Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

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.

First Belgian Report of Ertapenem Resistance in an ST11 Klebsiella Pneumoniae Strain Isolated from a Dog Carrying blaSCO-1 and blaDHA-1 Combined with Permeability Defects.

Klebsiella pneumoniae of sequence type (ST) 11 is a hyper-epidemic nosocomial clone, which is spreading worldwide among humans and emerging in pets. This is the first report, to the best of our knowledge, of multidrug-resistant (MDR) K. pneumoniae ST11 carrying blaSCO-1 and blaDHA-1, isolated from a four-month-old dog in Belgium. Antimicrobial susceptibility testing (AST) of the isolate, performed via broth microdilution following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, revealed resistance to eight different classes of antimicrobials, including carbapenems, in particular ertapenem, third-generation cephalosporins and fluoroquinolones. A hybrid approach, combining long- and short-read sequencing, was employed for in silico plasmid characterization, multi-locus sequence typing (MLST) and the identification and localization of antimicrobial resistance (AMR) and virulence-associated genes. Three plasmids were reconstructed from the whole-genome sequence (WGS) data: the conjugative IncFIB(K), the non-mobilizable IncR and the mobilizable but unconjugative ColRNAI. The IncFIB(K) plasmid carried the blaSCO-1 gene, whereas IncR carried blaDHA-1, both alongside several other antimicrobial resistance genes (ARGs). No virulence genes could be detected. Here, we suggest that the resistance to ertapenem associated with susceptibility to imipenem and meropenem in K. pneumoniae could be related to the presence of blaSCO-1 and blaDHA-1, combined with permeability defects caused by point mutations in an outer membrane porin (OmpK37). The presence of the blaSCO-1 gene on a conjugative IncFIB(K) plasmid is worrisome as it can increase the risk of transmission to humans, to animals and to the environment.

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.

The Isolation of Culturable Bacteria in Ixodes ricinus Ticks of a Belgian Peri-Urban Forest Uncovers Opportunistic Bacteria Potentially Important for Public Health.

Most bacteria found in ticks are not pathogenic to humans but coexist as endosymbionts and may have effects on tick fitness and pathogen transmission. In this study, we cultured and isolated 78 bacteria from 954 Ixodes ricinus ticks collected in 7 sites of a Belgian peri-urban forest. Most isolated species were non-pathogenic environmental microorganisms, and were from the Firmicutes (69.23%), Actinobacteria (17.95%) and Proteobacteria (3.84%) phyla. One bacterium isolate was particularly noteworthy, Cedecea davisae, a rare opportunistic bacterium, naturally resistant to various antibiotics. It has never been isolated from ticks before and this isolated strain was resistant to ampicillin, cefoxitin and colistin. Although cultivable bacteria do not represent the complete tick microbiota, the sites presented variable bacterial compositions and diversities. This study is a first attempt to describe the culturable microbiota of ticks collected in Belgium. Further collections and analyses of ticks of different species, from various areas and using other bacterial identification methods would strengthen these results. However, they highlight the importance of ticks as potential sentinel for opportunistic bacteria of public health importance.

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.

Genomic Insights into Methicillin-Resistant Staphylococcus aureus spa Type t899 Isolates Belonging to Different Sequence Types.

Methicillin-resistant Staphylococcus aureus (MRSA) presenting spa type t899 is commonly associated with sequence type 9 (ST9) but is also increasingly linked to ST398. This study provides genomic insight into the diversity of t899 isolates using core genome multilocus sequence typing (cgMLST), single nucleotide polymorphism (SNP)-based phylogeny, and the description of selected antimicrobial resistance and virulence markers. The SNP-based phylogenic tree showed that isolates sharing the same spa type (t899) but different STs highly diverged in their core and accessory genomes, revealing discriminant antimicrobial resistance (AMR) and virulence markers. Our results highlighted the idea that in a surveillance context where only spa typing is used, an additional multiplex PCR for the detection of the tet(M), sak, and seg genes would be valuable in helping distinguish ST9 from ST398 isolates on a routine basis.IMPORTANCE This study showed the genetic diversity and population structure of S. aureus presenting the same spa type, t899, but belonging to different STs. Our findings revealed that these isolates vary deeply in their core and accessory genomes, contrary to what is regularly inferred from studies using spa typing only. Given that identical spa types can be associated with different STs and that spa typing only is not appropriate for S. aureus isolates that have undergone major recombination events which include the passage of the spa gene (such as in t899-positive MRSA), the combination of both MLST and spa typing methods is recommended. However, spa typing alone is still largely used in surveillance studies and basic characterization. Our data suggest that additional markers, such as tet(M), sak, and seg genes, could be implemented in an easy and inexpensive manner in order to identify S. aureus lineages with a higher accuracy.

Genome Sequences of Livestock-Associated Methicillin-Resistant Staphylococcus aureusspa Type t899 Strains Belonging to Three Different Sequence Types (ST398, ST9, and ST4034).

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an emerging MRSA lineage rapidly evolving in the community. In this report, we present the draft genome sequences of nine LA-MRSA strains. These strains were isolated from meat and a human nasal swab sample and belong to one unique spa type (t899), but to three different sequence types, ST398, ST9, and ST4034.

ST131 fimH22 Escherichia coli isolate with a blaCMY-2/IncI1/ST12 plasmid obtained from a patient with bloodstream infection: highly similar to E. coli isolates of broiler origin.

OBJECTIVES: This study compares the genome of an ST131 CMY-2-producing Escherichia coli isolate from a Danish patient with other ST131 CMY-2-producing E. coli isolates of both human and animal origin. METHODS: In 2016, an ST131 CMY-2-producing E. coli isolate (ESBL20160056) was obtained from a patient with a bloodstream infection. The genome of the ESBL20160056 isolate was compared with genomes from six ST131 CMY-2-producing E. coli isolates obtained from broiler meat imported to Denmark, 15 ST131 CMY-2-producing E. coli isolates obtained from Enterobase (http://enterobase.warwick.ac.uk) and two ST131 CMY-2-producing E. coli from European collaborators. The plasmid from ESBL20160056 was sequenced using a MinION Mk1B (Oxford Nanopore Technologies). RESULTS: The E. coli isolate from the Danish patient clustered together with 13 other fimH22 ST131 CMY-2-producing E. coli isolates in a distinct clade. The clade consisted of genomes from six E. coli isolates from humans collected in Denmark, Spain, Cambodia and the USA, six E. coli isolates obtained from broiler meat samples imported to Denmark from France, the Netherlands and Germany, and two E. coli isolates obtained from broilers in Belgium and Luxembourg. The 101.5 kb plasmid with blaCMY-2 from ESBL20160056 had an IncI1 replicon and belonged to ST12 using the plasmid MLST scheme. In total, 10 of the 14 ST131 E. coli isolates belonging to the fimH22 clade carried an IncI1 ST12 plasmid with blaCMY-2. CONCLUSIONS: From our data, it seems plausible that the ST131 fimH22 CMY-2-producing E. coli isolate obtained from the Danish patient could have a zoonotic broiler origin.

A liquid bead array for the identification and characterization of fljB-positive and fljB-negative monophasic variants of Salmonella Typhimurium.

Salmonella1,4,[5],12:i:- accounts currently for one of the most common serotypes observed worldwide. These isolates do not express the FljB flagellin and mostly derive from Salmonella Typhimurium. They are therefore termed Salmonella Typhimurium monophasic variants (STMV) and are considered of comparable public health risk. Since serological identification of the somatic and flagellar antigens of STMV is not sufficient to demonstrate relatedness with Salmonella Typhimurium, additional assays detecting genetic markers unique to Salmonella Typhimurium are required. In addition, identification of the mutations affecting expression of the flagellar gene fljB can be useful to support the monophasic character observed phenotypically. Finally, genetic subtyping of the various mono- and biphasic Salmonella Typhimurium clonal groups can facilitate their epidemiological follow-up. Here, we present a home-made liquid bead array able to fulfill these requirements. This array confirmed the monophasic character of 240 STMV isolates collected in Belgium during 2014-2015 and identified 10 genetic subtypes. Microevolution in and around the fljB locus linked to IS26 insertions is probably one of the driven force accounting for STMV population diversity. Thanks to its open design, other genetic signatures could later be merged to the assay to subtype additional STMV clonal groups and to detect rare mutations.

Extensive genetic variability linked to IS26 insertions in the fljB promoter region of atypical monophasic variants of Salmonella enterica serovar Typhimurium.

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:- and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.

Molecular typing of monophasic Salmonella 4,[5]:i:- strains isolated in Belgium (2008-2011).

To assess the distribution of Salmonella 4,[5]:i:- subtypes in the Belgian food chain and compare it to the subtypes associated with human infections, a molecular assessment was initiated. Two hundred fifty-three Salmonella isolates serotyped as 4,[5]:i:- during the period 2008-2011 in Belgium and originating from animal productions, food or human clinical samples were analysed by a specific duplex PCR. One hundred ninety-four isolates (76.7%) fit the profile of a S. Typhimurium monophasic variant as defined by the European Food Safety Authority. The other isolates possessed but did not express the phase II flagellin gene (23.3%). Multiple Locus Variable Number of Tandem Repeats Analysis (MLVA) revealed many but closely related profiles in the fljB-negative S. Typhimurium monophasic variant isolates. Some MLVA types were associated with both human and animal isolates but no unique source of human contamination could be demonstrated.

Methodologies for Salmonella enterica subsp. enterica subtyping: gold standards and alternatives.

For more than 80 years, subtyping of Salmonella enterica has been routinely performed by serotyping, a method in which surface antigens are identified based on agglutination reactions with specific antibodies. The serotyping scheme, which is continuously updated as new serovars are discovered, has generated over time a data set of the utmost significance, allowing long-term epidemiological surveillance of Salmonella in the food chain and in public health control. Conceptually, serotyping provides no information regarding the phyletic relationships inside the different Salmonella enterica subspecies. In epidemiological investigations, identification and tracking of salmonellosis outbreaks require the use of methods that can fingerprint the causative strains at a taxonomic level far more specific than the one achieved by serotyping. During the last 2 decades, alternative methods that could successfully identify the serovar of a given strain by probing its DNA have emerged, and molecular biology-based methods have been made available to address phylogeny and fingerprinting issues. At the same time, accredited diagnostics have become increasingly generalized, imposing stringent methodological requirements in terms of traceability and measurability. In these new contexts, the hand-crafted character of classical serotyping is being challenged, although it is widely accepted that classification into serovars should be maintained. This review summarizes and discusses modern typing methods, with a particular focus on those having potential as alternatives for classical serotyping or for subtyping Salmonella strains at a deeper level.