An in-house 45-plex array for the detection of antimicrobial resistance genes in Gram-positive bacteria.

Identifying antimicrobial resistance (AMR) genes and determining their occurrence in Gram-positive bacteria provide useful data to understand how resistance can be acquired and maintained in these bacteria. We describe an in-house bead array targeting AMR genes of Gram-positive bacteria and allowing their rapid detection all at once at a reduced cost. A total of 41 AMR probes were designed to target genes frequently associated with resistance to tetracycline, macrolides, lincosamides, streptogramins, pleuromutilins, phenicols, glycopeptides, aminoglycosides, diaminopyrimidines, oxazolidinones and particularly shared among Enterococcus and Staphylococcus spp. A collection of 124 enterococci and 62 staphylococci isolated from healthy livestock animals through the official Belgian AMR monitoring (2018-2020) was studied with this array from which a subsample was further investigated by whole-genome sequencing. The array detected AMR genes associated with phenotypic resistance for 93.0% and 89.2% of the individual resistant phenotypes in enterococci and staphylococci, respectively. Although linezolid is not used in veterinary medicine, linezolid-resistant isolates were detected. These were characterized by the presence of optrA and poxtA, providing cross-resistance to other antibiotics. Rarer, vancomycin resistance was conferred by the vanA or by the vanL cluster. Numerous resistance genes circulating among Enterococcus and Staphylococcus spp. were detected by this array allowing rapid screening of a large strain collection at an affordable cost. Our data stress the importance of interpreting AMR with caution and the complementarity of both phenotyping and genotyping methods. This array is now available to assess other One-Health AMR reservoirs.

The AMR-ARRAY: A modular bead array detecting ß-lactam, (fluoro) quinolone, colistin, aminoglycoside and macrolide resistance determinants in Gram-negative bacteria.

The aim of this study was to develop a highly multiplexed bead array to detect genes and/or mutations frequently associated with resistance to antimicrobials of the ß-lactam, (fluoro)quinolone, colistin, macrolide and aminoglycoside families in Enterobacteriaceae such as Escherichia coli, Shigella spp. and Salmonella spp. Ligase Chain Reaction and the Luminex® technology were combined in a 53-plex assay designed to target selected genetic markers with 3 internal controls. The AMR-ARRAY consistently detected resistance determinants as compared to phenotypically expressed resistance for 94.7% (856/904) of the assessed resistances. When compared to resistance profiles inferred from whole genome sequencing results, the AMR-ARRAY showed a selectivity and specificity of 99.3% and 100%, respectively. The strong features of the AMR-ARRAY are (i) its competitive cost, currently 18€/sample (ii) its wide analytical scope, currently 50 markers covering 5 antimicrobial families, (iii) its robust and user-friendly design consisting in a single-tube assay conducted in 4 successive steps (iv) its relatively short turnaround time, less than 8 h (v) its ability to detect allelic variability at critical SNPs (vi) its open access and easily upgradable design, with probes sequences, procedure and software source code freely available. The use of the AMR-ARRAY as a screening method in official antimicrobial resistance monitoring could improve the granularity of the collected data and pinpoint remarkable isolates harbouring unusual resistance determinants thereby enabling fit-for-purpose selection of isolates for Whole Genome analysis.

Large diversity of linezolid-resistant isolates discovered in food-producing animals through linezolid selective monitoring in Belgium in 2019.

BACKGROUND: Linezolid is a critically important antibiotic used to treat human infections caused by MRSA and VRE. While linezolid is not licensed for food-producing animals, linezolid-resistant (LR) isolates have been reported in European countries, including Belgium. OBJECTIVES: To: (i) assess LR occurrence in staphylococci and enterococci isolated from different Belgian food-producing animals in 2019 through selective monitoring; and (ii) investigate the genomes and relatedness of these isolates. METHODS: Faecal samples (n = 1325) and nasal swab samples (n = 148) were analysed with a protocol designed to select LR bacteria, including a 44-48 h incubation period. The presence of LR chromosomal mutations, transferable LR genes and their genetic organizations and other resistance genes, as well as LR isolate relatedness (from this study and the NCBI database) were assessed through WGS. RESULTS: The LR rate differed widely between animal host species, with the highest rates occurring in nasal samples from pigs and sows (25.7% and 20.5%, respectively) and faecal samples from veal calves (16.4%). WGS results showed that LR determinants are present in a large diversity of isolates circulating in the agricultural sector, with some isolates closely related to human isolates, posing a human health risk. CONCLUSIONS: LR dedicated monitoring with WGS analysis could help to better understand the spread of LR. Cross-selection of LR transferable genes through other antibiotic use should be considered in future action plans aimed at combatting antimicrobial resistance and in future objectives for the rational use of antibiotics in a One Health perspective.

Colistin resistance genes mcr-1 to mcr-5, including a case of triple occurrence (mcr-1, -3 and -5), in Escherichia coli isolates from faeces of healthy pigs, cattle and poultry in Belgium, 2012-2016.

Colistin is a last-resort antimicrobial used to treat infections caused by multidrug-resistant Gram-negative bacilli (MDR-GNB). The emergence of colistin resistance, particularly linked to mobile genetic elements including the mcr genes, is a major threat to the management of MDR-GNB infections. The aim of this study was to assess the presence of mcr genes in a collection of 40 colistin-resistant commensal Escherichia coli isolated from healthy pigs, cattle and poultry in Belgium between 2012 and 2016. All isolates carried at least one mcr gene. The genes mcr-1 to -5 were observed in this collection. Different replicons associated with mcr genes were identified, including IncHI2/IncHI2A associated with mcr-1, IncX4 associated with mcr-1 and mcr-2, and ColE10 associated with mcr-4. While the occurrence of multiple mcr genes in a single isolate has rarely been reported elsewhere, a triple occurrence (mcr-1, -3 and -5) was found in this study. All isolates were MDR and carried between one and nine different replicons. Seventeen different sequence types were observed among the 40 E. coli isolates. In conclusion, this study revealed the presence of a reservoir of mobile colistin resistance genes (mcr-1 to -5) observed during at least 5 years (2012-2016) in the commensal gut flora of pigs, cattle and poultry in Belgium.

The Proximity of Ribosomal Protein Genes to oriC Enhances Vibrio cholerae Fitness in the Absence of Multifork Replication.

Recent works suggest that bacterial gene order links chromosome structure to cell homeostasis. Comparative genomics showed that, in fast-growing bacteria, ribosomal protein genes (RP) locate near the replication origin (oriC). We recently showed that Vibrio cholerae employs this positional bias as a growth optimization strategy: under fast-growth conditions, multifork replication increases RP dosage and expression. However, RP location may provide advantages in a dosage-independent manner: for example, the physical proximity of the many ribosomal components, in the context of a crowded cytoplasm, may favor ribosome biogenesis. To uncover putative dosage-independent effects, we studied isogenic V. cholerae derivatives in which the major RP locus, S10-spc-α (S10), was relocated to alternative genomic positions. When bacteria grew fast, bacterial fitness was reduced according to the S10 relative distance to oriC The growth of wild-type V. cholerae could not be improved by additional copies of the locus, suggesting a physiologically optimized genomic location. Slow growth is expected to uncouple RP position from dosage, since multifork replication does not occur. Under these conditions, we detected a fitness impairment when S10 was far from oriC Deep sequencing followed by marker frequency analysis in the absence of multifork replication revealed an up to 30% S10 dosage reduction associated with its relocation that closely correlated with fitness alterations. Hence, the impact of S10 location goes beyond a growth optimization strategy during feast periods. RP location may be important during the whole life cycle of this pathogen.IMPORTANCE The role of gene order within the bacterial chromosome is poorly understood. In fast growers, the location of genes linked with the expression of genetic information (i.e., transcription and translation) is biased toward oriC It was proposed that the location of these genes helps to maximize their expression by recruiting multifork replication during fast growth. Our results show that such genomic positioning impacts cell fitness beyond fast-growth conditions, probably across the whole life cycle of fast growers. Thus, the genomic position of key highly expressed genes, such as RP, was finely tuned during the evolution of fast-growing bacteria and may also be important in slow growers. In the near future, many more genes whose genomic position impacts bacterial phenotype will be described. These studies will contribute to discovery the rules of genome organization and application of them for the design of synthetic chromosomes and the creation of artificial life forms.