On January 7, 2020, the World Health Organization (WHO) announced a novel coronavirus to be the cause of unclear pneumonia cases in China.…

Introduction: Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). Materials and Methods: Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1^∘C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed. Results: Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT. Discussion: We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.

Vancomycin-resistant enterococci (VRE) are relevant nosocomial pathogens with an increasing incidence in the last decades. Their transmission is optimal in the hospital setting, as it offers two potential, large reservoirs that are closely related: susceptible patients and their environment. Here we investigate the role of the hospital environment in the nosocomial transmission of VRE by establishing concrete links between contaminated surfaces and colonized/infected patients in outbreak and non-outbreak settings. Environmental and patient VRE isolates were collected between 2013 and 2019 and analyzed by whole-genome sequencing (WGS), subsequent multilocus sequence typing (MLST), and core genome (cg) MLST. Pairs of isolates differing in <3 alleles were rated as closely related, making a transmission likely. Fifty-three environmental VRE isolates were analyzed. MLST sequence types (ST) ST203 (50.0%), ST192 (21.3%), ST117 (17.3%), ST721 (8.8%), ST80 (2%), and ST1489 (0.7%) were detected, carrying the resistance determinants vanA (72.7%), vanB (24%), or both (3.3%). Of the 53 environmental isolates, 51 were found to form five clusters with genetically related patient isolates (n = 97 isolates). WGS confirms the role of the environment in the transmission dynamics of VRE in both the outbreak and non-outbreak settings, highlighting the importance of prevention and control of VRE spread.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that require effective infection control measures, representing a challenge for healthcare systems. This study aimed at identifying risk factors associated with prolonged VRE carriage and determining the rate of clearance that allows the discontinuation of contact precautions. During a 2-year study, screening was performed in patients with a history of VRE or at risk of becoming colonized. After bacterial identification and antibiotic susceptibility testing, glycopeptide resistance was confirmed by PCR. Isolates were compared via whole genome sequence-based typing. Risk factors were recorded, and follow-up screening was performed upon readmission, defining patients as long-term carriers if still colonized ≥10 weeks after first detection. Of 1059 patients positive for VRE, carriage status was assessed upon readmission in 463 patients. VRE was cleared in 56.4% of the cases. Risk factors associated with long-term persistence were hospital stays (frequency, length), hemato-oncological disease, systemic treatment with steroids, and use of antibiotics. No specific genotypic clustering was observed in patients with VRE clearance or persistence. VRE clearance is possibly underestimated. The identification of risk factors favoring long-term carriage may contribute to a targeted implementation of infection control measures upon readmission of patients with history of VRE.

Carbapenemase-producing bacteria are a growing issue worldwide. Most phenotypic detection methods are culture-based, requiring long incubation times. We present a phenotypic screening panel for detection of carbapenem non-susceptibility and differentiation of carbapenemase classes and AmpC, the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). It was validated on 7 reference strains and 20 challenge Enterobacterales isolates. Broth microdilution (BMD) and combination disk test (CDT) were also performed, as well as PCR as reference method. The panel based on the synergy between meropenem and carbapenemase inhibitors, determined by incubating these substances with bacterial suspension on a MALDI-TOF MS target and subsequently assessing bacterial growth on the target’s spots by MS. After 4 hours of incubation, DOT-MGA correctly identified KPC, MBL and OXA (100% agreement with PCR). Detection of AmpC coincided with BMD and CDT but agreement with PCR was low, not ruling out false negative PCR results. DOT-MGA delivered more accurate results than BMD and CDT in a significantly shorter time, allowing for detection of carbapenem non-susceptibility, MIC determination and carbapenemase differentiation in one step.