Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli.

The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to escape from the antibiotics' action, and this mechanism is predominantly adopted by microorganisms resistant to MLS antibiotics. ErmSF methyltransferase is one of the four gene products synthesized by Streptomyces fradiae NRRL 2338 to be resistant to its autogenous antibiotic, tylosin. In order to have a convenient source for the purification of milligram amounts, we expressed ErmSF in Escherichia coli using a T7 promoter-driven expression vector system, pET 23b, and the protein was expressed with a carboxy-terminal addition of six histidine residues in order to facilitate purification. Expression at 22 degrees C reduced the formation of insoluble aggregate, inclusion body, and resulted in accumulation of soluble hexahistidine-ErmSF up to 30% of total cell protein after 18 h. Metal-chelation chromatography yielded 126 mg of hexahistidine-ErmSF per liter of culture with a purity slightly greater than 95%. To examine the function of ErmSF in vivo and in vitro, its activity in E. coli (antibiotic susceptibility assay) andin vitro methyltransferase activity using in vitro-produced B. subtilis domain V, 434-, 257-, and 243-nt RNAs were investigated. The ErmSF in E. coli conferred resistance to erythromycin, whereas E. coli harboring an empty vector, pET23b, was susceptible. The purified recombinant protein successfully methylated domain V of 23S rRNA, which is known to contain all of the substrate elements recognized and to be methylated by erm proteins. However, the truncated substrates were methylated with decreased efficiencies. Almost all of domain V was monomethylated with less than 0.2 pM S-[methyl-(3)H]adenosylmethionine concentration. The roles of three structurally divided regions of domain V in recognition and methylation by ErmSF are proposed through kinetic studies using RNA substrates, in which each region is deleted, under the monomethylation condition. 2002 Elsevier Science (USA)

Benefit/risk ratio of antibiotic therapy

This publication evaluates critically the benefit/risk profile of several antibiotics currently at our disposal. The considered antibiotics are divided into pharmacological classes, because generally the drugs of the same class share the same adverse events. Moreover, the high therapeutic profile of the antibiotics puts them at the top of the safest drugs. Therefore the choice of an antibiotic is based above all on the evaluation of the patient and of the pathology to be treated, in terms of severity and possibility of achieving a response to treatment. An accurate anamnesis, the identification of the correct dosage and of the therapy duration minimise the potential risks of the chosen treatment. Detailed knowledge of the safety profile of these drugs is a further element in order for the antibiotic to perform at its best

Infective Endocarditis.

Despite improvements in antibiotic regimens, patients with infective endocarditis (IE) have a high risk of valve replacement and death. Effective initial treatment depends on two steps: 1) diagnosis of the infecting organism, enabling specific antibiotic therapy, and 2) complete characterization of the anatomic extent of infection. Identification of the infecting organism requires culturing of blood prior to the initiation of antibiotics. Whenever possible, at least three sets of blood cultures should be obtained over 6 to 24 hours and held for 4 weeks if necessary to detect unusual or fastidious organisms. Transesophageal echocardiography (TEE) is usually necessary either to confirm the diagnosis or, most importantly, to identify the local complications of infection, many of which mandate surgery. Despite widespread availability, TEE remains under-used, both for the prevention of unnecessary antibiotic therapy in patients at very low risk for the disease and for the recognition of patients likely to benefit from early surgery. The selection of optimal antibiotic therapy depends on microbiologic data to establish the sensitivities of the specific causative organism. Short courses of antibiotic therapy and outpatient administration of intravenous antibiotics are useful in selected cases

Recurrence of vancomycin-resistant Enterococcus stool colonization during antibiotic therapy.

OBJECTIVE: To test the hypothesis that antibiotic therapy may promote recurrence of vancomycin-resistant Enterococcus (VRE) stool colonization in patients who have previously had three consecutive negative stool cultures obtained at least 1 week apart. DESIGN: One-year prospective cohort study examining the effect of antibiotic therapy on recurrence and density of VRE stool colonization in patients who have cleared colonization. Pulsed-field gel electrophoresis (PFGE) was performed to determine whether recurrent VRE strains were the same clone as the previous colonizing strain. SETTING: A Department of Veterans Affairs medical center including an acute care hospital and nursing home. PATIENTS: All patients with at least one stool culture positive for VRE who subsequently had three consecutive negative stool cultures obtained at least 1 week apart. RESULTS: Of the 16 patients who cleared VRE colonization, 13 received antibiotic therapy during the study period. Eight (62%) of the 13 patients who received antibiotics developed recurrent high-density VRE stool colonization (range, 4.9 to 9.1 log10 colony-forming units per gram) during a course of therapy. Five patients had VRE strains available for PFGE analysis; recurrent strains were unrelated to the prior strain in 3 patients, closely related in 1 patient, and indistinguishable in 1 patient. CONCLUSIONS: Antibiotic therapy may be associated with recurrent high-density VRE stool colonization in many patients who have previously had three consecutive negative stool cultures. These patients should be screened for recurrent stool colonization when antibiotic therapy is administered

Modern management of non-chemotherapy drug-induced agranulocytosis: a monocentric cohort study of 90 cases and review of the literature.

BACKGROUND: The present study reports a monocentric experience of 90 drug-induced agranulocytosis cases and discusses their management, in particular the role of hematopoietic growth factors. METHODS: Data from 90 patients with drug-induced agranulocytosis who met the criteria of the IAAAS group and of Benichou and Solal-Celigny [Nouv Rev Fr Hematol 1993; 33: 257.] were retrospectively reviewed. All cases were extracted from a cohort study of the Hopitaux Universitaires de Strasbourg, France. Data were specifically analyzed with regard to the use of hematopoietic growth factors (in 42 patients). RESULTS: Mean patient age was 63 (range 17-95) years and the sex ratio (M/F) was 0.39. An underlying disease was present in 37% of the patients. Antibiotics (25%), antithyroid drugs (23%), and antiaggregative platelet agents (16%) were the most frequent causative drugs. Main clinical features included isolated fever (41%), septicemia or septic shock (31%), and pneumonia (10%). Mean neutrophil count was 0.13 (range 0-0.46)x10(9)/l. Outcome was favorable in 98% of patients. The mean durations of hematological recovery (neutrophil count over 1.5x10(9)/l), antibiotic therapy, and hospitalization was 8.5 (range 2-21) days, 9.2 (range 2-21) days, and 10.5 (range 3-23) days, respectively. All patients were treated with broad-spectrum antibiotics and 42 patients with hematopoietic growth factors. In these 42 patients, the mean durations for hematological recovery, antibiotic therapy, and hospitalization were significantly reduced at: 6.3 (range 2-16) days, 7.1 (range 2-16) days, and 9.1 (range 3-23) days, respectively (all P<0.05). CONCLUSIONS: The present study shows that new causative drugs are emerging (antibiotics, antithyroid, and antiaggregative platelet agents), that drug-induced agranulocytosis remains typically a serious accident with severe sepsis, and that modern management with broad spectrum antibiotics and hematopoietic growth factors may reduce the mortality

An investigation into the sensitivities of translocating bacteria to a prophylactic antibiotic regimen.

INTRODUCTION: It is well established that bacterial translocation is associated with a significant increase in septic morbidity. The purpose of this study was to determine the antibiotic sensitivities of translocating bacteria on the basis that this information may influence antibiotic prophylaxis in surgical patients. METHODS: Routine microbiological techniques were used to assess the antibiotic sensitivities of those bacteria cultured from a mesenteric lymph node harvested at laparotomy in a large series of patients. RESULTS: Culture of the mesenteric lymph nodes yielded growth in 51 out of a total of 447 patients studied (11.4%). The isolates from 40 patients, a total of 60 organisms, were available for sensitivity testing. The most common species grown was Escherichia coli (48% of isolates). Thirty-three patients (83%) grew organisms sensitive to the antibiotic prophylaxis used, but there was no significant difference in the incidence of postoperative septic complications between these patients and those in whom resistant bacteria were grown (39% versus 29%, P = 0.64 Fisher's Exact test mid P). CONCLUSIONS: The majority of translocating bacteria are sensitive to the prophylactic antibiotics commonly used in patients undergoing laparotomy. However, the occurrence of postoperative septic morbidity is independent of this variable

Influence of Pharmacokinetic and Pharmacodynamic Principles on Antibiotic Selection.

When evaluating the efficacy of antibiotics for the treatment of respiratory tract infections, such as community acquired pneumonia and acute exacerbations of chronic bronchitis, assessment of clinical cure may not be the most relevant parameter, as it may not be related to microbiological eradication or to the minimum inhibitory concentration (MIC) of the infecting pathogen. It is more relevant to study the efficacy of the antibiotic in eradicating the bacterial pathogen, because this is frequently related to both the MIC of the pathogen and the antibiotic dosage regimen. Pharmacodynamics correlates the concentration of antibiotic in the blood or at the infection site with its biological effect against the organism (bacteriological eradication). For beta-lactams, the pharmacodynamic parameter that best correlates with eradication is time (T) above MIC (T > MIC); for aminoglycosides and fluoroquinolones, it is the area under the curve at 24 hours (AUC(24))-to-MIC ratio (AUC(24)/MIC). Knowledge of pharmacodynamics allows optimum use of antibiotics; in vitro models, animal models, and retrospective and prospective clinical trials have shown that the use of such knowledge optimizes bacteriological eradication and enhances patient outcome. In the future, pharmacodynamic studies will be used not only to assess optimal ways for antibiotics to eradicate resistant pathogens, but also to investigate the ability of antibiotics to prevent the development of resistance on therapy and to eradicate pathogens from colonizing sites

Department of pharmacy-initiated program for streamlining empirical antibiotic therapy.

The outcome of a department of pharmacy-initiated "streamlining" study designed to promote cost-conscious modifications of empirically selected antibiotic therapy is described. Two hundred forty-one evaluable adult patients started on restricted-use antibiotics at this university-affiliated community private teaching hospital were enrolled in a 9-week prospective streamlining study. Patients were alternately assigned to a Control (i.e., no pharmacist-initiated streamlining recommendations offered based on culture and susceptibility reports) or a Pharmacist Intervention group (i.e., pharmacist offers recommendations to streamline therapy). A statistically significant greater number of patients had their empiric antibiotic treatment courses modified to more appropriate antibiotic choices after receipt of culture and susceptibility reports among private prescribers in the Pharmacist Intervention group (83%) than in the Control group (38%) (p = .006). Additionally, pharmacists were overall successful in gaining prescriber acceptance for 64% of recommended changes of empiric antibiotic treatment courses before the receipt of culture and susceptibility reports (e.g., dose and/or frequency changes). There was no program effect observed with respect to improved physician response to microbiologic data that would allow streamlining empirical antibiotic choices in the Housestaff (i.e., medical or surgical residents), or infectious disease consultant prescriber groups. Projected overall annual cost savings that would be achieved as a result of continued efforts by pharmacists directed at streamlining empirical "restricted" antibiotic regimens is approximately +40,000

Incidence and risk factors of oral antibiotic-associated diarrhea in an outpatient pediatric population.

BACKGROUND: Little information is available on the epidemiologic characteristics of antibiotic-associated diarrhea (AAD) in children. The authors' aim was to evaluate the incidence of AAD in an outpatient pediatric population and to identify risk factors. METHODS: Children aged 1 month to 15.4 years treated with oral antibiotics for a proven or suspected infection were enrolled from an ambulatory pediatric practice during an 11-month period. Parents recorded the daily frequency and characteristics of stools using a diary during the antibiotic treatment and for 1 week after it was stopped. An episode of diarrhea was defined by at least 3 soft or liquid stools/d for at least 2 consecutive days. Risk factors for AAD-age, type of antibiotic treatment, type of combined treatment, and site of infection-were analyzed. RESULTS: Of 650 children included, 11% had an episode of AAD, lasting a mean of 4.0 +/- 3.0 days, beginning a mean of 5.3 +/- 3.5 days after the start of antibiotic treatment. No child was hospitalized because of AAD. The incidence of AAD was higher in children less than 2 years (18%) than in those more than 2 years (3%; P < 0.0001). The incidence of AAD was particularly high after administration of certain antibiotics (amoxicillin/clavulanate, 23%; P = 0.003 compared with other antibiotics). The type of combined treatment and site of infection did not influence the onset of AAD. CONCLUSIONS: Antibiotic-associated diarrhea was common in these outpatient children, especially for those aged less than 2 years and after the prescription of certain antibiotics, particularly, the combination of amoxicillin/clavulanate

Effect of prophylactic antibiotics and incision type on the incidence of endophthalmitis after cataract surgery.

BACKGROUND: There is controversy as to the efficacy of various measures in the prophylaxis of endophthalmitis after cataract surgery. In addition, it has been suggested that clear-corneal incisions may convey an increased risk of postoperative infection. We performed a retrospective review to assess the effect of prophylactic antibiotics and incision type on the incidence of endophthalmitis after cataract surgery. METHODS: A retrospective chart review and surgeon survey were used to collect data for the 13,886 consecutive cataract operations performed between Sept. 1, 1994, and Jan. 31, 1998 by nine surgeons at a hospital-based surgical unit in Saskatoon. All cataract extractions were by phacoemulsification. All cases of endophthalmitis arising from the unit are managed at the hospital except in extenuating circumstances. We assessed the effect of preoperative administration of antibiotic drops, subconjunctival antibiotic injections at the conclusion of surgery and clear-corneal versus scleral tunnel incisions on the incidence of endophthalmitis by means of univariate and multivariate Poisson regression analysis. RESULTS: The incidence of postoperative endophthalmitis was significantly lower with subconjunctival antibiotic injections than without such injections (0.011% vs. 0.179%) (p = 0.009, odds ratio 16.23 [95% confidence interval 1.92 to 137.14]). The difference in the incidence of endophthalmitis with preoperative use of antibiotic drops (0.066%) and with no antibiotic drops preoperatively (0.115%) was not significant. Similarly, the difference in the incidence of endophthalmitis with clear-corneal (0.129%) and scleral tunnel (0.050%) incisions was not significant. INTERPRETATION: Our results suggest that prophylactic subconjunctival antibiotic injections at the conclusion of cataract surgery decrease the incidence of postoperative endophthalmitis