Etiology and antimicrobial susceptibility profiles of anaerobic bacteria isolated from clinical samples in a university hospital in Madrid, Spain.

BACKGROUND: The anaerobic infection management is usually based on empirical treatment because anaerobic culture techniques take a long time due to their fastidious nature. The aim of this study was to analyze the etiological profile of severe anaerobic infections and AST data from clinical anaerobic bacteria isolated in a tertiary hospital in Madrid (Spain). MATERIAL AND METHODS: A consecutive study was carried out over 19 months in Ramón y Cajal Universitary Hospital, Madrid. Clinical samples were processed in appropriate anaerobic media and incubated using Anoxomat system. Identification was performed by MALDI-TOF. AST were determined with gradient diffusion method using EUCAST (penicillin, co-amoxiclav, imipenem, clindamycine and metronidazole) or CLSI (cefoxitin) breakpoints. RESULTS: During the period of study, 503 anaerobic microorganisms isolated from 424 clinical samples were included. Twenty-six percent of the cultures were monomicrobial, while 70.0% also contained aerobic bacteria. The most common source of infection was abscesses (26%), while blood infections represented the 11%. Anaerobic gram-negative bacilli were predominant (41%), being Bacteroides fragilis (13%) the most prevalent overall; anaerobic gram-positive bacilli represented 35%, anaerobic gram-positive cocci 19% and anaerobic gram-negative cocci 5%. Metronidazole and imipenem were the most effective agents tested against anaerobic bacteria, while clindamycin presented higher resistance rates. CONCLUSION: Antimicrobial susceptibility surveillance of anaerobic bacteria should be performed to monitor changes in resistance patterns and to be able to optimize empiric antimicrobial treatment. Reliable species identification and quick reporting of results would guide clinicians to select the optimal antimicrobial therapy.

Direct antimicrobial susceptibility testing from the blood culture pellet obtained for MALDI-TOF identification of Enterobacterales and Pseudomonas aeruginosa.

To standardize the methodology for conducting direct antimicrobial susceptibility testing (AST) of Enterobacterales and Pseudomonas aeruginosa causing bacteremia from positive blood culture pellets. Two methods for processing positive blood cultures with Enterobacterales and P. aeruginosa were compared: a conventional method for identification and AST versus a direct method obtaining a pellet for both matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) identification and direct AST. A total of 157 (145 Enterobacterales, 12 P. aeruginosa) positive blood cultures were included. Microorganism identification showed 100% concordance between both methods at species and genus level. Definitive AST results were obtained 24 h earlier with the rapid method than the conventional one (p < 0.001). Of the 2814 MICs generated, there were discrepancies with respect to the conventional method in 47 (1.7%), 0.3% being very major (VME) and 1.3% major (ME) errors. Better results for AST were obtained when colony counts with the pellet were ≥ 105 cfu/ml. The essential agreement (EA) for antibiotics tested in Enterobacterales was at least 97%, except for ampicillin (95%). Regardless of colony count, the greatest discrepancies were observed for first/s-generation cephalosporins and aminoglycosides. In P. aeruginosa, EA was at least 92%, except for piperacillin-tazobactam (84%) and cefepime (76%). No VME occurred except for ceftazidime (8%). ME occurred in piperacillin/tazobactam (16%), ticarcillin, ceftazidime, tobramycin, amikacin, and colistin (8% each). Direct use of the blood culture pellet permits fast AST in bacteremia of Enterobacterales, enabling the clinicians to perform an early treatment adjustment. However, for Pseudomonas aeruginosa, the data needs expanding to improve the reliability of this technique.

Usefulness of EUCAST rapid antibiotic susceptibility breakpoints and screening cut-off values directly from blood cultures for the inference of ß-lactam resistance mechanisms in Enterobacterales.

Background: Reducing the turnaround time for reporting antimicrobial susceptibility testing (AST) results is important for adjusting empirical treatments and may impact clinical outcomes of septic patients, particularly in settings with high antimicrobial resistance. Disc diffusion could be useful for inferring ß-lactam resistance mechanisms. Objectives: To evaluate the usefulness of EUCAST rapid AST (RAST) disc diffusion breakpoints for the screening of resistance mechanisms (sRAST) and interpretive reading of resistance phenotypes to infer ESBL and carbapenemases production in Enterobacterales. Methods: Blood cultures were artificially spiked with Enterobacterales clinical isolates with well-characterized ß-lactam resistance mechanisms (n = 93), WT phenotypes (n = 26) and ATCC strains (n = 8). AST was performed by disc diffusion directly from blood cultures and inhibition zones were manually measured at 4, 6 and 8 h. To infer the presence of resistance mechanisms, EUCAST RAST breakpoints and screening cut-off values (sRAST) combined with the double-disc synergy test (DDS) for ESBLs or aztreonam susceptibility for carbapenemases detection were used. Results: DDS together with sRAST detected all ESBL producers as early as at 4 h incubation. Cefotaxime was the antibiotic with the highest discriminatory power. The suspicion of carbapenemase production by sRAST at 8 h was possible in 73% of Klebsiella pneumoniae and in 100% of Escherichia coli carbapenemase-producing isolates. Phenotypic analysis improves the detection of some low hydrolytic carbapenemases (OXA-48 or KPC-3 mutants). Conclusions: Early detection of ß-lactam resistance mechanisms directly from positive blood cultures was possible using sRAST together with the interpretive reading of antibiotic resistance phenotypes. Some carbapenemase types such as OXA-48 might be difficult to infer. Screening-positive isolates should be confirmed using an alternative technique.

Clinical and microbiological features of a cystic fibrosis patient chronically colonized with Pandoraea sputorum identified by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Clonal isolates identified as various nonfermentative Gram-negative bacilli over a 5-year period from sputum cultures of a 30-year-old cystic fibrosis patient were successfully reidentified as Pandoraea sputorum by combining 16S rRNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Decreased lung function improved after 1 year of azithromycin and inhaled 7%-hypertonic saline treatment.

Carbapenem Heteroresistance in VIM-1-producing Klebsiella pneumoniae isolates belonging to the same clone: consequences for routine susceptibility testing.

Susceptibility results with low reproducibility by the same or different methods have been observed for metallo-beta-lactamase (MBL)-producing Enterobacteriaceae. Eighteen VIM-1-producing Klebsiella pneumoniae isolates (one per patient) belonging to a single epidemic clone in our hospital (2005 to 2008) but with different susceptibilities to carbapenems were studied. Imipenem MICs ranged from 8 to >128 mg/liter by standard CLSI microdilution, from ≤1 to >8 mg/liter by the semiautomatic Wider system, and from 0.75 to >32 mg/liter by Etest. Meropenem MICs ranged from 0.5 to 128, ≤1 to >8, and 0.38 to >32 mg/liter, respectively. Ertapenem MICs by CLSI microdilution and Etest ranged from 1 to 64 and 0.75 to >32 mg/liter, respectively. The rates of essential agreement (±1 log(2) dilution) for imipenem and meropenem MICs between the Wider system and the reference microdilution method were 45% and 49%, respectively. Those between Etest and the reference microdilution method for imipenem, meropenem, and ertapenem MICs were 33%, 67%, and 84%. The rates of very major errors for the Wider system and Etest were 33% and 28% for imipenem and 25% and 75% for meropenem, respectively. Low MIC reproducibility was observed even when the same inoculum was used (differences up to 4-fold dilutions). Heteroresistance was suspected due to the presence of colonies in the Etest inhibition zone. It was confirmed by population analysis profiles of 4 isolates displaying different imipenem MICs, with the exception of an OmpK36-porin-deficient isolate that homogeneously expressed carbapenem resistance (MIC, >128 mg/liter). Low carbapenem MIC reproducibility could be due to the presence of resistant subpopulations and variable expression of the resistance mechanisms. Since carbapenem MICs are not good markers of MBL production, reliable and reproducible phenotypic methods are needed to detect the presence of this mechanism.

Is there a prognostic role for C-reactive protein in ischemic stroke?

OBJECTIVES: We investigated the relationship between C-reactive protein (CRP)-values in the acute phase of stroke and the risk of further fatal and non-fatal ischemic events. MATERIALS AND METHODS: We analysed 462 consecutive incident ischemic strokes. Patients were divided into two subgroups on the basis of a CRP cut-off level of 9 mg/l. Primary end points were any new vascular fatal and non-fatal event recorded during the follow-up period. RESULTS: During a follow-up of 2.27 years, in 132 patients occurred a primary end point. Patients with CRP values > or = 9 mg/l had more frequently primary end point. The hazard ratio (HR) for cardiovascular events was 3.59; 1.93 for cerebrovascular events; 7.43 for vascular deaths and 5.78 for death from any cause. Cox proportional hazard multivariate analysis identified CRP values > or = 9 (HR = 4.19, 95% CI: 1.85-9.50, P = 0.001), the lack of secondary prevention therapy at discharge (HR = 4.35, 95% CI: 1.87-10.1, P = 0.001), age >70 years (HR = 3.09, 95% CI: 1.04-9.24, P = 0.04) as independent predictors of fatal events. CONCLUSIONS: CRP levels > or = 9 mg/l, evaluated in incident ischemic stroke within 24 h, predict a higher risk of further ischemic events and mortality.

Frequency of Th1, Th2 and Th17 producing T lymphocytes in bronchoalveolar lavage of patients with systemic sclerosis.

OBJECTIVE: The pathophysiology of the lung fibrotic process in systemic sclerosis (SSc) is not fully elucidated. Since this pattern represents the leading cause of death in SSc, the knowledge of its actual pathophysiology is basic to prevent and stage pulmonary damage. In this study, we aimed to further investigate the relationship between the functional profiles of bronchoalveolar lavage (BAL) T cells and the pulmonary manifestation of the disease. METHODS: With this aim, we assessed the frequency of Th1, Th2 and Th17 producing T-lymphocytes and their effector cytokines in BAL of SSc patients without signs or symptoms of lung interstitial involvement (SScFib-) and with interstitial lung fibrosis (SScFib+). We also study as control groups: patients with usual interstitial pneumonia (UIP), patients with sarcoidosis and 9 healthy controls (NHCs). RESULTS: SScFib- showed an increase in BAL Th1/Th2 balance compared to NHCs, which was even higher than that observed in sarcoidosis. SScFib+ showed a shift towards a lower Th1/Th2 ratio as compared to SScFib-. The frequency of Th17 BAL T cells did not change among study groups. CONCLUSION: Our data confirm the Th1/Th2 imbalance hypothesis on the pathogenesis of interstitial fibrosis in SSc patients, and suggest a possible utility in the assessment of BAL Th1/Th2 ratio.

[Regulatory T cells in kidney transplant recipients]. / Cellule T-regolatorie nel trapianto di rene.

Immunosuppressive drugs are essential for the prevention of acute transplant rejection but some may not promote long-term tolerance. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes (Treg), particularly CD4+CD25+ T cells. The transcription factor forkhead box protein 3 (Foxp3) has been identified as a molecular marker for Treg cells. The direct effects of immunosuppressive drugs on CD4+CD25+ cells are uncertain. In the clinical setting, basiliximab used in the induction phase of immunosuppression effectively reduced the number of acute rejection episodes. We studied the effects of the most widely used immunosuppressive induction regimens including cyclosporine, mycophenolate mofetil, steroids, and anti-CD25 monoclonal antibody (basiliximab) on the capacity to regulate human Treg in vivo. Twenty first cadaveric kidney transplant recipients (14 men, 6 women) were enrolled in the study. Blood samples were collected before kidney transplant and after one month. Blood sampling was done immediately before the administration of immunosuppressive therapy after an overnight fast. None of the transplant recipients presented laboratory or clinical signs of infection or acute rejection. The number and percentage of CD4+CD25+ and Foxp3+ T cells were determined by fluorescence activated cell sorter (FACS) analysis. Our results showed absence of both CD4+CD25+ and CD4+CD25+ Foxp3+ T cells one month after transplant. Peripheral CD4+CD25-Foxp3+ T cells significantly decreased after transplant but did not disappear. These preliminary data suggest that immunosuppressive induction therapy with basiliximab completely suppresses CD4+CD25+ regulatory cells and significantly reduces the total number of Foxp3+ lymphocytes.

Mechanisms of action and antimicrobial activity of ceftobiprole.

Ceftobiprole, a novel last generation parenteral cephalosporin, has an extended spectrum of activity, notably against methicillin-resistant Staphylococcus aureus (MRSA), ampicillin-susceptible enterococci, penicillin-resistant pneumococci, Enterobacterales and susceptible Pseudomonas aeruginosa. It exerts an inhibitory action on essential peptidoglycan transpeptidases, interfering with cell wall synthesis. The inhibitory action of ceftobiprole through binding to abnormal PBPs like PBP2a in methicillin-resistant staphylococci and PBP2b and PBP2x in the case of ß-lactam-resistant pneumococci, ultimately leads to rapid bacterial cell death. In the case of Enterobacterales, ceftobiprole retains activity against narrow spectrum ß-lactamases but is hydrolysed by their extended-spectrum counterparts, overexpressed Amp C, and carbapenemases. It is also affected by certain efflux pumps from P. aeruginosa. For anaerobic bacteria, ceftobiprole is active against Gram-positive Clostridioides difficile and Peptococcus spp. and Gram-negative Fusobacterium nucleatum but not against Bacteroides group or other anaerobic Gram-negatives. In in vitro studies, a low propensity to select for resistant subpopulations has been demonstrated. Currently, ceftobiprole is approved for the treatment of community-acquired pneumonia and hospital-acquired pneumonia with the exception of ventilator-associated pneumonia. Ceftobiprole's place in therapy appears to lie mainly in its combined activity against Gram-positive organisms, such as S. aureus and S. pneumoniae alongside that against Gram-negative organisms such as P. aeruginosa.

Chemokine redundancy in BOS pathogenesis. A possible role also for the CC chemokines: MIP3-beta, MIP3-alpha, MDC and their specific receptors.

Bronchiolitis obliterans syndrome (BOS) is one of the most important factors limiting the long-term survival of lung transplant recipients (LTR), however its pathogenesis still remains unclear. We hypothesized that an increased production of certain specific proinflammatory mediators in the first post-transplant year would predispose to BOS. We retrospectively evaluated temporal kinetics of some CC chemokines that have not yet been evaluated, including CCL3/MIP1-alpha, CCL4/MIP1-beta, CCL17/TARC, CCL19/MIP3-beta, CCL20/MIP3-alpha, CCL22/MDC and CCL26/eotaxin, in broncho-alveolar lavage fluid (BAL-f) in the first post-transplant year in a cohort of 8 LTR before the development of BOS (pre-BOS LTR) and 8 LTR with long-term stable clinical conditions (stable LTR). Chemokine levels were assayed by means of a multiplex sandwich ELISA. Furthermore, for those ligands which resulted significantly predictive of BOS onset, we analyzed the expression of specific receptors (CCR) on BAL cells. The proportion of CCR-expressing BAL cells was assessed by flow cytometry. We demonstrated that MIP3-beta/CCL19, MIP3-alpha/CCL20, MDC/CCL22 levels at 6 months post-transplant significantly predicted BOS onset. In addition, the temporal behavior of these factors resulted significantly different in pre-BOS patients as compared to stable LTR. Finally the expression of CCR was documented on BAL lymphocytes and macrophages, and, in some cases, their expression was found to vary between the two groups. Within the complexity of the chemokine network, these three CCL factors could play an additive role in the pathogenesis of the inflammatory process leading to bronchiolar fibro-obliteration.

IRT and CMT beta-lactamases and inhibitor resistance.

Acquired resistance to penicillin-beta-lactamase inhibitor combinations in Escherichia coli is due to: (i) penicillinase hyperproduction due to the presence of the bla(TEM-1) gene in small multicopy plasmids or strong promoters; (ii) overproduction of constitutive AmpC cephalosporinase; and (iii) OXA-type and inhibitor-resistant TEM (IRT) beta-lactamases. IRT enzymes emerge via mutational events from TEM-1 or TEM-2 beta-lactamases that affect substrate affinity for beta-lactamase inhibitors. They are mainly isolated in urinary infections from community patients. Prevalence is variable, depending on geographical area, detection methods and potential selection pressure. These enzymes may evolve into complex mutants (CMT enzymes), which also confer resistance to extended-spectrum cephalosporins. CTX-M enzymes with the IRT phenotype have not been detected to date. New studies of IRT enzymes, including population structure, association with virulence traits and plasmid dispersion, are needed.

Changes in bacterial hospital epidemiology.

Antibiotics' use and prescription requires a profound review, as their inadequate administration has been one of the main forces leading to resistance as a result of overuse and misuse. Resistance is particularly challenging in nosocomial environments in which there has been a gradual change in bacterial epidemiology owing to the continuous increase of multi-drug-resistant isolates, which imply a threat to prevent and cure infections. Expertise at the time of using antibiotics, development of new diagnostic tools and the possibility of having new antimicrobials are required to stay ahead of evolving resistance. Moreover, surveillance is also relevant to monitor antimicrobial resistance.

Peripheral CD4+ CD25+ Treg cell expansion in lung transplant recipients is not affected by calcineurin inhibitors.

CD4+CD25+ regulatory T (Treg) cells have been shown to play a role in allograft tolerance and their peripheral counts vary according to the degree of graft acceptance in lung transplant recipients (LTR). Recent studies demonstrate that certain drugs might modulate generation, expansion and activity of Treg cells. Aim of this study was to evaluate the effect of therapeutic regimens used in our institution on peripheral CD4+CD25(high)CD69- Treg cell numbers in a group of 51 LTR with stable clinical conditions. They were treated with standard immunosuppression: calcineurin inhibitor (CNI)+azathioprine (AZA)+steroids (n=28) or with CNI+mycophenolate mofetil (MMF)+steroids (n=11) or with CNI+steroids (n=12). These stable LTR were compared with age-matched healthy controls (n=35) and with 19 LTR who developed bronchiolitis obliterans syndrome (BOS) and were treated analogously. Stable LTR showed higher peripheral Treg cell counts with respect to age-matched healthy controls (59.9+/-31.8/mul versus 42.1+/-16.9/mul, respectively; p<0.05). This increase was detectable in all patients treated with CNI either in association with AZA or MMF. During these treatments a significant expansion of Treg cell counts was detectable during acute rejection (AR) episodes (86.03+/-26.6/mul during AR versus 36.34+/-7.6 before AR; p<0,05). Moreover, the development of BOS was associated to a significant decrease of Treg cell counts irrespective to the immunosuppressive regimen used. In conclusion, therapeutic regimens based on CNI seem to allow a certain degree of peripheral Treg cell expansion in stable LTR.

Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review.

The genus Raoultella was excised from Klebsiella in 2001, but difficulties in its identification may have led to an underestimation of its incidence and uncertainty on its pathogenic role. Recently, clinical reports involving Raoultella have increased, probably through the introduction of mass-spectrometry in clinical microbiology laboratories and the development of accurate molecular techniques. We performed a retrospective analysis using our blood culture collection (2011-14) to identify Raoultella isolates that could have been erroneously reported as Klebsiella. PCR and gene sequencing of highly specific chromosomal class A ß-lactamase genes was established as the reference method, and compared with 16S rRNA and rpoß sequencing, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), MicroScan Walkaway system and API20E biochemical identification. MALDI-TOF and rpoß correctly identified all Raoultella isolates, whereas 16S rRNA provided inconclusive results, and MicroScan and API20E failed to detect this genus. The analysis of the clinical characteristics of all Raoultella bacteraemia cases reported in the literature supports the role of Raoultella as an opportunistic pathogen that causes biliary tract infections in elderly patients who suffer from some kind of malignancy or have undergone an invasive procedure. Two salient conclusions are that Raoultella shows tropism for the biliary tract and so its identification could help clinicians to suspect underlying biliary tract disease when bacteraemia occurs. Concomitantly, as most phenotypic identification systems are not optimized for the identification of Raoultella, the use of MALDI-TOF or additional phenotypic tests is recommended for the reliable identification of this genus.

Ex vivo evaluation of PPD-specific IFN-gamma or IL-5 secreting cells in the peripheral blood and lungs of patients with tuberculosis.

OBJECTIVES: To evaluate ex vivo purified protein derivative (PPD) specific Th1- and Th2-type functional responses in human tuberculosis (TB). DESIGN: IFN-gamma and IL-5 secreting cells were measured by a computer-assisted ELISPOT assay in the peripheral blood of patients with pulmonary TB, in patients with other respiratory diseases (control patients) and in tuberculin skin test negative or positive healthy controls. Moreover, the number of IFN-gamma or IL-5 spots was assessed in the bronchoalveolar lavage (BAL) cells of five patients with advanced TB and lung inflammation. RESULTS: The frequency of PPD-specific IFN-gamma secreting cells in TB patients was higher than in control patients and healthy subjects. Although the number of PPD-specific IL-5 spots was low, a trend towards a higher frequency was observed in the peripheral blood of TB patients. Patients with advanced TB and lung inflammation had an increased number of both PPD-specific IFN-gamma and IL-5 spots in BAL as compared to that in peripheral blood, but the IFN-gamma/IL-5 ratio was about two-fold lower. CONCLUSIONS: In human TB, the host response in the periphery is driven by a specific Th1-type cytokine response, whereas in the lungs of patients with advanced disease and lung inflammation, polarisation towards a Th2-like bias is observed.

The complexed structure and antimicrobial activity of a non-beta-lactam inhibitor of AmpC beta-lactamase.

Beta-lactamases are the major resistance mechanism to beta-lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to beta-lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non-beta-lactam inhibitors of beta-lactamases were investigated for complementarity to the structure of AmpC beta-lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo(b)thiophene-2-boronic acid (BZBTH2B), which inhibited AmpC with a Ki of 27 nM. This inhibitor is chemically dissimilar to beta-lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X-ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 A resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1-amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non-beta-lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against beta-lactams in conjunction with beta-lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many beta-lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure-based elaboration of this class of non-beta-lactam inhibitors.

An extended-spectrum AmpC-type beta-lactamase obtained by in vitro antibiotic selection.

A predictive approach was assayed to evaluate the possibility of mutant Amp-C beta-lactamase emergence with increased substrate spectrum (including new C-3' quaternary ammonium cephems). The ampC gene encoding the AmpC beta-lactamase from Enterobacter cloacae was cloned and expressed in an AmpC-defective strain of E. coli. After the AmpC containing strain was challenged with cefpirome, an ampC variant encoding an enzyme with increased resistance to cefpirome and cefepime was selected. In addition, this variant conferred increased resistance to penicillins and third generation cephalosporins. The complete nucleotide sequence of the gene was determined. The deduced peptide sequence showed a single change with respect to the wild-type gene: valine to glutamic acid at position 318 of the native protein (298 of the mature enzyme). The potential emergence and spread of this type of AmpC variants among pathogens should be considered.

The assessment of IFN-gamma and its regulatory cytokines in the plasma and bronchoalveolar lavage fluid of patients with active pulmonary tuberculosis.

OBJECTIVES: To assess the role of IFN-gamma and its regulatory cytokines in active pulmonary tuberculosis (TB). DESIGN: Cytokines were measured in the plasma of TB patients and healthy subjects with different risk for TB exposure. In addition, cytokine profile was assessed in the bronchoalveolar lavage fluid (BALf) of six TB patients and nine normal controls. RESULTS: Circulating IFN-gamma, IL-10 and IL-18 were higher in TB patients than in control groups. Plasma IL-12 levels were extremely variable, and no difference was observed among study groups. An inverse correlation between plasma IFN-gamma and IL-10 levels was found in TB patients. Furthermore, circulating IL-18 correlated with IL-10 but not with IFN-gamma levels. Finally, IFN-gamma, IL-18 and IL-12 were increased in the BALf of TB patients, whereas no difference was observed in IL-10 levels. CONCLUSIONS: In human TB, at least at certain disease stages, there is a differential compartmentalization of the IFN-gamma-regulatory factors IL-12 and IL-10, the former being concentrated in the lungs and the latter being present in peripheral circulation. In addition, our findings address more critically the role of IL-18 in the host response to tuberculosis infection in humans.

Effects of continuous or pulsed exposure to rifabutin and sparfloxacin on the intracellular growth of Staphylococcus aureus and Mycobacterium tuberculosis.

The time-kinetics of the intracellular bioactivity and intracellular post-antibiotic effect (PAE) of rifabutin and sparfloxacin against Staphylococcus aureus and Mycobacterium tuberculosis, grown in human monocytes, were evaluated. Intracellular bactericidal activity against staphylococci was shown in the presence of extracellular drug concentrations equal or superior to 1/10 plasma Cmax. The bactericidal activity of rifabutin was dependent on both its extracellular concentrations and the exposure time. In contrast, the pattern of the intracellular activity of sparfloxacin was characterized by a minimal concentration dependent killing. Both antibiotics (from 1/10 to the expected lung Cmax) showed intracellular bioactivity against M. tuberculosis H37Ra and H37Rv strains. A long intracellular PAE on staphylococci (>4 hours) was demonstrated when drugs were removed from the infected monocytes after 1 h treatment. Our findings suggest that rifabutin and sparfloxacin may be useful in the treatment of lower respiratory tract infections due to intracellular pathogens.

A survey of antimicrobial drug resistance in respiratory tract pathogens, isolated in a northern Italian teaching hospital between 1990 and 1999.

Drug susceptibility test results of respiratory tract pathogens, isolated from patients admitted to the Clinic of Respiratory Diseases of the IRCCS San Matteo Hospital, University of Pavia (Italy) between 1990 and 1999, were retrospectively evaluated. A total of 1366 bacterial isolates were collected, including 499 gram-positive and 867 gram-negative strains. In comparison to methicillin-susceptible Staphylococcus aureus, the methicillin-resistant strains (MRSA) showed high levels of resistance to many selected antibiotics, except for glycopeptides. Resistance rates to beta-lactams were high in both Pseudomonas aeruginosa and in the other gram-negative isolates, while aminoglycoside and ciprofloxacin resistance was less than 20%. Some pathogens became more resistant to selected antimicrobials during the observation period, including staphylococci to methicillin, MRSA to ciprofloxacin, P. aeruginosa isolates to imipenem and ciprofloxacin, and the other gram-negative strains to almost all drugs considered, with the exception of cefotaxime and cotrimoxazole.