lactamase wide spectrum), which can be verified by the increased resistance of Klebsiella pneumoniae to 3rd generation cephalosporins and cefepime. However, the first strains of Klebsiella pneumoniae carbapenemase-producing appeared in 2009, compromising the efficacy of carbapenems. The rates of resistance to ertapenem ranged from 16%, in 2005, to 40% in 2011. Another class of committed antibiotics was the fluoroquinolones; for ciprofloxacin, resistance rates ranged from 13% to 62%, in 2001 and 2011, respectively. Aminoglycosides exhibited oscillations of resistance during the period studied, reaching,in 2011, values of 56% and 30% for gentamicin and amikacin, respectively. Meanwhile, trimethoprim/sulfamethoxazole and piperacillin/tazobactam reached about 60%, in the same period. Therefore, knowing the antimicrobial resistance of Klebsiella pneumoniae strains is essential for proper treatment of patients and adoption of appropriate measures that aims infection control, and proper use of these drugs.

Tendência de resistência entre isolados clínicos de Staphylococcus aureus em um hospital universitário do norte do Paraná de 2002 a 2011 / Trends in antimicrobial resistance among clinical isolates of Staphylococcus aureus in a university hospital of the north of Paraná from 2002 through 2011

Staphylococcus aureus são patógenos com alta ocorrência em infecções hospitalares e comunitárias e têm grande capacidade de adquirir resistência. O objetivo deste estudo foi determinar o perfil de resistência aos antimicrobianos de S. aureus isolados no Hospital Universitário de Londrina de janeiro de 2002 a dezembro de 2011. A análise retrospectiva de 3.494 S. aureus foi realizada a partir de um banco de dados do setor de Microbiologia do Laboratório Clínico do Hospital Universitário de Londrina (HUL). Resistência aos antimicrobianos foi determinada de acordo com os critérios recomendados pelo Clinical Laboratory Standard Institute (CLSI-2011). Os maiores percentuais de resistência foram verificados para eritromicina (49,4%), oxacilina, clindamicina (41,8%) e ciprofloxacina (36,5%). Adicionalmente, ocorreu redução significativa nas taxas de resistência à gentamicina, e a sulfametoxazol-trimetoprim. Todas as cepas analisadas foram sensíveis à linezolide. Verificou-se que 40% apresentaram susceptibilidade reduzida à vancomicina. Estes dados revelaram uma provável mudança na epidemiologia de S. aureus na nossa região, o que pode trazer impacto no tratamento e controle da infecção por este agente etiológico.

Staphylococcus aureus are pathogens with high incidence of nosocomial infections and community and has great ability to acquire resistance. The objective of this study was to determine susceptibility to antimicrobial profiles of S. aureus isolated in Londrina University Hospital, from January 2002 through December 2011. The retrospective analysis of 3,494 S. aureus performed from a database of Clinical Laboratory Microbiology HU sector. Antibiotic resistance was judged according to the criteria recommended by the Clinical Laboratory Standards Institute. The highest percentages of resistance was founding to erythromycin (49.4%), clindamycin and oxacillin (41.8%) and ciprofloxacin (36.5%). In addition, there was a significant reduction in gentamicin resistance rates, and trimethoprim-sulfamethoxazol. All strains studied were susceptible to linezolid. We found that 40% showed reduced susceptibility to vancomycin. These data indicate a possible change in the epidemiology of S. aureus in our region, which can bring impact on the treatment and control of infection by this agent.

Enterococcus spp. resistentes à vancomicina: características clínicas efatores de risco / Vancomycin-resistant Enterococcus spp: clinical characteristics and risk factors

Enterococcus spp. resistentes à vancomicina (ERV) têm emergido como um patógeno multirresistente relevante e de etiologia potencialmente letal nas infecções associadas à assistência em saúde ao redor do mundo. Este estudo pretende mostrar epidemiologia e características clínicas de pacientes com ERV em um hospital do sul do Brasil. Um estudo retrospectivo foi conduzido no período de janeiro de 2005 a novembro de 2007 no Hospital Universitário de Londrina. Todos os pacientes com cultura clínica com ERV foram identificados e seu prontuário médico revisado. A presença de colonização foi avaliada através de culturas de swab retal e a identificação das amostras clínicas foi realizada pelo método automatizado MicroScan®. A média de idade dos pacientes foi de 54 anos. Trato urinário (68,0%) e corrente sanguínea (23,8%) foram os sítios mais frequentes, e a UTI apresentou se como setor de maior ocorrência (49,2%) das culturas positivas. E. faecium foi a espécie predominante, em 82,8% dos casos. Os fatores de risco observados foram a duração da internação (média de 58,2 dias), uso de antimicrobianos prévios e realização de procedimento invasivos, como o uso de cateter venoso central, sonda vesical e ventilação mecânica. Medidas de controle e culturas de vigilância são imprescindíveis no controle da disseminação do ERV. Os resultados obtidos no presente trabalho contribuem para uma melhor compreensão da dinâmica epidemiológica das infecções e da disseminação do ERV no Hospital Universitário de Londrina.

Vancomycin-resistant Enterococci (VRE) have emerged as a relevant multidrug-resistant pathogen and potencially lethal etiology of healthcare associated infections worldwide. This study intends to show the epidemiology and clinical characteristics of patients with VRE in a Hospital in South Brazil. A retrospective study was conducted from January 2005 to November 2007. A total of 122 VRE were identified in this period at the University Hospital of Londrina. All patients with VRE clinical culture have identified and their medical records have reviewed. The presence of colonization was evaluated through rectal swab cultures, and the species identification of clinical samples was performed by automated method MicroScan®. The mean age of patients was 54 years. Urinary tract (68.0%) and blood (23.8%) were the most frequent sites, and ICU was the largest sector of occurrence (49.2%). E. faecium was the predominant species, in 82.8% of cases.The risk factors presents were length of hospitalization (mean 58.2 days), previous use of antimicrobials and invasive procedure, such as use of central venous catheter, urinary catheter and mechanical ventilation. Control barriers and surveillance cultures are essential to prevent the VRE spread. The results obtained in this study contribute to a better understanding of the epidemiological dynamics of infections and the spread of VRE in University Hospital of Londrina.