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Abstract 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 bla CTX-M and bla DHA 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

ABSTRACT: Objectives: OCH class C β-lactamases have been reported in several species belonging to the Brucella genus that were formerly known as Ochrobactrum. Moreover, only one complete genome of Brucella pseudintermedia has been published. In this work, we describe the genome of a B. pseudintermedia strain possessing a new blaOCH gene that was isolated from Zophobas morio larvae. Methods: Hybrid whole-genome sequencing analysis (Illumina and Nanopore) was used to identify and characterise the strain (Ops-OCH-23). Phylogenetic analyses based on the 16S rRNA gene sequence and a core-genome alignment were performed to study the relationships among Ops-OCH-23 and deposited genomes. Moreover, all deposited blaOCH genes were compared to the one found in Ops-OCH-23. Results: Ops-OCH-23 showed a susceptibility profile consistent with the production of AmpC β-lactamase(s). Its genome consisted of two chromosomes, of which one carried the blaOCH gene. Such gene encoded a new class C OCH β-lactamase among the fifteen so far reported. Two plasmids (120-Kb and 59-Kb) without any associated antimicrobial resistance genes were also found. Analysis of 16S rRNA revealed that Ops-OCH-23 shared 100% homology with four deposited B. pseudintermedia strains. Moreover, the core-genome analysis indicated that the closest match (279 ΔSNVs) to Ops-OCH-23 was strain CTOTU49018 isolated from an urban environment in Germany in 2013. Conclusion: We described the second complete genome of a B. pseudintermedia that also encoded a new OCH β-lactamase variant. Overall, this report expands our knowledge regarding this rarely isolated Brucella species that have been reported so far only a few times in human sources.

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.

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 Gut colonization with multidrug-resistant Enterobacterales (MDR-Ent) has reached worrisome levels worldwide. In this context, Escherichia ruysiae is a recently described species mostly found in animals. However, its spread and impact on humans is poorly understood. A stool sample from a healthy individual living in India was screened for the presence of MDR-Ent using culture-based methods. Colonies were routinely identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and phenotypically characterized by broth microdilution. Illumina and Nanopore whole-genome sequencing (WGS) platforms were implemented to generate a complete assembly. E. ruysiae genomes deposited in international databases were used for a core genome phylogenetic analysis. An extended-spectrum β-lactamase (ESBL)-producing E. coli strain (S1-IND-07-A) was isolated from the stool. WGS confirmed that S1-IND-07-A was indeed E. ruysiae, belonged to sequence type 5792 (ST5792), core genome (cg) ST89059, serotype O13/O129−:H56-like, clade IV phylogroup, and possessed five virulence factors. A copy of blaCTX-M-15 and five other antimicrobial resistance genes (ARGs) were detected in a conjugative IncB/O/K/Z plasmid. A database search identified 70 further E. ruysiae strains from 16 countries (44, 15, and 11 strains isolated from animals, the environment, and humans, respectively). The core genome phylogeny revealed five major STs: ST6467, ST8084, ST2371, ST9287, and ST5792. Three out of the seventy strains possessed important ARGs: OTP1704 (blaCTX-M-14; ST6467), SN1013-18 (blaCTX-M-15; ST5792), and CE1758 (blaCMY-2; ST7531). These strains were of human, environmental, and wild animal origin, respectively. E. ruysiae may acquire clinically important ARGs and transmit them to other species. Due to its zoonotic potential, further efforts are needed to improve routine detection and surveillance across One Health settings. IMPORTANCE Escherichia ruysiae is a recently described species of the cryptic clades III and IV of the genus Escherichia and is commonly found in animals and the environment. This work highlights the zoonotic potential of E. ruysiae, as it has been shown to colonize the human intestinal tract. Importantly, E. ruysiae may be associated with conjugative plasmids carrying clinically relevant antibiotic resistance genes. Therefore, it is important to closely monitor this species. Overall, this study highlights the need for improved identification of Escherichia species and continued surveillance of zoonotic pathogens in One Health settings.

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.

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.

Objectives: Klebsiella michiganensis is an emerging pathogen. Like Klebsiella pneumoniae, this species is able to acquire antibiotic resistance genes (ARGs) via mobile genetic elements. In this context, K. michiganensis isolates producing carbapenemases of KPC, NDM, IMP and OXA-48-like types have already been reported. Here we characterised a strain (BD-50-Km) isolated from a rectal swab of a Turkish patient hospitalised in Switzerland. Methods: Species identification was initially performed using MALDI-TOF/MS. Antimicrobial susceptibility testing was done by the microdilution method. Whole-genome sequencing (WGS) was performed with both Illumina and Nanopore platforms and was used to confirm species identification, to characterise plasmids and to perform core-genome analyses. Results: BD-50-Km was initially identified as Klebsiella oxytoca and showed reduced susceptibility to imipenem. However, WGS indicated that the isolate was actually K. michiganensis. BD-50-Km carried the blaVIM-1 gene associated with a rare class 1 integron (In87) located on a pST1 196 kb IncC plasmid. This plasmid shares its backbone with many other IncC plasmids found in different species (including five K. michiganensis), but not the same In87 and the remaining region harbouring various ARGs. BD-50-Km belongs to the novel ST342. Moreover, core-genome analysis (single nucleotide variant analysis) showed that BD-50-Km was not closely related to any K. michiganensis strains deposited in NCBI (n = 212), including the 38 so far reported as possessing carbapenemase genes. Conclusion: This is the first report of a blaVIM-possessing K. michiganensis clinical isolate. The spread of plasmid-mediated VIM carbapenemases in this emerging pathogen represents an additional threat to our therapeutic armamentarium.

Objectives: We investigated the use of bacteriophages as a strategy to decolonize intestinal carriers of multidrug-resistant Escherichia coli. Methods: A fermentor was used as a continuous culture system for 48 h. Two different pools of faeces (studies I and II) obtained from volunteers were spiked with a CTX-M-15-producing ST131 E. coli (strain 4901.28) susceptible to bacteriophages and challenged with three doses of INTESTI Bacteriophage cocktail administered at 2, 6 and 10 h after the inoculum. Bacterial typing was performed by implementing microdilution panels, spot test, rep-PCR and whole-genome sequencing (including cgMLST and single-nucleotide variant analysis) obtained using Nanopore and Illumina platforms. Results: In study I, bacteriophages decreased the numbers of 4901.28 dramatically (≤101 CFU/mL after 6 h). In contrast, during study II, a phage-resistant mutant of 4901.28 persisted in the continuous culture (104 CFU/mL at 48 h). Whole-genome sequencing revealed the presence of two additional plasmids in the mutant as well as 11 single-nucleotide variants, including one chromosomal in a glycosyltransferase family 2 protein that is responsible for the transfer of sugars to polysaccharides and lipids. In both studies, the commensal E. coli population remained unchanged by the phage treatment maintaining itself at 108 CFU/mL. Conclusions: Our data indicates that bacteriophage cocktails may be implemented to decolonize some intestinal carriers. However, the individual microbiota composition may have an impact on the development of phage resistance. Mechanisms underlying this phenomenon are likely to be various and complex. Further in vivo studies and protein expression experiments are needed to confirm our observations and hypotheses.

Abstract Background Intensive medical care in companion animal clinics could pose a risk for the selection and dissemination of multidrug-resistant organisms (MDROs). Infection prevention and control (IPC) concepts are key measures to reduce the spread of MDROs, but data on IPC standards in companion animal clinics is sparse. The study assessed IPC standards in seven companion animal clinics and practices in Switzerland by structured IPC audits and combined results with environmental MDRO contamination and MDRO carriage of the personnel. Methods IPC audits were held between August 2018 and January 2019. The observations in 34 IPC areas were scored based on predefined criteria (not fulfilled/partially fulfilled/fulfilled = score 0/1/2). Environmental swabs and nasal and stool samples from veterinary personnel were tested for methicillin-resistant (MR) staphylococci and macrococci and for colistin-resistant, extended-spectrum β-lactamase- and carbapenemase-producing (CP) Enterobacterales (CPE). Species was identified by MALDI-TOF MS, antimicrobial resistance determined by microdilution and β-lactam resistance gene detection, and genetic relatedness assessed by REP−/ERIC-PCR and multilocus sequence typing. Results Of a maximum total IPC score of 68, the institutions reached a median (range) score of 33 (19–55). MDROs were detected in median (range) 8.2% (0–33.3%) of the sampling sites. Clinics with low IPC standards showed extensive environmental contamination, i.e. of intensive care units, consultation rooms and utensils. CPE were detected in two clinics; one of them showed extensive contamination with CP Klebsiella pneumoniae (ST11, bla OXA-48) and MR Staphylococcus pseudintermedius (ST551, mecA). Despite low IPC scores, environmental contamination with MDROs was low in primary opinion practices. Three employees were colonized with Escherichia coli ST131 (bla CTX-M-15, bla CTX-M-27, bla CTX-M-14). Two employees carried CP E. coli closely related to environmental (ST410, bla OXA-181) and patient-derived isolates (ST167, bla NDM-5). MR Staphylococcus aureus (ST225, mecA) and MR S. pseudintermedius (ST551, mecA) of the same sequence types and with similar resistance profiles were found in employees and the environment in two clinics. Conclusions The study indicates that IPC standards in companion animal clinics are variable and that insufficient IPC standards could contribute to the evolution of MDROs which can be transferred between the environment and working personnel. The implementation of IPC concepts in companion animal clinics should urgently be promoted.

Background: The emergence and spread of multidrug-resistant organisms (MDROs) represent a threat to human and animal health. Objectives: To assess duration of carriage of MDROs in dogs and cats presented to veterinary clinics/hospitals in Switzerland. To estimate prevalence, duration of and risk factors for MDRO carriage in their owners and the occurrence of co-carriage in owner-pet pairs. Methods: Prospective, longitudinal, observational study. Nasal swabs and fecal samples were collected from 50 owners of dogs and cats presented to 3 large veterinary hospitals, 1 medium-sized clinic and 1 practice. If pet or owner tested positive for a MDRO, follow-up samples were collected for up to 8 months. Methicillin-resistant (MR) Staphylococcus aureus, MR S. pseudintermedius, MR coagulase-negative staphylococci (MRCoNS), MR Macrococcus spp., cephalosporinase- and carbapenemase-producing (CP) Enterobacterales were isolated and further characterized by MALDI-TOF MS, microdilution, β-lactam resistance gene detection, REP/ERIC-PCR, multilocus sequence typing or whole-genome sequencing. Risk factors for MDRO carriage in owners were explored based on questionnaire-derived data. Results: Five out of 50 owners carried 3rd generation cephalosporin-resistant Enterobacterales (3GC-R-Ent.), and 5/50 MRCoNS. In 3 dogs and 4 cats carriage of 3GC-R-Ent. persisted for up to 136 days after discharge (median 99 days, IQR 83 days, range 36–136 days), in two cats isolates were carbapenem-resistant. Owner-pet co-carriage was not observed. No specific risk factors for MDRO carriage in owners were identified. Conclusions: After discharge from veterinary care, dogs and cats may carry 3GC-R-Ent. for prolonged time periods. Carriage of MDROs was common in owners, but pet-owner co-carriage of the same MDRO was not observed.

Background. Patients colonized with multiple species of carbapenemase-producing Enterobacterales (CPE) are increasingly observed. This phenomenon can be due to the high local prevalence of these pathogens, the presence of important host risk factors, and the great genetic promiscuity of some carbapenemase genes. Methods. We analyzed 4 CPE (Escherichia coli, Klebsiella pneumoniae, Providencia stuartii, Citrobacter sedlakii), 1 extended-spectrum cephalosporin-resistant K. pneumoniae (ESC-R-Kp), and 1 carbapenemase-producing Acinetobacter baumannii simultaneously isolated from a patient transferred from Macedonia. Susceptibility tests were performed using a microdilution MIC system. The complete genome sequences were obtained by using both short-read and long-read whole-genome sequencing technologies. Results. All CPE presented high-level resistance to all aminoglycosides due to the expression of the armA 16S rRNA methylase. In C. sedlakii and E. coli (ST69), both the carbapenemase blaNDM-1 and armA genes were located on an identical IncC plasmid of type 1a. The K. pneumoniae (ST268) and P. stuartii carried chromosomal blaNDM-1 and blaOXA-48, respectively, while the ESC-R-Kp (ST395) harbored a plasmid-located blaCTX-M-15. In the latter 3 isolates, armA-harboring IncC plasmids similar to plasmids found in C. sedlakii and E. coli were also detected. The A. baumannii strain possessed the blaOXA-40 carbapenemase gene. Conclusions. The characterization of the genetic organization of IncC-type plasmids harbored by 3 different species from the same patient offered insights into the evolution of these broad- host-range plasmids. Moreover, we characterized here the first complete genome sequence of a carbapenemase-producing C. sedlakii strain, providing a reference for future studies on this rarely reported species.

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.