Molecular analysis of NDM-1-producing enterobacterial isolates from Geneva, Switzerland

Objectives To analyse the mechanisms responsible for decreased susceptibility or resistance to carbapenems in several enterobacterial isolates recovered in 2009-10 in Geneva University Hospitals, Switzerland. Methods PCR and sequencing were used to identify β-lactamases, 16S RNA methylases and plasmid-mediated quinolone resistance genes. The transferable properties of the plasmids were analysed, as well as their plasmid type. The strains were typed by multilocus sequence typing. Results Three patients were found to be positive for NDM-1-producing enterobacterial isolates (one with Escherichia coli and Klebsiella pneumoniae, one with K. pneumoniae only and one with Proteus mirabilis), where NDM-1 stands for New Delhi metallo-β-lactamase-1. The blaNDM-1 carbapenemase gene was detected in all isolates in addition to genes encoding narrow-spectrum β-lactamases (TEM-1, SHV-11, OXA-1, OXA-9 and OXA-10), extended-spectrum β-lactamases (CTX-M-15, CMY-16 and CMY-30), ArmA and quinolone resistance determinants (Qnr). The blaNDM-1 gene was located on conjugative IncA/C- or IncF-type plasmids. Upstream of the blaNDM-1 gene, part of ISAba125, previously identified in NDM-1-negative Acinetobacter baumannii, was found. Downstream of the blaNDM-1 gene, variable sequences were found. Conclusions This work constitutes the first identification of NDM-1 producers in Switzerland. Interestingly, patients from whom these NDM-1-producing isolates were recovered had a link with the Indian subcontinent or the Balkan

Septic shock caused by Capnocytophaga canis after a cat scratch

Capnocytophaga canis is an uncommon cause of septic shock. Only three cases have been previously reported in the literature. In this article, we describe the case of a 70-year-old male admitted to the intensive care unit for septic shock of unknown origin. On day 2, one anaerobic bottle out of the two sets taken at admission turned positive with Gram-negative bacilli. The pathogen was identified by 16S rRNA gene as C. canis. The strain was characterized and compared with other clinical isolates of Capnocytophaga spp

Meropenem/colistin synergy testing for multidrug-resistant Acinetobacter baumannii strains by a two-dimensional gradient technique applicable in routine microbiology

Objectives Precise assessment of potential therapeutic synergy, antagonism or indifference between antimicrobial agents currently depends on time-consuming and hard-to-standardize in vitro chequerboard titration methods. We here present a method based on a novel two-dimensional antibiotic gradient technique named Xact™. Methods We used a test comprising a combination of perpendicular gradients of meropenem and colistin in a single quadrant. We compared test outcomes with those obtained with classical chequerboard microbroth dilution testing in a study involving 27 unique strains of multidrug-resistant Acinetobacter baumannii from diverse origins. Results We were able to demonstrate 92% concordance between the new technology and classical chequerboard titration using the A. baumannii collection. Two strains could not be analysed by Xact™ due to their out-of-range MIC of meropenem (>128 mg/L). Conclusions The new test was shown to be diagnostically useful, easy to implement and less labour intensive than the classical metho

Gram and acridine orange staining for diagnosis of septic arthritis in different patient populations

Purpose: The sensitivity of Gram staining is known to be suboptimal for the diagnosis of native joint septic arthritis. We lack information about the accuracy of Gram compared to other microscopic staining techniques for predicting infection in different patient populations. Methods: This was a cohort study with cost evaluations at the Orthopaedic Service of Geneva University Hospitals (January 1996-October 2012). Results: Among 500 episodes of arthritis (196 with immunosuppression, 227 with underlying arthroplasties and 69 with gout or other crystals in synovial fluid), Gram staining revealed pathogens in 146 episodes (146/500, 29%) or in 146 of the 400 culture-positive episodes (37%). Correlation between the Gram and acridine staining of the same sample was good (Spearman 0.85). Overall, the sensitivity, specificity, positive predictive value and negative predictive value of Gram stain for rapid diagnosis of septic arthritis was 0.37, 0.99, 0.99 and 0.28, respectively, compared to microbiological cultures. Quite similar values were recorded across the different patient subpopulations, in particular for sensitivity values that were 0.33 for patients with prosthetic joint infections, 0.40 for immunosuppressed patients, 0.36 for patients under antibiotic administration and 0.52 for patients with concomitant crystalline disease. Conclusions: The sensitivity of Gram or acridine orange staining for a rapid diagnosis of episodes of septic arthritis is suboptimal compared to microbiological culture, regardless of underlying conditions, immunosuppression or antibiotic therapy. The sensitivity in the presence of synovial fluid crystals is moderate. Acridine orange and Gram stains are equivalent

Regional spread of an atypical ESBL-producing Escherichia coli ST131H89 clone among different human and environmental reservoirs in Western Switzerland

We describe the inter-regional spread of a novel ESBL-producing Escherichia coli subclone (ST131H89) in long-term care facility residents, general population, and environmental water sources in Western Switzerland between 2017 and 2020. The study highlights the importance of molecular surveillance for tracking emerging antibiotic-resistant pathogens in healthcare and community settings

Household acquisition and transmission of extended-spectrum β-lactamase (ESBL) -producing Enterobacteriaceae after hospital discharge of ESBL-positive index patients

MODERN WP2 study group: Caroline Brossier, Elodie von Dach, Gesuele Renzi, Jacques Schrenzel, Stefanie Bunk, Siri Goepel, Florian Hölzl, Michael Eib, Ingo B.Autenrieth, Álvaro Pascual, Xavier Bertrand, Jelle Scharringa, Patrick Musicha.[Objectives] This study aimed to determine rates and risk factors of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) acquisition and transmission within households after hospital discharge of an ESBL-PE-positive index patient.[Methods] Two-year prospective cohort study in five European cities. Patients colonized with ESBL-producing Escherichia coli (ESBL-Ec) or Klebsiella pneumoniae (ESBL-Kp), and their household contacts were followed up for 4 months after hospital discharge of the index case. At each follow up, participants provided a faecal sample and personal information. ESBL-PE whole-genome sequences were compared using pairwise single nucleotide polymorphism-based analysis.[Results] We enrolled 71 index patients carrying ESBL-Ec (n = 45), ESBL-Kp (n = 20) or both (n = 6), and 102 household contacts. The incidence of any ESBL-PE acquisition among household members initially free of ESBL-PE was 1.9/100 participant-weeks at risk. Nineteen clonally related household transmissions occurred (case to contact: 13; contact to case: 6), with an overall rate of 1.18 transmissions/100 participant-weeks at risk. Most of the acquisition and transmission events occurred within the first 2 months after discharge. The rate of ESBL-Kp household transmission (1.16/100 participant-weeks) was higher than of ESBL-Ec (0.93/100 participant-weeks), whereas more acquisitions were noted for ESBL-Ec (1.06/100 participant-weeks) compared with ESBL-Kp (0.65/100 participant-weeks). Providing assistance for urinary and faecal excretion to the index case by household members increased the risk of ESBL-PE transmission (adjusted prevalence ratio 4.3; 95% CI 1.3–14.1).[Conclusions] ESBL-PE cases discharged from the hospital are an important source of ESBL-PE transmission within households. Most acquisition and transmission events occurred during the first 2 months after hospital discharge and were causally related to care activities at home, highlighting the importance of hygiene measures in community settings.[Clinical study registration] German Clinical Trials Register, DRKS-ID: DRKS00013250.This study was part of a Joint Programming Initiative on Antimicrobial Resistance collaborative research project, under the 2016 Joint Call framework (Transnational Research Projects on the Transmission Dynamics of Antibacterial Resistance). It received funding from the following national research agencies: Instituto de Salud Carlos III (grant no. AC16/00076), Netherlands Organization for Health Research and Development (grant no. AC681055), Swiss National Science Foundation (grant no. 40AR40-173608), German Federal Ministry of Education and Research (grant no. 01KI1830) and Agence Nationale de la Recherche (grant no. ANR-16-JPEC-0007-03). As part of a separate research project, Marlieke de Kraker has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement nos 115523, 115620 and 115737 (Combatting Bacterial Resistance in Europe projects (COMBACTE)), resources of which are composed of financial contribution from the European Union's 7th Framework Programme (FP7/2007±2013) and the European Federation of Pharmaceutical Industries and associations companies' in-kind contribution. Also, Elena Salamanca, Mercedes Delgado and Jesús Rodríguez-Baño received support for research from by the Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0001), co-financed by the European Development Regional Fund ‘A way to achieve Europe’, Operative Programme Intelligence Growth 2014-2020.Peer reviewe

Total Laboratory Automation in Clinical Bacteriology: A Technology to Improve Patient Care

At a time when diagnostic bacteriological testing has become more complex and its associated costs are steadily increasing, the expected benefits of TLA cannot just consist in a simple transposition of the traditional manual procedures used to process clinical specimens. In contrast, automation should drive a fundamental change in the laboratory workflow and challenge users to reconsider all the approaches currently used in the diagnostic work-up (from receiving samples to communicating the results to the prescribing physicians). This thesis fits several purposes: 1. to highlight key issues faced when shifting to Total Laboratory Automation (TLA) 2. to illustrate the selected implementation steps without discontinuing the routine diagnostic service 3. to discuss the impact of TLA on various aspects following implementation: a- project management and change management b- training and adherence of our Technical Support team (technologists and biologists) c- managing the quality of the operational processes d- adjusting the workflow for efficiency e- assessing the impact of TLA on turnaround-times f- providing an outlook of the challenges following the implementation of TLA and an overview of the upcoming studies 4. to illustrate the implementation of a fully automated solution for antimicrobial disk diffusion susceptibility testing, and the clinical validation study performed before the implementation of this approach in our routine 5. to discuss the upcoming validation project of the EUCAST rapid AST using TLA, and its impact on the early adjustments of antimicrobials by physician

Imipenem Heteroresistance in Nontypeable Haemophilus influenzae

In the post-vaccine era, the nontypeable (i.e. nonencapsulated) Haemophilus influenzae (NTHi) revealed as an opportunistic pathogen causing and exacerbating multiple upper and lower respiratory tract diseases. Worryingly, several studies in various post-vaccine populations have observed a steadily increase of NTHi invasive incidence rates and antibiotic resistance. In this thesis work, we provide evidence indicating that altered penicillin-binding protein 3 (PBP3), slowed drug influx and direct efflux regulation contribute to the development of imipenem hetero-resistance in NTHi. We then established that the enhancement of imipenem-heteroresistant NTHi susceptibility to imipenem under heat stress conditions depends largely on the expression levels of PBP1b, AcrAB-TolC, and OmpP2, indicating again the role of the same pathways. Finally, we characterized the mechanisms of resistance to fluoroquinolones and macrolides in H. influenzae; assessed the extent of the AcrAB-TolC-mediated imipenem resistance; and defined a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae by using whole-genome sequencing

Total Laboratory Automation for Rapid Detection and Identification of Microorganisms and Their Antimicrobial Resistance Profiles

At a time when diagnostic bacteriological testing procedures have become more complex and their associated costs are steadily increasing, the expected benefits of Total laboratory automation (TLA) cannot just be a simple transposition of the traditional manual procedures used to process clinical specimens. In contrast, automation should drive a fundamental change in the laboratory workflow and prompt users to reconsider all the approaches currently used in the diagnostic work-up including the accurate identification of pathogens and the antimicrobial susceptibility testing methods. This review describes the impact of TLA in the laboratory efficiency improvement, as well as a new fully automated solution for AST by disk diffusion testing, and summarizes the evidence that implementing these methods can impact clinical outcomes.</p

Diagnostic de la gastroentérite bactérienne

The etiologic agents of acute gastroenteritis are diverse. The diagnosis of bacterial pathogens is particularly challenging given the large amount of vastly diverse indigenous gastrointestinal organisms present in stool. Multiple methods must be used by the clinical microbiology laboratories to diagnose the cause of acute gastroenteritis, including bacterial cultures, ELISA, and microscopy. Due to the limitations of conventional methods, there is still room for improvement in the detection of pathogens by using the molecular methods. This paper discusses these different diagnostic approaches and limitations