Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 ß-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem.

While Extended-Spectrum ß-Lactamases (ESBL) and AmpC ß-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC ß-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC ß-lactamase, and the plasmid-mediated CMY-2 ß-lactamase. Bla CMY-2-like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like ß-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli.

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics.

Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography-mass spectrometry (LC-MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC-MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC-MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.

Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

Successful High-Dosage Monotherapy of Tigecycline in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Background: Recent scientific reports on the use of high dose tigecycline monotherapy as a "drug of last resort" warrant further research into the use of this regimen for the treatment of severe multidrug-resistant, Gram-negative bacterial infections. In the current study, the therapeutic efficacy of tigecycline monotherapy was investigated and compared to meropenem monotherapy in a newly developed rat model of fatal lobar pneumonia-septicemia. Methods: A Klebsiella pneumoniae producing extended-spectrum ß-lactamase (ESBL) and an isogenic variant producing K. pneumoniae carbapenemase (KPC) were used in the study. Both strains were tested for their in vitro antibiotic susceptibility and used to induce pneumonia-septicemia in rats, which was characterized using disease progression parameters. Therapy with tigecycline or meropenem was initiated at the moment that rats suffered from progressive infection and was administered 12-hourly over 10 days. The pharmacokinetics of meropenem were determined in infected rats. Results: In rats with ESBL pneumonia-septicemia, the minimum dosage of meropenem achieving survival of all rats was 25 mg/kg/day. However, in rats with KPC pneumonia-septicemia, this meropenem dosage was unsuccessful. In contrast, all rats with KPC pneumonia-septicemia were successfully cured by administration of high-dose tigecycline monotherapy of 25 mg/kg/day (i.e., the minimum tigecycline dosage achieving 100% survival of rats with ESBL pneumonia-septicemia in a previous study). Conclusions: The current study supports recent literature recommending high-dose tigecycline as a last resort regimen for the treatment of severe multidrug-resistant bacterial infections. The use of ESBL- and KPC-producing K. pneumoniae strains in the current rat model of pneumonia-septicemia enables further investigation, helping provide supporting data for follow-up clinical trials in patients suffering from severe multidrug-resistant bacterial respiratory infections.

Cefpirome Treatment Results in Limited Selection of Stable Derepressed Enterobacter cloacae Mutants in the Intestinal Flora of Rats Treated for an Experimental Klebsiella pneumoniae Pulmonary Infection.

Background: Fourth-generation cephalosporins have been developed to improve their potency, that is, low minimal inhibitory concentrations (MICs) and to prevent resistance selection of derepressed AmpC-producing mutants in comparison to third-generation cephalosporins as ceftazidime. Objectives: We investigated the role of the administered cefpirome dose on the efficacy of treatment of a Klebsiella pneumoniae lung infection as well as in the selection of resistant Enterobacter cloacae isolates in the intestines of rats treated for a K. pneumoniae lung infection. Materials and Methods: Rats with K. pneumoniae lung infection received therapy with cefpirome doses of 0.4 to 50 mg/kg/day b.i.d. for 18 days. Resistance selection in intestinal E. cloacae was monitored during 43 days. Mutants were checked for ß-lactamase activity, mutations in their structural ampC gene, ampD gene, and omp39-40 gene. Results: A 45% and 100% rat survival rate was obtained by administration of 3.1 and 12.5 mg/kg b.i.d. of cefpirome. A significant correlation was demonstrated in the reduction of the susceptible E. cloacae isolates with %fT>MIC at days 7, 14, 22, and 29. Cefpirome E. cloacae mutants, with increased cefpirome MICs, were obtained in only four rats. Conclusions: The treatment with cefpirome resulted in less selection of derepressed mutants in comparison to ceftazidime as shown by their low number per gram of feces and in a limited number of animals.

Antimicrobial activity of two novel antimicrobial peptides AA139 and SET-M33 against clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles.

Colistin is an antimicrobial peptide (AMP) used as a drug of last resort, although plasmid-mediated colistin resistance (MCR) has been reported. AA139 and SET-M33 are novel AMPs currently in development for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. As many AMPs have a similar mode of action to colistin, potentially leading to cross-resistance, the antimicrobial activity of AA139 and SET-M33 was investigated against a collection of 50 clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles, including colistin-resistant strains. The collection was genotypically characterised and susceptibility to clinically relevant antibiotics was determined. Susceptibility to AA139 and SET-M33 did not differ among the collection despite differences in underlying mechanisms of resistance or susceptibility to colistin. For three colistin-susceptible and three colistin-resistant strains with distinct MDR profiles as well as an additional MCR-producing strain, the bactericidal activity of AA139, SET-M33 and colistin during 24 h of exposure was examined. Following 24 h of exposure to AA139, SET-M33 or colistin, the seven strains were tested for changes in susceptibility to the respective AMPs. AA139 and SET-M33 showed a concentration-dependent bactericidal effect irrespective of bacterial susceptibility to colistin. Exposure to low colistin concentrations resulted in the development of colistin resistance in colistin-susceptible strains, whereas susceptibility to AA139 and SET-M33 following exposure to the respective AMPs was maintained. The two novel AMPs remained effective against colistin-resistant strains and may be promising novel drugs for the treatment of clinically and genotypically diverse MDR K. pneumoniae infections, including infections associated with colistin-resistant bacteria.

Chitinase Induction Prior to Caspofungin Treatment of Experimental Invasive Aspergillosis in Neutropenic Rats Does Not Enhance Survival.

Host chitinases, chitotriosidase and acidic mammalian chitinase (AMCase), improved the antifungal activity of caspofungin (CAS) against Aspergillus fumigatus in vitro These chitinases are not constitutively expressed in the lung. Here, we investigated whether chitosan derivatives were able to induce chitinase activity in the lungs of neutropenic rats and, if so, whether these chitinases were able to prolong survival of rats with invasive pulmonary aspergillosis (IPA) or of rats with IPA and treated with CAS. An oligosaccharide-lactate chitosan (OLC) derivative was instilled in the left lung of neutropenic rats to induce chitotriosidase and AMCase activities. Rats instilled with OLC or with phosphate-buffered saline (PBS) were subsequently infected with A. fumigatus and then treated with suboptimal doses of CAS. Survival, histopathology, and galactomannan indexes were determined. Instillation of OLC resulted in chitotriosidase and AMCase activities. However, instillation of OLC did not prolong rat survival when rats were subsequently challenged with A. fumigatus In 5 of 7 rats instilled with OLC, the fungal foci in the lungs were smaller than those in rats instilled with PBS. Instillation of OLC did not significantly enhance the survival of neutropenic rats challenged with A. fumigatus and treated with a suboptimal dosage of CAS. Chitotriosidase and AMCase activities can be induced with OLC, but the presence of active chitinases in the lung did not prevent the development of IPA or significantly enhance the therapeutic outcome of CAS treatment.

Addition of 17-(allylamino)-17-demethoxygeldanamycin to a suboptimal caspofungin treatment regimen in neutropenic rats with invasive pulmonary aspergillosis delays the time to death but does not enhance the overall therapeutic efficacy.

Caspofungin (CAS) which is used as salvage therapy in patients with invasive pulmonary aspergillosis (IPA) inhibits the 1,3-ß-D-glucan synthesis in Aspergillus fumigatus. Inhibiting 1,3-ß-D-glucan synthesis induces a stress response and in an invertebrate model it was demonstrated that inhibiting this response with geldamycin enhanced the therapeutic efficacy of CAS. Since geldamycin itself is toxic to mammalians, the therapeutic efficacy of combining geldamycin with CAS was not studied in rodent models. Therefore in this study we investigated if the geldamycin derivate 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was able to enhance the therapeutic efficacy of CAS in vitro and in our IPA model in transiently neutropenic rats. In vitro we confirmed the earlier demonstrated synergy between 17-AAG and CAS in ten A. fumigatus isolates. In vivo we treated A. fumigatus infected neutropenic rats with a sub-optimal dose of 0.75 mg/kg/day CAS and 1 mg/kg/day 17-AAG for ten days. Survival was monitored for 21 days after fungal inoculation. It appeared that the addition 17-AAG delayed death but did not improve overall survival of rats with IPA. Increasing the doses of 17-AAG was not possible due to hepatic toxicity. This study underlines the need to develop less toxic and more fungal specific geldamycin derivatives and the need to test such drugs not only in invertebrate models but also in mammalian models.

Evaluation of different pretreatment protocols to detect accurately clinical carbapenemase-producing Enterobacteriaceae by MALDI-TOF.

OBJECTIVES: Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications. METHODS: The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the ß-lactam common structural motif, which can be detected using MALDI-TOF MS. RESULTS: A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed. CONCLUSIONS: Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.

Tigecycline Potentiates Clarithromycin Activity against Mycobacterium avium In Vitro.

Thein vitroactivities of clarithromycin and tigecycline alone and in combination againstMycobacterium aviumwere assessed. The activity of clarithromycin was time dependent, highly variable, and often resulted in clarithromycin resistance. Tigecycline showed concentration-dependent activity, and mycobacterial killing could only be achieved at high concentrations. Tigecycline enhanced clarithromycin activity againstM. aviumand prevented clarithromycin resistance. Whether there is clinical usefulness of tigecycline in the treatment ofM. aviuminfections needs further study.

Colistin as a potentiator of anti-TB drug activity against Mycobacterium tuberculosis.

OBJECTIVES: The mycobacterial cell wall is an effective permeability barrier that limits intracellular concentrations of anti-TB drugs and hampers the success of treatment. We hypothesized that colistin might enhance the efficacy of anti-TB drugs by increasing mycobacterial cell wall permeability. In this study, we investigated the additional effect of colistin on the activity of anti-TB drugs against Mycobacterium tuberculosis in vitro. METHODS: The concentration-dependent and time-dependent killing activity of isoniazid, rifampicin or amikacin alone or in combination with colistin against M. tuberculosis H37Rv was determined. Mycobacterial populations with both high and low metabolic activity were studied, and these were characterized by increasing or steady levels of ATP, respectively. RESULTS: With exposure to a single drug, striking differences in anti-TB drug activity were observed when the two mycobacterial populations were compared. The addition of colistin to isoniazid and amikacin resulted in sterilization of the mycobacterial load, but only in the M. tuberculosis population with high metabolic activity. The emergence of isoniazid and amikacin resistance was completely prevented by the addition of colistin. CONCLUSIONS: The results of this study emphasize the importance of investigating mycobacterial populations with both high and low metabolic activity when evaluating the efficacy of anti-TB drugs in vitro. This is the first study showing that colistin potentiates the activity of isoniazid and amikacin against M. tuberculosis and prevents the emergence of resistance to anti-TB drugs. These results form the basis for further studies on the applicability of colistin as a potentiator of anti-TB drugs.

Enhancement of in vitro activity of tuberculosis drugs by addition of thioridazine is not reflected by improved in vivo therapeutic efficacy.

OBJECTIVES: Assessment of the activity of thioridazine towards Mycobacterium tuberculosis (Mtb), in vitro and in vivo as a single drug and in combination with tuberculosis (TB) drugs. METHODS: The in vitro activity of thioridazine as single drug or in combination with TB drugs was assessed in terms of MIC and by use of the time-kill kinetics assay. Various Mtb strains among which the Beijing genotype strain BE-1585 were included. In vivo, mice with TB induced by BE-1585 were treated with a TB drug regimen with thioridazine during 13 weeks. Therapeutic efficacy was assessed by the change in mycobacterial load in the lung, spleen and liver during treatment and 13 weeks post-treatment. RESULTS: In vitro, thioridazine showed a concentration-dependent and time-dependent bactericidal activity towards both actively-replicating and slowly-replicating Mtb. Thioridazine at high concentrations could enhance the activity of isoniazid and rifampicin, and in case of isoniazid resulted in elimination of mycobacteria and prevention of isoniazid-resistant mutants. Thioridazine had no added value in combination with moxifloxacin or amikacin. In mice with TB, thioridazine was poorly tolerated, limiting the maximum tolerated dose (MTD). The addition of thioridazine at the MTD to an isoniazid-rifampicin-pyrazinamide regimen for 13 weeks did not result in enhanced therapeutic efficacy. CONCLUSIONS: Thioridazine is bactericidal towards Mtb in vitro, irrespective the mycobacterial growth rate and results in enhanced activity of the standard regimen. The in vitro activity of thioridazine in potentiating isoniazid and rifampicin is not reflected by improved therapeutic efficacy in a murine TB-model.

Enhancement of in vitro activity of tuberculosis drugs by addition of thioridazine is not reflected by improved in vivo therapeutic efficacy.

OBJECTIVES: Assessment of the activity of thioridazine towards Mycobacterium tuberculosis (Mtb), in vitro and in vivo as a single drug and in combination with tuberculosis (TB) drugs. METHODS: The in vitro activity of thioridazine as single drug or in combination with TB drugs was assessed in terms of MIC and by use of the time-kill kinetics assay. Various Mtb strains among which the Beijing genotype strain BE-1585 were included. In vivo, mice with TB induced by BE-1585 were treated with a TB drug regimen with thioridazine during 13 weeks. Therapeutic efficacy was assessed by the change in mycobacterial load in the lung, spleen and liver during treatment and 13 weeks post-treatment. RESULTS: In vitro, thioridazine showed a concentration-dependent and time-dependent bactericidal activity towards both actively-replicating and slowly-replicating Mtb. Thioridazine at high concentrations could enhance the activity of isoniazid and rifampicin, and in case of isoniazid resulted in elimination of mycobacteria and prevention of isoniazid-resistant mutants. Thioridazine had no added value in combination with moxifloxacin or amikacin. In mice with TB, thioridazine was poorly tolerated, limiting the maximum tolerated dose (MTD). The addition of thioridazine at the MTD to an isoniazid-rifampicin-pyrazinamide regimen for 13 weeks did not result in enhanced therapeutic efficacy. CONCLUSIONS: Thioridazine is bactericidal towards Mtb in vitro, irrespective the mycobacterial growth rate and results in enhanced activity of the standard regimen. The in vitro activity of thioridazine in potentiating isoniazid and rifampicin is not reflected by improved therapeutic efficacy in a murine TB-model.

Evidence supporting a role for mammalian chitinases in efficacy of caspofungin against experimental aspergillosis in immunocompromised rats.

OBJECTIVES: Caspofungin, currently used as salvage therapy for invasive pulmonary aspergillosis (IPA), strangely only causes morphological changes in fungal growth in vitro but does not inhibit the growth. In vivo it has good efficacy. Therefore the question arises how this in vivo activity is reached. Caspofungin is known to increase the amount of chitin in the fungal cell wall. Mammals produce two chitinases, chitotriosidase and AMCase, which can hydrolyse chitin. We hypothesized that the mammalian chitinases play a role in the in vivo efficacy of caspofungin. METHODS: In order to determine the role of chitotriosidase and AMCase in IPA, both chitinases were measured in rats which did or did not receive caspofungin treatment. In order to understand the role of each chitinase in the breakdown of the caspofungin-exposed cells, we also exposed caspofungin treated fungi to recombinant enzymes in vitro. RESULTS: IPA in immunocompromised rats caused a dramatic increase in chitinase activity. This increase in chitinase activity was still noted when rats were treated with caspofungin. In vitro, it was demonstrated that the action of both chitinases were needed to lyse the fungal cell wall upon caspofungin exposure. CONCLUSION: Caspofungin seemed to alter the cell wall in such a way that the two chitinases, when combined, could lyse the fungal cell wall and assisted in clearing the fungal pathogen. We also found that both chitinases combined had a direct effect on the fungus in vitro.

Optimization of the rifampin dosage to improve the therapeutic efficacy in tuberculosis treatment using a murine model.

RATIONALE: The dosage of 10 mg/kg/d rifampin, as currently used in the treatment of tuberculosis (TB), is not an optimal dose. Shortening of treatment duration might be achievable using an increased rifampin dose. OBJECTIVES: Determination of optimal rifampin dosage in mice, resulting in maximum therapeutic effect and without adverse effects. Assessment of associated pharmacokinetic parameters and pharmacokinetic/pharmacodynamic indices. METHODS: A murine TB infection using a Beijing genotype Mycobacterium tuberculosis strain was established by intratracheal bacterial instillation followed by proper inhalation, while keeping mice in a vertical position. We assessed dose-dependent activity of rifampin in single-drug treatment during 3 weeks. The maximum tolerated dosage, pharmacokinetic parameters, and pharmacokinetic/pharmacodynamic index were determined. Therapeutic efficacy of a range of rifampin (R) dosages added to a regimen of isoniazid (H) and pyrazinamide (Z) was assessed. MEASUREMENTS AND MAIN RESULTS: Maximum tolerated dosage of rifampin in the murine TB was 160 mg/kg/d. Pharmacokinetic measurement in HR(10)Z and HR(160)Z therapy regimens showed for rifampin a C(max) of 16.2 and 157.3 mg/L, an AUC(0-24h) of 132 and 1,782 h·mg/L, and AUC(0-24h)/minimum inhibitory concentration ratios of 528 and 7129, respectively. A clear dose-effect correlation was observed for rifampin after 3-week single-drug treatment. Administration of HR(80)Z allowed 9-week treatment duration to be effective without relapse of infection. CONCLUSIONS: Our findings indicate that the currently used rifampin dosage in the therapy of TB is too low. In our murine TB model a rifampin dosage of 80 mg/kg/d enabled a significant reduction in therapy duration without adverse effects.

Distinctive cytokines as biomarkers predicting fatal outcome of severe Staphylococcus aureus bacteremia in mice.

Invasive Staphylococcus aureus infections are frequently associated with bacteraemia. To support clinical decisions on antibiotic therapy, there is an urgent need for reliable markers as predictors of infection outcome. In the present study in mice, bacteraemia was established by intravenous inoculation of a clinical S. aureus isolate at the LD50 inoculum. As potential biomarkers for fatal outcome, blood culture (qualitative and quantitative), serum levels of C-reactive protein (CRP), as well as 31 selected cytokines and chemokines were assessed during the first three days of infection. A positive S. aureus blood culture, the quantitative blood culture, CRP levels, and levels of eight cytokines were indicative for the presence of S. aureus bacteraemia. However, only tumor necrosis factor (TNF) α, interleukin (IL) 1α, and keratinocyte chemoattractant (KC; a functional homologue of human IL-8) were each significantly elevated in eventually non-surviving infected mice versus eventually surviving infected mice. In severe S. aureus bacteraemia in mice, TNF-α, IL-1α, and KC are biomarkers predicting fatal outcome of infection. KC was a biomarker elevated irrespective the progression of infection, which is very interesting regarding clinical application in view of the heterogeneity of patients experiencing bacteraemia in this respect.

The therapeutic effect of tigecycline, unlike that of Ceftazidime, is not influenced by whether the Klebsiella pneumoniae strain produces extended-spectrum ß-lactamases in experimental pneumonia in rats.

The efficacies of tigecycline and ceftazidime against fatal pneumonia in rats caused by an extended-spectrum ß-lactamase (ESBL)-positive Klebsiella pneumoniae strain or its wild-type (WT) progenitor were compared. Ceftazidime at 12.5 or 50 mg/kg of body weight twice daily (b.i.d.) was effective (50% or 100% rat survival) in pneumonia caused by the WT isolate but unsuccessful (100% rat mortality) in pneumonia caused by the ESBL-positive variant. In contrast, tigecycline at 6.25, 12.5, or 25 mg/kg b.i.d. showed dosage-dependent efficacy up to 100% rat survival irrespective of the ESBL character of the infecting organism.

Relapse of tuberculosis versus primary tuberculosis; course, pathogenesis and therapy in mice.

Relapse of tuberculosis (TB) is defined as re-emergence of clinical symptoms after stopping anti-TB treatment, while this treatment appeared effective initially. Relapse of TB can occur in patients that are therapy-compliant, but the risk of relapse is dramatically increased when patients are non-compliant. Additionally, the probability of antibiotic resistance is higher in those patients who have a relapse of TB and thus longer treatment is recommended. Further insight in the pathogenesis of relapsing TB could provide a basis for future treatment improvement. In the present study, using a murine TB model, we assessed the differences between primary TB and relapse of TB in terms of mycobacterial load in infected organs, (immuno-) histopathology, and plasma cytokine concentrations. Compared to primary TB, in relapse of TB we observed a lower mycobacterial load in lung, spleen and liver at the phase of established infection. Also the levels of TNF-α, IFN-γ, IL-6, MIG/CXCL9, IP-10/CXCL10 and IL-17 were significantly lower. It was observed that in relapse of TB memory Th-1 cells were locally and systemically expanded and congregated in the lung, permitting an efficient control of Mtb growth. Treatment response in relapse of TB is as good as the treatment response in primary TB; thereby no supportive evidence could be given for the recommended longer treatment duration in case of relapse of TB.

Rifampicin-induced transcriptome response in rifampicin-resistant Mycobacterium tuberculosis.

Tuberculosis (TB) is still a major life-threatening infectious disease, within which especially the rise of multidrug resistant TB (MDR-TB) is currently worrying. This study focuses on mechanisms of development of rifampicin resistance, since rifampicin seems to play an important role in the development of MDR-TB. To provide further insight in rifampicin resistance, we performed a genome-wide transcriptional profile analysis for Mycobacterium tuberculosis (M. tuberculosis) using microarray technology and qRT-PCR analysis. We exposed a rifampicin-susceptible H37Rv wild type (H37Rv-WT) and a rifampicin-resistant progeny H37Rv strain with a H526Y mutation in the rpoB gene (H37Rv-H526Y) to several concentrations of rifampicin, to define the effect of rifampicin on the transcription profile. Our study showed that there are resistance-dependant differences in response between both M. tuberculosis strains. Gene clusters associated with efflux, transport and virulence were altered in the rifampicin-resistant H37Rv mutant compared to the rifampicin-susceptible H37Rv-WT strain after exposure to rifampicin. We conclude that the small gene cluster Rv0559c-Rv0560c in the H37Rv-H526Y strain was remarkably up-regulated in the microarray analysis and qRT-PCR results and appeared to be dependent on rifampicin concentration and time of exposure. Therefore this study suggests that Rv0559c and Rv0560c play a pivotal role in rifampicin resistance of M. tuberculosis. Further investigation of Rv0559c and Rv0560c is needed to reveal function and mechanism of both genes that were triggered upon rifampicin exposure.

Consequences of noncompliance for therapy efficacy and emergence of resistance in murine tuberculosis caused by the Beijing genotype of Mycobacterium tuberculosis.

Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, we showed the association between the Beijing genotype and an increased mutation frequency for rifampin resistance. In this study, we use a Beijing genotype strain and an East-African/Indian genotype strain to investigate with our mouse TB model whether the higher mutation frequency observed in a Beijing genotype strain is associated with treatment failure particularly during noncompliance therapy. Both genotype strains showed high virulence in comparison to that of M. tuberculosis strain H37Rv, resulting in a highly progressive infection with a rapid lethal outcome in untreated mice. Compliance treatment was effective without relapse of TB irrespective of the infecting strain, showing similar decreases in the mycobacterial load in infected organs and similar histopathological changes. Noncompliance treatment, simulated by a reduced duration and dosing frequency, resulted in a relapse of infection. Relapse rates were correlated with the level of noncompliance and were identical for Beijing infection and East African/Indian infection. However, only in Beijing-infected mice, isoniazid-resistant mutants were selected at the highest level of noncompliance. This is in line with the substantial selection of isoniazid-resistant mutants in vitro in a wide isoniazid concentration window observed for the Beijing strain and not for the EAI strain. These results suggest that genotype diversity of M. tuberculosis may be involved in emergence of resistance and indicates that genotype-tailor-made treatment should be investigated.