Highlights • Decades of colistin use to treat Enterobacteriaceae infections in animals. • Moderate to high occurrence of colistin resistance in pathogenic animal strains. • Low resistance to colistin ...in indicator E. coli in Europe compared with Asia. • Some Salmonella serovars exhibit a degree of intrinsic resistance. • Resistance in Salmonella depends on percentages of intrinsically resistant strains.
Resistance to antibiotics is escalating and threatening humans and animals worldwide. Different countries have legislated or promoted the ban of antibiotics as growth promoters in livestock and ...aquaculture to reduce this phenomenon. Therefore, to improve animal growth and reproduction performance and to control multiple bacterial infections, there is a potential to use probiotics as non-antibiotic growth promoters. Lactic acid bacteria (LAB) offer various advantages as potential probiotics and can be considered as alternatives to antibiotics during food-animal production. LAB are safe microorganisms with abilities to produce different inhibitory compounds such as bacteriocins, organic acids as lactic acid, hydrogen peroxide, diacetyl, and carbon dioxide. LAB can inhibit harmful microorganisms with their arsenal, or through competitive exclusion mechanism based on competition for binding sites and nutrients. LAB endowed with specific enzymatic functions (amylase, protease…) can improve nutrients acquisition as well as animal immune system stimulation. This review aimed at underlining the benefits and inputs from LAB as potential alternatives to antibiotics in poultry, pigs, ruminants, and aquaculture production.
Abstract
Objectives
WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based ...AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output.
Methods
The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins.
Results
Genotype–phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype–phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance.
Conclusions
WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.
CNCM I-5369, formerly
CNCM I-5369, produces bacteriocins that are remarkably active against Gram-negative bacteria, among which is the
carrying
-1 gene that is involved in resistance to colistin. ...These bacteriocins present in the culture supernatant of the producing strain were extracted and semi-purified. The fraction containing these active bacteriocins was designated as E20. Further, E20 was loaded onto alginate nanoparticles (Alg NPs), leading to a highly active nano-antibiotics formulation named hereafter Alg NPs/E20. The amount of E20 adsorbed on the alginate nanoparticles was 12 wt.%, according to high-performance liquid chromatography (HPLC) analysis. The minimal inhibitory concentration (MIC) values obtained with E20 ranged from 250 to 2000 μg/mL, whilst those recorded for Alg NPs/E20 were comprised between 2 and 4 μg/mL, which allowed them to gain up to 500-fold in the anti-
activity. The damages caused by E20 and/or Alg NPs/E20 on the cytology of the target bacteria were characterized by transmission electron microscopy (TEM) imaging and the quantification of intracellular proteins released following treatment of the target bacteria with these antimicrobials. Thus, loading these bacteriocins on Alg NPs appeared to improve their activity, and the resulting nano-antibiotics stand as a promising drug delivery system.
Lactobacillus paracasei CNCM I-5369 isolated from a traditional Algerian dairy product produces extracellular inhibitory substances, namely, bacteriocins, which are active against a panel of ...pathogenic Escherichia coli strains. This activity was observed only at a narrow pH 4.5-5, and resulted to be heat stable and sensitive to the action of proteolytic enzymes, which indicate a proteinaceous nature. This new strain has a genome of 2,752,975 bp, with a 46.6% G + C ratio and contains at least 2664 coding sequences. The Bagel software analysis identified five open reading frames (ORFs) that are translated to new class II bacteriocin. Each ORF was cloned in frame with a His-tag tail and expressed in E. coli BL21 (DE3) (pLysS) strain. Of note, each fusion protein carrying any of these ORFs at the C- or N-terminal position resulted to be active against E. coli 184 strain used as target organism. This manuscript reports the first multi-bacteriocinogenic strain producing five new class II bacteriocins with activity against Gram-negative bacilli (GNB), namely, E. coli. Heterologous expression and activity of each new class II bacteriocin were demonstrated.
Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal ...and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.
Campylobacter was detected in 76% of broiler meat products collected in retail outlets during a monitoring plan carried out in France throughout 2009. Campylobacter jejuni was the most prevalent ...species (64.7% of products being contaminated). The 175 C. jejuni isolates collected were characterized. MLST typing results confirmed substantial genetic diversity as the 175 C. jejuni isolates generated 76 sequence types (STs). The ST-21, ST-45 and ST-464 complexes predominated accounting for 43% of all isolates.
A class-specific PCR to screen the sialylated lipooligosaccharide (LOS) locus classes A, B and C showed that 50.3% of the C. jejuni isolates harbored sialylated LOS.
The antimicrobial resistance profiles established using a subset of 97 isolates showed that resistance to tetracycline was the most common (53.6%), followed with ciprofloxacin and nalidixic acid (32.9%, and 32.0% respectively). All the tested isolates were susceptible to erythromycin, chloramphenicol and gentamicin.
Clear associations were demonstrated between certain clonal complexes and LOS locus classes and between certain clonal complexes and antimicrobial resistance.
This work paints a representative picture of C. jejuni isolated from poultry products circulating in France, providing data on STs, LOS locus classes and antibiotic resistance profiles in isolates recovered from products directly available to the consumer.
•76% of the French chicken meat was contaminated by Campylobacter.•The C. jejuni population was genetically diverse and the ST-21 complex predominated.•More than 25% of the isolates were potentially neuropathic with LOS class A or B.•Isolates were susceptible to erythromycin, but more than 30% were ciprofloxacin-resistant.•ST-21 complex isolates had mostly LOS class C and a high proportion was ciprofloxacin-resistant.
We studied a collection of 79 colistin-resistant
isolates isolated from diseased pigs in France between 2009 and 2013. We determined a number of phenotypic and genetic characters using broth ...microdilution to characterize their antimicrobial susceptibility. We performed pulse field gel electrophoresis (PFGE) to assess their genetic diversity and assign them to phylogroups. High-throughput real-time PCR micro-array was used to screen for a selection of genetic markers of virulence, and PCR and sequencing of the main recognized resistance genes allowed us to investigate the mechanisms of colistin resistance. Results showed that isolates belonged to several phylogroups and most had a unique PFGE profile. More than 50% of the isolates were also resistant to sulfonamides, trimethoprim, tetracycline, ampicillin or chloramphenicol. The
gene was detected in 70 out of 79 isolates and was transferred by conjugation in 33 of them, sometimes together with resistance to sulfonamides, trimethoprim, tetracycline, ampicillin, chloramphenicol, cefotaxime, or gentamicin. Mutations in the amino-acid sequences of proteins MgrB, PhoP, PhoQ, PmrB, but not PmrA, were detected in isolates with or without the
gene. More than one-third of the isolates harbored the F18, F4,
, or
virulence markers. In conclusion, although most isolates had a unique PFGE profile, a few particular combinations of phylogenetic groups, virulence genes and mutations in the sequenced genes involved in colistin resistance were identified on a number of occasions, suggesting the persistence of certain isolates over several years.
Introduction
Within the 2007–2014 programme for the surveillance of antimicrobial resistance (AMR) in livestock in France,
mcr-1
prevalence average in commensal
Escherichia coli
was found to be 5.9% ...in turkeys and 1.8% in broilers, indicating that mobile colistin resistance had spread in farm animals. In 2017, the French national Ecoantibio2 plan was established to tackle AMR in veterinary medicine, with the objective of a 50% reduction in exposure to colistin in farm animals within 5 years (from 2014–2015 to 2020). Our objective was to update data concerning the prevalence and molecular epidemiology of colistin resistance, in consideration of colistin sales in poultry production in France.
Methods
Antimicrobial susceptibility of commensal
E. coli
isolated from broilers and turkeys at slaughterhouse was determined by broth micro-dilution. The
mcr
genes were screened by polymerase chain reaction (PCR). Whole genome sequencing (WGS) was used to investigate the genetic diversity of colistin-resistant isolates. Transformation experiments enabled identification of the
mcr
-bearing plasmid replicon types. The correlation between prevalence of colistin resistance and colistin usage data was explored statistically.
Results and discussion
In 2020, in France, the resistance prevalence to colistin in poultry production was 3% in turkeys and 1% in broilers, showing a significant highly positive correlation with a −68% decrease of poultry exposure to colistin since 2014. Only the
mcr-1
gene was detected among the colistin-resistant
E. coli
. More than 80% of isolates are multi-drug resistant with 40% of isolates originating from turkeys and 44% originating from broilers co-resistant to the critically important antimicrobial ciprofloxacin. Most of the strains had no clonal relationship. The
mcr
gene was located in different plasmid types, carrying various other AMR genes. The decrease in colistin resistance among poultry in France can be considered a positive outcome of the national action plans for reduced colistin usage.
Proposals to update the harmonised monitoring and reporting of antimicrobial resistance (AMR) from a public health perspective in Salmonella, Campylobacter coli, Campylobacter jejuni, ...Escherichia coli, Enterococcus faecalis, Enterococcus faecium and methicillin‐resistant Staphylococcus aureus (MRSA) from food‐producing animals and derived meat in the EU are presented in this report, accounting for recent trends in AMR, data collection needs and new scientific developments. Phenotypic monitoring of AMR in bacterial isolates, using microdilution methods for testing susceptibility and interpreting resistance using epidemiological cut‐off values is reinforced, including further characterisation of those isolates of E. coli and Salmonella showing resistance to extended‐spectrum cephalosporins and carbapenems, as well as the specific monitoring of ESBL/AmpC/carbapenemase‐producing E. coli. Combinations of bacterial species, food‐producing animals and meat, as well as antimicrobial panels have been reviewed and adapted, where deemed necessary. Considering differing sample sizes, numerical simulations have been performed to evaluate the related statistical power available for assessing occurrence and temporal trends in resistance, with a predetermined accuracy, to support the choice of harmonised sample size. Randomised sampling procedures, based on a generic proportionate stratified sampling process, have been reviewed and reinforced. Proposals to improve the harmonisation of monitoring of prevalence, genetic diversity and AMR in MRSA are presented. It is suggested to complement routine monitoring with specific cross‐sectional surveys on MRSA in pigs and on AMR in bacteria from seafood and the environment. Whole genome sequencing (WGS) of isolates obtained from the specific monitoring of ESBL/AmpC/carbapenemase‐producing E. coli is strongly advocated to be implemented, on a voluntary basis, over the validity period of the next legislation, with possible mandatory implementation by the end of the period; the gene sequences encoding for ESBL/AmpC/carbapenemases being reported to EFSA. Harmonised protocols for WGS analysis/interpretation and external quality assurance programmes are planned to be provided by the EU‐Reference Laboratory on AMR.
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