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Isolation of Bacteroides corrodens and Eikenella corrodens from human clinical specimens. Comparative study of incidence and methods of identification.

In the course of a systematic search for Bacteroides corrodents and Eikenella corrodens in clinical specimens submitted for microbiological analysis, 61% of the specimens from anal abscesses, 6% of the vaginal specimens and none of the pharyngeal specimens yielded B. corrodens, whereas E. corrodens was recovered from only 9% of the pharyngeal specimens. Some characteristics were found to be useful in differentiating between the two species: B. corrodens strains were strictly anaerobic, cytochrome-oxidase-negative, urease-positive and gelatinase-positive; they were sensitive to lincomycin but resistant to vancomycin. E. corrodens strains on the other hand were facultatively anaerobic, oxidase-positive, urease-negative and gelatinase-negative; they were resistant to lincomycin but sensitive to vancomycin. The pathogenicity of the two species was difficult to assess as in most cases they were recovered from mixed cultures.

Anti-Bacterial Agents↗

Genetics of drug resistance in methicillin-resistant Staphylococcus aureus from Australian hospitals.

The drug-resistance determinants in methicillin-resistant Staphylococcus aureus (MRSA) from three different hospitals in Eastern Australia have been examined. With one exception, all the isolates had chromosomal determinants for penicillinase and resistance to cadmium (Cd), mercury (Hg), phenyl mercuric acetate, methicillin, tetracycline, erythromycin, lincomycin and low level streptomycin. The strain which was the exception differed in that it did not have chromosomal resistance to Cd and lincomycin. In addition, the strains often contained plasmids which belonged to one of three categories: a small cryptic plasmid of either c. 1.4 Mdal, c. 1.7 Mdal or c. 1.9 Mdal; a chloramphenicol resistance plasmid of c. 2.8 Mdal; and a gentamicin resistance plasmid within the range of c. 15.3 to c. 28.5 Mdal. The predominant gentamicin-resistant plasmid in isolates from two hospitals had a molecular weight of c. 18 Mdal, whereas the isolates from the third hospital had a plasmid of molecular weight c. 15.3 Mdal. The only other gentamicin resistance plasmids detected were associated with penicillinase determinants. In one isolate, this corresponded to a plasmid of c. 19.6 Mdal and in the other to a plasmid of c. 28.5 Mdal. These results indicate that MRSAs which are prevalent in Eastern Australian hospitals are substantially different in the location of their drug resistance determinants to earlier strains reported in the literature.

Australia↗

Toxic and genotoxic evaluation of six antibiotics on non-target organisms.

The ecotoxicity of the following six antibiotics on aquatic organisms was investigated: Erythromycin, Oxytetracyclin, Sulfamethoxazole, Ofloxacin, Lincomycin and Clarithromycin. Bioassays were performed on bacteria, algae, rotifers, microcrustaceans and fish to assess acute and chronic toxicity, while SOS Chromotest and Ames test were used to detect the genotoxic potential of the investigated drugs. For risk assessment, the environmental impact was calculated by MEC/PNEC ratio using the available data from the literature regarding their occurrence in the aquatic environment and the toxicity data obtained from the bioassays performed. The ecotoxicological results showed that acute toxicity was in the order of mg/L while, for the chronic data the antibiotics were bioactive at concentrations in the order of microg/L, mainly for the algae. Drugs investigated were one or two order of magnitude less active against rotifers and crustaceans. Ofloxacin was the only genotoxic compound and Sulfamethoxazole, Ofloxacin and Lincomycin were mutagenic. As for environmental risk, the macrolides were found to be the most harmful for the aquatic environment.

Animals↗

Occurrence of antibiotics in hospital, residential, and dairy effluent, municipal wastewater, and the Rio Grande in New Mexico.

This study had three objectives: 1) determine occurrence of antibiotics in effluent from hospitals, residential facilities, and dairies, and in municipal wastewater 2) determine antibiotic removal at a large wastewater treatment plant (WWTP) in Albuquerque, NM, and 3) determine concentrations of antibiotics in the Rio Grande, which receives wastewater from the Albuquerque WWTP. Twenty-three samples of wastewater and 3 samples of Rio Grande water were analyzed for the presence of 11 antibiotics. Fifty-eight percent of samples had at least one antibiotic present while 25% had three or more. Hospital effluent had detections of sulfamethoxazole, trimethoprim, ciprofloxacin, ofloxacin, lincomycin, and penicillin G, with 4 of 5 hospital samples having at least one antibiotic detected and 3 having four or more. At the residential sampling sites, ofloxacin was found in effluent from assisted living and retirement facilities, while the student dormitory had no detects. Only lincomycin was detected in dairy effluent (in 2 of 8 samples, at 700 and 6600 ng/L). Municipal wastewater had detections of sulfamethoxazole, trimethoprim, ciprofloxacin, and ofloxacin, with 4 of 6 samples having at least one antibiotic present and 3 having 3 or more. The relatively high concentrations (up to 35,500 ng/L) of ofloxacin found in hospital and residential effluent may be of concern due to potential genotoxic effects and development of antibiotic resistance. At the Albuquerque WWTP, both raw wastewater and treated effluent had detections of sulfamethoxazole, trimethoprim, and ofloxacin, at concentrations ranging from 110 to 470 ng/L. However, concentrations in treated effluent were reduced by 20% to 77%. No antibiotics were detected in the Rio Grande upstream of the Albuquerque WWTP discharge, and only one antibiotic, sulfamethoxazole, was detected in the Rio Grande (300 ng/L) below the WWTP.

Anti-Bacterial Agents↗

Antimicrobial resistance and resistance genes in Staphylococcus aureus strains from rabbits.

Fifty-six Staphylococcus aureus isolates recovered between 1998 and 2003 from 31 rabbit farms with and without problems of chronic staphylococcosis, were screened for resistance to enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, penicillin and tetracyclines using the agar dilution test. For penicillin, a disk diffusion test was also performed. The detection of tetP(B), tet(K), tet(L), tet(M), tet(O), tet(T), tet(W), erm(A), erm(B), erm(C) and mec(A) genes was done via a PCR assay. Four isolates showed resistance to erythromycin and lincomycin. These isolates were positive for the erm(C) gene in the PCR. Eleven strains were resistant to tetracyclines and all harboured the tet(K) gene. In the agar dilution test, five isolates showed resistance to penicillin, whereas in the disk diffusion test 12 isolates showed resistance. None of these 12 resistant isolates carried the mec(A) gene. Only one strain showed resistance to gentamicin, and all strains were susceptible to enrofloxacin and neomycin. This study demonstrates that resistance to antimicrobial agents in S. aureus isolates originating from rabbits is relatively rare compared to resistance in S. aureus isolates originating from other animals and humans.

Animals↗

Antibiotic depression of evoked and spontaneous responses of opossum distal colonic muscularis mucosae in vitro: a factor in antibiotic-associated colitis?

Certain antibiotics depress both skeletal neuromuscular transmission and intestinal neuroeffector transmission. Impaired intestinal motility may facilitate the proliferation of the bacterium Clostridium difficle and thus lead to the development of antibiotic-associated pseudomembranous colitis. Many antibiotics accumulate in the colonic lumen at concentrations several times their associated blood levels. This study examined whether certain of these could interfere with colonic muscularis mucosal movement in vitro, using tissue from opossum distal colon as a model. At concentrations approximating those in the colonic lumen, ampicillin, clindamycin, erythromycin, and lincomycin depressed tone and spontaneous contractions of the muscularis mucosae. Clindamycin, gentamicin, kanamycin, and lincomycin abolished electrically evoked contractions but only gentamicin and kanamycin could abolish the ensuing relaxation. Vancomycin potentiated the response of the muscularis mucosae to acetylcholine; erythromycin and clindamycin depressed it. Antibiotic-induced depression of colonic muscularis mucosal movement may contribute to the development of antibiotic-associated pseudomembranous colitis.

Animals↗

Epidemic infection by a gentamicin-resistant Staphylococcus aureus in three hospitals.

A Staphylococcus aureus strain, resistant to aminoglycosides, including gentamicin and tobramycin, and to penicillin, lincomycin, and other antibiotics, caused an outbreak of hospital infection in a surgical ward which spread to the intensive-therapy unit and to two other hospitals. 21 patients were colonised and 9 of these had overt clinical infection. Administration of antibiotics to which the staphylococcus was resistant was associated with colonisation. The use of gentamicin, tobramycin, and lincomycin in these hospitals has increased as has the occurrence of bacterial resistance to these agents. An antibiotic policy to control their use is essential to preserve their value in life-threatening infections by bacteria resistant to other agents.

Acetyltransferases↗

Decline of the hospital Staphylococcus? Incidence of multiresistant Staph. aureus in three Birmingham hospitals.

The prevalance of antibiotic-resistant strains of Staphylococcus aureus was studied in three Birmingham hospitals. In a general hospital periodic surveys showed a progressive decline in the proportions of patients with Staph. aureus in their noses which were resistant to tetracycline, erythromycin, and kanamycin. This change was associated with a progressive reduction in the use of tetracycline without an overall reduction in the use of antibiotics. There was no similar decline in resistance of staphylococci isolated in a hospital for skin diseases. In a burns unit there was a sudden large reduction during September, 1978, in the proportions of Staph, aureus from burns which were resistant to tetracycline, methicillin, cephaloridine, erythromycin, lincomycin, novobiocin, gentamicin, and kanamycin, and in the proportions of multiresistant strains (resistant to penicillin, tetracycline, erythromycin, kanamycin, methicillin, novobiocin, cephaloridine, and lincomycin). This change was associated with a reduction in the number of patients and in the use of antibiotics; tetracycline was not in use except during one month of the study. Strains of Staph. aureus resistant to these antibiotics became common again in the burns unit when a larger number of patients were admitted and more antibiotics were used in the wards.

Anti-Bacterial Agents↗

Novel antibiotic-resistance markers in pGK12-derived vectors for Borrelia burgdorferi.

Extension of molecular genetics studies in Borrelia burgdorferi has been hampered by a lack of a variety of antibiotic resistance selective markers. Such markers are critical for isolation of B. burgdorferi strains with multiple mutants, for complementation with different cloning vectors, and for methods such as negative selection and reporter genes. To remedy this lack, resistance to various antibiotics of non-infectious (B31, 297) and infectious (N40) B. burgdorferi strains was examined and vectors incorporating appropriate antibiotic resistance genes as selective markers were developed. Minimal inhibitory concentrations for growth of B. burgdorferi on plates and in liquid media for aminoglycosides (kanamycin, gentamycin, sisomycin, amikacin, spectinomycin, neomycin), macrolides-lincosamids (erythromycin, lincomycin), coumarin derivatives (coumermycin A(1), novobiocin), glycopeptides (vancomycin, ristocetin), peptides (bacitracin, cycloserine), and chloramphenicol were found to differ significantly. There were also striking differences in resistance to these antibiotics between non-infectious and infectious B. burgdorferi strains. Antibiotic-resistance genes aph(3')-IIIa from Streptococcus faecalis, aad9 from Staphylococcus aureus Tn554, linA' from Staphylococcus aureus, and aac(3)-VIa from Enterobacter cloacae (conferring resistance to kanamycin, spectinomycin, lincomycin, and gentamycin/sisomycin, respectively) were subcloned either with their own promoters or under the control of the B. burgdorferi flaB promoter into pGK12 or its derivative pED1 to develop new cloning vectors for B. burgdorferi with the rationale that the absence of homologous regions between derived recombinant plasmids lacking the flaB promoter and the B. burgdorferi genome would permit avoidance of possible recombination with target DNA. Resistance to the corresponding antibiotic was conferred by vectors containing aph(3')-IIIa, aad9, linA' or aac(3)-VIa whether under the control of their own promoters or under the control of the flaB promoter. We conclude that these markers can be used for genetic study of B. burgdorferi and suggest they will be an important addition to the previously used coumermycin A(1), erythromycin and kanamycin in these studies.

Anti-Bacterial Agents↗

Antimicrobial susceptibility testing of Australian isolates of Brachyspira hyodysenteriae using a new broth dilution method.

The antimicrobial susceptibilities of 76 field isolates of Brachyspira hyodysenteriae from different states of Australia were tested in a newly developed broth dilution procedure. The antimicrobial agents used were tiamulin, valnemulin, tylosin, erythromycin, lincomycin and clindamycin. The results from the broth dilution susceptibility testing of 39 of the isolates were compared with results obtained for the same isolates using the agar dilution method. Amongst the isolates tested by broth dilution, 17 were from three farms and had been collected over a number of years. Their pulsed field gel electrophoresis pattern previously had been determined. The broth dilution technique was simple to use, less labor intensive than agar dilution, and gave clear end points. The results obtained using the two methods generally corresponded well, although in a few cases the MIC obtained by broth dilution were lower than those with agar dilution. For the 76 isolates tested by broth dilution, the MIC(90) (mg/l) was: tiamulin, 1; valnemulin, 0.5; tylosin>256; erythromycin>256; lincomycin, 64 and clindamycin, 16. Only minor differences in susceptibility patterns were found amongst isolates from different Australian states. Over all the isolates, and also amongst the isolates obtained from different years on the three farms, there was no trend for the susceptibility of the isolates to alter with time.

Animals↗

Identification and antimicrobial susceptibility of Staphylococcus chromogenes isolates from intramammary infections of dairy cows.

Staphylococcus chromogenes is a highly prevalent species in subclinical mastitis with a well-established impact on somatic cell count. Few data are available on its antimicrobial susceptibility. The objective of this study was three-fold: (1) to evaluate simple identification tests by comparing them with a genomic method; (2) to determine minimal inhibitory concentrations (MICs) of different antibiotics; (3) to search for the presence of important resistance mechanisms and resistance-determining genes.Seventy-three staphylococcal strains, all collected on different dairy farms, were tentatively identified as S. chromogenes based on their lack of hemolysis and their characteristic intermediate DNase activity. The identification of 70 strains was confirmed as S. chromogenes by tRNA intergenic spacer PCR (tRNA PCR). Three strains were identified as S. sciuri, a species that is naturally cloxacillin- and lincomycin-resistant. All 70 S. chromogenes strains were found to be normally susceptible to neomycin, gentamicin, erythromycin, enrofloxacin, and to penicillinase-stable penicillins and cephalosporins, represented in this study by cloxacillin. The latter result was confirmed by the absence of the mecA gene in each of 13 strains in which this gene was searched for. Twenty-seven (38%) strains were penicillinase producers. Three lincomycin-resistant S. chromogenes strains were found to carry the linA gene. It was concluded that S. chromogenes can be identified reliably in routine mastitis bacteriology, and that the only resistance of importance is against penicillinase-susceptible penicillins.

Animals↗

In-vitro antimicrobial susceptibility of Clostridium perfringens from commercial turkey and broiler chicken origin.

The minimum inhibitory concentrations (MIC) of eight antibiotics and two anticoccidial agents were determined for Clostridium perfringens strains isolated from 26 commercial broiler farms and 22 commercial turkey farms. Isolates were obtained from the intestines of birds on the farm or as the processing plant using standard culture and identification techniques. The microbroth dilution test was used to determine the MIC for each compound. Most isolates from chickens had MICs in the range of 2-16 mg/L for tilmicosin, tylosin and virginiamycin, whereas the MICs for avilamycin, avoparcin, monensin, narasin and penicillin were < or = 1 mg/L. Most strains from chickens had high MICs (> or = 64 mg/L) and appeared to be resistant to bacitracin and lincomycin. Most turkey isolates had MICs in the range of 2-16 mg/L for bacitracin, tilmicosin, tylosin and virginiamycin, with strains exhibiting MICs < or = 1 mg/L for avilamycin, avoparcin, monensin, narasin and penicillin. Several turkey isolates had MICs > or = 64 mg/L to lincomycin. No attempt was made to associate farm usage of a particular antibiotic to the antibiograms.

Animals↗

Serological characterization and antimicrobial susceptibility of Streptococcus suis isolates from diagnostic samples in Denmark during 1995 and 1996.

At the Danish Veterinary Laboratory Streptococcus suis infections in pigs were diagnosed in 114 cases in 1995 and in 151 cases in 1996. Isolates were serotyped using specific antisera against type 1 through 28 and a total of 67 cases from 1995 and 113 cases in 1996 were tested for resistance to 11 antimicrobial agents. The majority of cases were lung diseases (57%), followed by septicaemia (16%), meningitis (15%) and endocarditis (8%). Almost 96% of the isolates could be typed using the 28 antisera. The most common serotype was serotype 2 (29%), followed by serotype 7 (17%), and serotypes 3, 4 and 8 (9-10%). The remaining serotypes were observed in frequencies of less than 5%. Serotype 7 was more commonly isolated from septicaemia than the other serotypes. Serotype 2 was more commonly isolated from pigs older than 4 weeks compared to the other serotypes. Most isolates were susceptible to amoxycillin + clavulanate, ampicillin, ceftiofur, enrofloxacin, penicillin, spectinomycin, tiamulin and trimethoprim + sulphadiazine. A high frequency (> 30%) of resistance to tetracycline was observed. Among isolates of serotype 2, 9.7% were resistant to lincomycin and 12.9% to spiramycin. Among other serotypes 56.8% were resistant to lincomycin and spiramycin. The differences in susceptibility between isolates of serotype 2 and the other serotypes were statistically significant. Compared to a previous Danish study the distribution of serotypes of S. suis causing infections among pigs in Denmark has changed during the last 15 years.

Age Factors↗

Antibiotic resistance genes in coagulase-negative staphylococci isolated from food.

Coagulase-negative staphylococci were isolated from different raw milk cheeses and raw meat products and screened for their antibiotic resistances. They were identified as Staphylococcus xylosus, S. lentus, S. caprae, S. epidemidis and S. haemolyticus. The most frequent resistances found were those to chloramphenicol, tetracycline, erythromycin and lincomycin. They have been characterized on the molecular level. The chloramphenicol resistance genes were localized in several S. xylosus and S. caprae on plasmids with sizes ranging from 3.8-kb to 4.3-kb and were identified as chloramphenicol acetyltransferase (cat). All the tetracycline resistant strains were identified as S. xylosus and harboured a 4.4-kb plasmid carrying the tetracycline efflux resistance gene (tetK). The two erythromycin/lincomycin resistant S. caprae and S. epidermidis strains did not hybridize with the MLSB resistance genes ermAM, ermA, ermB and ermC. Three erythromycin resistant Staphylococcus sp. strains harboured an erythromycin efflux resistance gene (msr) localized twice on a 18-kb plasmid and once on the chromosome. A S. haemolyticus strain showing resistance to both lincomycin and clindamycin harboured a linA gene-carrying 2.2-kb plasmid. Further resistances to gentamicin, penicillin and kanamycin were less frequently observed and yet not characterized on a molecular level.

Animals↗

Liquid chromatography method for separation of clindamycin from related substances.

A reversed-phase liquid chromatography method has been developed for the separation of clindamycin from 7-epiclindamycin, clindamycin B, lincomycin, lincomycin B, 7-epilincomycin and other impurities of unknown identity. The method uses a Hypersil ODS, 5 microm, 250 x 4.6 mm i.d. column maintained at 45 degrees C. The mobile phase comprises acetonitrile phosphate buffer (1.35% v/v phosphoric acid, adjusted to pH 6.0 with ammonium hydroxide)-water (35:40:25, v/v) at a flow rate of 1.0 ml/min. UV detection is performed at 210 nm. The method was tested on several C-18 columns and showed good robustness. Robustness was further evaluated by performing a full-fraction factorial design experiment. The method showed good selectivity, linearity, and repeatability. It is also suitable for analysis of clindamycin formulations.

Anti-Bacterial Agents↗

Oxidation of antibacterial molecules by aqueous ozone: moiety-specific reaction kinetics and application to ozone-based wastewater treatment.

Ozone and hydroxyl radical (*OH) reaction kinetics were measured for 14 antibacterial compounds from nine structural families, to determine whether municipal wastewater ozonation is likely to result in selective oxidation of these compounds' biochemically essential moieties. Each substrate is oxidized by ozone with an apparent second-order rate constant, k''(O3,app) > 1 x 10(3) M(-1) s(-1), at pH 7, with the exception of N(4)-acetylsulfamethoxazole (K''(O3,app) is 2.5 x 102 M(-1) s(-1)). k''(O3,app) values (pH 7) for macrolides, sulfamethoxazole, trimethoprim, tetracycline, vancomycin, and amikacin appear to correspond directly to oxidation of biochemically essential moieties. Initial reactions of ozone with N(4)-acetylsulfamethoxazole, fluoroquinolones, lincomycin, and beta-lactams do not lead to appreciable oxidation of biochemically essential moieties. However, ozone oxidizes these moieties within fluoroquinolones and lincomycin via slower reactions. Measured k''(O3,app) values and second-order *OH rate constants, k''(*OH,app) were utilized to characterize pollutant losses during ozonation of secondary municipal wastewater effluent. These losses were dependent on k''(O3,app), but independent of k''(*OH,app). Ozone doses > or =3 mg/L yielded > or =99% depletion of fast-reacting substrates (K''(O3,app) > 5 x 10(4) M(-1) s(-1)) at pH 7.7. Ten substrates reacted predominantly with ozone; only four were oxidized predominantly by .OH. These results indicate that many antibacterial compounds will be oxidized in wastewater via moiety-specific reactions with ozone.

Anti-Bacterial Agents↗

[Current data on antibiotic resistance of the most important bovine mastitis pathogens in Switzerland].

100 strains of Staphylococcus aureus (S. aureus), 100 strains of coagulase-negative staphylococci (CNS), 100 strains of Streptococcus spp. and 100 strains of Escherichia coli (E. coli), isolated from bovine mastitis milk samples between November 2002 and April 2003, were tested for their sensitivity to various antibiotics by means of the agar diffusion method. The antibiotics were chosen on the basis of their licenses for intramammary application in Switzerland (www.vetpharm.unizh.ch). 91% of the S. aureus strains were sensitive to all the antibiotics tested. Only 9% of the strains were resistant to Penicillin G and 7% to Ampicillin. 53% of the CNS strains were sensitive to all the antibiotics tested. 31% exhibited resistance to Penicillin G, 26% to Ampicillin, 16% to Cloxacillin and 14% to Lincomycin. 30% of the Streptococcus spp. strains were sensitive to all the antibiotics tested. 4% were resistant to Penicillin G, 4% to Amoxicillin/clavulanic acid, 1% to Cefoperazone, 2% to Cefquinome, 35% to Neomycin, 22% to Gentamicin, 61% to Kanamy-cin and 11% to Lincomycin. 43% of the strains showed multiple resistance. 79% of the E. coli strains were sensitive to all the antibiotics tested. 20% exhibited resistance to Ampicillin, 9% to Neomycin and 10% to Kanamycin. A comparison of the own results with data of other authors in Switzerland shows no important changes in the resistance situation during the last 20 years. With the exception of two strains (Streptococcus spp.), all tested isolates were sensible against Cefquinome.

Animals↗

Whole-genome sequencing reveals hidden antimicrobial resistance genes in phenotypically susceptible probiotic candidate lactic acid bacteria.

Phenotypic assays commonly used to evaluate probiotic safety may fail to detect clinically relevant antimicrobial resistance (AMR), potentially allowing genetically concerning strains to appear acceptable based on MIC testing alone. To explore this issue, we applied whole-genome sequencing (WGS) to three lactic acid bacteria (LAB) isolates previously identified as probiotic candidates based on acid and bile tolerance, antagonism against enteric pathogens, and biofilm formation in vitro: Lactiplantibacillus plantarum L25F and L22F (from pigs) and Ligilactobacillus salivarius AF2319 (from a chicken). Genome annotation identified extensive repertoires of probiotic-associated genes (46-47 per strain) linked to stress tolerance, adhesion, immunomodulation, and quorum sensing, supporting functional potential. The two L. plantarum strains exhibited broader predicted metabolic capacities than L. salivarius AF2319. However, genomic analysis revealed acquired AMR genes with complex genotype-phenotype relationships not fully apparent from phenotypic testing. The L. plantarum strains harbored lnu(A) (99.79% identity) on extrachromosomal DNA, conferring the L-phenotype (lincomycin resistance, clindamycin susceptibility); clindamycin MICs (1&#xa0;mg/L) were concordant with this genotype, though lincomycin MICs were not determined. L. salivarius AF2319 carried tet(M), tet(L), and erm(C) (99.48%, 99.49%, and 99.45% identity by ResFinder, respectively) on extrachromosomal DNA; notably, the erythromycin MIC (1&#xa0;mg/L) was precisely at the EFSA breakpoint (&#x2264;&#x2009;1&#xa0;mg/L), representing borderline genotype-phenotype discordance potentially due to silent gene expression. Under current EFSA QPS criteria, these acquired ARGs would preclude all three strains from approval as probiotic feed additives despite favorable functional profiles, underscoring the indispensable role of WGS-based AMR gene detection in modern probiotic safety evaluation.

Probiotics↗