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.

Effectiveness of three different walking prescription durations on total physical activity in normal- and overweight women.

OBJECTIVE: While there is a dose-response relationship between physical activity (PA) and health benefit, little is known about the effectiveness of different PA prescriptions on total daily PA. AIM: To test, under real-life conditions and using an objective, non-invasive measurement technique (accelerometry), the effect of prescribing additional physical activity (walking only) of different durations (30, 60 and 90 min/day) on compliance (to the activity prescribed) and compensation (to total daily PA). Participants in each group were prescribed 5 sessions of walking per week over 4 weeks. METHODS: 55 normal-weight and overweight women (mean BMI 25 ± 5 kg/m(2), height 165 ± 1 cm, weight 68 ± 2 kg and mean age 27 ± 1 years) were randomly assigned to 3 prescription groups: 30, 60 or 90 min/day PA. RESULTS: Walking duration resulted in an almost linear increase in the number of steps per day during the prescription period from an average of about 10,000 steps per day for the 30-min prescription to about 14,000 for the 90-min prescription. Compliance was excellent for the 30-min prescription but decreased significantly with 60-min and 90-min prescriptions. In parallel, degree of compensation subsequent to exercise increased progressively as length of prescription increased. CONCLUSION: A 30-min prescription of extra walking 5 times per week was well tolerated. However, in order to increase total PA further, much more than 60 min of walking may need to be prescribed in the majority of individuals. While total exercise 'volume' increased with prescriptions longer than 30 min, compliance to the prescription decreased and greater compensation was evident. © 2014 S. Karger GmbH, Freiburg.

Reduction in the use of diagnostic tests in infants with risk factors for early-onset neonatal sepsis does not delay antibiotic treatment.

Despite a low positive predictive value, diagnostic tests such as complete blood count (CBC) and C-reactive protein (CRP) are commonly used to evaluate whether infants with risk factors for early-onset neonatal sepsis (EOS) should be treated with antibiotics. We investigated the impact of implementing a protocol aiming at reducing the number of diagnostic tests in infants with risk factors for EOS in order to compare the diagnostic performance of repeated clinical examination with CBC and CRP measurement. The primary outcome was the time between birth and the first dose of antibiotics in infants treated for suspected EOS. Among the 11,503 infants born at ≥35 weeks during the study period, 222 were treated with antibiotics for suspected EOS. The proportion of infants receiving antibiotics for suspected EOS was 2.1% and 1.7% before and after the change of protocol (p = 0.09). Reduction of diagnostic tests was associated with earlier antibiotic treatment in infants treated for suspected EOS (hazard ratio 1.58; 95% confidence interval [CI] 1.20-2.07; p <0.001), and in infants with neonatal infection (hazard ratio 2.20; 95% CI 1.19-4.06; p = 0.01). There was no difference in the duration of hospital stay nor in the proportion of infants requiring respiratory or cardiovascular support before and after the change of protocol. Reduction of diagnostic tests such as CBC and CRP does not delay initiation of antibiotic treatment in infants with suspected EOS. The importance of clinical examination in infants with risk factors for EOS should be emphasised.

Adult native septic arthritis: a review of 10 years of experience and lessons for empirical antibiotic therapy.

Objectives To review the epidemiology of native septic arthritis to establish local guidelines for empirical antibiotic therapy as part of an antibiotic stewardship programme. Methods We conducted a 10 year retrospective study based on positive synovial fluid cultures and discharge diagnosis of septic arthritis in adult patients. Microbiology results and medical records were reviewed. Results Between 1999 and 2008, we identified 233 episodes of septic arthritis. The predominant causative pathogens were methicillin-susceptible Staphylococcus aureus (MSSA) and streptococci (respectively, 44.6% and 14.2% of cases). Only 11 cases (4.7%) of methicillin-resistant S. aureus (MRSA) arthritis were diagnosed, among which 5 (45.5%) occurred in known carriers. For large-joint infections, amoxicillin/clavulanate or cefuroxime would have been appropriate in 84.5% of cases. MRSA and Mycobacterium tuberculosis would have been the most frequent pathogens that would not have been covered. In contrast, amoxicillin/clavulanate would have been appropriate for only 75.3% of small-joint infections (82.6% if diabetics are excluded). MRSA and Pseudomonas aeruginosa would have been the main pathogens not covered. Piperacillin/tazobactam would have been appropriate in 93.8% of cases (P < 0.01 versus amoxicillin/clavulanate). This statistically significant advantage is lost after exclusion of diabetics (P = 0.19). Conclusions Amoxicillin/clavulanate or cefuroxime would be adequate for empirical coverage of large-joint septic arthritis in our area. A broad-spectrum antibiotic would be significantly superior for small-joint infections in diabetics. Systematic coverage of MRSA is not justified, but should be considered for known carriers. These recommendations are applicable to our local setting. They might also apply to hospitals sharing the same epidemiology.

Antibiotic susceptibility of Neochlamydia hartmanellae and Parachlamydia acanthamoebae in amoebae.

Parachlamydia acanthamoebae and Neochlamydia hartmanellae are Chlamydia-related bacteria naturally infecting free-living amoebae. These strict intracellular bacteria might represent emerging pathogens. Recent studies report an association with lower respiratory tract infections, especially with pneumonia where they have been identified as a potential causative agent in 1-2% of cases. In this study, we defined the antibiotic susceptibility of N. hartmanellae, two strains of P. acanthamoebae and two yet unclassified Parachlamydiaceae strains using a quantitative approach. We confirmed the results obtained earlier for P. acanthamoebae strain Bn9 in an observational study. Macrolides (MICs < 0.06-0.5 μg/ml), rifampicin (MICs 0.25-2) and doxycycline (2-4 μg/ml) were active against P. acanthamoebae strains and Neochlamydia. All strains were resistant to amoxicillin, ceftriaxone and imipenem (MIC ≥32 μg/ml). Similarly to other Chlamydia-related bacteria, all investigated Parachlamydiaceae were resistant to quinolones (MICs ≥ 16 μg/ml). Therefore, we recommend a treatment with macrolides for Parachlamydia-associated pneumonia.

Chronic kidney disease in type 2 diabetic patients followed-up by primary care physicians in Switzerland: prevalence and prescription of antidiabetic drugs.

QUESTION UNDER STUDY: The aim of this study was to assess the prevalence of chronic kidney disease (CKD) among type 2 diabetic patients in primary care settings in Switzerland, and to analyse the prescription of antidiabetic drugs in CKD according to the prevailing recommendations. METHODS: In this cross-sectional study, each participating physician was asked to introduce anonymously in a web database the data from up to 15 consecutive diabetic patients attending her/his office between December 2013 and June 2014. Demographic, clinical and biochemical data were analysed. CKD was classified with the KDIGO nomenclature based on estimated glomerular filtration rate (eGFR) and urinary albumin/creatinine ratio. RESULTS: A total of 1 359 patients (mean age 66.5 ± 12.4 years) were included by 109 primary care physicians. CKD stages 3a, 3b and 4 were present in 13.9%, 6.1%, and 2.4% of patients, respectively. Only 30.6% of patients had an entry for urinary albumin/creatinine ratio. Among them, 35.6% were in CKD stage A2, and 4.1% in stage A3. Despite prevailing limitations, metformin and sulfonylureas were prescribed in 53.9% and 16.5%, respectively, of patients with advanced CKD (eGFR <30 ml/min). More than a third of patients were on a dipeptidyl-peptidase-4 inhibitor across all CKD stages. Insulin use increased progressively from 26.8% in CKD stage 1-2 to 50% in stage 4. CONCLUSIONS: CKD is frequent in patients with type 2 diabetes attending Swiss primary care practices, with CKD stage 3 and 4 affecting 22.4% of cases. This emphasizes the importance of routine screening of diabetic nephropathy based on both eGFR and urinary albumin/creatinine ratio, the latter being largely underused by primary care physicians. A careful individual drug risk/benefit balance assessment is mandatory to avoid the frequently observed inappropriate prescription of antidiabetic drugs in CKD patients.