Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes

ABSTRACT Purpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM) and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the β-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure

Mucoidy, Quorum Sensing, Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung

Modified Spot CAMP Test: A rapid, inexpensive and accurate method for identification of group B streptococci Prueba de CAMP por spot modificada: Un método rápido, preciso y económico para la identificación de estreptococos grupo B

A rapid modified spot CAMP test using 183 clinical isolates of &#946; haemolytic streptococci was compared with the standard CAMP test described by Christie et al. The scheme of biochemical identification and serological confirmation was taken as reference method. The sensitivity of both tests was 100%, and the specificity of the rapid and standard tests was 96.8% and 88.9% respectively. The modified spot CAMP test is a rapid, inexpensive and accurate method for the identification of group B streptococci, and is more specific than the standard CAMP test.<br>En este estudio se comparó los resultados de una prueba de CAMP por spot modificada en 20 minutos y la prueba de CAMP original descripta por Christie et al usada para la identificación de Streptococcus agalactiae. Se analizaron 183 aislamientos de estreptococos &#946; hemolíticos, tomando como método de referencia el esquema tradicional de identificación bioquímica y confirmación serológica. La sensibilidad de ambas pruebas fue del 100% y la especificidad de la prueba rápida y la estándar fue de 96,8% y 88,9% respectivamente. La prueba de CAMP por spot modificada es un método rápido, económico y seguro para la identificación de estreptococos del grupo B y posee mayor especificidad que la prueba original

Pharmacodynamic assessment of Amoxicillin-Sulbactam against Acinetobacter baumannii: searching the optimal dose and infusion time through a human ex-vivo model

Amoxicillin-sulbactam (AMX-SUL) is an aminopenicillin/ß-lactamase inhibitor combination currently available in 29 countries and may be a suitable option for treating infections caused by Acinetobacter spp. Thus, we sought to search the optimal dosing strategy for this formulation through an ex vivo pharmacodynamic human model against Acinetobacter baumanniii. Four volunteers were randomized to receive alternatively a single dose AMX-SUL infused both either over 30 min or 3h at the following ratios (g/g): 1/0.5; 1/1, and 0/2. Time-kill studies were performed with the 0-, 0.5-, 2-, 4-, 6- and 8-h sera after dose against a clinical isolate of A. baumannii (sulbactam MIC, 4µg/mL). Bactericidal activity (i.e. a mean decrease >3 log10 CFU/mL in the viable cell counts from the initial inoculum) was displayed by the 0.5- and the 2-h sera after dose for all formulations. The 4-h sera proved inhibitory with the AMX-SUL 1g/1g formulation, albeit a trend to regrowth was observed after 24-h incubation. With the AMX-SUL 0g/2g dose, the 4-h sera proved almost bactericidal activity (i.e. a mean decrease of 2.4 log10 CFU/mL in the viable cell counts from the initial inoculum), whereas the 6-h sera was inhibitory, with a trend to regrowth after 24-h incubation. When infused over 3h, AMX-SUL 1g/0.5g and 1g/1g, bactericidal activity was displayed by the 0.5-, 2- and the 4-h sera after dose and the 6-h sera proved inhibitory with the AMX-SUL 1g/1g formulation. The present study, albeit preliminary, might give a rationale for the dosing strategy to treat infections caused by A. baumannii with sulbactam, either alone or combined with amoxicillin. A 2-g sulbactam dose seems to be optimal to be infused over 30 min with a 6-h dosing interval. When infused over 3h, AMX-SUL 1g/1g given every 6h or 8h seems a suitable dosing schedule

Comparative In Vitro Activity of Tigecycline Against Bacteria Recovered from Clinical Specimens in Latin America

Q4Q2144-152the present study was performed to evaluate the in vitroactivity of tigecycline in comparison to other agents against isolates recovered from patients hospitalized in latin American. Organisms were collected in 47 clinical laboratories from 4 countries of latin America between November 2005 and October 2006 and were tested by using disk diffusion method as described by the ClSi. A total of 7966 isolates were assessed. tigecycline proved highly active against staphylococci and enterococci (>99% suscep- tibility). imipenem was the most active agent against Escherichia coli (100% suscep- tibility), followed by tigecycline, 98.6% susceptibility. Resistance to cefotaxime in this species was 15.3%. Global tigecycline susceptibility of Klebsiella species was 90.2%, but the susceptibility rate was significantly slower in Venezuela (82%) than in Ar- gentina, Colombia and Chile (93%) (p<0.01). Global cefotaxime resistance to Kleb- siellaspp. was 32.2% and carbapenem resistance was detected in all countries. by adopting a susceptible breakpoint ≥ 16mm, 91.3% of the Acinetobacter isolates proved susceptible to tigecycline. Results from the present study suggest that tigecy- cline may be a suitable option in latin America, a region where multidrug resistance seems to be a dramatic, increasing problem and new antimicrobial choices are urgently needed

Actividad &#8220;in vitro&#8221; de 10 antimicrobianos frente a bacterias anaerobias: Estudio multicéntrico, 1999-2002 &#8220;In vitro&#8221; activity of ten antimicrobial agents against anaerobic bacteria. A collaborative study, 1999-2002

Se evaluó la actividad de ampicilina, ampicilina-sulbactama, cefoxitina, ceftriaxona, imipenem, piperacilina, piperacilina-tazobactama, clindamicina, metronidazol y azitromicina frente a 166 cepas de bacterias anaerobias aisladas en 8 hospitales de Buenos Aires. Se estudiaron: Bacteroides grupo fragilis (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), otros clostridios (12) y cocos gram-positivos (22). Las CIMs se determinaron usando el método patrón de dilución en agar recomendado por el NCCLS, documento M11-A5. Los antibióticos más activos fueron metronidazol y piperacilina-tazobactama que exhibieron valores de CIM90£ 2 µg/ml y £ 4 µg/ml frente a los microorganismos gram-negativos y £ 2 µg/ml y £ 8 µg/ml frente a los microorganismos gram-positivos, respectivamente. Entre los b-lactámicos el orden de actividad frente a bacilos gram-negativos fue: imipenem > piperacilina > cefoxitina > ceftriaxona > ampicilina. En gram-positivos la actividad decreciente fue: piperacilina> imipenem > cefoxitina > ceftriaxona > ampicilina. La mayoría de las especies estudiadas mostraron distintos niveles de resistencia con clindamicina y azitromicina. Sin embargo, el 90% de las cepas de Fusobacterium nucleatum y Por-phyromonas spp. fue inhibido por una concentración de 0,125 µg/ml de clindamicina y azitromicina, respectivamente.<br>The antimicrobial activity of ampicillin, ampicillin-sulbactam, cefoxitin, ceftriaxone, imipenem, piperacillin, piperacillin-tazobactam, clindamycin, metronidazole, and azitromycin was assesed against 166 strains of anaerobic bacteria recovered from eight hospitals in Buenos Aires. The strains studied were Bacteroidesfragilis group (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), other clostridia (12), and gram-positive cocci (22). The MICs were determined by the agar dilution method according to NCCLS document M11-A5. Metronidazole and piperacillin-tazobactam were the most active antimicrobial agents tested and exhibited MIC90values of £ 2 µg/ml and £ 4 µg/ml against gram-negative organisms, and £ 2 µg/ml, and £ 8 µg/ml against gram-positive organisms, respectively. Among b-lactams the activity against gram-negative rods was in the following order: imipenem> piperacillin > cefoxitin > ceftriaxone > ampicillin. Among the gram-positive bacteria the decreased activity was: piperacillin> imipenem> cefoxitin > ceftriaxone > ampicillin. The majority of the species studied showed different degrees of resistance to clindamycin and azitromycin. Nevertheless, 90% of Fusobacterium nucleatum and Porphyromonas spp. isolates were inhibited by 0.125 mg/ml of clindamycin and azitromycin, respectively

Susceptibility trends of Bacteroides fragilis group isolates from Buenos Aires, Argentina Tendencias en el perfil de sensibilidad de aislamientos del grupo Bacteroides fragilis obtenidos en Buenos Aires, Argentina

The aim of this study was to analyze the susceptibility trends to seven antibiotics of Bacteroides fragilis group isolates based on three survey studies performed by the Committee of Anaerobic Bacteria between 1989 and 2002. Fifty three, 82 and 65 B. fragilis group isolates were collected during each period. The antimicrobial agents included were: ampicillin, ampicillin-sulbactam (2:1), cefoxitin, piperacillin, imipenem, clindamycin, and metronidazole. Minimal inhibitory concentrations (MICs) were determined according to the reference agar dilution method described by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). The most active antibiotics for B. fragilis and non- B. fragilis species throughout the three periods were: imipenem with 99.1 and 100% of activity, respectively, and metronidazole with 100% of activity. The susceptibility to ampicillin-sulbactam showed a decrease, from 100% to 90.3% and to 82.4 % in the last period, for both B. fragilis and non-B. fragilis species, respectively. The overall susceptibility rates for cefoxitin, piperacillin, and clindamycin were significantly different between B. fragilis and non-B. fragilis species (84.2% vs. 56.5%; 85.9% vs. 66.7% and 88.8% vs. 64.7%, respectively, p< 0.05). Cefoxitin was the antibiotic that showed more variations as regards periods and species. The susceptibility rates for clindamycin were low, about 60%, for non-B. fragilis species during the last two periods. The variations observed in the susceptibility patterns of the B. fragilis group isolates emphasize the need to continue monitoring the emergence of resistance in order to guide the election of the most appropriate antibiotic therapy scheme for anaerobic infections.<br>El objetivo de este estudio fue evaluar las variaciones en el perfil de sensibilidad frente a siete antimicrobianos de aislamientos del grupo Bacteroides fragilis, mediante el análisis de tres relevamientos realizados por la Subcomisión de Bacterias Anaerobias de la Asociación Argentina de Microbiología (años 1989-1991, 1996-1998 y 1999-2002). En los citados períodos se recolectaron 53, 82 y 65 aislamientos del grupo B. fragilis. Se evaluó la actividad de: ampicilina, ampicilina-sulbactama (2:1), cefoxitina, piperacilina, imipenem, clindamicina y metronidazol. La concentración inhibitoria mínima (CIM) se determinó utilizando el método de dilución en agar, según las normas del Clinical and Laboratory Standards Institute (CLSI, anteriormente NCCLS). En los tres períodos considerados, los antibióticos más activos frente a aislamientos de la especie B. fragilis como así también frente a aislamientos pertenecientes a otras especies del grupo B. fragilis fueron imipenem, con 99,1 y 100% de actividad, respectivamente, y metronidazol, con 100% de actividad. Con ampicilina-sulbactama se observó a lo largo del tiempo una disminución de la sensibilidad, desde el 100% en el primer período hasta un 90,3 y 82,4% en el último, para B. fragilis y para especies del grupo distintas de B. fragilis, respectivamente. Cuando se consideraron los tres períodos juntos, se observaron diferencias significativas entre la especie B. fragilis y los restantes aislamientos del grupo para cefoxitina, piperacilina y clindamicina (84,2% vs. 56,5%; 85,9% vs. 66,7% and 88,8% vs. 64,7%, respectivamente, p< 0.05). Cefoxitina fue el antibiótico que mostró mayores variaciones a través del tiempo y entre especies. Las tasas de sensibilidad a clindamicina fueron bajas (alrededor del 60%) entre los aislamientos no pertenecientes a la especie B. fragilis durante los últimos dos períodos. Las variaciones observadas en los perfiles de sensibilidad del grupo B. fragilis muestran la necesidad de vigilar periódicamente la emergencia de resistencia a los antimicrobianos, a fin de orientar el tratamiento de las infecciones por bacterias anaerobias

Distribution of the number of mutations among <i>mucA</i>, <i>lasR</i> and <i>mexZ</i> genes in hypermutator and nonmutator isolates.

<p>The number of mutations occurring in <i>mucA</i> (blue bars), <i>lasR</i> (green bars) and <i>mexZ</i> (red bars) is expressed as a percentage respect to the total number of mutations found in hypermutator and nonmutator isolates. Above the bars, the total number of mutations per isolate for the hypermutator and nonmutator subpopulations is indicated.</p