Caracterização molecular e fenotípica de Klebsiella spp. produtoras de NDM / Molecular and phenotypic characterization of Klebsiella spp. NDM producers

O perfil de resistência, que algumas das espécies do complexo Klebsiella pneumoniae podem expressar, representa uma grande ameaça à saúde humana, particularmente quando resistentes aos carbapenêmicos, que são amplamente utilizados no tratamento de infecções graves em pacientes hospitalizados. O principal mecanismo de resistência aos carbapenêmicos é a produção de carbapenemases, particularmente dos tipos KPC e NDM. Um dos compostos desenvolvidos para o tratamento de infecções causadas por cepas produtoras de KPC é a combinação ceftazidimaavibactam (CAZ-AVI), mas que não tem atividade inibitória sobre metalo-betalactamases, a exemplo das NDMs. Os objetivos deste trabalho foram determinar a frequência das espécies do complexo K. pneumoniae e da coprodução de KPC, avaliar a clonalidade dos isolados, a sensibilidade ao aztreonam-avibactam (ATM-AVI), o desempenho do disco de meropenem (MEM) com inibidores para detecção de coprodução de NDM e KPC e desenvolver um teste de triagem para prever a sensibilidade ao ATM-AVI. Um total de 113 isolados do complexo K. pneumoniae produtoras de NDM ou coprodutoras de NDM e KPC, provenientes da coleção de bactérias do Grupo Fleury, coletadas períodos pré e pós início do uso de CAZ-AVI no Brasil, foram utilizadas neste estudo. A identificação da espécie e a presença dos genes blaNDM e blaKPC foi confirmada por PCR multiplex. A clonalidade dos isolados foi avaliada por eletroforese em campos pulsados (PFGE) após clivagem com XbaI. A produção de carbapenemases foi confirmada utilizando-se o teste Blue Carba. O desempenho dos discos de meropenem e CAZ-AVI contendo um ou mais inibidores de carbapenemases foi comparado com o teste molecular. A pré-difusão combinada foi realizada pré-incubando-se o ágar não inoculado com disco de CAZ-AVI, e a seguir aplicando-se o inóculo bacteriano e um disco de ATM após remover o disco de CAZ-AVI. Após incubação, os halos foram aferidos e correlacionados com a concentração inibitória mínima para ATM-AVI. As CIMs para ATM e ATM-AVI foram determinadas segundo o EUCAST. A identificação das espécies por PCR evidenciou as seguintes frequências: K. pneumoniae 75,2% (n=85); K. quasipneumoniae 16,8% (n=19), e K. variicola 8% (n=9). Uma fração de 12,4% (n=14) dos isolados apresentaram os genes blaNDM e blaKPC e 87,6% (n=99) apenas blaNDM. A análise dos perfis de PFGE de K. pneumoniae evidenciou a presença de cinco grupos clonais predominantes. Isolados do principal grupo clonal Ap (n=15) foram detectados nas cidades de São Paulo e Porto Alegre durante todo o período analisado. O grupo clonal Lp foi detectado nas cidades de São Paulo e Recife em 2019. Os dois principais grupos clonais no período pré-CAZ-AVI continham maior número de isolados do que aqueles no período de uso do CAZ-AVI. Os perfis de PFGE de K. quasipneumoniae evidenciaram quatro grupos clonais predominantes, e presentes apenas no estado de São Paulo, com persistência do grupo clonal Aq desde 2017. Quanto à K. variicola, foram observados dois grupos clonais predominantes Av e Bv, o primeiro presente apenas em São Paulo desde 2018 e o segundo em Porto Alegre apenas em 2019. Calculando-se a diferença entre os diâmetros de halo do disco MEM contendo EDTA e ácido fenilborônico (AFB) e o maior dos halos obtidos para MEM com EDTA ou AFB, observou-se que todos os isolados com coexpressão de KPC e NDM apresentaram diferença ≥ 5 mm. Uma fração de 42,3% dos isolados positivos apenas para blaNDM apresentaram sensibilidade para ATM (CIM ≤ 4 mg/L). Todos os isolados testados apresentaram CIM para ATM-AVI ≤ 1/4 mg/L, sendo a CIM90 0,125/4 mg/l. No teste de pré-difusão combinada, o menor diâmetro de halo obtido foi de 23 mm. A espécie predominante na amostragem foi K. pneumoniae. A disseminação clonal, observada neste estudo, contrasta com a diversidade clonal descrita em outros locais do mundo para produtores de NDM, exceto Grécia e China. Considerando os pontos de corte atuais para ATM, é provável que haja resposta clínica adequada no uso de ATM-AVI no tratamento de infecções causadas por isolados produtores de NDM e coprodutores de KPC e NDM. Utilizando-se o valor de corte de ≤ 5 mm para a diferença entre halos de inibição, de MEM com AFB e EDTA e o segundo maior halo com inibidor, a sensibilidade foi de 100% e a especificidade foi de 96,1,0%. O método de pré-difusão com CAZ-AVI e ATM é um método simples e o diâmetro ≥ 23 mm tem excelente correlação com a CIM para ATM-AVI ≤ 1/4 mg/L

The resistance profile, which some species of the Klebsiella pneumoniae complex may express, represent a great threat to human health, particularly when resistant to carbapenems, which are widely used in the treatment of severe infections in hospitalized patients. The main mechanism of resistance to carbapenems is the production of carbapenemases, particularly KPCs and NDMs. One of the compounds developed for the treatment of infections caused by KPC-producing strains is the combination ceftazidime-avibactam (CAZ-AVI), but which has no inhibitory activity on metallobetalactamases, as is the case for NDMs. The objectives of this work were to determine the frequency of K. pneumoniae complex species and KPC co-production, evaluate the clonality of isolates, the susceptibility to aztreonam-avibactam (ATM-AVI), the performance of meropenem (MEM) disks with inhibitors for detecting NDM co-production and KPC and develop a screening test to predict sensitivity to ATM-AVI. A total of 113 NDM-producing or NDM and KPC co-producing K. pneumoniae complexes, from the Fleury Group's bacteria collection, collected in the pre- and post-starting periods of CAZ-AVI use in Brazil, were used in this study. Species identification and the presence of the blaNDM and blaKPC genes were confirmed by multiplex PCR. The clonality of the isolates was evaluated by pulsed field electrophoresis (PFGE) after cleavage with XbaI. Carbapenemase production was confirmed using the Blue Carba test. The performance of MEM and CAZ-AVI disks containing one or more carbapenemase inhibitors was compared with the molecular test. Combined pre-diffusion was performed by preincubating the uninoculated agar with a CAZ-AVI disk, and then applying the bacterial inoculum and na ATM disk after removal of the CAZ-AVI disk. After incubation, halos were measured and correlated with the minimum inhibitory concentration (MIC) for ATM-AVI. ATM and ATM-AVI MICs were determined according to EUCAST. The identification of species by PCR evidenced the following frequencies: K. pneumoniae 75.2% (n=85); K. quasipneumoniae 16.8% (n=19), and K. variicola 8% (n=9). A fraction of 12.4% (n=14) of the isolates had the blaNDM and blaKPC genes and 87.6% (n=99) had only blaNDM. The analysis of the PFGE profiles of K. pneumoniae evidenced the presence of five predominant clonal groups. Isolates from the main clonal group Ap (n=16) were detected in the cities of São Paulo and Porto Alegre throughout the analyzed period. The clonal group Lp was detected in the cities of São Paulo and Recife 2019. The PFGE profiles of K. quasipneumoniae showed four predominant clonal groups, present only in the state of São Paulo, with persistence of the clonal group Aq since 2017. As for K. variicola, two predominant clonal groups Av and Bv were observed, the first present only in São Paulo since 2018 and the second in Porto Alegre only in 2019. Calculating the difference between the inhibition zone diameters of the MEM disk containing EDTA and phenylboronic acid (AFB) and the largest of the inhibition zone diameters obtained for MEM with EDTA or AFB, it was observed that all isolates with co-expression of KPC and NDM showed a difference 5 ≥mm. A fraction of 42.3% of isolates positive only for blaNDM showed sensitivity to ATM (MIC ≤ 4 mg/L). All tested isolates presented MIC for ATM-AVI ≤ 1/4 mg/L, being the MIC90 0.125/4 mg/l. In the combined pre-diffusion test, the smallest inhibition zone diameter obtained was 23 mm. The predominant species in the sample was K. pneumoniae, but a significant fraction of the other species in the complex was also observed in the sample. The clonal spread observed in this study contrasts with the clonal diversity described elsewhere in the world for NDM-producing isolates, except Greece and China. Considering the current cut-off points for ATM, it is likely that there is an adequate clinical response in the use of ATM-AVI in infections caused by NDM-producing and KPC-NDM co-producing isolates in Brazil. Using the cutoff value of 5 mm for the difference between inhibition zones, of MEM with AFB and EDTA and the second largest zone of MEM with inhibitor, the sensitivity was 100% and the specificity was 96.1%. The pre-diffusion method with CAZ-AVI and ATM is a simple method and the diameter ≥ 23 mm has excellent correlation with the MIC for ATM-AVI ≤ 1/4 mg/L

Investigación traslacional en microbioma (InTraMic): un área de interés común y creciente en el IMTIB, Hospital Italiano e Instituto Universitario / Translational research in microbiome (InTraMic): an area of common and growing interest at IMTIB, Hospital Italiano and Instituto Universitario

Se estima que aproximadamente 100 trillones de microorganismos (incluidos bacterias, virus y hongos) residen en el intestino humano adulto y que el total del material genético del microbioma es 100 veces superior al del genoma humano. Esta comunidad, conocida como microbioma se adquiere al momento del nacimiento a través de la flora comensal de la piel, vagina y heces de la madre y se mantiene relativamente estable a partir de los dos años desempeñando un papel crítico tanto en el estado de salud como en la enfermedad. El desarrollo de nuevas tecnologías, como los secuenciadores de próxima generación (NGS), permiten actualmente realizar un estudio mucho más preciso de ella que en décadas pasadas cuando se limitaba a su cultivo. Si bien esto ha llevado a un crecimiento exponencial en las publicaciones, los datos sobre las poblaciones Latinoamérica son casi inexistentes. La investigación traslacional en microbioma (InTraMic) es una de las líneas que se desarrollan en el Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). Esta se inició en 2018 con la línea de cáncer colorrectal (CCR) en una colaboración con el Colorectal Cancer Research Group del Leeds Institute of Medical Research en el proyecto Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents. A fines de 2019 se cumplió el objetivo de comprobar la factibilidad de la recolección, envío y análisis de muestras de MBF en 5 continentes, incluyendo muestras provenientes de la Argentina, Chile, India y Vietnam. Luego de haber participado de capacitaciones en Inglaterra, se ha cumplido con el objetivo de la etapa piloto, logrando efectivizar la recolección, envío y análisis metagenómico a partir de la secuenciación de la región V4 del ARNr 16S. En 2019, la línea de enfermedad de hígado graso no alcohólico se sumó a la InTraMic iniciando una caracterización piloto en el marco de una colaboración con el laboratorio Novartis. Los resultados de ese estudio, así como el de cáncer colorrectal, están siendo enviados a publicación. En 2020, con la incorporación de la línea de trasplante alogénico de células progenitoras hematopoyéticas, fue presentado un proyecto para un subsidio del CONICET que ha superado la primera etapa de evaluación. En el presente artículo se brinda una actualización sobre la caracterización taxonómica de microbioma y se describen las líneas de investigación en curso. (AU)

It is estimated that approximately 100 trillion microorganisms (including bacteria, viruses, and fungi) reside in the adult human intestine, and that the total genetic material of the microbiome is 100 times greater than that of the human genome. This community, known as the microbiome, is acquired at birth through the commensal flora of the mother's skin, vagina, and feces and remains relatively stable after two years, playing a critical role in both the state of health and in disease. The development of new technologies, such as next-generation sequencers (NGS), currently allow for a much more precise study of it than in past decades when it was limited to cultivation. Although this has led to exponential growth in publications, data on Latin American populations is almost non-existent. Translational research in microbiome (InTraMic) is one of the lines developed at the Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). This started in 2018 with the Colorectal Cancer Line (CRC) in a collaboration with the Colorectal Cancer Research Group of the Leeds Institute of Medical Research in the project "Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents". At the end of 2019, the objective of verifying the feasibility of collecting, sending and analyzing MBF samples on 5 continents, including samples from Argentina, Chile, India and Vietnam, was met. After having participated in training in England, the objective of the pilot stage has been met, achieving the collection, delivery and metagenomic analysis from the sequencing of the V4 region of the 16S rRNA. In 2019, the non-alcoholic fatty liver disease line joined InTraMic, initiating a pilot characterization in the framework of a collaboration with the Novartis laboratory. The results of that study, as well as that of colorectal cancer, are being published. In 2020, with the incorporation of the allogeneic hematopoietic stem cell transplantation line, a project was presented for a grant from the CONICET that has passed the first stage of evaluation. This article provides an update on the taxonomic characterization of the microbiome and describes the lines of ongoing research. (AU)

Culture-Positive Spontaneous Ascitic Infection in Patients with Acute Decompensated Cirrhosis: Multidrug-Resistant Pathogens and Antibiotic Strategies

Differences in Antimicrobial Resistance Phenotypes by the Group of CTX-M Extended-Spectrum β-Lactamase / 대한임상미생물학회지

BACKGROUND: Escherichia coli and Klebsiella pneumoniae clinical isolates producing CTX-M extendedspectrum β-lactamases (ESBLs) were assessed for antimicrobial resistance phenotypes varied by group of enzymes. METHODS: A total of 1,338 blood isolates, including 959 E. coli and 379 K. pneumoniae, were studied. All the strains were collected between January and July 2017 from eight general hospitals in South Korea. The species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibilities were determined by disk diffusion methods and ESBL phenotypes by double-disk synergy tests using disks containing cefotaxime, ceftazidime, cefepime, aztreonam, and clavulanic acid (CA). The genes for β-lactamases were identified by PCR and sequencing. RESULTS: Of total microbes, 31.6% (303/959) E. coli and 24.0% (91/379) K. pneumoniae were resistant to cefotaxime and 28.1% (269/959) E. coli and 20.1% (76/379) K. pneumoniae were CTX-M-type ESBL producers. Among the detected CTX-M ESBLs, 58.0% (156/269) in E. coli and 86.8% (66/76) in K. pneumoniae belonged to group 1, 46.8% (126/269) in E. coli and 14.5% (11/76) in K. pneumoniae were group 9. Ten E. coli and one K. pneumoniae isolates co-produced both groups of CTX-M ESBL. The group 1 CTX-M producers had a higher level of resistance to cefotaxime, ceftazidime, cefepime, and aztreonam and exhibited stronger synergistic activities when combined with CA compared to group 9. CONCLUSION: ESBL phenotypes differ by CTX-M ESBL group and phenotype testing with drugs including 4th generation cephalosporins and monobactams is critical for screening CTX-M-producers with better sensitivity.

Biological and Genetic Characteristics of Clinically Isolated Enterobacter cloacae with Multidrug Resistance

BACKGROUND: From January 2014 to December 2015, 69 clones of Enterobacter cloacae showing multidrug resistance to six classes of antimicrobial agents were collected from two medical centers in Korea. METHODS: Minimum inhibitory concentrations were determined using the E-test method, and 17 genes were detected using polymerase chain reaction (PCR). The epidemiological relatedness of the strains was identified using repetitive element sequence-based PCR and multilocus sequence typing. RESULTS: The 69 E. cloacae clones produced extended spectrum β lactamase (ESBL) and AmpC and showed multidrug resistance to cefotaxime, ceftazidime, and aztreonam. We identified the following sequence types: ST56 of type VI for ESBL SHV (N=12, 17.4%); ST53, ST114, ST113, and ST550 of types I, IV, VI, and VII, respectively, for CTX-M (N=11, 15.9%); and ST668 of type III for the carbapenemase NDM gene (N=1, 1.5%). The AmpC DHA gene (N=2, 2.89%) was confirmed as ST134, although its type was not identified, whereas EBC (MIR/ACT; N=18, 26.1%) was identified as ST53, ST24, ST41, ST114, ST442, ST446, ST484, and ST550 of types V, I, III, IV, VII, and VI, respectively. The formed subclasses were bla CTX-M-3 and bla CTX-M-22 by CTX-M-1, bla CTX-M-9 and bla CTX-M-125 by CTX-M-9, bla DHA-1 by DHA, and bla MIR-7 and bla ACT-15,17,18,25,27,28 by EBC (MIR/ACT). CONCLUSIONS: There were no epidemiological relationships between the gene products and the occurrence of resistance among the strains.

Changing trends in clinical characteristics and antibiotic susceptibility of Klebsiella pneumoniae bacteremia

BACKGROUND/AIMS: Klebsiella pneumoniae is second most common organism of gram-negative bacteremia in Korea and one of the most common cause of urinary tract infection, and intra-abdominal infection. METHODS: We compared clinical and microbiological characteristics about K. pneumoniae bacteremia in a tertiary hospital between 10 years. Group A is who had K. pneumoniae bacteremia at least one time from January 2004 to December 2005. Group B is from January 2012 to December 2013. We also analyzed antibiotic resistance, clinical manifestation of the K. pneumoniae bacteremia divided into community-acquired infections, healthcare associated infections, and nosocomial infections. RESULTS: The resistance for ampicillin, aztreonam, cefazolin, and cefotaxime significantly increased compared to 10 years ago. Extended spectrum β-lactamase positivity surged from 4.3% to 19.6%. Ten years ago, 1st, 2nd cephalosporin, and aminoglycoside were used more as empirical antibiotics. But these days, empirical antibiotics were broad spectrum such as 3rd and 4th cephalosporin. In treatment outcome, acute kidney injury decreased from 47.5% to 28.7%, and mortality decreased from 48.9% to 33.2%. In community-acquired infections, there was similar in antimicrobial resistance and mortality. In healthcare-associated and nosocomial infections, there was significantly increasing in antibiotic resistance, decreasing in mortality, and acute kidney injury. CONCLUSIONS: In community-acquired infections, broader antibiotics were more used than 10 years ago despite of similar antimicrobial resistance. When K. pneumoniae bacteremia is suspected, we recommend to use the narrow spectrum antibiotics as initial therapy if there are no healthcare-associated risk factors, because the antibiotic resistance is similar to 10 years ago in community-acquired infections.

Association between Beta-lactam Antibiotic Resistance and Virulence Factors in AmpC Producing Clinical Strains of P. aeruginosa

OBJECTIVES: The purpose of this study was to determine the presence of IMP and OXA genes in clinical strains of Pseudomonas aeruginosa (P. aeruginosa) that are carriers of the ampC gene. METHODS: In this study, 105 clinical isolates of P. aeruginosa were collected. Antibiotic resistance patterns were determined using the disk diffusion method. The strains carrying AmpC enzymes were characterized by a combination disk method. Multiplex-PCR was used to identify resistance and virulence genes, chi-square test was used to determine the relationship between variables. RESULTS: Among 105 isolates of P. aeruginosa, the highest antibiotic resistance was to cefotaxime and aztreonam, and the least resistance was to colictin and ceftazidime. There were 49 isolates (46.66%) that showed an AmpC phenotype. In addition, the frequencies of the resistance genes were; OXA48 gene 85.2%, OXA199, 139 3.8%, OXA23 3.8%, OXA2 66.6%, OXA10 3.8%, OXA51 85.2% and OXA58 3.8%. The IMP27 gene was detected in 9 isolates (8.57%) and the IMP3.34 was detected in 11 isolates (10.47%). Other genes detected included; lasR (17.1%), lasB (18%) and lasA (26.6%). There was a significant relationship between virulence factors and the OX and IMP genes (p ≤ 0.05). CONCLUSION: The relationship between antibiotic resistance and virulence factors observed in this study could play an important role in outbreaks associated with P. aeruginosa infections.

Informe de evaluación científica basada en la evidencia disponible: fibrosis quística / Scientific evaluation report based on available evidence: cystic fibrosis

INTRODUCCIÓN: La fibrosis quística (FQ) es la enfermedad autosómica recesiva más frecuente entre las poblaciones caucásicas, con una frecuencia de 1 en 2000 a 3000 nacidos vivos. Esta patología es producida por la mutación del gen que codifica la proteína reguladora de la conductancia transmembrana de la FQ (CFTR), existiendo en la actualidad más de 1.400 mutaciones que pueden determinarla. El defecto de la proteína provoca un trastorno del transporte de cloro y sodio por las células de los epitelios, generándose un gran espesamiento de las secreciones, que daña los epitelios secretores, siendo los principales órganos afectados el pulmón, páncreas, hígado, la piel, el aparato reproductor masculino y otros. Los síntomas y signos habituales de presentación incluyen infección pulmonar persistente, insuficiencia pancreática y niveles elevados de cloruro de sudor. Sin embargo, muchos pacientes presentan síntomas leves o atípicos y los clínicos deben permanecer alertas. No se realiza evaluación de los fármacos descritos, en conformidad con los artículos 6º y 9º del Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con Sistema de Protección Financiera, según lo establecido en los artículos 7°y 8° de la ley N°20.850, aprobado por el decreto N°13 de 2017 del Ministerio de Salud. TECNOLOGÍAS SANITARIAS ANALIZADAS: Ivacaftor; combinación Lumacaftor/Ivacaftor, Aztreonam nebulizador. ALTERNATIVAS DISPONIBLES: No existe una cura para la fibrosis quística, pero existen muchos tratamientos que pueden aliviar los síntomas y reducir las complicaciones. Terapia farmacológica: - Antibióticos para tratar y prevenir las infecciones pulmonares. -Medicamentos antiinflamatorios para reducir la hinchazón de las vías respiratorias de los pulmones. -Medicamentos que aflojen la mucosidad para ayudar a expulsarla con la tos, lo cual puede mejorar la función pulmonar. -Medicamentos inhalados llamados broncodilatadores que pueden ayudar a mantener abiertas las vías respiratorias mediante la relajación de los músculos que rodean los bronquios. -Enzimas pancreáticas por vía oral para ayudar a que el tubo digestivo absorba nutrientes. -Para las personas con fibrosis quística que tienen determinadas mutaciones genéticas, los médicos podrían recomendar un nuevo medicamento llamado ivacaftor. Fisioterapia y rehabilitación pulmonar: La fisioterapia y la rehabilitación pulmonar ayudan a ablandar la mucosidad espesa de los pulmones facilitando su expulsión mediante la tos y a mejorar la función pulmonar. Cirugía: Por último, se recomienda en algunos casos tratamiento quirúrgico que pueden variar desde la extracción de pólipos nasales, cirugía intestinal para eliminar la obstrucción hasta el trasplante de pulmón en casos graves. CONCLUSIÓNES: El siguiente informe no continúa en evaluación y, por lo tanto, no conforma parte del informe de evaluación entregado a la comisión de recomendación priorizada, debido a que los tratamientos a evaluar no se encuentran registrados en el país ni tampoco han sido presentados a registro, faltando menos de 1 año para la dictación del próximo decreto.

Antimicrobial Susceptibility and Clonal Distribution of the Blood Isolates of Pseudomonas aeruginosa from Two Korean Hospitals

An increasing prevalence of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) causes a serious therapeutic problem in clinical setting. This study investigated the antimicrobial susceptibility, resistance mechanisms against aminoglycosides, and molecular epidemiology of 76 blood isolates of P. aeruginosa from two Korean hospitals. Thirty-four isolates were susceptible to all 13 antimicrobial agents tested, whereas 28 isolates showed a MDR or extensively drug-resistant phenotype. There was a significant difference in resistance rates of P. aeruginosa isolates against aztreonam, piperacillin-tazobactam, imipenem, meropenem, ciprofloxacin, and norfloxacin between two hospitals. Genes for aminoglycoside-modifying enzymes (AMEs), including aphA6 (n = 14), aadB (n = 11), aacA4 (n = 8), and aphA1 (n = 1), and 16S rRNA methylase armA (n = 6) were detected in 26 P. aeruginosa isolates resistant to aminoglycosides. There was no significant difference in carriage of genes for AME and 16S rRNA methylase between two hospitals, but aacA4 and aphA1 were specifically detected in P. aeruginosa isolates from one hospital. Seventy-six P. aeruginosa isolates were classified into 55 pulsotypes at similarity value of 0.85, and 31 and 24 pulsotypes were specifically detected in each hospital. This study demonstrates that differences in antimicrobial susceptibility of P. aeruginosa isolates between two hospitals are possibly due to the presence of diverse clones specific in each hospital.

Pitfalls of the Clinical and Laboratory Standards Institute's Revised Breakpoints on Interpretation of the Cephalosporin Susceptibility of an Extended-Spectrum beta-lactamase Producing Klebsiella pneumoniae: Analysis of a 2010 Nationwide Proficiency Surve / 임상검사와정도관리

BACKGROUND: In 2010, the Clinical and Laboratory Standards Institute (CLSI) revised the minimum inhibitory concentration (MIC) breakpoints of cephalosporins and aztreonam to exempt extended-spectrum beta-lactamase (ESBL) confirmatory tests for Enterobacteriaceae. However, the CLSI did not change the MIC breakpoint of cefepime. Here, a proficiency survey of a strain of ESBL-producing Klebsiella pneumoniae was analyzed for MIC distribution and interpretation of cephalosporins and aztreonam. METHODS: The survey strain, K. pneumoniae, which produced SHV-18, was distributed to 170 clinical laboratories as 1 of 5 presumptive clinical specimens through the proficiency survey of the clinical microbiology division of the Korean Association of Quality Assurance for Clinical Laboratories (KAQACL). MIC, zone diameter of inhibition (ZDI), and interpretation of tested antimicrobials, methods of antimicrobial susceptibility testing (AST), and ESBL confirmatory results were collected. RESULTS: According to the revised breakpoints of the 2010 CLSI guidelines, MIC results indicated resistance to aztreonam in 100%, cefepime in 5.5%, cefotaxime in 20%, ceftazidime in 100%, and ceftriaxone in 100% of samples by broth microdilution methods. ZDI results also indicated resistance to aztreonam in 75%, cefepime in 0%, cefotaxime in 66.7%, ceftazidime in 100%, and ceftriaxone in 80% of samples by disk diffusion method. Ninety (75.6%) participants performed an ESBL confirmatory test, and 89 (98.9%) reported ESBL-positive tests. Of the 55 laboratories that tested the susceptibility of cefepime, 50 (90.9%) self-reported to be "resistant" because of ESBL-positive results. CONCLUSIONS: In conclusion, susceptibility testing of ESBL producers against certain cephalosporins is not reliable enough to apply the revised breakpoints presented in the 2010 CLSI guidelines. It is therefore necessary to reach a consensus for interpretation of ASTs of ESBL producers in Korea. Ideally, clinicians should be provided two interpretations based on both the revised breakpoints and ESBL confirmatory testing.

An unexpected similarity between antibiotic-resistant NDM-1 and beta-lactamase II from Erythrobacter litoralis

NDM-1 (New Delhi metallo-beta-lactamase) gene encodes a metallo-beta-lactamase (MBL) with high carbapenemase activity, which makes the host bacterial strain easily dispatch the last-resort antibiotics known as carbapenems and cause global concern. Here we present the bioinformatics data showing an unexpected similarity between NDM-1 and beta-lactamase II from Erythrobacter litoralis, a marine microbial isolate. We have further expressed these two mature proteins in E. coli cells, both of which present as a monomer with a molecular mass of 25 kDa. Antimicrobial susceptibility assay reveals that they share similar substrate specificities and are sensitive to aztreonam and tigecycline. The conformational change accompanied with the zinc binding visualized by nuclear magnetic resonance, Zn(2+)-bound NDM-1, adopts at least some stable tertiary structure in contrast to the metal-free protein. Our work implies a close evolutionary relationship between antibiotic resistance genes in environmental reservoir and in the clinic, challenging the antimicrobial resistance monitoring.

Comparison of Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing Breakpoints for beta-Lactams in Enterobacteriaceae Producing Extended-Spectrum beta-Lactamases and/or Plasmid-Mediated AmpC beta-Lacta / 대한임상미생물학회지

BACKGROUND: In 2010, the Clinical and Laboratory Standards Institute (CLSI) revised breakpoints for cephalosporins and carbapenems and indicated that extended-spectrum beta-lactamase (ESBL) testing is no longer necessary for Enterobacteriaceae. We compared the results of the CLSI 2010 and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints for Enterobacteriaceae producing ESBL and/or plasmid-mediated AmpC beta-lactamase (PABL). METHODS: A total of 94 well-characterized clinical isolates of Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Shigella spp., Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, and Serratia marcescens were analyzed. Of them, 57 were ESBL producers, 24 were PABL producers, and 13 were ESBL plus PABL co-producers. Broth microdilution MIC tests were performed for cefotaxime, ceftazidime, aztreonam, cefepime, and imipenem. RESULTS: Among the 94 isolates containing ESBL and/or PABL, the number of isolates that were susceptible to cefotaxime, ceftazidime, aztreonam, cefepime, and imipenem according to the CLSI 2010 vs. the EUCAST breakpoints were 4 (4.3%) vs. 4 (4.3%); 26 (27.7%) vs. 8 (8.5%); 37 (39.4%) vs. 14 (14.9%); 71 (75.5%) vs. 31 (33.0%); and 76 (80.9%) vs. 90 (95.7%), respectively. Of the 18 isolates that were not susceptible to imipenem according to the CLSI 2010 breakpoints, 13 isolates (72.2%) were P. mirabilis. CONCLUSION: The CLSI 2010 MIC breakpoints without tests to detect ESBL and/or PABL for Enterobacteriaceae could be unreliable. Thus, special tests for ESBLs and AmpC beta-lactamases are required to detect the resistance mechanisms involved.

Antibiotic susceptibility and imaging findings of the causative microorganisms responsible for acute urinary tract infection in children: a five-year single center study / 소아과

PURPOSE: We studied the differences in the antibiotic susceptibilities of the microorganisms that causeing urinary tract infections (UTI) in children to obtain useful information on appropriate drug selection for childhood UTI. METHODS: We retrospectively analyzed the antibiotic susceptibilities of 429 microorganisms isolated from 900 patients diagnosed with UTI in the Department of Pediatrics, Chungbuk National University Hospital, from 2003 to 2008. RESULTS: The most common causative microorganisms for UTI were Escherichia coli (81.4%), Klebsiella pneumoniae (8.4%), Enterobacter spp. (1.7%), and Proteus spp. (0.4%). E. coli showed relatively high susceptibility as compared to imipenem (100%), amikacin (97.7%), aztreonam (97.9%), cefepime (97.7%), and ceftriaxone (97.1%), while it showed relatively low susceptibility to gentamicin (GM) (79.0%), trimethoprim/sulfamethoxazole (TMP/SMX) (68.7%), ampicillin/sulbactam (33.0%), and ampicillin (AMP) (28.6%). There were no significant differences in the image findings for causative microorganisms. CONCLUSION: Gram-negative organisms showed high susceptibility to amikacin and third-generation cephalosporins, and low susceptibility to AMP, GM, and TMP/SMX. Therefore, the use of AMP or TMP/SMX as the first choice in empirical and prophylactic treatment of childhood UTI in Korea should be reconsidered and investigated further.

Characterization of Acinetobacter baumannii Co-producing Carbapenemases OXA-23 and OXA-66, and armA 16S Ribosomal RNA Methylase at a University Hospital in South Korea / 대한임상미생물학회지

BACKGROUND: In the present study, the resistance mechanisms against carbapenems and aminoglycosides for 23 strains of multi-drug-resistant Acinetobacter baumannii isolated at a university hospital were investigated. METHODS: The minimal inhibitory concentrations (MICs) were determined via broth microdilution or Etest. The genes encoding OXA-type carbapenemases and 16S rRNA methylase were identified using multiplex PCR, and the amplified products were sequenced. Conjugation experiments were conducted, and an epidemiologic study was performed using enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS: In the isolates, the MICs of the tested aminoglycosides, including arbekacin, were >1024 microg/mL; the MICs of aztreonam, cefepime, ceftazidime, and ciprofloxacin ranged from 64 to 128 microg/mL; and the MICs of carbapenem ranged from 32 to 64 microg/mL, as determined through the broth microdilution test. According to the E-test, the MICs of ampicillin/sulbactam and colistin were 8 and 0.25 to 0.38 microg/mL, respectively. Sequence analysis confirmed that all of the isolates expressed carbapenemases OXA-23 and OXA-66, as well as armA 16S rRNA methylase. In addition, ISAba1 was identified upstream of the gene encoding OXA-23. OXA-23 and armA were not transferred to Escherichia coli J53 cells in the transconjugation experiments. ERIC-PCR molecular fingerprinting produced a single pattern in all cases. CONCLUSION: The co-production of OXA-23 and armA 16S rRNA methylase may be attributed to the multidrug resistance of the A. baumannii isolates in the present study. Stricter surveillance and more rapid detection are necessary to prevent the spread of this type of resistance in the future.

Causas de cambio de tratamiento en un grupo de pacientes VIH/SIDA cubanos / Causes of change in treatment in a group of HIV/AIDS Cuban patients

La infección por virus de inmunodeficiencia humana (VIH) es uno de los mayores problemas de salud actuales, se estima que más de 40 millones de personas están infectadas en el mundo. Se realizó un estudio descriptivo de corte transversal en 40 pacientes VIH/SIDA pertenecientes al servicio de Medicina del IPK con el propósito de caracterizar las causas del cambio de tratamiento antirretroviral y los tipos de reacciones adversas presentadas con este tratamiento en un grupo de pacientes VIH/SIDA..Los pacientes recibieron diferentes esquemas de antirretrovirales entre los que predominaron los siguientes: 3TC, d4T e Indinavir (57,5 por ciento), seguido de 3TC, AZT e Indinavir (22,5 por ciento). Las causas más frecuentes de cambio de tratamiento fueron las reacciones adversas y la mala adherencia al tratamiento. Entre las personas que están recibiendo terapia contra el VIH, existe una tendencia cada vez más frecuente de abandonar o cambiar la terapia. Las causas de estos cambios y del abandono de las terapias suelen estar relacionadas con los efectos secundarios, la fatiga del tratamiento, la fase de la infección por VIH en que se encuentra el paciente, y factores relativos a su estilo de vida. Las causas más frecuentes de cambio de tratamiento fueron las reacciones adversas, seguidas de la mala adherencia al tratamiento antirretroviral. Las reacciones adversas más frecuentes en el grupo de estudio fueron los vómitos y los trastornos digestivos respectivamente

The HIV virus infection is one of the major current problems of health estimating that more than 40 millions of persons are infected at world level. A cross-sectional and descriptive study was conducted in 40 HIV/AIDS patients from the Tropical Medicine Institute service to characterize the causes of the change in antiretroviruses treatment and the types of adverse reactions related to this treatment in a group of HIV/AIDS patients. Patients received different antiretroviruses schemes with predominance of 3TC, d4T and Indinarir (57,5 percent), followed by 3TC, AZT and Indinavir (22,5 percent). The more frequent causes of change of this treatment were the adverse reactions and a poor adherence to it. Among the persons with therapy HIV there is a more and more frequent trend to give up or to change of therapy. The causes of these changes and the leaving the therapy are related to side effects, treatment fatigue, and the HIV infection phase in patient and relative factors related to the lifestyle. The more frequent causes of the change of treatment were the adverse reactions followed by a poor adherence to antiretroviruses treatment. The more frequent adverse reactions were vomiting and digestive disorders, respectively

Antimicrobial resistant pattern of staphylococcus aureus strains isolated from pediatric patients in Jordan

Development of resistance to antimicrobial agents by staphylococci is a major public health problem. Therefore, a retrospective study was conducted to determine the rate of resistance to antibiotics by S. aureus strains isolated from pediatric patients. A total of 472 different clinical specimens of pediatric patients were investigated. A high resistance of 96.7%, to aztreonam followed by 95.2% to imipenem, and 85.8% to cephalexin by Staphylococcus aureus strains was recorded. Low resistance rate was also recorded for vancomycin [1.4%] followed by cephalothin [9%],clindamycin [153%],tobramycin [15.7%],amikacin [18.1%] and ciprofloxacin [18.6%]. Aztreonam, imipenem, and most of [beta-lactam antibiotics were found to be ineffective at in vitro inhibition of the S. aureus of pediatric origin. S. aureus infections could be effectively treated with vancomycin, cephalothin, clindamycin, tobramycin, amikacin and ciprofloxacin. The high resistance rate to most of beta-lactam antibiotics studied could be attributed to their prevailing usage and abuse in the area of study

In Vitro Effects of Combined Antibiotics against Multidrug-resistant Pseudomonas aeruginosa / 대한내과학회지

BACKGROUND/AIMS: The inhibitory effects of the combination of beta-lactam with ciprofloxacin or amikacin against clinical isolates of multidrug-resistant Pseudomonas aeruginosa were evaluated. METHODS: This study examined ten isolates with variable levels of resistance to ceftazidime, cefepime, piperacillin/tazobactam, meropenem, ciprofloxacin, and amikacin. The efficacy of the combined antibiotics was studied using a checkerboard method or in vitro killing assay. RESULTS: The combination of ceftazidime, cefepime, aztreonam, piperacillin-tazobactam, or meropenem with amikacin showed synergistic effects for all of the strains regardless of the minimum inhibitory concentration (MIC) of amikacin, but combination with ciprofloxacin showed a synergistic effect for the isolate with a low MIC of ciprofloxacin by the checkerboard method. The isolates with a high MIC of ciprofloxacin showed an indifferent effect in combination with beta-lactam and ciprofloxacin. The in vitro killing assay showed that meropenem with ciprofloxacin acted synergistically for the isolates with a MIC of 16 microgram/mL of ciprofloxacin. However, amikacin showed synergistic effects with meropenem for the isolates with high-level resistance against amikacin, i.e., up to an MIC of 128 microgram/mL. Contrary to the checkerboard method results, no synergy was observed for the combination of ceftazidime/piperacillin-tazobactam and amikacin. CONCLUSIONS: Meropenem with amikacin can be the first choice for infections caused by multidrug-resistant P. aeruginosa when the level of resistance is not known.

Tigeciclina versus vancomycin más aztreonam en el tratamiento de infecciones complicadas de piel y tejidos blandos: Experiencia en Latinoamérica / Tigecycline as effective as vancomycin plus aztreonam in the treatment of complicated skin and skin structure infections: Experience in Latin America

BACKGROUND: Treating complicated skin and skin structure infections (cSSSIs) can be challenging. Tigecycline was compared to vancomycin/aztreonam in patients with cSSSIs in a multinational trial; this article reports on the Latin American (LA) population. METHODS: Patients were randomly assigned to receive tigecycline or vancomycin/ aztreonam. Primary endpoint was clinical cure rate at test-of-cure (TOC). Several secondary endpoints and safety were also assessed. RESULTS: A subtotal of 167 LA patients from the multinational trial (N = 573) received ≥ 1 dose of study drug. At TOC, cure rates were similar between tigecycline and vancomycin/aztreonam in the clinically evaluable population.) Noninferiority of tigecycline could not be demonstrated (insufficient sample sizes). Tigecycline-treated patients had higher incidences of nausea, vomiting, anorexia; vancomycin/aztreonam-treated patients had higher incidences of pruritus and rash. CONCLUSIONS: Efficacy results in the LA population were consistent with the multinational study suggesting that tigecycline is noninferior to vancomycin/aztreonam in treating patients with cSSSI.

INTRODUCCIÓN: El tratamiento de infecciones complicadas de piel y tejidos blandos (ICPTB) puede representar un desafío. Se comparó la eficacia de tigeciclina versus vancomicina/aztreonam en pacientes con ICPTB en un estudio multicéntrico; este artículo se refiere a la experiencia en Latinoamérica (LA). MÉTODO: Se asignaron, en forma randomizada, los pacientes a dos grupos de tratamiento: tigeciclina o vancomicina/aztreonam. La meta a evaluar (outcome) primaria fue la curación clínica, denominada test de curación (TC). Se establecieron, además, metas secundarias y la evaluación de seguridad del fármaco. RESULTADOS: Un subtotal de 167 pacientes procedentes de LA, de un estudio multinacional que incluyó 573 pacientes, recibieron ≥ 1 dosis del fármaco en estudio. Al TC, los porcentajes de curación fueron similares entre tigeciclina y vanco-micina/aztreonam en los pacientes clínicamente evaluables). La no inferioridad de tigeciclina no pudo ser demostrada (tamaño de muestra insuficiente). Los pacientes tratados con tigeciclina tuvieron mayor incidencia de náuseas, vómitos y anorexia; los pacientes que recibieron vancomicina/aztreonam tuvieron mayor incidencia de prurito y rash. CONCLUSIONES: Los resultados de eficacia en LA fueron consistentes con el estudio multinacional sugiriendo que tigeciclina no es inferior a vancomicina/aztreonam en el tratamiento de pacientes con ICPTB.

A Case of Multidrug-Resistant Salmonella enterica Serovar Typhi Treated with a Bench to Bedside Approach

We report a relapsed case of a 25 year-old man with multi-drug resistant Salmonella serovar Typhi (MDRST) bacteremia who had recently returned from travel in India. Due to unresponsiveness to ciprofloxacin and ceftriaxone, we examined the strain's resistance to quinolones and extended-spectrum beta-lactamases (ESBLs). The strain had a single gyrA mutation at codon 83 (Ser83Phe), which explains its decreased susceptibility to fluoroquinolone and resistance to nalidixic acid. In the screening tests of ESBLs, TEM-1 was positive, which is beta-lactamase but not ESBL. The patient was finally successfully treated with meropenem and aztreonam. In the presence of clinical unresponsiveness despite favorable sensitivity tests, further laboratory evaluations are needed, which should include studies of genes related to antibiotic resistance and ESBLs. In addition, further prospective trials should be done about the possible inclusion of antibiotics not yet mentioned in the current guidelines. With MDRST on the rise worldwide, the most optimal and effective line of antibiotic defense needs to be devised.