Comparative phylogenomics of extended-spectrum beta-lactamase-producing Escherichia coli revealed a wide diversity of clones and plasmids in Spanish chicken meat

International audienceAnimal food products are important sources of zoonotic agents, increasing the risk of exposure to antibioticresistant bacteria from farm to fork. Therefore, we aimed to detect and fully characterise Extended-Spectrum Beta-Lactamase (ESBL)-producing E. coli from the poultry sector in a One Health approach. From December 2021 to March 2022, 48 chicken meat samples were collected from 16 establishments in La Rioja (Northern Spain). Antibiotic susceptibility testing was assessed by the disk-diffusion method. Forty E. coli isolates were recovered from 33 of the 48 chicken meat samples tested (68.8%) when plated on MacConkey-agar. In addition, six ESBL-E. coli (6/48, 12.5%) were obtained on cefotaxime-supplemented MacConkey-agar, which were Whole-Genome Sequenced. A large diversity of clones and ESBL genes was observed, namely ST1140-E/bla CTX-M-32 (n = 1), ST752-A/bla TEM-52 (n = 1), ST117-B2/bla CTX-M-1 /bla SHV-12 (n = 2), ST10-A/bla SHV-12 (n = 1) and ST223-B1/ bla SHV-12 (n = 1). Three IncI1-plasmids (pST3-CC3) were found carrying the bla SHV-12 /bla CTX-M-1 /bla CTX-M-32 genes in two genetic environments: i) IS26-smc-glpR-bla SHV-12 -IS26; and ii) wbuC-bla CTX-M-32 /bla CTX-M-1 -ISEcp1. The bla TEM-52 gene was carried on a P1-like phage-plasmid flanked by an IS4-mediated composite transposon. An IncHI2 plasmid harboured a bla SHV-12 gene flanked by an IS26-mediated composite transposon but also additional genes conferring resistance to aminoglycosides, chloramphenicol, and sulphonamides. To analyse the cross-sectoral relatedness of our ESBL-E. coli isolates, our six genomes were mapped with publicly available genomes (n = 2588) related to the STs detected, revealing that one of our genomes (X3078-ST117) displayed strong similarities (34-40 allelic differences) with few genomes belonging to ST117 from the poultry sector from Germany and USA. This study demonstrated that the proportion of ESBL-E. coli is still high in chicken meat in Spain. In addition, the ST117 clone and the IncI1-bla CTX-M-1-32 /bla SHV-12 plasmids might represent successful clones and plasmids adapted to the chicken host

A Surveillance Study of Culturable and Antimicrobial-Resistant Bacteria in Two Urban WWTPs in Northern Spain

Background/Objectives: Wastewater treatment plants (WWTPs) are hotspots for the spread of antimicrobial resistance into the environment. This study aimed to estimate the proportion of clinically relevant antimicrobial-resistant bacteria in two Spanish urban WWTPs, located in the region of La Rioja (Spain); Methods: Ninety-four samples (48 water/46 sludge) were collected and streaked on ten different selective media, in order to recover the culturable bacterial diversity with relevant resistance phenotypes: Extended-Spectrum -Lactamase-producing Escherichia coli/Klebsiella pneumoniae (ESBL-Ec/Kp), Carbapenem-resistant Enterobacteriaceae (CR-E), Methicillin-resistant Staphylococcus aureus (MRSA), and Vancomycin-resistant Enterococcus faecium/faecalis (VR-E. faecium/faecalis). Isolates were identified by MALDI-TOF and were tested for antimicrobial susceptibility using the disk diffusion method. The confirmation of ESBL production was performed by the double-disk test; Results: A total of 914 isolates were recovered (31 genera and 90 species). Isolates with clinically relevant resistance phenotypes such as ESBL-Ec/Kp and CR-E were recovered in the effluent (0.4 × 1004.8 × 101 CFU/mL) and organic amendment samples (1.01016.0 × 102 CFU/mL), which are discharged to surface waters/agricultural fields. We reported the presence of VR-E. faecium in non-treated sludge and in the digested sludge samples (1.3 × 1011 × 103 CFU/mL). MRSA was also recovered, but only in low abundance in the effluent (0.2 × 101 CFU/mL); Conclusions: This study highlights the need for improved wastewater technologies and stricter regulations on the use of amendment sludge in agriculture. In addition, regular monitoring and surveillance of WWTPs are critical for early detection and the mitigation of risks associated with the spread of antimicrobial resistance

Bacteriocin-Producing Staphylococci and Mammaliicocci Strains for Agro-Food and Public Health Applications with Relevance of Micrococcin P1

Antimicrobial-producing strains and their bacteriocins hold great promise for the control of bacterial diseases, being an attractive alternative to antibiotics. Thus, the aim of this study was to evaluate the inhibitory activity of 15 bacteriocin-producing staphylococci and mammaliicocci (BP-S/M) strains and their pre-purified extracts with butanol (BT) against a collection of 27 harmful or zoonotic strains (including Gram-positive/-negative bacteria and molds) with relevance in the public health and agro-food fields. These indicators (excluding Gram-negative strains) were grouped into seven categories based on their potential application areas: dairy livestock mastitis, avian pathogen zoonoses, swine zoonoses, food safety, aquaculture, wine making, and mushroom cultivation. In addition, cross-immunity assays between the BP-S/M strains were carried out to identify potential strain combinations to enhance their activity against pathogens. Finally, the hemolytic and gelatinase activities were tested in the BP-S/M strains. A strong inhibitory capacity of the BP-S/M strains was verified against relevant Gram-positive indicators, such as methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, and Clostridium perfringens, among others, while no activity was detected against Gram-negative ones. Interestingly, several BT extracts inhibited the two mold indicators included in this study as representants of mushroom pathogens. The Micrococcin P1 producer Staphylococcus hominis C5835 (>60% of indicators were intensively inhibited by all the methods) can be proposed as a potential candidate for the control of bacterial diseases in the aforementioned categories alone or in combination with other BP-S/M strains (mainly with Staphylococcus warneri X2969). In this regard, five potential combinations of BP-S/M strains that enhanced their activity against specific pathogens were detected

Nitrogen metabolic profile of Lactococcus lactis subsp. cremoris strains under stress conditions

Lactococcus lactis subsp. cremoris MG1363 is an international prototype for genetic and physiological studies in lactic acid bacteria. The aim of this study was to determine and evaluate changes in the production of extracellular metabolites generated in aminoacidic routes by the species L. lactis subsp. cremoris in response to the stress due to the presence of ethanol in the culture medium. We analyzed aminoacids generated by the strain L. lactis subsp. cremoris MG1363 and three mutants: MGargR, MGahrC and MGargRahrC under different culture conditions. Results showed that L. lactis subsp. cremoris used arginine to grow in the presence of ethanol in the medium, using the ADI pathway and producing ornithine as final product of degradation, concluding that L. lactis subsp. cremoris utilized the ADI arginine degradation pathway as a response to the ethanol stress, producing ornithine as the final metabolic product

Evaluation of the double-zone hemolysis (DZH) test for the detection of livestock-associated methicillin-resistant Staphylococcus aureus

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), such as clonal-complex (CC)398, are of clinical relevance due to their multi-drug resistance profiles, adding to the overall burden of MRSA in humans. The objective was to evaluate the double-zone hemolysis (DZH) test as a simple and reliable method for detecting LA-MRSA in the clinical microbiology laboratory. S. aureus isolates assigned to CC398 (n = 183; 152 MRSA/31 methicillin-susceptible S. aureus [MSSA]), CC1 (n = 44; MRSA), and other CCs (n = 144; 94 MRSA/50 MSSA) were investigated. These isolates were screened for DZH on sheep blood agar plates after incubation at 37°C for 24 h. Identification of the scn (human adaptation marker) and hlb genes (encoding hemolysin, intact or truncated) was performed by PCR. The positive and negative predictive values (PPV and NPV), sensitivity (SS), and specificity (SP) of the DZH test were determined. The DZH-positive phenotype was observed in 94.7%, 25%, and 6.4% of MRSA-CC398, MRSA-CC1, and MRSA of other lineages, respectively. Moreover, the DZH-positive phenotype was identified in 9.7% of MSSA-CC398 isolates but not in other MSSA lineages. All 164 DZH-positive isolates carried hlb intact, and 99.4% was scn negative, suggesting an animal origin. Of the 207 DZH-negative isolates, 99.5% was scn positive (indicating human adaptation), and 95.2% possessed a truncated hlb gene. The PPV/NPV/SS/SP values (in %) of the DZH test were as follows: detection of (i) LA-MRSA-CC398: (87.8/96.1/94.7/90.9); (ii) LA-MRSA-CC398/CC1 scn negative: (94.5/100/100/95.8); and (iii) S. aureus scn negative: (99.4/99.5/99.4/99.5). The DZH is a reliable strategy to detect and distinguish LA-MRSA in the clinical microbiology laboratory and is recommended as an adjunct diagnostic tes