Antimicrobial Activity of an Fmoc-Plantaricin 149 Derivative Peptide against Multidrug-Resistant Bacteria.

Antimicrobial resistance poses a major threat to public health. Given the paucity of novel antimicrobials to treat resistant infections, the emergence of multidrug-resistant bacteria renewed interest in antimicrobial peptides as potential therapeutics. This study designed a new analog of the antimicrobial peptide Plantaricin 149 (Pln149-PEP20) based on previous Fmoc-peptides. The minimal inhibitory concentrations of Pln149-PEP20 were determined for 60 bacteria of different species and resistance profiles, ranging from 1 mg/L to 128 mg/L for Gram-positive bacteria and 16 to 512 mg/L for Gram-negative. Furthermore, Pln149-PEP20 demonstrated excellent bactericidal activity within one hour. To determine the propensity to develop resistance to Pln149-PEP20, a directed-evolution in vitro experiment was performed. Whole-genome sequencing of selected mutants with increased MICs and wild-type isolates revealed that most mutations were concentrated in genes associated with membrane metabolism, indicating the most likely target of Pln149-PEP20. Synchrotron radiation circular dichroism showed how this molecule disturbs the membranes, suggesting a carpet mode of interaction. Membrane depolarization and transmission electron microscopy assays supported these two hypotheses, although a secondary intracellular mechanism of action is possible. The molecule studied in this research has the potential to be used as a novel antimicrobial therapy, although further modifications and optimization remain possible.

Community-genotype methicillin-resistant Staphylococcus aureus skin and soft tissue infections in Latin America: a systematic review.

BACKGROUND: Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) emerged in the 1990s as a global community pathogen primarily involved in skin and soft tissue infections (SSTIs) and pneumonia. To date, the CG-MRSA SSTI burden in Latin America (LA) has not been assessed. OBJECTIVE: The main objective of this study was to report the rate and genotypes of community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) causing community-onset skin and soft tissue infections (CO-SSTIs) in LA over the last two decades. In addition, this research determined relevant data related to SSTIs due to CG-MRSA, including risk factors, other invasive diseases, and mortality. DATA SOURCES: Relevant literature was searched and extracted from five major databases: Embase, PubMed, LILACS, SciELO, and Web of Science. METHODS: A systematic review was performed, and a narrative review was constructed. RESULTS: An analysis of 11 studies identified epidemiological data across LA, with Argentina presenting the highest percentage of SSTIs caused by CG-MRSA (88%). Other countries had rates of CG-MRSA infection ranging from 0 to 51%. Brazil had one of the lowest rates of CG-MRSA SSTI (4.5-25%). In Argentina, being younger than 50 years of age and having purulent lesions were predictive factors for CG-MRSA CO-SSTIs. In addition, the predominant genetic lineages in LA belonged to sequence types 8, 30, and 5 (ST8, ST30, and ST5). CONCLUSION: There are significant regional differences in the rates of CG-MRSA causing CO-SSTIs. It is not possible to conclude whether or not CG-MRSA CO-SSTIs resulted in more severe SSTI presentations or in a higher mortality rate.

Community-genotype methicillin-resistant Staphylococcus aureus skin and soft tissue infections in Latin America: a systematic review

ABSTRACT Background: Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) emerged in the 1990s as a global community pathogen primarily involved in skin and soft tissue infections (SSTIs) and pneumonia. To date, the CG-MRSA SSTI burden in Latin America (LA) has not been assessed. Objective: The main objective of this study was to report the rate and genotypes of community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) causing community-onset skin and soft tissue infections (CO-SSTIs) in LA over the last two decades. In addition, this research determined relevant data related to SSTIs due to CG-MRSA, including risk factors, other invasive diseases, and mortality. Data sources: Relevant literature was searched and extracted from five major databases: Embase, PubMed, LILACS, SciELO, and Web of Science. Methods: A systematic review was performed, and a narrative review was constructed. Results: An analysis of 11 studies identified epidemiological data across LA, with Argentina presenting the highest percentage of SSTIs caused by CG-MRSA (88%). Other countries had rates of CG-MRSA infection ranging from 0 to 51%. Brazil had one of the lowest rates of CG-MRSA SSTI (4.5-25%). In Argentina, being younger than 50 years of age and having purulent lesions were predictive factors for CG-MRSA CO-SSTIs. In addition, the predominant genetic lineages in LA belonged to sequence types 8, 30, and 5 (ST8, ST30, and ST5). Conclusion: There are significant regional differences in the rates of CG-MRSA causing CO-SSTIs. It is not possible to conclude whether or not CG-MRSA CO-SSTIs resulted in more severe SSTI presentations or in a higher mortality rate.

Characterization and distribution of viridans group streptococci isolated from infectious endophthalmitis and keratitis / Caracterização e distribuição de estreptococos do grupo viridans isolados de endoftalmite infecciosa e ceratite

ABSTRACT Purpose: The aims of this study were to characterize alpha-hemolytic streptococci among isolates from cases of infectious endophthalmitis and keratitis and to determine their distributions. Methods: The sample included 27 and 35 nonduplicated isolates of alpha-hemolytic streptococci recovered from patients with infectious endophthalmitis (2002-2013) and keratitis (2008-2013), respectively. Isolates were identified by the optochin susceptibility and bile solubility tests, using a biochemical identification system. The minimum inhibitory concentration was determined by the broth microdilution method. Molecular identification was performed by analyses of three constitutive genes and the complementary multilocus sequence. The molecular epidemiology of Streptococcus pneumoniae was investigated using multilocus sequence typing, and the presence of the capsular polysaccharide-encoding gene was assessed using conventional polymerase chain reaction. Outcomes were evaluated using the patients' medical records. Results: Phenotypic tests differentiated S. pneumoniae from other alpha-hemolytic streptococci, consistent with later molecular identifications. Streptococcus oralis was significantly prevalent among the endophthalmitis isolates, as was S. pneumoniae in the keratitis isolates. High levels of susceptibility to antibiotics were observed, including vancomycin, cephalosporins, and fluoroquinolones. High genetic variability was detected among the 19 S. pneumoniae strains, with 15 predicted to be encapsulated. The medical records of patients with infectious endophthalmitis were reviewed (n=15/27; 56%), and final visual acuity was assessed in 12 cases (44%). Many patients progressed to a final visual acuity state of "no light perception" (6/12; 50%), "light perception" (3/12; 25%), or "hand motion" (1/12; 8%). The medical records of patients with infectious keratitis were also reviewed (n=24/35; 69%), and final visual acuity was assessed in 18 cases (51%). Similarly, most patients progressed to a final visual acuity state of "no light perception" (6/18; 33%), "light perception" (1/18; 6%), or "hand motion" (6/18; 33%). Overall, the majority of patients progressed to a final visual acuity state of "no light perception" (12/30), "light perception" (4/30), or "hand motion" (7/30). Conclusions: The distribution of alpha-hemolytic streptococci in ocular infections suggested the presence of a species-specific tissue tropism. The prognoses of patients with ocular streptococcal infections were highly unfavorable, and antibiotic resistance did not contribute to the unfavorable clinical progressions and poor outcomes.

RESUMO Objetivo: O objetivo deste estudo foi caracterizar os estreptococos alfa-hemolíticos isolados de endoftalmite infecciosa e ceratite e determinar sua distribuição. Métodos: A amostra incluiu 27 e 35 isolados não-duplicados de estreptococos alfa-hemolíticos recuperados de pacientes com endoftalmite infecciosa (2002-2013) e ceratite (2008-2013), respectivamente. Os isolados foram identificados pelos testes de suscetibilidade à optoquina e bile solubilidade, utilizando um sistema de identificação bioquímica. A concentração inibitória mínima foi determinada pelo método de microdiluição em caldo. A identificação molecular foi realizada pela análise de três genes constitutivos e análise complementar de sequências multilocus. A epidemiologia molecular do Streptococcus pneumoniae foi investigada por tipagem de sequência multilocus, e a presença do gene codificador do polissacarídeo capsular foi avaliada por reação em cadeia da polymerase convencional. Os resultados foram avaliados utilizando os prontuários médicos dos pacientes. Resultados: Os testes fenotípicos diferenciaram S. pneumoniae dos outros estreptococos alpha-hemolíticos, consistentes com identificações moleculares posteriores. S. oralis foi significativamente prevalente entre os isolados de endoftalmite, assim como S. pneumoniae nos isolados de ceratite. Foram observados altos níveis de suscetibilidade a antibióticos, incluindo vancomicina, cefalosporinas e fluoroquinolonas. Alta variabilidade genética foi detectada entre as 19 cepas de S. pneumoniae, com 15 previstas para serem encapsuladas. Os prontuários médicos dos pacientes com endoftalmite infecciosa foram revisados (n=15/27; 56%), e a acuidade visual final foi avaliada em 12 casos (44%). Muitos pacientes evoluiram para um estado final de acuidade visual de "sem percepção luminosa" (6/12; 50%), "percepção luminosa" (3/12; 25%) ou "movimentos de mãos" (1/12; 8%). Também foram revisados os prontuários médicos dos pacientes com ceratite infecciosa (n=24/35; 69%), e a acuidade visual final foi avaliada em 18 casos (51%). Da mesma foram, a maioria dos pacientes evoluiu para um estado final de acuidade visual de "sem percepção luminosa" (6/18; 33%), "percepção luminosa" (1/18; 6%) ou "movimentos de mãos" (6/18; 33%). No geral, a maioria dos pacientes evoluiu para um estado final de acuidade visual de "sem percepção luminosa" (12/30), "percepção luminosa" (4/30) ou "movimentos de mãos" (7/30). Conclusões: A distribuição de estreptococos alfa-hemolíticos nas infecções oculares sugeriu a presença de um tropismo de tecido específico da espécie. Os prognósticos dos pacientes com infeções oculares por estreptococos foram altamente desfavoráveis e a resistência a antibióticos contribuiu não para as progressões clínicas des­favoráveis e os maus resultados.

Characterization and distribution of viridans group streptococci isolated from infectious endophthalmitis and keratitis.

PURPOSE: The aims of this study were to characterize alpha-hemolytic streptococci among isolates from cases of infectious endophthalmitis and keratitis and to determine their distributions. METHODS: The sample included 27 and 35 nonduplicated isolates of alpha-hemolytic streptococci recovered from patients with infectious endophthalmitis (2002-2013) and keratitis (2008-2013), respectively. Isolates were identified by the optochin susceptibility and bile solubility tests, using a biochemical identification system. The minimum inhibitory concentration was determined by the broth microdilution method. Molecular identification was performed by analyses of three constitutive genes and the complementary multilocus sequence. The molecular epidemiology of Streptococcus pneumoniae was investigated using multilocus sequence typing, and the presence of the capsular polysaccharide-encoding gene was assessed using conventional polymerase chain reaction. Outcomes were evaluated using the patients' medical records. RESULTS: Phenotypic tests differentiated S. pneumoniae from other alpha-hemolytic streptococci, consistent with later molecular identifications. Streptococcus oralis was significantly prevalent among the endophthalmitis isolates, as was S. pneumoniae in the keratitis isolates. High levels of susceptibility to antibiotics were observed, including vancomycin, cephalosporins, and fluoroquinolones. High genetic variability was detected among the 19 S. pneumoniae strains, with 15 predicted to be encapsulated. The medical records of patients with infectious endophthalmitis were reviewed (n=15/27; 56%), and final visual acuity was assessed in 12 cases (44%). Many patients progressed to a final visual acuity state of "no light perception" (6/12; 50%), "light perception" (3/12; 25%), or "hand motion" (1/12; 8%). The medical records of patients with infectious keratitis were also reviewed (n=24/35; 69%), and final visual acuity was assessed in 18 cases (51%). Similarly, most patients progressed to a final visual acuity state of "no light perception" (6/18; 33%), "light perception" (1/18; 6%), or "hand motion" (6/18; 33%). Overall, the majority of patients progressed to a final visual acuity state of "no light perception" (12/30), "light perception" (4/30), or "hand motion" (7/30). CONCLUSIONS: The distribution of alpha-hemolytic streptococci in ocular infections suggested the presence of a species-specific tissue tropism. The prognoses of patients with ocular streptococcal infections were highly unfavorable, and antibiotic resistance did not contribute to the unfavorable clinical progressions and poor outcomes.

Detection of herpes simplex-1 and -2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis.

OBJECTIVE:: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. METHODS:: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. RESULTS:: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. CONCLUSIONS:: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.

Detection of herpes simplex-1 and -2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis / Detecção de vírus herpes simplex tipo 1 e 2 e varicella zoster por reação em cadeia de polimerase em tempo real em córneas de pacientes com ceratites bacterianas

ABSTRACT Objective: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. Methods: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. Results: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. Conclusions: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.

RESUMO Objetivo: Ceratites bacterianas ocorrem mundialmente e apesar dos novos desenvolvimentos permanece como uma condição que pode levar à cegueira. Avaliar a presença de herpes simples (-1 e -2) e vírus varicella zoster (VZV) por reação em cadeia quantitativa de polimerase em tempo real (qPCR) em raspados corneanos de pacientes com ceratite bacteriana. Métodos: Sessenta e cinco pacientes com ceratite infecciosa foram submetidos a raspados corneanos estudados para gram, Giemsa, cultura e qPCR (grupo de estudo). Foram avaliados fatores de risco e epidemiológicos. O grupo controle foi composto por 25 casos de úlcera dendrítica típica por herpes analisados por qPCR. Resultados: Do grupo de estudo (n=65), nove pacientes (13,8%) apresentaram cultura, qPCR e raspado negativos. Cinquenta e seis (86,2%) pacientes apresentaram cultura positiva, 51 para bacteria, 4 para fungo e 1 para ameba. A qPCR identificou 10 pacientes do grupo de cultura positiva para bactéria que também foram positivos para vírus, um VZV e 9 para HSV-1. Dos 25 pacientes que compunham o grupo controle, 21 apresentaram qPCR positivo para HSV-1. Conclusão: Herpes pode estar presente em pacientes com úlceras de córnea bacterianas e a qPCR pode ser útil na sua detecção.

DNA extraction methods for panbacterial and panfungal PCR detection in intraocular fluids.

PURPOSE: Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification. MATERIAL AND METHODS: Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out. RESULTS: The column-based extraction method was the most time-effective, achieving DNA detection limits ≥10(2) and 10(3 )CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4). CONCLUSIONS: The column-based extraction method for very small inocula in small volume samples (50-100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

Demonstration of plasmid-mediated drug resistance in Mycobacterium abscessus.

Plasmid-mediated kanamycin resistance was detected in a strain of Mycobacterium abscessus subsp. bolletii responsible for a nationwide epidemic of surgical infections in Brazil. The plasmid did not influence susceptibility to tobramycin, streptomycin, trimethoprim-sulfamethoxazole, clarithromycin, or ciprofloxacin. Plasmid-mediated drug resistance has not been described so far in mycobacteria.

Prevalence and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus in ocular infections / Prevalência e suscetibilidade antibiótica de Staphylococcus aureus resistente à meticilina em infecções oculares

PURPOSE: To study the prevalence of methicillin-resistant Staphylococcus aureus among S. aureus ocular infections in a tertiary health center in Brazil and compare antibiotic susceptibility patterns between MRSA and methicillin-susceptible S. aureus isolates. METHODS: Electronic records from the ocular microbiology laboratory of the Universidade Federal de São Paulo were retrospectively reviewed. During a 10-year period (between January 2000 and December 2009) all conjunctivitis, keratitis, and endophthalmitis cases with a positive culture for S. aureus were identified. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. RESULTS: Five hundred sixty-six S. aureus isolates were identified; of those, 56 (9.9%) were resistant to methicillin. Throughout the 10-year period, Staphylococcus aureus showed a significant increasing trend from 7.55% to 16.18% among overall S. aurues infections (p=0.001) and from 3.7% to 13.16% in conjunctivitis (p=0.001). Conversely, we did not observe the same trend among those with keratitis (p=0.38). Staphylococcus aureus isolates showed higher resistance rates to tobramycin, gentamicin, ciprofloxacin, gatifloxacin, and moxifloxacin when compared with S. aureus isolates (p< 0.001). All cases were susceptible to vancomycin. CONCLUSION: We observed an increasing trend in the overall prevalence of Staphylococcus aureus ocular infections and statistically significant higher resistance rates to commonly used antibiotics compared to Staphylococcus aureus. Our data supports the need for constant bacterial surveillance and should be taken into consideration before initiating empiric treatment of ocular infections.

OBJETIVO: Estudar a prevalência do Staphylococcus aureus resistente à meticilina nas infecções oculares causadas por S. aureus em um centro de saúde terciário no Brasil e comparar o perfil de suscetibilidade antimicrobiana entre as cepas de Staphylococcus aureus resistente à meticilina e S. aureus susceptível à meticilina MÉTODOS: Foi realizada uma análise retrospectiva dos arquivos do laboratório de microbiologia ocular da Universidade Federal de São Paulo e selecionados todos os casos de conjuntivite, ceratite e endoftalmite com cultivo positivo para S. aureus, durante um período de 10 anos (entre janeiro de 2000 e dezembro de 2009). Foi avaliada a prevalência de Staphylococcus aureus resistente à meticilina e comparado o perfil de susceptibilidade antimicrobiano dos Staphylococcus aureus resistente à meticilina e S. aureus susceptível à meticilina. RESULTADOS: Quinhentos e sessenta e seis isolados de S. aureus foram identificados. Desses, 56 (9,9%) apresentaram resistência à meticilina. Durante o período de 10 anos estudado, Staphylococcus aureus resistente à meticilina mostrou uma tendência significante de aumento de 7,6% para 16,2% entre as infecções oculares causadas por S. aureus em geral (p=0,001) e de 3,7% para 13,2% nas conjuntivites (p=0,001). A mesma tendência não foi observada entre as amostras de ceratite (p=0,38). Os isolados de Staphylococcus aureus resistente à meticilina mostraram maiores taxas de resistência à tobramicina, gentamicina, ciprofloxacino, gatifloxacino e moxifloxacino em comparação com os isolados de S. aureus susceptível à meticilina (p<0,001). Todos os casos foram susceptíveis à vancomicina. CONCLUSÃO:Foi observada uma tendência de aumento na prevalência do Staphylococcus aureus resistente à meticilina nas infecções oculares causadas por S. aureus, bem como taxas de resistência significantemente maiores aos antibióticos comumente utilizados na prática oftalmológica. Nossos dados alertam para a necessidade de constante vigilância de resistência bacteriana a antimicrobianos e devem ser considerados na eleição do tratamento empírico das infecções oculares.

Prevalence and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus in ocular infections.

PURPOSE: To study the prevalence of methicillin-resistant Staphylococcus aureus among S. aureus ocular infections in a tertiary health center in Brazil and compare antibiotic susceptibility patterns between MRSA and methicillin-susceptible S. aureus isolates. METHODS: Electronic records from the ocular microbiology laboratory of the Universidade Federal de São Paulo were retrospectively reviewed. During a 10-year period (between January 2000 and December 2009) all conjunctivitis, keratitis, and endophthalmitis cases with a positive culture for S. aureus were identified. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. RESULTS: Five hundred sixty-six S. aureus isolates were identified; of those, 56 (9.9%) were resistant to methicillin. Throughout the 10-year period, Staphylococcus aureus showed a significant increasing trend from 7.55% to 16.18% among overall S. aurues infections (p=0.001) and from 3.7% to 13.16% in conjunctivitis (p=0.001). Conversely, we did not observe the same trend among those with keratitis (p=0.38). Staphylococcus aureus isolates showed higher resistance rates to tobramycin, gentamicin, ciprofloxacin, gatifloxacin, and moxifloxacin when compared with S. aureus isolates (p< 0.001). All cases were susceptible to vancomycin. CONCLUSION: We observed an increasing trend in the overall prevalence of Staphylococcus aureus ocular infections and statistically significant higher resistance rates to commonly used antibiotics compared to Staphylococcus aureus. Our data supports the need for constant bacterial surveillance and should be taken into consideration before initiating empiric treatment of ocular infections.

Bactericidal effect of S-nitrosothiols against clinical isolates from keratitis.

BACKGROUND: The purpose of this study was to evaluate the antimicrobial activity of two nitric oxide donors, ie, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC), against clinical isolates from patients with infectious keratitis. METHODS: Reference broth microdilution assays were performed to determine the minimum inhibitory and bactericidal concentrations for GSNO and SNAC against four American Type Culture Collection strains and 52 clinical isolates from patients with infectious keratitis as follows: 14 (26.9%) Pseudomonas species; 13 (25.0%) coagulase-negative Staphylococci; 10 (19.2%) Staphylococcus aureus; nine (17.3%) Serratia marcescens; and six (11.5%) Enterobacter aerogenes. Sterility control and bacterial growth control were also performed. RESULTS: SNAC showed lower minimum inhibitory and bactericidal concentrations than GSNO for all clinical isolates from patients with infectious keratitis. For Gram-positive bacteria, mean minimum inhibitory and bactericidal concentrations were 2.1 ± 1.3 and 8.6 ± 3.8 mM for SNAC and 4.6 ± 3.2 and 21.5 ± 12.5 mM for GSNO (P < 0.01). For Gram-negative bacteria, mean minimum inhibitory and bactericidal concentrations were 3.3 ± 1.4 and 6.1 ± 3.4 mM for SNAC and 12.4 ± 5.4 and 26.5 ± 10.1 mM for GSNO (P < 0.01). The minimum bactericidal to inhibitory concentration ratio was ≤8 in 100% of all isolates tested for SNAC and in 94.2% tested for GSNO. CONCLUSIONS: SNAC and GSNO had effective inhibitory and bactericidal effects against bacterial isolates from keratitis. SNAC showed greater antimicrobial activity than GSNO against all bacteria. Gram-positive bacteria were more susceptible to the inhibitory and bactericidal effects of the S-nitrosothiols.

Pseudomonas aeruginosa endophthalmitis after penetrating keratoplasty transmitted from the same donor to two recipients confirmed by pulsed-field gel electrophoresis.

Two devastating cases of multidrug-resistant Pseudomonas aeruginosa endophthalmitis after keratoplasty as the result of transmission from the same donor were confirmed by pulsed-field gel electrophoresis. Strategies for preventing donor-to-host transmission, such as the use of antimicrobial agents of greater efficacy and better methods for detecting microorganisms in preservation medium, could minimize this type of transmission.

Real-time polymerase chain reaction test to discriminate between contamination and intraocular infection after cataract surgery.

PURPOSE: To determine the usefulness of real-time polymerase chain reaction (PCR) assays in the diagnosis of postoperative bacterial endophthalmitis in clinically diagnosed infectious cases and to test for bacterial DNA in control samples collected from noninfected eyes. SETTING: Federal University of São Paulo, Brazil. DESIGN: Evaluation of diagnostic test or technology. METHODS: This study comprised patients with clinically diagnosed infectious endophthalmitis after cataract surgery and vitreous samples (from noninflamed eyes obtained through vitrectomy) and aqueous samples (at end of phacoemulsification) from control patients at a single university setting. Universal and gram-specific real-time PCR, Gram staining, and culture were performed. Sensitivity and cycle thresholds were determined. Clinical and microbiologic data were also assessed. RESULTS: The study evaluated 11 patients with infectious endophthalmitis (9 vitreous and 7 aqueous samples), 12 control vitreous samples, and 50 control aqueous samples. Gram and culture identified 80% and 75%, respectively, of patients with infectious endophthalmitis. Real-time PCR assays were positive in 91% of patients with a clinical diagnosis of endophthalmitis using aqueous samples, vitreous samples, or both. None of the 12 vitreous controls were positive by PCR. Two aqueous control samples were positive by real-time PCR. The cycle threshold cutoff value was 36 for universal PCR (sensitivity 93.8%; specificity 100%) and 38 for gram-specific PCR (sensitivity 93.8%; specificity 100%). Gram-positive microorganisms prevailed, and visual acuity varied according to the causative bacteria. CONCLUSIONS: Real-time PCR provided fast and accurate diagnosis of bacterial endophthalmitis. As a quantitative technique, it may be useful in distinguishing between contamination and infection based on the cycle thresholds value.

Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays.

PURPOSE: To develop and apply real-time PCR protocols to the detection and classification of the Gram status of bacterial pathogens in aqueous and vitreous humor collected from clinically suspected intraocular infections. METHODS: The analytical specificity of two PCR assays, SYBR Green 16S rDNA-Based Universal PCR (SGRU-PCR), and a Multiplex Gram-Specific TaqMan-Based PCR (MGST-PCR), was determined with 31 clinically important pathogens, including 20 Gram-positive and 11 Gram-negative. Analytical sensitivity was determined with a 10-fold dilution of Staphylococcus epidermidis and Escherichia coli DNA. Assays were further tested on aqueous (n = 10) and vitreous humor (n = 11) samples collected from patients with clinically diagnosed intraocular infections. RESULTS: DNA was amplified from all control bacterial isolates when using SGRU-PCR. MGST-PCR correctly classified the Gram status of all these isolates. The SGRU-PCR limit of detection of S. epidermidis and E. coli DNA was 100 fg/µL (E = 0.82 and 0.86; r(2) = 0.99) and for MGST-PCR, 1 pg/µL (E = 0.66 and 0.70; r(2) = 0.99. For clinical intraocular samples, positivity of culture was 47.6% and for real-time PCR assays, 95.2%. Gram classification was achieved in 100% of MGST-PCR-positive samples. Among microbiologically negative samples, real-time PCR assays were positive in 90% of cases. The false-positive rate in control aqueous was 3.2%, and control samples of vitreous were negative. CONCLUSIONS: The real-time PCR assays demonstrated good correlation, with culture-proven RESULTS: With the use of these methods, bacterial detection was improved from 47.6% to 95.3%, demonstrating them to be sensitive, rapid tests for diagnosis of bacterial endophthalmitis.

Incidence of endophthalmitis after cataract surgery (2002-2008) at a Brazilian university-hospital / Incidência de endoftalmite após cirurgia de catarata (2002-2008) num hospital universitário brasileiro

Purpose: To report on the incidence, diagnostic technique, and microbiological features of endophthalmitis at a university-setting in Brazil. Methods: All cases of presumed postoperative endophthalmitis from 2002 to 2008 at a teaching-hospital were included. Main data assessed were: number of cataract surgeries performed, incidence of endophthalmitis, microbiological outcome (aqueous and/or vitreous culture and Gram staining), and antimicrobial susceptibility testing of the positive cases. Results: Seventy-three eyes of 73 patients (43 females and 30 males) developed endophthalmitis after 24,590 cataract surgeries. The incidence decreased from 0.49 percent in 2003 to 0.17 percent in 2006 and stabilized afterwards. Coagulase negative Staphylococci (CoNS) and Streptococcus viridans (56.5 percent and 15 percent, respectively) were the most common bacterial isolates. Culture and Gram stain were negative in 36.9 percent. CoNS presented susceptibility rates of 80 percent-sensitivity to oxacillin, 90 percent to fourth-generation quinolones and 100 percent to vancomycin. Conclusions: The rate of endophthalmitis, diagnostic ability of conventional laboratory investigation, microbial isolates and antibiotic susceptibility are in accordance with other findings of the literature. Despite using prophylactic antibiotic drops, it was possible to identify cases that were susceptible to the antibiotics topically applied.

Objetivo: Relatar incidência, técnica diagnóstica e características microbiológicas de endoftalmite numa instituição universitária no Brasil. Métodos: Todos os casos de endoftalmite pós-operatória presumida de 2002 a 2008 foram incluídos. Os principais dados avaliados foram: número de cirurgias de catarata realizadas, incidência de endoftalmite, resultado microbiológico (bacterioscopia e cultura de aquoso e vítreo) e teste de sensibilidade antibiótica dos casos positivos. Resultados: Setenta e três olhos de 73 pacientes (43 do sexo feminino e 30 do masculino) desenvolveram endoftalmite após 24.590 cirurgias de catarata. A incidência reduziu de 0,49 por cento em 2003 para 0,17 por cento em 2006 e estabilizou-se depois disso. Staphylococcus coagulase-negativa (SCoN) e Streptococcus viridans (56.5 por cento e 15 por cento, respectivamente) foram os isolados bacterianos mais comuns. Cultura e bacterioscopia foram negativas em 36,9 por cento. SCoN apresentou taxas de sensibilidade de 80 por cento à oxacilina, 90 por cento às quinolonas de quarta geração e 100 por cento à vancomicina. Conclusões: A taxa de endoftalmite, a capacidade diagnóstica das técnicas laboratoriais convencionais, os microrganismos isolados e a sensibilidade aos antibióticos estão em acordo com outros achados na literatura. Apesar do uso profilático de colírio antibiótico, foi possível identificar casos de infecção em que as bactérias eram sensíveis aos antibióticos usados topicamente.

Incidence of endophthalmitis after cataract surgery (2002-2008) at a Brazilian university-hospital.

PURPOSE: To report on the incidence, diagnostic technique, and microbiological features of endophthalmitis at a university-setting in Brazil. METHODS: All cases of presumed postoperative endophthalmitis from 2002 to 2008 at a teaching-hospital were included. Main data assessed were: number of cataract surgeries performed, incidence of endophthalmitis, microbiological outcome (aqueous and/or vitreous culture and Gram staining), and antimicrobial susceptibility testing of the positive cases. RESULTS: Seventy-three eyes of 73 patients (43 females and 30 males) developed endophthalmitis after 24,590 cataract surgeries. The incidence decreased from 0.49% in 2003 to 0.17% in 2006 and stabilized afterwards. Coagulase negative Staphylococci (CoNS) and Streptococcus viridans (56.5% and 15%, respectively) were the most common bacterial isolates. Culture and Gram stain were negative in 36.9%. CoNS presented susceptibility rates of 80%-sensitivity to oxacillin, 90% to fourth-generation quinolones and 100% to vancomycin. CONCLUSIONS: The rate of endophthalmitis, diagnostic ability of conventional laboratory investigation, microbial isolates and antibiotic susceptibility are in accordance with other findings of the literature. Despite using prophylactic antibiotic drops, it was possible to identify cases that were susceptible to the antibiotics topically applied.

Molecular biology applied to the laboratory diagnosis of bacterial endophthalmitis.

Bacterial endophthalmitis is a serious but uncommon intraocular infection which frequently results in vision loss. Early diagnosis and appropriate therapy are associated with better visual outcome. Conventional microbiological methods are currently used for microbiological characterization of eyes with suspected endophthalmitis. However, the sensitivity of bacterial detection from aqueous and vitreous humor using microbiology techniques is poor, and time-consuming to confirm the results. The application of molecular methods enhances significantly laboratory confirmation of bacterial endophthalmitis, demanding a shorter time to draw a definitive result and thereby promoting the early initiation of a more specific therapy to limit the empirical or unnecessary use of broad-spectrum antibiotics. PCR-based techniques, including post-PCR methods such RFLP, DNA probe hybridization and DNA sequencing have been successfully used for the diagnostic elucidation of clinically suspected bacterial endophthalmitis cases, showing promising application in the routine practice of ocular microbiology laboratories.

Molecular biology applied to the laboratory diagnosis of bacterial endophthalmitis / Biologia molecular aplicada ao diagnóstico laboratorial de endoftalmite bacteriana

Bacterial endophthalmitis is a serious but uncommon intraocular infection which frequently results in vision loss. Early diagnosis and appropriate therapy are associated with better visual outcome. Conventional microbiological methods are currently used for microbiological characterization of eyes with suspected endophthalmitis. However, the sensitivity of bacterial detection from aqueous and vitreous humor using microbiology techniques is poor, and time-consuming to confirm the results. The application of molecular methods enhances significantly laboratory confirmation of bacterial endophthalmitis, demanding a shorter time to draw a definitive result and thereby promoting the early initiation of a more specific therapy to limit the empirical or unnecessary use of broad-spectrum antibiotics. PCR-based techniques, including post-PCR methods such RFLP, DNA probe hybridization and DNA sequencing have been successfully used for the diagnostic elucidation of clinically suspected bacterial endophthalmitis cases, showing promising application in the routine practice of ocular microbiology laboratories.

Endoftalmite bacteriana é uma infecção intraocular grave, mas de baixa frequência, podendo resultar em grande prejuízo visual. O diagnóstico precoce e a rápida instituição de terapia adequada estão associadas a um melhor prognóstico da doença. Os métodos microbiológicos convencionais são utilizados rotineiramente para caracterização microbiológica de olhos com suspeita de endoftalmite. No entanto, a sensibilidade de detecção bacteriana em amostras de humor aquoso e vítreo utilizando técnicas microbiológicas é baixa, além de demandar um maior tempo para a confirmação dos resultados. A utilização de métodos moleculares aumenta significativamente os casos de endoftalmite bacteriana confirmados laboratorialmente, com tempo menor para a liberação de um resultado definitivo, auxiliando assim a instituição precoce de uma terapia mais específica, limitando o uso empírico ou desnecessário de antibióticos de amplo espectro. A técnica de PCR e outras metodologias para análises pós-PCR como, RFLP, hibridização com sondas e sequenciamento do DNA, tem sido utilizadas com sucesso para elucidação diagnóstica em casos com suspeita clínica de endoftalmite bacteriana, demonstrando promissora aplicação para a rotina dos laboratórios de microbiologia ocular.

[Culture proven bacterial endophthalmitis: a 6-year review]. / Endoftalmites bacterianas com culturas positivas: uma revisão de 6 anos.

PURPOSE: To assess the distribution of microorganisms isolated from patients with bacterial endophthalmitis and their antimicrobial susceptibility. METHODS: Retrospective analysis of medical and microbiological records of patients with suspected diagnosis of endophthalmitis and bacterial culture-proven at the Department of Ophthalmology, UNIFESP, between January 1 2000 and December 31 2005. RESULTS: 153 (33.9%) of 451 patients showed positive bacterial culture. A total of 155 microorganisms were isolated, 79.35% were gram-positive and 20.65% gram-negative. Staphylococcus (CoNS) (41.94%) were the most frequently isolated. The antimicrobial susceptibility for gram-negative microorganisms was as follows: amikacin 87.10%, tobramycin 80.65%, ciprofloxacin 96.67%, levofloxacin, gatifloxacin and moxifloxacin 100%, ceftazidime 85.0%, and gentamicin 80.65%. Vancomycin sensitivity among gram-positive microorganisms was 100%. S. aureus and CoNS showed 83.33% of susceptibility to oxacillin, 89.61% to ciprofloxacin and 100% to gatifloxacin and moxifloxacin. The main acquisition mechanism was postoperative (60.65%). CONCLUSION: We detected a low sensitivity of vitreous/aqueous culture for the etiologic diagnosis of endophthalmitis. The empiric antimicrobial therapy or prophylaxis should be active against gram-positive bacteria, particularly staphylococci. Surveillance studies of bacterial resistance are important for a better utilization of antimicrobials in this clinical setting.