New methods for detecting antibiotic resistant bacteria and preventing their spread

Methicillin resistant Staphylococcus aureus (MRSA) bacteria have emerged in the early 1980's in numerous health care institutions around the world. The main transmission mechanism within hospitals and healthcare facilities is through the hands of health care workers. Resistant to several antibiotics, the MRSA is one of the most feared pathogens in the hospital setting since it is very difficult to eradicate with the standard treatments. There are still a limited number of anti-MRSA antibiotics but the first cases of resistance to these compounds have already been reported and their frequency is likely to increase in the coming years. Every year, the MRSA infections result in major human and financial costs, due to the high associated mortality and expenses related to the required care. Measures towards a faster detection of resistant bacteria and establishment of appropriate antibiotic treatment parameters are fundamental. Also as part as infection prevention, diminution of bacteria present on the commonly touched surfaces could also limit the spread and selection of antibiotic resistant bacteria. During my thesis, projects were developed around MRSA and antibiotic resistance investigation using innovative technologies. The thesis was subdivided in three main parts with the use of atomic force microscopy AFM for antibiotic resistance detection in part 1, the importance of the bacterial inoculum size in the selection of antibiotic resistance in part 2 and the testing of antimicrobial surfaces creating by sputtering copper onto polyester in part 3. In part 1 the AFM was used two different ways, first for the measurement of stiffness (elasticity) of bacteria and second as a nanosensor for antibiotic susceptibility testing. The stiffness of MRSA with different susceptibility profiles to vancomycin was investigated using the stiffness tomography mode of the AFM and results have demonstrated and increased stiffness in the vancomycin resistant strains that also paralleled with increased thickness of the bacterial cell wall. Parts of the AFM were also used to build a new antibiotic susceptibility-testing device. This nano sensor was able to measure vibrations emitted from living bacteria that ceased definitively upon antibiotic exposure to which they were susceptible but restarted after antibiotic removal to which they were resistant, allowing in a matter of minute the assessment of antibiotic susceptibility determination. In part 2 the inoculum effect (IE) of vancomycin, daptomycin and linezolid and its importance in antibiotic resistance selection was investigated with MRSA during a 15 days of cycling experiment. Results indicated that a high bacterial inoculum and a prolonged antibiotic exposure were two key factors in the in vitro antibiotic resistance selection in MRSA and should be taken into consideration when choosing the drug treatment. Finally in part 3 bactericidal textile surfaces were investigated against MRSA. Polyesters coated after 160 seconds of copper sputtering have demonstrated a high bactericidal activity reducing the bacterial load of at least 3 logio after one hour of contact. -- Au cours des dernières décennies, des bactéries multirésistantes aux antibiotiques (BMR) ont émergé dans les hôpitaux du monde entier. Depuis lors, le nombre de BMR et la prévalence des infections liées aux soins (IAS) continuent de croître et sont associés à une augmentation des taux de morbidité et de mortalité ainsi qu'à des coûts élevés. De plus, le nombre de résistance à différentes classes d'antibiotiques a également augmenté parmi les BMR, limitant ainsi les options thérapeutiques disponibles lorsqu'elles ont liées a des infections. Des mesures visant une détection plus rapide des bactéries résistantes ainsi que l'établissement des paramètres de traitement antibiotiques adéquats sont primordiales lors d'infections déjà présentes. Dans une optique de prévention, la diminution des bactéries présentes sur les surfaces communément touchées pourrait aussi freiner la dissémination et l'évolution des bactéries résistantes. Durant ma thèse, différents projets incluant des nouvelles technologies et évoluant autour de la résistance antibiotique ont été traités. Des nouvelles technologies telles que le microscope à force atomique (AFM) et la pulvérisation cathodique de cuivre (PCC) ont été utilisées, et le Staphylococcus aureus résistant à la méticilline (SARM) a été la principale BMR étudiée. Deux grandes lignes de recherche ont été développées; la première visant à détecter la résistance antibiotique plus rapidement avec l'AFM et la seconde visant à prévenir la dissémination des BMR avec des surfaces crées grâce à la PCC. L'AFM a tout d'abord été utilisé en tant que microscope à sonde locale afin d'investiguer la résistance à la vancomycine chez les SARMs. Les résultats ont démontré que la rigidité de la paroi augmentait avec la résistance à la vancomycine et que celle-ci corrélait aussi avec une augmentation de l'épaisseur des parois, vérifiée grâce à la microscopie électronique. Des parties d'un AFM ont été ensuite utilisées afin de créer un nouveau dispositif de test de sensibilité aux antibiotiques, un nanocapteur. Ce nanocapteur mesure des vibrations produites par les bactéries vivantes. Après l'ajout d'antibiotique, les vibrations cessent définitivement chez les bactéries sensibles à l'antibiotique. En revanche pour les bactéries résistantes, les vibrations reprennent après le retrait de l'antibiotique dans le milieu permettant ainsi, en l'espace de minutes de détecter la sensibilité de la bactérie à un antibiotique. La PCC a été utilisée afin de créer des surfaces bactéricides pour la prévention de la viabilité des BMR sur des surfaces inertes. Des polyesters finement recouverts de cuivre (Cu), connu pour ses propriétés bactéricides, ont été produits et testés contre des SARMs. Une méthode de détection de viabilité des bactéries sur ces surfaces a été mise au point, et les polyesters obtenus après 160 secondes de pulvérisation au Cu ont démontré une excellente activité bactéricide, diminuant la charge bactérienne d'au moins 3 logio après une heure de contact. En conclusion, l'utilisation de nouvelles technologies nous a permis d'évoluer vers de méthodes de détection de la résistance antibiotique plus rapides ainsi que vers le développement d'un nouveau type de surface bactéricide, dans le but d'améliorer le diagnostic et la gestion des BMR.

Analysis of the interacting components between larval Schistosoma mansoni and schistosome-susceptible and resistant Biomphalaria glabrata

Cell-free hemolymph (serum) and hemocytes from Schistosoma mansoni-susceptible (PR albino M-line) and resistant (10-R2) strains of Biomphalaria glabrata were compared by SDS-PAGE, immunoblotting and radioiodination. Whole serum of both snail strains is dominated by hemoglobin (Hb) (MW = 160 Kd). SDS-PAGE. of Hb-depleted serum indicated that the 10-R2 strain has dominant polypeptides in the 50 to 30 Kd range whereas PR albino snails have few low MW proteins. Antibodies raised to whole PR albino and 10-R2 serum, and the 160 Kd (Hb) band reacted similarly in immunoblot assays. Analysis of hemocytes revealed that 10-R2 snails have a surface-exposed protein at about 80 Kd which is not present on PR albino hemocytes. An examination of primary cultured sporocysts indicated the presence of four major surface proteins (40, 50, 55, 70 Kd) and two minor surface-exposed polypeptides (92, 170 Kd). Antibodies raised against live, intact sporocysts reacted almost exclusively with sporocyst-surface proteins when tested by immunoblotting.

Electronic compliance monitoring in resistant hypertension: the basis for rational therapeutic decisions.

OBJECTIVE: Incomplete compliance is one of several possible causes of uncontrolled hypertension. Yet, non-compliance remains largely unrecognized and is falsely interpreted as treatment resistance, because it is difficult to confirm or exclude objectively. The goal of this study was to evaluate the potential benefits of electronic monitoring of drug compliance in the management of patients with resistant hypertension. METHODS: Forty-one hypertensive patients resistant to a three-drug regimen (average blood pressure 156/ 106 +/- 23/11 mmHg, mean +/- SD) were studied prospectively. They were informed that for the next 2 months, their presently prescribed drugs would be provided in electronic monitors, without any change in treatment, so as to provide the treating physician with a measure of their compliance. Thereafter, patients were offered the possibility of prolonging the monitoring of compliance for another 2 month period, during which treatment was adapted if necessary. RESULTS: Monitoring of compliance alone was associated with a significant improvement of blood pressure at 2 months (145/97 +/- 20/15 mmHg, P < 0.01). During monitoring, blood pressure was normalized (systolic < 140 mmHg or diastolic < 90 mmHg) in one-third of the patients and insufficient compliance was unmasked in another 20%. When analysed according to tertiles of compliance, patients with the lowest compliance exhibited significantly higher achieved diastolic blood pressures (P = 0.04). In 30 patients, compliance was monitored up to 4 months and drug therapy was adapted whenever necessary. In these patients, a further significant decrease in blood pressure was obtained (from 150/100 +/- 18/15 to 143/94 +/- 22/11 mmHg, P = 0.04/0.02). CONCLUSIONS: These results suggest that objective monitoring of compliance using electronic devices may be a useful step in the management of patients with refractory hypertension, as it enables physicians to take rational decisions based on reliable and objective data of drug compliance and hence to improve blood pressure control.

HIV-1 drug resistance transmission networks in southwest Switzerland.

To determine viral subtypes and resistance mutations to antiretroviral treatment (ART) in untreated HIV-1 acutely infected subjects from Southwest Switzerland. Clinical samples were obtained from the HIV primary infection cohort from Lausanne. Briefly, pol gene was amplified by nested PCR and sequenced to generate a 1?kb sequence spanning protease and reverse transcriptase key protein regions. Nucleotide sequences were used to assess viral genotype and ART resistance mutations. Blood specimens and medical information were obtained from 30 patients. Main viral subtypes corresponded to clade B, CRF02_AG, and F1. Resistant mutations to PIs consisted of L10V and accessory mutations 16E and 60E present in all F1 clades. The NNRTI major resistant mutation 103N was detected in all F1 viruses and in other 2 clades. Additionally, we identified F1 sequences from other 6 HIV infected and untreated individuals from Southwest Switzerland, harboring nucleotide motifs and resistance mutations to ART as observed in the F1 strains from the cohort. These data reveal a high transmission rate (16.6%) for NNRTI resistant mutation 103N in a cohort of HIV acute infection. Three of the 5 resistant strains were F1 clades closely related to other F1 isolates from HIV-1 infection untreated patients also coming from Southwest Switzerland. Overall, we provide strong evidence towards an HIV-1 resistant transmission network in Southwest Switzerland. These findings have relevant implications for the local molecular mapping of HIV-1 and future ART surveillance studies in the region.

Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: a multicohort European case-control study using centralized ultrasensitive 454 pyrosequencing.

OBJECTIVES: It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART. METHODS: This Europe-wide case-control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%-25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression. RESULTS: Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35-5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76-6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12-5.18, P = 0.024). A dose-effect relationship between virological failure and mutational load was found. CONCLUSIONS: Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based ART.

Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy.

Autophagy or "self eating" is frequently activated in tumor cells treated with chemotherapy or irradiation. Whether autophagy represents a survival mechanism or rather contributes to cell death remains controversial. To address this issue, the role of autophagy in radiosensitive and radioresistant human cancer cell lines in response to gamma-irradiation was examined. We found irradiation-induced accumulation of autophagosomes accompanied by strong mRNA induction of the autophagy-related genes beclin 1, atg3, atg4b, atg4c, atg5, and atg12 in each cell line. Transduction of specific target-siRNAs led to down-regulation of these genes for up to 8 days as shown by reverse transcription-PCR and Western blot analysis. Blockade of each autophagy-related gene was associated with strongly diminished accumulation of autophagosomes after irradiation. As shown by clonogenic survival, the majority of inhibited autophagy-related genes, each alone or combined, resulted in sensitization of resistant carcinoma cells to radiation, whereas untreated resistant cells but not sensitive cells survived better when autophagy was inhibited. Similarly, radiosensitization or the opposite was observed in different sensitive carcinoma cells and upon inhibition of different autophagy genes. Mutant p53 had no effect on accumulation of autophagosomes but slightly increased clonogenic survival, as expected, because mutated p53 protects cells by conferring resistance to apoptosis. In our system, short-time inhibition of autophagy along with radiotherapy lead to enhanced cytotoxicity of radiotherapy in resistant cancer cells.

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/lOJ) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.

Severe and steroid-resistant Crohn's disease.

Patients with moderate to severe disease and patients with steroid-refractory or steroid-dependent disease differ in their management, as the latter groups usually include patients with less acute situations. Systemic corticosteroids represent the mainstay of the management of moderate to severe disease and remain the first-line therapy in this setting. Infliximab is the choice alternative for patients who do not respond to steroids or in whom steroids are contraindicated. Purine analogues, methotrexate and infliximab have shown efficacy in achieving steroid-free remission in patients with steroid-refractory or -dependent disease. Other fast-acting immunosuppressors showed little benefit. Surgery may be indicated in this setting. Nataluzimab may prove useful in patients refractory to infliximab.

Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the diseases. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9 and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative morbility (Mr) or the quantitative amount (intensity) of the antigenic determinats. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patient's sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared. These results indicate that isolates of L. amazonensis are not antigenically identical (homogeneous) and that the immune responses (antibodies) observed among infected patients are heterogeneous.

Efficacy of trovafloxacin in treatment of experimental staphylococcal or streptococcal endocarditis.

The efficacy of trovafloxacin against Staphylococcus aureus and viridans group streptococci was investigated in vitro and in an experimental model of endocarditis. The MICs at which trovafloxacin and ciprofloxacin inhibited 90% of clinical isolates of such bacteria (MIC90s) were (i) 0.03 and 2 mg/liter, respectively, for 30 ciprofloxacin-susceptible S. aureus isolates, (ii) 32 and 128 mg/liter, respectively, for 20 ciprofloxacin-resistant S. aureus isolates, and (iii) 0.25 and 8 mg/liter, respectively, for 28 viridans group streptococci. Rats with aortic vegetations were infected with either of two ciprofloxacin-susceptible but methicillin-resistant S. aureus strains (strains COL and P8), one penicillin-susceptible Streptococcus sanguis strain, or one penicillin-resistant Streptococcus mitis strain. Rats were treated for 3 or 5 days with doses that resulted in kinetics that simulated those achieved in humans with trovafloxacin (200 mg orally once a day), ciprofloxacin (750 mg orally twice a day), vancomycin (1 g intravenously twice a day), or ceftriaxone (2 g intravenously once a day). Against the staphylococci, the activities of both trovafloxacin and ciprofloxacin were equivalent to that of vancomycin, and treatment of endocarditis with these drugs was successful (P &lt; 0.05). However, ciprofloxacin selected for resistant derivatives in vitro and in vivo, whereas trovafloxacin was 10 to 100 times less prone than ciprofloxacin to select for resistance in vitro and did not select for resistance in vivo. Against the two streptococcal isolates, trovafloxacin significantly (P &lt; 0.05) decreased bacterial counts in the vegetations but was less effective than the control drug, ceftriaxone. Thus, a simulated oral dose of trovafloxacin (200 mg per day) was effective against ciprofloxacin-susceptible staphylococci and was less likely than ciprofloxacin to select for resistance. The simulated oral dose of trovafloxacin also had some activity against streptococcal endocarditis, but optimal treatment of infections caused by such organisms might require higher doses of the drug.