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Finding strategies for decolonizing gut carriers of multidrug-resistant Escherichia coli (MDR-Ec) is a public-health priority. In this context, novel approaches should be validated in preclinical in vivo gut colonization models before being translated to humans. However, the use of mice presents limitations. Here, we used for the first time Zophobas morio larvae to design a new model of intestinal colonization (28-days duration, T28). Three hyperepidemic MDR-Ec producing extended-spectrum β-lactamases (ESBLs) or carbapenemases were administered via contaminated food to larvae for the first 7 days (T7): Ec-4901.28 (ST131, CTX-M-15), Ec-042 (ST410, OXA-181) and Ec-050 (ST167, NDM-5). Growth curve analyses showed that larvae became rapidly colonized with all strains (T7, ~106–7 CFU/mL), but bacterial load remained high after the removal of contaminated food only in Ec-4901.28 and Ec-042 (T28, ~103–4 CFU/mL). Moreover, larvae receiving a force-feeding treatment with INTESTI bacteriophage cocktail (on T7 and T10 via gauge needle) were decolonized by Ec-4901.28 (INTESTI-susceptible); however, Ec-042 and Ec-050 (INTESTI-resistant) did not. Initial microbiota (before administering contaminated food) was very rich of bacterial genera (e.g., Lactococcus, Enterococcus, Spiroplasma), but patterns were heterogeneous (Shannon diversity index: range 1.1–2.7) and diverse to each other (Bray–Curtis dissimilarity index ≥30%). However, when larvae were challenged with the MDR-Ec with or without administering bacteriophages the microbiota showed a non-significant reduction of the diversity during the 28-day experiments. In conclusion, the Z. morio larvae model promises to be a feasible and high-throughput approach to study novel gut decolonization strategies for MDR-Ec reducing the number of subsequent confirmatory mammalian experiments.

ABSTRACT: Objectives: Core-genome single nucleotide variant (cgSNV) analysis represents a powerful tool for epidemiological investigations of multidrug-resistant (MDR) bacteria. However, cgSNV thresholds to confirm whether isolates are the same clone are not formally defined. Methods: We implemented hybrid whole-genome sequencing to study the genomic changes of four MDR isolates belonging to hyperepidemic sequence types (STs) during 20 propagation steps (T20) on MacConkey and CHROMID(R) ESBL plates. The following strains were analyzed: Klebsiella pneumoniae AE-2247421 (OXA-48/NDM-1-producing, ST101), K. pneumoniae MCL-2017-2 (CTX-M-15-producing, ST307), Escherichia coli Ec-042 (OXA-181-producing, ST410), and E. coli Ec-050 (NDM-5-producing, ST167). The genome assembly at T5 and T20 was compared to that at time point zero (T0) and to two reference genomes. Results: At T20, AE-2247421 lost the IncL blaOXA-48-carrying plasmid when grown on CHROMID(R) ESBL plates, while a large fragment encompassing blaNDM-1 was lost from its IncC plasmid when grown on both plates. In contrast, no structural changes were noted for the other three strains. Regarding the cgSNVs, the following results were obtained at T5 and T20 (ranges considering the different agar plates and reference genomes): AE-2247421 (1–8 and 2–12 cgSNVs), MCL-2017-2 (both 1–2 cgSNVs), Ec-042 (both 0 cgSNVs), and Ec-050 (0–6 and 0–9 cgSNVs). Conclusion: We showed that structural changes and accumulation of cgSNVs can occur in few propagation steps under laboratory conditions. These changes might also arise in the clinical context in a short time, especially under antibiotics treatment. This phenomenon should be carefully considered because it might affect the final interpretation of epidemiological genomic analyses.

We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESC-R-Ent) in 42 volunteers. Both methods were performed using native and pre-enriched (broth supplemented with cefuroxime) stools. Native culture screening on CHROMID® ESBL plates resulted in 17 positive samples, whereas the pre-enriched culture (gold-standard) identified 23 carriers. Overall, 26 ESC-R-Ent strains (24 Escherichia coli) were identified: 25 CTX-M and 3 DHA-1 producers (2 co-producing CTX-Ms). Using the SMS on native stool (“native SMS”) with thresholds ≥60% for both identity and coverage, only 7 of the 23 pre-enriched culture-positive samples resulted positive for blaCTX-M/blaDHA genes (native SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 59.0%; specificity 100%). Moreover, an average of 31.5 and 24.6 antimicrobial resistance genes (ARGs) were detected in the 23 pre-enriched culture-positive and the 19 negative samples, respectively. When the pre-enriched SMS was implemented, more blaCTX-M/blaDHA genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 78.3%; specificity 75.0%). In addition, the pre-enriched SMS identified on average 38.6 ARGs/sample, whereas for the corresponding native SMS it was 29.4 ARGs/sample. Notably, stools resulting false-negative by using the native SMS had lower concentrations of ESC-R-Ent (average: ~105 vs. ~107 CFU/g) and E. coli classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of blaCTX-M/blaDHA genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-Ent. However, its performance was not comparable to the pre-enriched culture-based approach.

ABSTRACT: Background: The spread of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) strains belonging to high-risk sequence types (STs) is a concern. For Switzerland, national data about the molecular features (especially the STs) of CP-Kp of human origin is not available. In veterinary clinics, ST11 and ST307 blaOXA-48-possessing K. pneumoniae strains have been recently reported. Methods: We analysed a collection of 285 K. pneumoniae genomes (170 were CP-Kp) isolated in Switzerland from human and non-human sources during 2006–2020. Whole-genome sequencing, core genome phylogenies and public databases were used to present a detailed overview regarding carbapenemases, STs and plasmids. Results: The top five STs were (main carbapenemase gene) ST512 (blaKPC-3), ST258 (blaKPC-2) and ST101 (blaOXA-48), consisting of strains of human origin only, and ST11 (blaOXA-48) and ST307 (blaOXA-48) strains isolated from human, animal and environmental sources. However, during 2016–2020, the main STs for CP-Kp were ST11 (17.6%), ST307 and ST101 (both 14.7%), whereas ST258 (5.9%) and ST512 (4.4%) significantly declined. Most carbapenemase genes were carried on plasmids already described. Core genome analysis revealed that ST11 K. pneumoniae of animal and human origin were closely related, whereas those of ST307 were distant. Conclusions: We described, for the first time, the features of the CP-Kp circulating in Switzerland in human and non-human settings. Our genomic analysis revealed that the emerging high-risk ST11 and ST307 lineages were often isolated from non-human settings. This study provided a baseline for further whole-genome sequencing-based One-Health surveillance of CP-Kp and emphasized the need for metadata to track dissemination routes between the different settings.

ABSTRACT: Objectives: Patients hospitalised abroad can become colonised with multidrug-resistant bacteria and import them to their home countries. In this study, we characterised an OXA-484 carbapenemase-producing Escherichia coli strain from a Swiss patient infected by SARS-CoV-2 and repatriated from India. Methods: At admission to Switzerland (April 2021), the patient undertook a nasopharyngeal swab to search for SARS-CoV-2 and a rectal swab to detect multidrug-resistant bacteria. Both SARS-CoV-2 and E. coli isolates were whole-genome sequenced and analysed for phylogenetic relatedness. Results: The patient was infected with the SARS-CoV-2 B.1.617.2 lineage (VOC Delta), a lineage that began to be reported across Switzerland at that time. He was also colonised with a sequence type 410 (ST410) E. coli strain (L3452210II) producing OXA-484, a single amino acid variant of OXA-181. The blaOXA-484 gene was carried by a 51.5 kb IncX3 plasmid identical to those described in blaOXA-181-harbouring ST410 E. coli strains. Core genome analysis showed that L3452210II was identical (ΔSNV ≤23) to two ST410 OXA-484 producers recently reported in Qatar and Germany, but differed from other ST410 OXA-181 producers reported worldwide. Conclusion: The patient was infected by an emerging SARS-CoV-2 variant and also imported an E. coli producing OXA-484, an OXA-48-like carbapenemase not yet reported in Switzerland. The genetic background of L3452210II indicated that blaOXA-484 shared the same plasmid as blaOXA-181, but its bacterial host differed from most of the pandemic OXA-181-producing ST410 strains reported previously. This case description underlines that the COVID-19 crisis can contribute to the worldwide spread of emerging carbapenemase producers.

Antimicrobial resistant (AMR) Enterobacterales are widely distributed among the healthy population of the Indochinese peninsula, including Laos. However, the local reservoir of these pathogens are currently not known and possible sources such as agricultural settings and food have rarely been analyzed. In this work, we investigated the extended-spectrum cephalosporin- (ESC-) and colistin-resistant Escherichia coli strains (CST-R-Ec) isolated from the gut of local people, feces of poultry, and from chicken meat (60 samples each group) in Laos. Whole-genome sequencing (WGS) analysis based on both short- and long-read sequencing approaches were implemented. The following prevalence of ESC-R-Ec and CST-R-Ec were recorded, respectively: local people (70 and 15%), poultry (20 and 23.3%), and chicken meat (21.7 and 13.3%). Core-genome analysis, coupled with sequence type (ST)/core-genome ST (cgST) definitions, indicated that no common AMR-Ec clones were spreading among the different settings. ESC-R-Ec mostly possessed blaCTX–M–15 and blaCTX–M–55 associated to ISEcp1 or IS26. The majority of CST-R-Ec carried mcr-1 on IncX4, IncI2, IncP1, and IncHI1 plasmids similar or identical to those described worldwide; strains with chromosomal mcr-1 or possessing plasmid-mediated mcr-3 were also found. These results indicate a high prevalence of AMR-Ec in the local population, poultry, and chicken meat. While we did not observe the same clones among the three settings, most of the blaCTX–Ms and mcr-1/-3 were associated with mobile-genetic elements, indicating that horizontal gene transfer may play an important role in the dissemination of AMR-Ec in Laos. More studies should be planned to better understand the extent and dynamics of this phenomenon.