Emergence of quinolone-resistant, topoisomerase-mutant Brucella after treatment with fluoroquinolones in a macrophage experimental infection model

AIM: of the study To determine the activity of fluoroquinolones (FQ) and the selection of FQ-resistant mutants in a macrophage experimental infection model (MEIM). MATERIAL AND METHODS: Canine macrophages were inoculated with Brucella melitensis ATCC 23457 (WT), achieving intracellular counts of around 105 CFU/mL. Cell cultures were incubated in the presence of ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MOX), and doxycycline (DOX). After cell lysis, surviving microorganisms were plated for count purposes, and plated onto antibiotics-containing media for mutant selection. Topoisomerases mutations were detected by PCR and sequencing. RESULTS: Bacterial counts after cell lysis were 14.3% (CIP), 65.3% (LEV), and 75% (MOX) lower compared to the control. Quinolone-resistant mutants emerged in cell cultures containing CIP and LEV with a frequency of around 0.5 × 10-3. All mutants showed an Ala87Val change in GyrA. Mutants had FQs MICs around 10 × WT. The ability of these mutants for infecting new macrophages and the intracellular lysis after antibiotic exposure did not change significantly. No 2nd step FQ-resistant mutants were selected from 1st step mutants. CONCLUSIONS: Intracellular activity of FQs is low against WT and gyrA-mutant Brucella. FQs easily select gyrA mutants in MEIM. The ability of mutants for infecting new macrophages remains unchanged. In this MEIM, 2nd step mutants do not emerge

OBJETIVO: del estudio Conocer la actividad de fluoroquinolonas (FQ) y la selección de mutantes resistentes (MR) a FQ en un modelo experimental de infección en macrófagos. MATERIAL Y MÉTODOS: Se inocularon macrófagos de origen canino con la cepa tipo Brucella melitensis ATCC 23457 (CT) hasta alcanzar recuentos intracelulares de aproximadamente 105 UFC/mL. Los cultivos celulares fueron incubados en presencia de ciprofloxacino, levofloxacino, moxifloxacino y doxiciclina. Una vez lisadas las células, los microorganismos supervivientes fueron sembrados en placas sin antibiótico para recuento, y en medios de cultivo conteniendo antimicrobianos para selección de MR. Los MR a topoisomerasas se caracterizaron mediante PCR y secuenciación. RESULTADOS: Los recuentos de microorganismos supervivientes tras el tratamiento con FQ y la lisis celular fueron: 14,3% ciprofloxacino, 65,3% levofloxacino y 75% moxifloxacino con respecto al control. Se seleccionaron MR a FQ en los cultivos celulares que contenían ciprofloxacino y levofloxacino, con una frecuencia en torno a 0,5 × 10-3. Todos los MR seleccionados portaban un cambio Ala87Val en gyrA, y mostraban CIM de FQ en torno a 10x la de la CT. La capacidad de estos MR para infectar nuevos macrófagos y su lisis intracelular tras tratamiento antibiótico no se modificaron de manera significativa con respecto a la CT. Usando la misma metodología, no se seleccionaron MR de segundo nivel a partir de los MR de primer nivel obtenidos. CONCLUSIONES: La actividad intracelular de FQ frente a Brucella es baja, tanto frente a la CT como frente a mutantes con cambios en gyrA. Las FQ seleccionan con facilidad mutantes con cambios en gyrA en este modelo experimental de infección en macrófagos. La capacidad de estos mutantes para infectar nuevos macrófagos permanece intacta. En este modelo experimental de infección en macrófagos no se observó la selección de mutantes de segundo nivel

Emergence of quinolone-resistant, topoisomerase-mutant Brucella after treatment with fluoroquinolones in a macrophage experimental infection model.

AIM OF THE STUDY: To determine the activity of fluoroquinolones (FQ) and the selection of FQ-resistant mutants in a macrophage experimental infection model (MEIM). MATERIAL AND METHODS: Canine macrophages were inoculated with Brucella melitensis ATCC 23457 (WT), achieving intracellular counts of around 105 CFU/mL. Cell cultures were incubated in the presence of ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MOX), and doxycycline (DOX). After cell lysis, surviving microorganisms were plated for count purposes, and plated onto antibiotics-containing media for mutant selection. Topoisomerases mutations were detected by PCR and sequencing. RESULTS: Bacterial counts after cell lysis were 14.3% (CIP), 65.3% (LEV), and 75% (MOX) lower compared to the control. Quinolone-resistant mutants emerged in cell cultures containing CIP and LEV with a frequency of around 0.5×10(-3). All mutants showed an Ala87Val change in GyrA. Mutants had FQs MICs around 10×WT. The ability of these mutants for infecting new macrophages and the intracellular lysis after antibiotic exposure did not change significantly. No 2nd step FQ-resistant mutants were selected from 1st step mutants. CONCLUSIONS: Intracellular activity of FQs is low against WT and gyrA-mutant Brucella. FQs easily select gyrA mutants in MEIM. The ability of mutants for infecting new macrophages remains unchanged. In this MEIM, 2nd step mutants do not emerge.

Profilaxis antifúngica en pacientes oncohematológicos: revisión de la bibliografía médica y recomendaciones / Antifungal prophylaxis in oncohematologic patients: Literature review and recommendations

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Multicenter study evaluating the role of enterococci in secondary bacterial peritonitis.

A 1-year prospective multicenter study was performed to explore the significance of the presence of enterococci in cultures of peritoneal fluid from patients with secondary bacterial peritonitis in seven Spanish hospitals. The clinical records of patients with positive peritoneal fluid cultures were reviewed and distributed into cases (patients with cultures yielding enterococci) and controls (patients with cultures not yielding enterococci). Of a total of 158 records, 38 (24.1%) were cases and 120 (75.9%) were controls. The percentages or the scores (cases versus controls) for the variables included in the multivariate analysis were as follows: age of >50 years, 89.5% versus 68.3%; malignancy, 39.5% versus 18.3%; chronic obstructive pulmonary disease (COPD), 15.8% versus 4.2%; postoperative peritonitis, 55.3% versus 30.1%; nosocomial onset, 57.9% versus 34.2%; a higher Charlson comorbidity index, 3.29 +/- 3.38 versus 1.84 +/- 2.31; APACHE II score, 10.71 +/- 4.37 versus 8.76 +/- 5.49; ultimately or rapidly fatal disease, 63.2% versus 34.8%; need for surgical reintervention, 36.1% versus 15.1%; and admission to an intensive care unit, 45.9% versus 30.8%. In the multivariate analysis, enterococci were associated only with postoperative peritonitis (P = 0.009; odds ratio [OR] = 5.0; 95% confidence interval [CI] = 1.49 to 16.80), a higher Charlson comorbidity index (P = 0.002; OR = 1.30; 95% CI = 1.11 to 1.54), and COPD (P = 0.046; OR = 6.50; 95% CI = 1.04 to 40.73). The results of this study showed that enterococci were associated with comorbidity. An association with mortality could not be demonstrated.

Tratamiento de las infecciones fúngicas en pacientes con neoplasias hematológicas / Treatment of fungal infections in patients with hematologic neoplasia

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Hepatitis B reactivation in a hepatitis B surface antigen-negative patient after allogeneic bone marrow transplant: successful treatment with lamivudine and seroconversion.

Hepatitis B reactivation in hepatitis B surface antigen (HBsAg)-negative and anti-HBsAg antibodies-positive patients is an infrequent complication of chemotherapy, usually with fatal evolution. Here we report an HBsAg-negative patient with a myelodysplastic syndrome, who developed hepatitis B reactivation after chemotherapy and evolved favorably after lamivudine treatment, allowing seroconversion.

Fluoroquinolone-resistant Brucella melitensis mutants obtained in vitro.

Three fluoroquinolone-resistant Brucella melitensis mutants were obtained after successive passages on norfloxacin-containing agar plates. The original strain was B. melitensis biovar 2 ATCC 23457. Mutants emerged at frequencies ranging between 1 x 10(-6) and 3 x 10(-8). The three mutants showed a gyrA Ala71-->Ser mutation as well as insertion of Ala340. No mutations were observed in the parC and gyrB genes. Minimum inhibitory concentrations (MICs) increased by four- to eight-fold from the wild-type to the first-step mutant for norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin. MICs of the second-step mutant increased significantly only for norfloxacin and those for the third-step mutant for norfloxacin and ciprofloxacin, suggesting the involvement of some efflux mechanism.

Tratamiento antimicrobiano de la agudización de la EPOC: Documento de Consenso 2007 / Antimicrobial treatment of exacerbation in Chronic Obstructive Pulmonary Disease: 2007 Consensus Statement

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Prevalence of clinical isolates of Escherichia coli and Klebsiella spp. producing multiple extended-spectrum beta-lactamases.

Eleven thousand two hundred seventy-two Escherichia coli, 1109 Klebsiella pneumoniae, 1124 Salmonella enterica, and 602 Klebsiella oxytoca unrelated clinical isolates were obtained between 2001 and 2004 in a university hospital in Salamanca, Spain. One hundred thirteen E. coli (1%), 32 K. pneumoniae (2.9%), 4 K. oxytoca (0.66%), and 5 S. enterica (0.44%) isolates produced extended-spectrum beta-lactamases (ESBLs). We obtained 42.2% of the ESBL-producing isolates from outpatients and 57.8% from inpatients. The most commonly detected ESBLs were CTX-M 14 (43.5% of ESBL-producing isolates), TEM-116 (22.1%), and SHV-2 (15.6%). A CTX-M 27-producing E. coli is 1st reported in Spain in this study. Two (20 isolates, 13%) or 3 (7 isolates, 4.5%) ESBLs were produced by 17.5% of ESBL-producing isolates (27 isolates). The most frequent combinations were CTX-M 14 + TEM-116 (5.7%), SHV-12 + TEM-116 (2.6%), and SHV-2 + CTX-M 14 + TEM-116 (2.6%). Clonal diversity was high even between isolates producing the same combinations of 2 or 3 beta-lactamases.

Salmonella enterica serovar Enteritidis producing a TEM-52 beta-lactamase: first report in Spain.

TEM-52 is an extended-spectrum beta-lactamase (ESBL) that is being increasingly reported in enterobacteria. In Spain, TEM-52 had been reported before only in Escherichia coli. Here we report the first finding of a TEM-52 ESBL in Salmonella in Spain, associated to a Tn3 transposon.

Activity of meropenem and comparators against Pseudomonas aeruginosa and Acinetobacter spp. isolated in the MYSTIC Program, 2002-2004.

This study examines the susceptibilities of meropenem and other broad-spectrum antimicrobials tested against bacterial isolates collected from hospitalized patients during 2002-2004 from worldwide medical centers participating in the Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program. The in vitro activity of meropenem and 5 comparator antimicrobial agents was assessed against Pseudomonas aeruginosa and Acinetobacter spp. Generally, the susceptibility of Australasian and North American isolates was higher than that of the European and South American isolates. The rank order of activity of the antimicrobial agents tested against a worldwide collection of P. aeruginosa was piperacillin/tazobactam (77.7% susceptible) > meropenem (75.4%) > ceftazidime (70.0%) > imipenem (69.7%) > gentamicin (66.1%) > ciprofloxacin (62.0%). Against a worldwide collection of Acinetobacter spp. meropenem (76.1% susceptible) was the most active compound followed by imipenem (74.7%) > gentamicin (51.9%) > ciprofloxacin (40.5%) > piperacillin/tazobactam (39.8%) > ceftazidime (38.1%). The carbapenems appear to be a valuable option for the treatment of serious nosocomial infections caused by P. aeruginosa or Acinetobacter spp. over a broad geographical region.

Risk factors associated with colonization by pneumococci with reduced susceptibility to fluoroquinolones in adult outpatients.

We developed a case-control study in order to identify risk factors associated with pharyngeal colonization by Streptococcus pneumoniae with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC, > or =4 microg/ml). A total of 400 patients were studied for colonization by quinolone-nonsusceptible S. pneumoniae (QNSP) isolates and risk factors for this colonization. Isolate susceptibility was determined by the agar dilution method. Forty patients were colonized by QNSP (case patients), and 360 patients were not colonized by QNSP (control patients). The MIC range of ciprofloxacin for QNSP isolates was 4 to 8 microg/ml. No isolates were resistant to levofloxacin and moxifloxacin. Risk factors significantly associated with QNSP colonization, according to univariate analysis, were recent hospitalizations (odds ratio [OR], 3.43; 95% confidence interval [CI], 1.6 to 7.2; P < 0.01) and prior exposure to fluoroquinolones (OR, 6.04; 95% CI, 3.0 to 12.0; P < 0.01). Other factors such as chronic obstructive pulmonary disease (OR, 1.94; 95% CI; 0.7 to 5.0), prior exposure to penicillins (OR, 1,68; 95% CI, 0.8 to 3.3) and prior exposure to macrolides (OR 2; 95% CI, 0.6 to 6.2) were more frequent among patients colonized with QNSP, but there was no statistical significance. Multivariate analysis showed that exposure to fluoroquinolones was the only independent factor associated with colonization by QNSP (OR, 4.2; 95% CI, 1.8 to 9.4; P < 0.01). Throat colonization by QNSP is becoming frequent, though most of these isolates (all the isolates in this case) remain susceptible to newer fluoroquinolones. Previous treatment with fluoroquinolones seems to be the main risk factor associated with colonization by QNSP.

Streptococcus pyogenes pharyngeal isolates with reduced susceptibility to ciprofloxacin in Spain: mechanisms of resistance and clonal diversity.

A survey of emm gene sequences and an analysis of the pulsed-field electrophoretic profiles of 30 Streptococcus pyogenes isolates with reduced susceptibilities to ciprofloxacin detected the prevalence of isolates with emm type 6 and considerable genetic diversity among isolates. The mechanism of ciprofloxacin resistance in these isolates was based on point mutations in topoisomerase IV subunit C encoded by parC, mainly replacement of serine-79 by alanine.

[Seroprevalence of antibodies against Treponema pallidum, Toxoplasma gondii, rubella virus, hepatitis B and C virus, and HIV in pregnant women]. / Seroprevalencia de anticuerpos frente a Treponema pallidum, Toxoplasma gondii, virus de la rubéola, virus de la hepatitis B y C y VIH en mujeres gestantes.

INTRODUCTION: The prevalence of antibodies against Treponema pallidum, Toxoplasma gondii, rubella virus, hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) was investigated in pregnant women. METHODS: With the use of several serological methods in samples from women who had their first obstetric visit in 2001, we studied the prevalence of serum antibodies against T. pallidum, T. gondii, rubella virus, HBV and HCV in 2,929 pregnant women, and anti-HIV antibodies in the 1,349 women agreeing to this test. RESULTS: Antibodies against T. pallidum were not detected in any case. HBsAg was found in 11 patients (0.4%), six of whom (54.5%) were not aware of their condition. The presence of anti-rubella antibodies was almost universal (99.95%). In the total population, 18.8% of patients had anti-T. gondii antibodies; only one had a serological profile suggesting acute toxoplasmosis. Among the 1,349 women studied, anti-HIV antibodies were detected in two intravenous drug abusers who were aware of their condition. Anti-HCV antibodies were found in 0.4% of the series, and 36.4% of the HCV-positive patients had no knowledge of their condition. CONCLUSIONS: Active infection by T. pallidum in pregnant women in Spain is currently exceptional. The level of immunization against rubella virus is excellent. Seropositivity to T. gondii is lower than rates reported in earlier studies. The prevalence of HBsAg and anti-HCV antibodies is around 0.4%, and seropositive status is often discovered in routine serological studies performed during pregnancy. HIV seropositivity is low, and the pregnant women included in this study were aware of their condition.

In vitro activity of older and newer fluoroquinolones against efflux-mediated high-level ciprofloxacin-resistant Streptococcus pneumoniae.

The effect of high-level efflux activity on the MICs of fluoroquinolones against Streptococcus pneumoniae in the absence of topoisomerase mutations leading to fluoroquinolones resistance was investigated. A S. pneumoniae ATCC 46619-derived strain with high-level efflux activity was obtained (SP-25A). Both the parent and obtained strains were tested against efflux substrates acriflavine (Acr) and ethidium bromide (EtBr), and against norfloxacin (NFX), ciprofloxacin (CFX), levofloxacin (LFX), moxifloxacin (MFX), trovafloxacin (TVX) and sitafloxacin (SFX), in presence and absence of the efflux pump inhibitor reserpine. gyrA, gyrB, parC and parE QRDR genes were amplified by PCR and sequenced. MICs of NFX and CFX against SP-25A were 64-fold higher than parent strain MICs (256 mg/L versus 4 mg/L and 64 mg/L versus 1mg/L, respectively). MIC of LFX increased from 1 to 4 mg/L and MICs of MFX, TVX and SFX remained virtually unchanged (0.1-0.2 mg/L). MICs of Acr and EtBr against SP-25A were 8- and 16-fold higher than against parent strains. In both cases, reserpine reverted MICs to the parent strain values (1 and 0.2 mg/L). Only parE showed two mutations leading to a Pro(454) --> Ser and Glu(443) changes, which have previously been shown not to lead to significant fluoroquinolones MIC increases. SP-25A showed a significant increase of MICs of the hydrophilic fluoroquinolones, apparently derived only from efflux activity. Efflux activity, at these high levels, can lead to high-level resistance to older hydrophilic fluoroquinolones, but does affect newer fluoroquinolones such as moxifloxacin, trovafloxacin and sitafloxacin.