Is the direct quantitation of antibiotics in agar by high-performance liquid chromatography useful?

The direct quantification of antibiotics in agar allows one to study the quality of the agar matrix, the kinetics of diffusion and the bacteria-antibiotic interaction. Mueller-Hinton agar (MHA) plates from three manufacturers were tested using HPLC and the disc diffusion test of ceftazidime (CAZ). Notable differences in the chromatographic profiles of MHA plate extracts from OXOID, DID and Becton Dickinson (BD) were shown, with a higher CAZ concentration after 24 h a 6 mm in BD P. aeruginosa inoculated plates (5.1 +/- 1.7 micrograms/ml, n = 6) vs. OXOID and DID (1.6 +/- 0.3 micrograms/ml, n = 12). BD plates gave also a different inhibition zone diameter (26 +/- 0.5 mm, n = 3) with respect to DID and OXOID (29 +/- 0.5 mm, n = 3).

Experimental gut-derived endotoxaemia and bacteraemia are reduced by systemic administration of monoclonal anti-LPS antibodies.

This study aimed to investigate the effects and mechanisms of action of systemic administration of monoclonal antibodies, anti-endotoxin (HA-1A), in an animal model of gut-origin sepsis. In the first experiment, Balb/c mice were transfused with allogeneic blood (C3H/HeJ mice). Five days post-transfusion the animals were gavaged with 1 x 10(9) Escherichia coli and randomized into three groups (n = 22 each) to receive a sham burn (SB group) or a 20 per cent TBSA thermal injury, immediately followed by the systemic administration of monoclonal antibodies (3 mg/kg) (HA-1A group) or aliquots of sterile saline (Control group). The animal survival rate was observed for 10 days postburn. In the second experiment transfusion and burn injury were reproduced but the mice (n = 8/group) were gavaged with 10(9) E.coli labelled with 111indium oxine. Four hours after the burn the mesenteric lymph nodes, liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of translocation of labelled bacteria measured by the residual radioactivity in the organs. Circulating endotoxin levels were determined by limulus assay. The mortality rate of the HA-1A group (9 per cent) was similar to the SB group (0 per cent) and significantly lower than the control group (59 per cent) (P < 0.05). Both plasma endotoxin levels and degree of bacterial translocation in all extraintestinal tissues were significantly lower (by approximately 50 per cent) in the HA-1A group than in the control group (P < 0.05). Systemic administration of HA-1A exerts a beneficial effect by reducing the circulating levels of endotoxin and by increasing the gut barrier function to translocating microorganisms.

Surveillance of legionellosis within a hospital in northern Italy: May 1998 to September 1999.

The incidence of legionella infection in Europe overall in 1997 was 3.9 cases per million population, with the lowest rates reported by Malta and Norway and the highest by Denmark. Ninety cases were reported in Italy (1.5 cases/million), 20% of which were

[Comparative evaluation of a new pregnancy test]. / Valutazione comparativa di un nuovo test di gravidanza.

PIP: A comparison was made between the results offered by a new pregnancy test (Event Test Slide) and 3 other immunologic methods. 2 tube and 2 slide methods were employed on 500 urine samples. In spite of a slight preference for the tube test, it is felt that the slide method, especially the new Event Test Slide, is equally valid. The agreement between results was never less than 99%. An attempt was also made to detect a series of possible interfering substances. Agreement is expressed with the view that 2 different methods should be used in the execution of a pregnancy test. (author's)^ieng

[Prevention and control of Legionella infection in the hospital environment]. / Prevenzione e controllo dell'infezione da Legionella in ambiente ospedaliero.

An outbreak of nosocomial legionnaires' disease in a hospital of Northern Italy is described, together with the epidemiological survey and the control measures adopted. Two patients developed Legionella pneumophila (serogroup 1) pneumonia, one (immunodepressed) died. The Task Group organised by the Health Service excluded other previous nosocomial infections, and made controls on patients and personnel of at risk units (all negative). An intensive programme of environmental sampling and educational activities on personnel have been carried out. The environmental surveillance revealed that the centralised hot water distribution system of the hospital was colonised with Legionella. Shock heating and hyperchlorination of water were applied, which reduced the number of contaminated sites short term, but recolonisation took place two months later. We underline the difficulties encountered to control Legionella by active surveillance of water quality; once the system is contamined, Legionella eradication may be difficult and expensive, and cases of hospital-acquired legionnaieres' disease are likely to occur.

Quantification of gentamicin in Mueller-Hinton agar by high-performance liquid chromatography.

The aim of this study was to optimise a method for gentamicin determination in an agar matrix and to investigate if and how agar composition can affect the gentamicin diffusion kinetics during the agar diffusion tests for antibiotics sensitivity. Gentamicin was separated by RP-HPLC and detected at 365 nm after pre-column derivatization with 1-fluoro-2,4-dinitrobenzene. Recovery (> or = 79%), linearity (r2 > or = 0.997) and sensitivity (1 microg/ml) were assessed using four different agar matrices. The kinetics of gentamicin diffusion tested on BioMerieux and DID manufacturers' products showed in uninoculated agar plates significant differences that were even more pronounced in the presence of Pseudomonas aeruginosa metabolism.

Evaluation of ceftazidime concentration released in agar from an E test strip.

The aim of this study was to evaluate, using high-performance liquid chromatography, the concentration of ceftazidime in agar released from an E test strip, sampling at the edge of the strip at different points (1, 2, 4, 8, 16, 32, 64, and 128 microg/ml) at 6, 15, and 24 h after its deposition on uninoculated plates. From 6 to 24 h, the ceftazidime concentration in agar increased at the graduations 1, 2, and 4 microg/ml (+140, +82, and +58%, respectively), remained fairly constant at 8 microg/ml (-1.9%), and decreased at 16, 32, 64, and 128 microg/ml (-25, -44, -36, and -58%, respectively). In the 6-24 h range, the ceftazidime concentrations between 16 and 1 microg/ml were +/-1 serial dilution of the values reported on the strip, confirming the accuracy of the E test in agar.

[Streptococcus pyogenes toxic shock. A clinical case]. / Shock tossiemico da Streptococcus pyogenes. Caso clinico.

Toxic shock-like syndrome (TSLS) due to Streptococcus pyogenes has been recently reported in both children and adults. This syndrome is characterized by hypotension or shock, fever, multiorgan system involvement and death in 30 to 60% of patients. This syndrome closely resembles the more frequent staphylococcal TSLS. Only one case of TSLS caused by streptococcus has been reported, up to now, in our Country. We describe a second case of fatal streptococcus pyogenes TSLS in a 64-year-old man, in which the site of infection was in the soft tissues. The illness was characterized by rapid progression of shock, erythematous rash, multisystem organ involvement and finally death. Clinicians must be aware of the presentation of this disease as its incidence appears to be increasing.

Ceftazidime kinetics of diffusion in inoculated agar plates.

The ceftazidime concentration in agar plates inoculated with Pseudomonas aeruginosa was determined by high-performance liquid chromatography at fixed points (3, 6, 9, 12, and 15 mm) from the disk center and at fixed times (2, 4, 6, 16, and 24 h) to study the antibiotic kinetics of diffusion. A statistical difference between the concentrations determined in the presence of microorganisms and in uninoculated plates after 16 and 24 h was evidenced and was probably ascribable to the drug hydrolysis carried out by the induced beta-lactamase.

[Preliminary considerations for blood culture (author's transl)]. / Considerazioni preliminari all'esecuzione dell'emocoltura.

Some procedures are examined that more affect blood culture reliability. Basic recommendations concern the indications for blood cultures and the training of personnel who have to collect the specimens aseptically. The following steps are considered: informations required; skin disinfection; procedures of blood collection as anticoagulant to be used, volume of blood to be drawn, dilution in culture medium; time and number of cultures to be collected.

Pharmacokinetics and tolerance of a new penem antibiotic, FCE 22101, in healthy volunteers after a single intravenous dose.

The clinical tolerance and pharmacokinetics of FCE 22101 (sodium (5R, 6S)-6-[(1R)-hydroxyethyl]-2-carbamoyloxymethyl-2-penem-3-carboxylate), a new penem antibiotic, have been studied after giving a single i.v. dose of 4 mg.kg-1 to ten healthy male volunteers. The pharmacokinetics was estimated according to a two-compartment open model. The peak plasma concentration (Cmax) was 15.5 (1.08) micrograms.ml-1, mean (SEM). FCE 22101 was rapidly cleared from the systemic circulation [t 1/2 lambda z = 44.2 (4.2) min; CL = 7.21 (0.47) ml.kg-1.min-1]. The mean apparent volume of distribution at steady-state was 246 (16.9) ml.kg-1. The mean residence time relative to the 10 min infusion was 39.4 (1.5) min. Urinary recovery of FCE 22101 showed wide inter-subject variation, ranging from 10.2 to 53.6% of the dose. No subject complained of adverse effects.

Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.

[Polycenter evaluation of the break-point system]. / Valutazione policentrica del sistema break-point.

Several research center have been set up to evaluate the system that deals with sensitivity to microbes under Sensititre break-point. The study has been broken down as follows: the break-point system was compared with the agar diffusion according to Bauer et al., using 1180 strains of fast-growing Gram-negative bacteria; a limited number of strains (176) have been used to compare the Sensititre break-point and the Sensititre MIC; results have been obtained testing 448 strains processed by break-point with correct inoculum and with simplified inoculum, from a colony; an investigation has been carried out on the time and cost of the break-point functioning. Having taken the Bauer system and others are compared them with the break-point, it was seen that their total agreement was 90.3% with 2% of major disagreement. The total major disagreement between Sensititre MIC and Sensititre break-point was 2.7%. The total major disagreement of the latter was largely the result of cephalotin (21%) on the Escherichia coli strains. An initial research centre has been formed to try to trow light upon the origins of such disagreements and we are now pleased to report back their initial findings. The preparation and reading of a test with the Bauer system and others takes about 18 minutes and costs 5600 Lit; a break-point test takes 10 minutes and costs 4500 Lit.

[Bacteremia and specific antibody response. IgG and IgM specificity in the evaluation of positivity in blood cultures]. / Batteriemia e risposta anticorpale specifica. IgG e IgM specifiche nella valutazione della "positività" delle emocolture.

Specific serum IgG and IgM directed against blood culture isolated bacteria have been determined in patients with positive blood culture by the indirect immunofluorescence and passive hemagglutination methods in order to distinguish the true positive blood cultures due to bacteremia from those due to contamination. 45 (16%) out of 280 blood cultures examined during the period 1/1/1980-30/1/1981 gave positive isolations: 25 cases (55.5%) were due to gram-negative bacteria and the remaining 20 cases (44.5) to gram-positive bacteria: among these there were 9 cases (20% of the total positive blood cultures) with isolation of micrococci. No positive blood culture for anaerobic bacteria were observed during that time interval. 96% of patients from whose blood cultures gram-negative bacteria had been isolated showed high titres of serum specific antibodies with both the indirect immunofluorescence and the passive hemagglutination methods. Only 40% of the cases whose blood cultures gave isolation of gram-positive bacteria showed high titres of serum specific IgG. The remaining 60% that did not show presence of serum specific antibodies included 9 cases of positive blood culture for micrococci (well known as contaminants) and 2 cases for streptococcus (in two of these there had been a mixed flora isolation). Specific IgM antibodies at significant level were also present in 76% of patients with positive blood culture for gram-negative bacteria and in 40% of patients with positive blood culture for gram-positive bacteria. All patients whose cultures gave isolation of micrococci showed absence of specific IgM. The observed good correspondence between isolation of contaminant bacteria on one hand and the absence of serum specific antibodies on the other, and vice versa between isolation of pathogenic bacteria (either obligate or opportunistic) an one hand and the presence of high level of specific antibodies on the other suggests that the search of specific immune response in patients with positive blood culture might constitute a good criterion (in addition to the classical criteria) for distinguishing true positive blood cultures from contaminated blood cultures.

Mycobacterium tuberculosis exploits the CD95/CD95 ligand system of gammadelta T cells to cause apoptosis.

Vgamma9/Vdelta2+ T cells specifically recognize Mycobacterium tuberculosis in vitro and are precociously recruited in early mycobacterial lesions. Even if gammadelta T cells are only fortuitously detected in granulomas or bronchoalveolar lavages of patients with active pulmonary tuberculosis, a role in shaping the mature alphabeta T cell response against M. tuberculosis is substantiated. Here we provide a molecular explanation for this paradox: the engagement of the gammadelta TCR by mycobacterial antigens induced the expression of CD95 ligand (CD95L) by chronically activated CD95+/CD95L- gammadelta T lymphocytes. The receptor was functional, as CD95/CD95L interaction triggered the bystander death of CD95+ cells by apoptosis. Cell death was abolished by CD95-blocking antibodies. The transient accumulation at the site of infection of CD95L+ gammadelta lymphocytes, capable of interacting with CD95+ leukocytes attracted by the response towards the pathogen, may determine the characteristics of the ensuing granulomatous disease.