{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T04:55:16Z","timestamp":1792040116427,"version":"4.4.0"},"reference-count":44,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,4,30]],"date-time":"2025-04-30T00:00:00Z","timestamp":1745971200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>Lactobacillaceae is a large family of bacteria from which probiotic strains often originate. Microorganisms used as feed additives in the EU must meet a number of formal criteria, some of which concern antimicrobial susceptibility. In this study, we determined the susceptibility of 19 reference strains and 121 wild-type strains of Lactobacillaceae to aminoglycoside antibiotics using the broth microdilution method based on the ISO 10932:2010\/IDF 223:2010 standard. Strains were categorized as resistant or susceptible according to European Food Safety Authority (EFSA) guidelines. Resistance genes were detected by whole genome sequence (WGS) analysis or by PCR. The MICs read after 48 h of incubation showed that 36.8% of reference strains were resistant to kanamycin, 26.3% to streptomycin, and 5.3% to gentamicin, with no aminoglycoside resistance genes detected in any genome. As many as 93.2% of field isolates of Ligilactobacillus salivarius, 85% of Ligilactobacillus agilis, and 58.8% of Lactiplantibacillus plantarum were classified as resistant to kanamycin, with the aac(6)-Ie-aph(2)-Ia gene detected only in two isolates. In six of 12 streptomycin-resistant strains, the ant(6)-Ia gene was identified, which usually coexisted with the spw gene. Three isolates with high neomycin MICs harbored the ant(4\u2032)-Ia gene. In Lactobacillus gallinarum strain LMG 9435, characterized by streptomycin MIC value &gt; 1024 \u00b5g\/mL, a potential resistance-causing mutation in the rpsL gene (Lys56 \u2192 Arg) was detected. The results of the study indicate that some genera of Lactobacillaceae, in particular L. salivarius and L. agilis, exhibit natural resistance to aminoglycoside antibiotics, mainly kanamycin. Therefore, there is a need to update the EFSA guidelines on antimicrobial susceptibility testing of Lactobacillaceae, so that strains lacking resistance genes and\/or chromosomal mutations are not considered to be resistant.<\/jats:p>","DOI":"10.3390\/life15050732","type":"journal-article","created":{"date-parts":[[2025,5,4]],"date-time":"2025-05-04T20:42:37Z","timestamp":1746391357000},"page":"732","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Susceptibility of Lactobacillaceae Strains to Aminoglycoside Antibiotics in the Light of EFSA Guidelines"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5456-9652","authenticated-orcid":false,"given":"Marta","family":"Dec","sequence":"first","affiliation":[{"name":"Department of Veterinary Prevention and Avian Diseases, University of Life Sciences in Lublin, 20-612 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4844-7031","authenticated-orcid":false,"given":"Klaudia","family":"Herman-Ostrzy\u017cek","sequence":"additional","affiliation":[{"name":"Department of Veterinary Prevention and Avian Diseases, University of Life Sciences in Lublin, 20-612 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0758-5190","authenticated-orcid":false,"given":"Aldert","family":"Zomer","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands"},{"name":"WHO Collaborating Centre for Reference and Research on Campylobacter and Antimicrobial Resistance from a One Health Perspective\/WOAH Reference Laboratory for Campylobacteriosis, 3584 CL Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8245-1646","authenticated-orcid":false,"given":"Renata","family":"Urban-Chmiel","sequence":"additional","affiliation":[{"name":"Department of Veterinary Prevention and Avian Diseases, University of Life Sciences in Lublin, 20-612 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Zhang, H., and Cai, Y. (2014). Phylogenesis and evolution of lactic acid bacteria. Lactic Acid Bacteria, Springer.","DOI":"10.1007\/978-94-017-8841-0"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e10941","DOI":"10.7717\/peerj.10941","article-title":"Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture","volume":"9","author":"Gilroy","year":"2021","journal-title":"PeerJ"},{"key":"ref_3","unstructured":"(2024, December 27). NCBI Database, Available online: https:\/\/www.ncbi.nlm.nih.gov\/taxonomy."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2782","DOI":"10.1099\/ijsem.0.004107","article-title":"taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae","volume":"70","author":"Zheng","year":"2020","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Bhogoju, S., and Nahashon, S. (2022). Recent Advances in Probiotic Application in Animal Health and Nutrition: A Review. Agriculture, 12.","DOI":"10.3390\/agriculture12020304"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"EFSA (2018). Guidance on the characterisation of microorganisms used as feed additives or as production organisms. EFSA J., 16, 5206.","DOI":"10.2903\/j.efsa.2018.5206"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"8982","DOI":"10.1128\/AEM.71.12.8982-8986.2005","article-title":"Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria","volume":"71","author":"Klare","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_8","first-page":"394","article-title":"Effects of inoculum size and incubation time on broth microdilution susceptibility testing of lactic acid bacteria","volume":"5","author":"Lindmark","year":"2007","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1089\/mdr.2013.0189","article-title":"Comparison of the CLSI guideline and ISO\/IDF standard for antimicrobial susceptibility testing of Lactobacilli","volume":"20","author":"Mayrhofer","year":"2014","journal-title":"Microb. Drug Resist."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"\u0141epecka, A., Szyma\u0144ski, P., Rutkowska, S., Iwanowska, K., and Ko\u0142o\u017cyn-Krajewska, D. (2021). The Influence of Environmental Conditions on the Antagonistic Activity of Lactic Acid Bacteria Isolated from Fermented Meat Products. Foods, 10.","DOI":"10.3390\/foods10102267"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Tscherne, A., Mantel, E., Boskani, T., Budniak, S., Elschner, M., Fasanella, A., Feruglio, S.L., Galante, D., Giske, C.G., and Grunow, R. (2022). Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method. Microorganisms, 10.","DOI":"10.3390\/microorganisms10071470"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1093\/jac\/3.suppl_C.47","article-title":"Chemical and physical factors influencing methicillin resistance of Staphylococcus aureus and Staphylococcus epidermidis","volume":"3","author":"Sabath","year":"1977","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1016\/j.ijantimicag.2018.10.023","article-title":"Cysteine induces resistance of lactobacilli to erythromycin and azithromycin","volume":"53","author":"Cabrera","year":"2019","journal-title":"Int. J. Antimicrob. Agents."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e01738-18","DOI":"10.1128\/AEM.01738-18","article-title":"Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp.","volume":"85","author":"Campedelli","year":"2018","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"N\u00f8hr-Meldgaard, K., Struve, C., Ingmer, H., Koza, A., Al-Nakeeb, K., and Agers\u00f8, Y. (2023). Antimicrobial susceptibility testing and tentative epidemiological cut-off values for Lactobacillaceae family species intended for ingestion. Front. Antibiot., 2.","DOI":"10.3389\/frabi.2023.1162636"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Stefa\u0144ska, I., Kwiecie\u0144, E., J\u00f3\u017awiak-Piasecka, K., Garbowska, M., Binek, M., and Rzewuska, M. (2021). Antimicrobial Susceptibility of Lactic Acid Bacteria Strains of Potential Use as Feed Additives\u2014The Basic Safety and Usefulness Criterion. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.687071"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.ijfoodmicro.2010.08.024","article-title":"Antibiotic susceptibility of members of the Lactobacillus acidophilus group using broth microdilution and molecular identification of their resistance determinants","volume":"144","author":"Mayrhofer","year":"2010","journal-title":"Int. J. Food Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"102251","DOI":"10.1016\/j.anaerobe.2020.102251","article-title":"Phenotypic and genotypic antimicrobial resistance profiles of fecal lactobacilli from domesticated pigeons in Poland","volume":"65","author":"Dec","year":"2020","journal-title":"Anaerobe."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Dec, M., Nowaczek, A., St\u0119pie\u0144-Py\u015bniak, D., Wawrzykowski, J., and Urban-Chmiel, R. (2018). Identification and antibiotic susceptibility of lactobacilli isolated from turkeys. BMC Microbiol., 18.","DOI":"10.1186\/s12866-018-1269-6"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/s13099-017-0203-z","article-title":"Assessment of antibiotic susceptibility in Lactobacillus isolates from chickens","volume":"9","author":"Dec","year":"2017","journal-title":"Gut Pathog."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.resmic.2014.02.003","article-title":"Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis","volume":"165","author":"Dec","year":"2014","journal-title":"Res. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Dec, M., Puchalski, A., Urban-Chmiel, R., and Wernicki, A. (2016). 16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry. BMC Microbiol., 16.","DOI":"10.1186\/s12866-016-0732-5"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1038\/s41587-019-0072-8","article-title":"Assembly of long, error-prone reads using repeat graphs","volume":"37","author":"Kolmogorov","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Huang, Y.T., Liu, P.Y., and Shih, P.W. (2021). Homopolish: A method for the removal of systematic errors in nanopore sequencing by homologous polishing. Genome Biol., 22.","DOI":"10.1186\/s13059-021-02282-6"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2068","DOI":"10.1093\/bioinformatics\/btu153","article-title":"Prokka: Rapid prokaryotic genome annotation","volume":"30","author":"Seemann","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_26","unstructured":"(2010). Milk and Milk Products\u2014Determination of the Minimal Inhibitory Concentration (Mic) of Antibiotics Applicable to Bifidobacteria and Non-Enterococcal Lactic Acid Bacteria (LAB) (Standard No. ISO 10932:2010 (IDF 223:2010)). Available online: https:\/\/www.iso.org\/standard\/46434.html."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3491","DOI":"10.1093\/jac\/dkaa345","article-title":"ResFinder 4.0 for predictions of phenotypes from genotypes","volume":"75","author":"Bortolaia","year":"2020","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_28","first-page":"D517","article-title":"CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database","volume":"48","author":"Alcock","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_29","unstructured":"Seemann, T. (2025, January 15). Abricate, Github. Available online: https:\/\/github.com\/tseemann\/abricate."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1423","DOI":"10.1128\/AAC.47.4.1423-1426.2003","article-title":"Multiplex PCR for detection of aminoglycoside resistance genes in enterococci","volume":"47","author":"Vakulenko","year":"2003","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Dec, M., Nowak, T., Webster, J., and W\u00f3dz, K. (2024). Serotypes, Antimicrobial Susceptibility, and Potential Mechanisms of Resistance Gene Transfer in Erysipelothrix rhusiopathiae Strains from Waterfowl in Poland. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms252212192"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0168-1605(02)00254-4","article-title":"Susceptibility of Lactobacillus spp. to antimicrobial agents","volume":"82","author":"Danielsen","year":"2003","journal-title":"Int. J. Food Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1128\/AEM.02105-06","article-title":"Antibiotic resistances of starter and probiotic strains of lactic acid bacteria","volume":"73","author":"Hummel","year":"2007","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_34","unstructured":"(2024). Performance Standards for Antimicrobial Susceptibility Testing CLSI M100 Includes Updated Tables for the Clinical and Laboratory Standards Institute Antimicrobial Susceptibility Testing Standards CLSI M02, M07, and M11. A CLSI Supplement for Global Application (Standard No. CLSI Document M100-Ed34)."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2867","DOI":"10.3168\/jds.2017-13323","article-title":"Whole-genome sequencing reveals the mechanisms for evolution of streptomycin resistance in Lactobacillus plantarum","volume":"101","author":"Zhang","year":"2018","journal-title":"J. Dairy Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1007\/978-1-0716-4096-8_2","article-title":"Introduction of Spontaneous Mutations Using Streptomycin as a Method for Lactic Acid Bacteria Breeding","volume":"2851","author":"Namai","year":"2024","journal-title":"Methods Mol. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Sirichoat, A., Fl\u00f3rez, A.B., V\u00e1zquez, L., Buppasiri, P., Panya, M., Lulitanond, V., and Mayo, B. (2020). Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21072594"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Dai, R., He, J., Zha, X., Wang, Y., Zhang, X., Gao, H., Yang, X., Li, J., Xin, Y., and Wang, Y. (2021). A novel mechanism of streptomycin resistance in Yersinia pestis: Mutation in the rpsL gene. PLoS Negl. Trop. Dis., 15.","DOI":"10.1371\/journal.pntd.0009324"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1030","DOI":"10.1099\/mic.0.034595-0","article-title":"Mutations in rpsL that confer streptomycin resistance show pleiotropic effects on virulence and the production of a carbapenem antibiotic in Erwinia carotovora","volume":"156","author":"Barnard","year":"2010","journal-title":"Microbiology"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1016\/j.ijmm.2013.07.006","article-title":"The contribution of common rpsL mutations in Escherichia coli to sensitivity to ribosome targeting antibiotics","volume":"303","author":"Pelchovich","year":"2013","journal-title":"Int. J. Med. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.jgar.2017.01.005","article-title":"Frequency of rrs and rpsL mutations in streptomycin-resistant Mycobacterium tuberculosis isolates from Iranian patients","volume":"9","author":"Khosravi","year":"2017","journal-title":"J. Glob. Antimicrob. Resist."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"341","DOI":"10.11144\/Javeriana.SC25-2.eopo","article-title":"Effect of pH on the growth of three lactic acid bacteria strains isolated from sour cream","volume":"25","year":"2020","journal-title":"Univ. Sci."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1007\/BF00164706","article-title":"Influence of pH and initial lactate concentration on the growth of Lactobacillus plantarum","volume":"36","author":"Giraud","year":"1991","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Kerek, \u00c1., Ecsedi, B.G., Szab\u00f3, \u00c1., Szimr\u00f3k, Z., Paliczn\u00e9 Kust\u00e1n, B., Jerzsele, \u00c1., and Nagy, G. (2024). Stability Studies of the Dilution Series of Different Antibiotic Stock Solutions in Culture Medium Incubated at 37 \u00b0C. Antibiotics, 13.","DOI":"10.3390\/antibiotics13060549"}],"container-title":["Life"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-1729\/15\/5\/732\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:25:28Z","timestamp":1760030728000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-1729\/15\/5\/732"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,30]]},"references-count":44,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,5]]}},"alternative-id":["life15050732"],"URL":"https:\/\/doi.org\/10.3390\/life15050732","relation":{},"ISSN":["2075-1729"],"issn-type":[{"value":"2075-1729","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,30]]}}}