Andrea Endimiani’s research while affiliated with National Institute of Allergy and Infectious Diseases, National Institutes of Health and other places

Klebsiella oxytoca complex ( Ko C) are important nosocomial pathogens that can be reservoirs of transmissible extended-spectrum β-lactamase (ESBL) genes. Therefore, it is essential for clinical microbiology laboratories to distinguish between Ko C producing ESBLs (ESBL- Ko C) and those hyperproducing the natural OXY-type β-lactamases (hOXY- Ko C). We investigated the abilities of VITEK 2 with and without using the Advanced Expert System (AES) to detect ESBL producers among 44 well-characterized Ko C strains (including 11 ESBL- Ko C and 21 hOXY- Ko C). VITEK 2/AES showed 100% sensitivity (Se) and 64.7% specificity (Sp), whereas the VITEK 2 coupled by the Clinical Laboratory Standards Institute (CLSI) ESBL confirmatory tests (ESBL-CTs; i.e., disk-combination tests) showed 100% Se and 97.5% Sp to detect ESBL- Ko C. We also analyzed Ko C-specific screening cutoffs for ceftriaxone (CRO), cefpodoxime (CPD), ceftazidime (CAZ), cefotaxime (CTX), and aztreonam (ATM) to negate unnecessary ESBL-CTs. As a result, we propose the following screening cutoffs (minimum inhibitory concentration [MIC] and inhibition zone diameter): CRO, >4 µg/mL and ≤16 mm; CPD, >4 µg/mL and ≤10 mm; CAZ, >1 µg/mL and ≤22 mm (European Committee on Antimicrobial Susceptibility Testing [EUCAST] disk)/≤30 mm (CLSI disk); CTX, >4 µg/mL and ≤12 mm (EUCAST disk)/≤22 mm (CLSI disk); ATM, >1 µg/mL and ≤28 mm. Notably, all suggested cutoffs could assure 100% Se and high Sp/positive predictive values for our 44 Ko C strains. In conclusion, the AES performed poorly, while VITEK 2 with the CLSI ESBL-CTs yielded a reliable methodology to distinguish ESBL- Ko C from hOXY- Ko C. This study also proposed revised screening cutoffs for detecting ESBL- Ko C and reducing the unnecessary use of ESBL-CTs. IMPORTANCE Species within the Klebsiella oxytoca complex ( Ko C) are emerging clinical pathogens of increasing concern. These bacteria can acquire plasmid-mediated ESBL genes, seriously complicating antibiotic treatment and overall management of infected patients. Differentiating ESBL-producing from non-ESBL-producing Ko C isolates is therefore crucial. However, this task presents significant challenges for clinical laboratories. In this work, we showed that the automated VITEK 2 system equipped with its AES fails to differentiate the two groups of Ko C isolates. In contrast, VITEK 2 alone followed by the ESBL screen and phenotypic confirmatory tests provides accurate differentiation. Since this latter approach increases the diagnostic workload, we also proposed new screening cutoffs for key cephalosporins that may reduce the current high number of unnecessary confirmatory tests.

Ambler class A IMI enzymes are minor carbapenemases primarily associated with the Enterobacter cloacae complex. Screening soil surfaces of a well-visited public garden revealed an IMI-6-producing Enterobacter asburiae ST657 near a trash can. The strain harboured a 163-kb conjugative IncFII(Yp) plasmid containing blaIMI−6 and putative virulence genes. WGS comparative analysis with other clinical and non-clinical E. asburiae ST657 showed that it was genetically related to strains from a patient in France (62 ΔSNPs) and from retail salad in Switzerland (79 ΔSNPs). This finding suggests possible trans-sectoral dissemination of IMI-producing bacteria raising concerns since they could further spread into the community.

Living in high-endemic regions increases the risk of intestinal colonization by multidrug-resistant Enterobacterales (MDR-Ent). This study investigated Swiss expatriates residing abroad (≥ 3 months) to assess their colonization status upon returning to Switzerland. Selective culture-based methods were implemented to detect third-generation cephalosporins- (3GC-R), carbapenems- (CR), and colistin-resistant (COL-R) strains. Whole-genome sequencing was used to characterize antimicrobial resistance genes, sequence type (ST), and phylogroup of MDR-Ent. Epidemiological data were analyzed using uni- and multivariable models to identify risk factors, providing crude and adjusted odds ratios (ORs). Among 196 participants living across Africa, Asia, the Americas, and Europe, the overall MDR-Ent colonization prevalence was 42.9%. Continent of residence emerged as a significant risk factor (p = 0.04) for colonization: Africa (adjusted OR = 3.4, 95% CI 1.0–11.0) and Asia (adjusted OR = 4.7, 95% CI 1.5–15.0). Extended-spectrum β-lactamase-producing Escherichia coli (Ec) was the most frequent isolated species (n = 107 out of 119 Ent). Most 3GC-R-Ec possessed blaCTX−M genes (n = 89; 83.2%) and pandemic lineages were frequent (e.g., ST69 and ST131, n = 18). No CR-Ent were detected, but some COL-R strains (n = 18; of which 15 Ec) harbored the mcr-1.1 gene. Expatriates represent an understudied population at risk of MDR-Ent colonization. This population may contribute to the importation and potential dissemination of dangerous bacteria into low-prevalence countries, as shown in this Swiss study, warranting further investigation and surveillance.

Klebsiella pneumoniae (Kp) is a Gram-negative pathogen responsible for both hospital- and community-acquired infections. Kp is classified into 2 distinct pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). First described in Taiwan in 1986, hvKp are highly pathogenic and characterized by unique phenotypic and genotypic traits. The hypermucoviscous (hmv) phenotype, generally marked by overproduction of the capsule, is often associated with hvKp, although recent studies show that some cKp strains may also have this characteristic. Furthermore, hvKp can cause severe community-acquired infections in healthy people and have been associated with metastatic infections such as liver abscess, meningitis, and endophthalmitis. HvKp are increasingly being reported in hospital-acquired settings, complicating treatment strategies. In particular, while hvKp have historically been antibiotic-susceptible, multidrug-resistant (MDR) strains have emerged and pose a significant public health threat. The combination of high virulence and limited antibiotic options demands further research into virulence mechanisms and rapid identification methods. This review discusses the epidemiology of hvKp and their virulence factors, highlighting the importance of phenotypic and non-phenotypic tests, including next-generation molecular diagnostics, for the early detection of hvKp.

Acinetobacter baumannii causes hospital-acquired infections in human patients with compromised immune system. Strains associated to nosocomial infections are often resistant to carbapenems and belong to few international clones (IC1-11). A. baumannii strains have been found in extra-hospital sources including food products. While molecular epidemiology of A. baumannii is well described in hospital settings, extra-hospital settings remain poorly investigated. In the frame of two screening campaigns for the presence of Gram-negative bacteria in retailed raw meat, we collected 70 A. baumannii isolates. To investigate if there was a genetic link between food isolates and those causing infections in humans, a core-genome pyMLST analysis was conducted including genomes from different sources as well as representatives of the IC1-11 (n = 224) retrieved from the NCBI database. Strains from raw meat were genetically diverse with 49 sequence types present among the 70 isolates. The core-genome phylogenetic analysis demonstrated that some A. baumannii strains from raw meat shared high genomic similarity with strains associated to human infections carrying carbapenem-resistance genes and belonging to IC11 and other clonal complexes (CC) that are emerging globally, like CC33. Strains from raw meat were able to acquire genes conferring carbapenem-resistance in vitro. If A. baumannii cannot be considered as a food-borne pathogen, colonization of raw meat can favor the propagation of this species in the community, facilitating the entrance of novel clones in the hospital environment. Once entering hospital settings, susceptible clones could turn into multidrug-resistant lineages under strong selective pressure. To avoid this risk, accurate hands and kitchen utensils hygiene should be recommended to all those in contact with raw meat.

The increasing prevalence of gut colonization with CTX-M extended-spectrum β-lactamase- and/or DHA plasmid-mediated AmpC-producing Escherichia coli is a concern. Here, we evaluate Nanopore-shotgun metagenomic sequencing (Nanopore-SMS) latest V14 chemistry to detect blaCTX-M and blaDHA genes from healthy stools. We test 25 paired samples characterized with culture-based methods (native and pre-enriched). Antimicrobial resistant genes (ARGs) are detected from reads and meta-assembled genomes (MAGs) to determine their associated genetic environments (AGEs). Sensitivity and specificity of native Nanopore-SMS are 61.1% and 100%, compared to 81.5% and 75% for pre-enriched Nanopore-SMS, respectively. Native Nanopore-SMS identifies only one sample with an AGE, whereas pre-enriched Nanopore-SMS recognizes 9/18 plasmids and 5/9 E. coli chromosomes. Pre-enriched Nanopore-SMS identifies more ARGs than native Nanopore-SMS (p < 0.001). Notably, blaCTX-Ms and blaDHAs AGEs (plasmid and chromosomes) are identified within 1 hour of sequencing. Furthermore, microbiota analyses show that pre-enriched Nanopore-SMS results in more E. coli classified reads (47% vs. 3.1%), higher differential abundance (5.69 log2 fold) and lower Shannon diversity index (p < 0.0001). Nanopore-SMS has the potential to be used for intestinal colonization screening. However, sample pre-enrichment is necessary to increase sensitivity. Further computational improvements are needed to reduce the turnaround time for clinical applications.

Leminorella grimontii strain LG-KP-E1-2-T0 was isolated from Zophobas morio larvae. It showed a susceptibility phenotype compatible with the expression of an inducible extended-spectrum β-lactamase. The presence of a chromosomal bla gene encoding for the class A GRI-1 β-lactamase was revealed by whole-genome sequencing. GRI-1 shared the highest amino acid identity with RIC-1 and OXY-type β-lactamases (76–80%). Analysis of six further publicly-available L. grimontii draft genomes deposited in NCBI revealed that bla GRI−1 was always present. Core-genome analysis indicated that LG-KP-E1-2-T0 was unique from other strains. We provided the first complete genome of L. grimontii and new insights on its chromosomal β-lactamases.

We present the complete genome sequence of Pseudomonas canadensis. The strain (Pcan-CK-23) was isolated from Zophobas morio (superworm) larvae. The genome consisted of a 6,424,469 bp chromosome with a GC content of 60.3% and 5,973 genes. Pcan-CK-23 can be used as a reference genome for further studies with P. canadensis.