Genotypic Antimicrobial Resistance Profiles Research Articles

Multidrug resistant Vibrio spp. identified from mussels farmed for human consumption in Central Italy.

This study investigated phenotypic and genotypic antimicrobial resistance profiles of Vibrio strains identified from Mytilus galloprovincialis farmed for human consumption in the Adriatic Sea Central Italy. A total of 475 mussels (M. galloprovincialis) were involved in the present study, and culture-dependent microbiological methods permitted to identify a total of 50 Vibrio strains that were tested for antibiotic susceptibility followed by the genetic determinant detections. Antibiograms showed resistance against ampicillin (36.0%), amoxicillin-clavulanic acid (30.0%), gentamycin (14.0%), and imipenem (18.0%). Biomolecular assays amplified a total of 264 antibiotic resistance genes harbored by both susceptible and resistant Vibrio species. Among resistance genes, aacC2 (62.0%) and aadA (58.0%) for aminoglycosides, blaTEM (54.0%) for beta-lactams, qnrS (24.0%) for quinolones, tetD (66.0%) for tetracyclines, and vanB (60.0%) for glycopeptides were mainly amplified by PCR assays. Vibrio genus is involved in the antibiotic resistance phenomenon diffusion in the aquatic environments, as demonstrated by the harboring of many genetic determinants representing a kind of genetic "dark world".

Phenotypic and genotypic characterization of antimicrobial resistance profiles in Salmonella isolated from waterfowl in 2002-2005 and 2018-2020 in Sichuan, China.

Salmonella enterica is a widespread foodborne pathogen with concerning antimicrobial resistance (AMR). Waterfowl are a major source of Salmonella transmission, but there are few systematic studies on Salmonella prevalence in waterfowl species. In this study, 126 Salmonella isolates (65 collected in 2018–2020 and 61 collected in 2002–2005) were obtained from waterfowl samples in Sichuan, China. Their serotypes, pulsed-field gel electrophoresis (PFGE) types, and phenotypic and genotypic AMR profiles were systematically examined. The isolates were distributed in 7 serotypes, including serovars Enteritidis (46.0%), Potsdam (27.8%), Montevideo (7.9%), Cerro (6.3%), Typhimurium (4.8%), Kottbus (4.0%) and Apeyeme (3.2%). Their PFGE characteristics were diverse; all isolates were distributed in four groups (cutoff value: 60.0%) and 20 clusters (cutoff value: 80.0%). Moreover, all isolates were multidrug resistant, and high rates of AMR to lincomycin (100.0%), rifampicin (100.0%), sulfadiazine (93.7%), erythromycin (89.7%), ciprofloxacin (81.0%), and gentamicin (75.4%) were observed. Finally, 49 isolates were subjected to whole-genome sequencing, and a wide variety of AMR genes were found, including multiple efflux pump genes and specific resistance genes. Interestingly, the tet(A)/tet(B) and catII resistance genes were detected in only isolates obtained in the first collection period, while the gyrA (S83F, D87N and D87G) and gyrB (E466D) mutations were detected at higher frequencies in the isolates obtained in the second collection period, supporting the findings that isolates from different periods exhibited different patterns of resistance to tetracycline, chloramphenicol and nalidixic acid. In addition, various incompatible plasmid replicon fragments were detected, including Col440I, Col440II, IncFIB, IncFII, IncX1, IncX9, IncI1-I and IncI2, which may contribute to the horizontal transmission of AMR genes and provide competitive advantages. In summary, we demonstrated that the Salmonella isolates prevalent in Sichuan waterfowl farms exhibited diverse serotypes, multiple AMR phenotypes and genotypes, and AMR changes over time, indicating their potential risks to public health.

Revealing antimicrobial resistance profile of the novel probiotic candidate Faecalibacterium prausnitzii DSM 17677

Faecalibacterium prausnitzii, a resident anaerobic bacterium commonly found in healthy gut microbiota, has been proposed as a next generation probiotic with high potential for application in food matrices and pharmaceutical formulations. Despite its recognized health benefits, detailed information regarding its antimicrobial susceptibility profile is still lacking. However, this information is crucial to determine its safety, since the absence of acquired antimicrobial resistance is required to qualify a probiotic candidate as safe for human and animal consumption. Herein, the antimicrobial susceptibility profile of F. prausnitzii DSM 17677 strain was evaluated by integrating both phenotypic and in silico data. Phenotypic antimicrobial susceptibility was evaluated by determining minimum inhibitory concentrations of 9 antimicrobials using broth microdilution and E-test® methods. Also, the whole genome of F. prausnitzii DSM 17677 was analysed, using several databases and bioinformatics tools, to identify possible antibiotic resistance genes (ARG), genomic islands (GI) and mobile genetic elements (MGE). With exception of erythromycin, the same classification (susceptible or resistant) was obtained in both broth microdilution and E-test® methods. Phenotypic resistance to ampicillin, gentamycin, kanamycin and streptomycin were detected, which was supported by the genomic context. Other ARG were also identified but they seem not to be expressed under the tested conditions. F. prausnitzii DSM 17677 genome contains 24 annotated genes putatively involved in resistance against the following classes of antimicrobials: aminoglycosides (such as gentamycin, kanamycin and streptomycin), macrolides (such as erythromycin), tetracyclines and lincosamides. The presence of putative ARG conferring resistance to β-lactams could only be detected using a broader homology search. The majority of these genes are not encoded within GI or MGE and no plasmids were reported for this strain. Despite the fact that most genes are related with general resistance mechanisms, a streptomycin-specific ARG poses the only potential concern identified. This specific ARG is encoded within a GI and a MGE, meaning that it could have been laterally acquired and might be transferred to other bacteria present in the same environment. Thus, our findings provide relevant insights regarding the phenotypic and genotypic antimicrobial resistance profiles of the probiotic candidate F. prausnitzii DSM 17677.

Analysis of the phenotypic and genotypic antimicrobial resistance profiles of clinically significant enterococci isolated in the Provincial Specialist Hospital in Lublin, Poland

Abstract The increasing significance of enterococci as healthcare-associated pathogens can be linked to their limited susceptibility to antibiotics. In this study, phenotypic and genotypic resistance profiles of 35 [n=18 E. faecium (Efm); n=17 E. faecalis (Efs)] invasive isolates cultured from hospitalized patients were analysed. Phenotypic identification was verified by the multiplex PCR targeting the 16S rDNA and the ddl genes encoding for the Efs and Efm – specific ligases. Antimicrobial susceptibility was determined using the disc diffusion method and E-tests. The high-level streptomycin resistance (HLSR), high-level gentamicin resistance (HLGR) and glycopeptide resistance was verified by amplification of the ant(6)-Ia, aac(6’)-Ie-aph(2’’)-Ia, as well as vanA and vanB genes, respectively. More than 70% of all isolates were cultured from patients in the Intensive Care and Internal Medicine Units. Blood was the predominant (77%) site of isolation. All Efm isolates were resistant to ampicillin, imipenem, and norfloxacin; 17 isolates demonstrated high-level aminoglycoside resistance (HLAR), including 27.7% with HLSR, 38.8% with HLGR and 27.7% with both phenotypes. HLAR was also common in Efs (HLSR>70%, HLGR>50%), followed by norfloxacin (64.7%) and ampicillin (11.7%) resistance. The ant(6)-Ia and aac(6’)-Ie-aph(2’’)-Ia genes were detected in >90% of the HLSR and HLGR isolates, respectively. Glycopeptide resistance was detected in 4 (22.2%) Efm isolates and mediated by the vanA gene. 19 (54.3%) isolates were multidrug resistant, including 17 (89.5%) Efm. All isolates were susceptible to linezolid. The study constitutes a contribution to the analysis of enterococcal antimicrobial resistance in Polish hospitals. The monitoring of enterococcal prevalence and antimicrobial resistance is crucial to control and prevent infections.

Klebsiella pneumoniae in vegetable products: antimicrobial resistance and consumer health risk

Abstract Background Klebsiella pneumoniae (Kp) is considered one of the major causes of human hospital infections. Moreover, Kp is often present in several food products and it could be a potential health hazard for the consumers through the increase of Kp antimicrobial resistance (AMR) to the main routinely used antibiotics classes. The aim of this study was to evaluate the AMR profile of Kp strains isolated from vegetable food products. Methods The whole genome sequences of 10 Kp strains, previously isolated from carrots (n. 4), salads (n. 3), tomatoes (n. 2) and pumpkin (n. 1) samples, were obtained by next generation sequencing technique, using Illumina platform. Phenotypic and genotypic AMR profiles, with particular attention to β-lactams, quinolones and aminoglycosides resistances, were acquired by both agar diffusion and micro-dilution susceptibility testing and uploading the Kp sequences to Institut Pasteur platform (https://bigsdb.pasteur.fr/), respectively. Results Most of Kp strains (9 of 10) resulted susceptible to the main antibiotics tested and few AMR genes were detected in silico besides the native blaSHV, blaOKP and blaLEN. Only one Kp harboured blaAMPC and blaCMY, showing resistance to 5 β-lactams (amoxicillin/clavulanate, cefoxitin, cefpodoxime, cefpodoxime/clavulanate, ceftobiprole). Regarding the quinolone resistances, all the Kp strains carried gyr and par genes. No aminoglycoside resistance genes were highlighted in the 10 Kp strains studied. Conclusions These preliminary results showed the presence, in vegetable foods, mainly in ready to eat raw products, of Kp strains, harbouring no or few AMR genes. Good hygiene practices should be properly applied during the entire food processing chain, to avoid hazardous implications for the consumer. Further studies are needed in order to: analyse more vegetable samples, deeply evaluate the phenotypic and genotypic AMR profile of the Kp strains and their possible transmission from food products to consumer. Key messages Klebsiella pneumoniae is often isolated in food, as ready to eat vegetables, and it could be a potential public health risk due to the antimicrobial resistance increase to main antibiotics. The presence of Klebsiella pneumoniae in food implies the correct application of good hygiene practices in the entire food processing chain, in order to avoid dangerous effects for consumers.

Molecular and phenotypic characterization of avian pathogenic Escherichia coli isolated from commercial broilers and native chickens

Many studies have examined avian pathogenic Escherichia coli (APEC) from commercial broilers but few have examined isolates from native chickens. This study compared APEC isolates from commercial broilers and native chickens in regard to the phylogenetic group and the phenotypic and genotypic antimicrobial resistance profiles. From 100 suspect colibacillosis cases in both commercial broilers and native chickens, a total of 90 broiler isolates and 42 native chicken isolates were identified as E. coli by biochemical tests. Phylogenetic grouping revealed that 90 broiler APEC isolates belonged to A group (5.56%), B1 group (22.22%), B2 group (31.11%), and D group (41.11%). The 42 native chicken APEC isolates belonged to A group (35.71%), B1 group (26.19%), B2 group (30.95%), and D group (7.14%). The difference in the allocation to groups A and D of the 2 isolate types was significant (P < 0.05). The APEC broiler isolates had a significantly higher multidrug-resistant (MDR) rate (80%) than the native chicken isolates (14.29%) (P < 0.05). The APEC broiler isolates demonstrated significantly higher resistance rates than the native chicken isolates for amoxicillin (98.89%; 78.57% respectively), chloramphenicol (42.2%; 9.5%), enrofloxacin (68.9%; 7.1%), gentamicin (11.1%; 0%), nalidixic acid (72.2%; 7.1%), sulfamethoxazole + trimethoprim (45.6%; 2.4%), and tetracycline (88.9%; 76.2%) (P < 0.05). The APEC broiler isolates had a significantly higher presence compared with the native chicken isolates of the following resistance genes:- by blaTEM (43.3%; 21.4%, respectively), cml-A (34.4%; 2.4%), tetA (76.7%; 40.5%), tetB (26.7%; 0%), sul2 (23.3%; 14.3%), and dhfrI (13.3%; 0%) (P < 0.05). The qnrB and qnrS genes were detected (12.16%; 72.97% respectively), in the APEC broiler isolates resistant to nalidixic acid and/or enrofloxacin while only qnrS genes was detected in all 3 APEC native chicken isolates. Regarding the point mutations of gyrA and parC, all isolates were positive to gyrA83S, gyrA87D, gyrA87L, gyrA87NY, parC80S and parC80I except that gyrA83S was not present in 20 APEC broiler isolates. Antimicrobial stewardship programs should be targeted at the backyard poultry sector as well as the commercial poultry sector.

Characterization of beta-lactam-resistant Escherichia coli from Australian fruit bats indicates anthropogenic origins.

Antimicrobial-resistant Escherichia coli , particularly those resistant to critically important antimicrobials, are increasingly reported in wildlife. The dissemination of antimicrobial-resistant bacteria to wildlife indicates the far-reaching impact of selective pressures imposed by humans on bacteria through misuse of antimicrobials. The grey-headed flying fox (GHFF; Pteropus poliocephalus), a fruit bat endemic to eastern Australia, commonly inhabits urban environments and encounters human microbial pollution. To determine if GHFF have acquired human-associated bacteria, faecal samples from wild GHFF (n=287) and captive GHFF undergoing rehabilitation following illness or injury (n=31) were cultured to detect beta-lactam-resistant E. coli . Antimicrobial susceptibility testing, PCR and whole genome sequencing were used to determine phenotypic and genotypic antimicrobial resistance profiles, strain type and virulence factor profiles. Overall, 3.8 % of GHFF carried amoxicillin-resistant E. coli (wild 3.5 % and captive 6.5 %), with 38.5 % of the 13 GHFF E. coli isolates exhibiting multidrug resistance. Carbapenem (bla NDM-5) and fluoroquinolone resistance were detected in one E. coli isolate, and two isolates were resistant to third-generation cephalosporins (bla CTX-M-27 and ampC). Resistance to tetracycline and trimethoprim plus sulfamethoxazole were detected in 69.2% and 30.8 % of isolates respectively. Class 1 integrons, a genetic determinant of resistance, were detected in 38.5 % of isolates. Nine of the GHFF isolates (69.2 %) harboured extraintestinal virulence factors. Phylogenetic analysis placed the 13 GHFF isolates in lineages associated with humans and/or domestic animals. Three isolates were human-associated extraintestinal pathogenic E. coli (ST10 O89:H9, ST73 and ST394) and seven isolates belonged to lineages associated with extraintestinal disease in both humans and domestic animals (ST88, ST117, ST131, ST155 complex, ST398 and ST1850). This study provides evidence of anthropogenic multidrug-resistant and pathogenic E. coli transmission to wildlife, further demonstrating the necessity for incorporating wildlife surveillance within the One Health approach to managing antimicrobial resistance.

Genomic features and antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli strains isolated from food in Chile.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes severe illness in humans, often associated with foodborne outbreaks. Antimicrobial resistance among foodborne E.coli has increased over the last decades becoming a public health issue. In this study, the presence and features of STEC were investigated in samples of meat, seafood, vegetables and ready-to-eat street-vended food collected in Chile, using a genomic and microbiological approach. Phenotypic and genotypic antimicrobial resistance profiles were determined, and serotype, phylogroup, sequence type (ST) and phylogenomics were predicted using bioinformatic tools. Three thousand three hundred samples collected in 2019 were screened, of which 18 were positive for STEC strains (0.5%), with stx2a (61.1%) being the predominant stx subtype. The presence of the virulence genes lpfA (100%), iha and ehaA (94.4%), and ehxA, hlyA and saa (83.3%) was confirmed among the STEC strains; the Locus of adhesion and autoaggregation (LAA) was predicted in 14 (77.8%) strains. Strains displayed resistance to colistin (100%), and intermediate resistance to enrofloxacin (11.1%) and chloramphenicol (5.6%). In this regard, mutations in the two-component regulatory system genes pmrA (S29G), pmrB (D283G) and phoP (I44L), and the presence of the qnrB19 gene were confirmed. STEC strains belonged to ST11231 (38.9%), ST297 and ST58 (16.7% each), and ST1635, ST11232, ST446, ST442 and ST54 (5.6% each), and the most frequently detected serotypes were O113:H21 (44.4%), O130:H11 and O116:H21 (16.7% each), and O174:H21 (11.1%). Strains belonging to the international ST58 showed genomic relatedness with worldwide strains from human and non-human sources. Our study reports for the first time the genomic profile of STEC strains isolated from food in Chile, highlighting the presence of international clones and sequence types commonly associated with human infections in different geographical regions, as well as the convergence of virulence and resistance in STEC lineages circulating in this country.

Phenotypic and genotypic antimicrobial resistance profiles of fecal lactobacilli from domesticated pigeons in Poland

Lactobacillus species play an important role in the host and although they are non-pathogenic, they could act as reservoirs for antibiotic resistance genes, with the potential risk of transfer to other bacteria inhabiting the gastrointestinal tract. The aim of this study was to identify Lactobacillus species derived from feces of domesticated pigeons and to characterize their phenotypic and genotypic antimicrobial resistance (AMR) profiles. A total of 57 Lactobacillus isolates were classified into six species using the MALDI-TOF technique and 16S rDNA restriction analysis. Strains of L. ingluviei (31%), L. salivarius (28%) and L. agilis (23%) were the dominant species isolated. Determination of antimicrobial susceptibility by the microdilution broth method showed widespread resistance to kanamycin (89%), tetracycline (84%), streptomycin (63%), and enrofloxacin (37%). Less than 30% of the isolates were resistant to erythromycin, lincosamides, gentamycin, chloramphenicol and vancomycin. Over half (51%) of the lactobacilli were classified as multidrug resistant. Tet genes were detected in 79% of isolates; the lnuA, cat, ermB, ermC, ant(6)-Ia, ant(4’)-Ia, and int-Tn genes were found at a lower frequency. Sequence analysis of the quinolone resistance-determining region (QRDR)of the gyrA gene showed that fluoroquinolone resistance in lactobacilli was the result of a mutation that lead to a change in the amino acid sequence (Ser83→Tyr/Leu/Phe). Domesticated pigeons could be a reservoir for AMR Lactobacillus strains and AMR genes.

Isolation and molecular characterization of Salmonellaenterica and Escherichiacolifrom poultry samples

Innumerable foodborne pathogens including Salmonella and Escherichia coli pose a serious threat to human health and food safety. As perishable foods, poultry products are considered one of the most common sources of foodborne pathogens including Salmonella and E. coli due to transmission of drug resistance, dissemination of organisms, and cross-contamination. In our study, phenotypic and genotypic characterizations of Salmonella enterica and E. coli isolated from packaged raw chicken products were carried out. Samples belonging to different commercial brands were collected in Ankara in 2015. Among 15 out of 19 Salmonella enterica subsp. enterica strains isolated from different and/or same poultry samples were found as Infantis serotype, while 4 of them were identified as Enteritidis serotype by pulsed-field gel electrophoresis (PFGE) footprints. In addition, 19 out of 40 samples gave positive results for E. coli. In addition, PFGE types of Salmonella Infantis isolates were detected as PT 08, 45, and 50. Furthermore, multilocus sequence typing types of the samples were identified as ST 32. Results of the phenotypic and genotypic antimicrobial resistance profiles of Salmonella Infantis and E. coli isolates revealed considerable resistance to nalidixic acid, tetracycline, streptomycin, sulfisoxazole and trimethoprim/sulfamethoxazole. On the other hand, 3 E. coli isolates showed antibiotic susceptibility. All in all, this study might enlighten some molecular features of Salmonella and E. coli isolated from chicken products in Turkey.

Genomic surveillance links livestock production with the emergence and spread of multi-drug resistant non-typhoidal Salmonella in Mexico.

Multi-drug resistant (MDR) non-typhoidal Salmonella (NTS) is increasingly common worldwide. While food animals are thought to contribute to the growing antimicrobial resistance (AMR) problem, limited data is documenting this relationship, especially in low and middle-income countries (LMIC). Herein, we aimed to assess the role of non-clinical NTS of bovine origin as reservoirs of AMR genes of human clinical significance. We evaluated the phenotypic and genotypic AMR profiles in a set of 44 bovine-associated NTS. For comparative purposes, we also included genotypic AMR data of additional isolates from Mexico (n = 1,067) that are publicly available. The most frequent AMR phenotypes in our isolates involved tetracycline (40/44), trimethoprim-sulfamethoxazole (26/44), chloramphenicol (19/44), ampicillin (18/44), streptomycin (16/44), and carbenicillin (13/44), while nearly 70% of the strains were MDR. These phenotypes were correlated with a widespread distribution of AMR genes (i.e. tetA, aadA, dfrA12, dfrA17, sul1, sul2, bla-TEM-1, blaCARB-2) against multiple antibiotic classes, with some of them contributed by plasmids and/or class-1 integrons. We observed different AMR genotypes for betalactams and tetracycline resistance, providing evidence of convergent evolution and adaptive AMR. The probability of MDR genotype occurrence was higher in meat-associated isolates than in those from other sources (odds ratio 11.2, 95% confidence interval 4.5-27.9, P < 0.0001). The study shows that beef cattle are a significant source of MDR NTS in Mexico, highlighting the role of animal production on the emergence and spread of MDR Salmonella in LMIC.

Phenotypic and Genotypic Antimicrobial Resistance Profiles of Escherichia coli O157 Isolates from Cattle in Cameroon

Phenotypic and Genotypic Antimicrobial Resistance Profiles of Escherichia coli O157 Isolates from Cattle in Cameroon

Phenotypic and genotypic antimicrobial resistance of staphylococci from bovine milk

The aim of this study was to examine phenotypic and genotypic antimicrobial resistance of staphylococci from milk samples from cows with subclinical and clinical mastitis and from cows without mastitis symptoms to methicillin, tetracyclines, macrolides and lincosamides (ML). Of 207 strains, including 34 S. aureus and 173 coagulase-negative staphylococci (CNS), 11 (6.4%) CNS strains were phenotypically resistant to methicillin. The mecA gene was detected by PCR only in two S. xylosus strains and one strain of S. epidermidis and S. simulans. No methicillin-resistant S. aureus strains were observed. In methicillin-resistant strains with mecA, gene resistance to other investigated antibiotics was not observed. Phenotypic resistance to tetracycline was detected in 11.0% of CNS strains and 47.4% of them carried the tetK gene. Of 173 CNS strains studied, 27 (15.6%) were resistant to at least one ML antibiotic. The resistance gene ermC was detected in 55.5% of the 27 ML-resistant strains. The ermA and ermB genes were detected in 14.8% and 11.1% of ML-resistant CNS strains, respectively. Antimicrobial resistance to methicillin, tetracyclines and macrolides was detected more frequently in staphylococcal strains from clinical mastitis compared to animals with subclinical symptoms and without mastitis, while the resistance to lincosamides showed a similar frequency in all groups of cows. In conclusion, CNS species from bovine milk differ in phenotypic and genotypic antimicrobial resistance profiles, and the use of PCR technique alone for the detection of methicillin, macrolide, lincosamide and tetyracycline resistance in CNS from cattle is not reliable.

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine milk samples

The aim of this study was to examine whether antimicrobial resistance profiles of coagulase-negative Staphylococcus (CNS) species isolated from milk of dairy cows differed between bacterial species, and to compare results obtained by phenotypic and genotypic profiling of resistance to penicillin, oxacillin and macrolide–lincosamide (ML) antibiotics. Of 170 CNS isolates, 83 (48.8%) were phenotypically susceptible to all antimicrobial agents tested in minimum inhibitory concentration (MIC) assays, 40.6% expressed resistance to a single compound or a single class of compounds, and 10.6% to multiple drug classes. Nine percent, 68%, 19%, 4% and 1% of isolates were negative for all resistance genes tested by PCR or positive for one, two, three or four resistance genes, respectively. Phenotypic resistance and detection of resistance genes other than blaZ were relatively rare in Staphylococcus chromogenes, which was the most common CNS species (36% of 170 genotypically identified isolates). In Staphylococcus epidermidis, which was the second most common CNS species (14% of isolates), phenotypic penicillin resistance was significantly more common than in other CNS species. Almost half of the S. epidermidis isolates carried multiple resistance genes and 30% carried the methicillin resistance gene mecA. Survival analysis using MIC values showed significant associations between phenotypic and genotypic resistance profiles. We conclude that CNS species from bovine milk differ significantly in phenotypic and genotypic antimicrobial resistance profiles, which has implications for treatment and management decisions.

Phenotypic and Genotypic Antimicrobial Resistance Profiles ofCampylobacter jejuniIsolated from Cattle, Sheep, and Free-Range Poultry Faeces

Minimum inhibitory concentrations (MIC) of 13 antimicrobial agents were determined by broth microdilution for 72 Campylobacter jejuni strains from livestock. Twenty-three (31.9%) isolates were fully susceptible; all isolates were susceptible to erythromycin, chloramphenicol, streptomycin, gentamicin, sulfamethoxazole, and meropenem, and all but one to kanamycin. Resistance to quinolones was highest (52.8%), reaching similar values among poultry, dairy cattle, and sheep, but lower in beef cattle. Resistance to tetracyclines (48.6%) was mainly associated to dairy cattle and β-lactams (26.4%) to poultry. Multidrug resistance was mainly detected in dairy cattle (28.6%) and poultry (21.0%), whereas beef cattle had the highest percentage of fully susceptible isolates. Two real-time PCR assays to detect point mutations associated to quinolone (C257T in the gyrA gene) and macrolide (A2075G in the 23S rRNA genes) resistance were developed and validated on these strains. The analysis of a further set of 88 isolates by real-time PCR confirmed the absence of macrolide resistance and demonstrated the reproducibility and processability of the assay.

Heterogeneity among Virulence and Antimicrobial Resistance Gene Profiles of Extraintestinal Escherichia coli Isolates of Animal and Human Origin

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates collected from different infected animals and from human patients with extraintestinal infections in 2001 were characterized for their phenotypic and genotypic antimicrobial resistance profiles, genotypes, and key virulence factors. Among the 10 antimicrobial agents tested, resistance to ampicillin, tetracycline, and sulfonamides was most frequent. Multiresistant strains were found in both the animal and the human groups of isolates. Resistance gene distribution was assessed by colony hybridization. Similar antibiotic resistance patterns could be observed in the animal and the human isolates. Although some resistance genes, such as bla(TEM), sulI, and sulII, were equally represented in the animal and human ExPEC isolates, differences in the distributions of tetracycline [tet(D)], chloramphenicol (catI, catIII, and floR), and trimethoprim (dhfrI, dhfrV, dhfrVII, and dhfrXIII) resistance genes were observed between the animal and the human isolates. Approximately one-third of the ExPEC isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained aminoglycoside (aadA1, aadA2, aadA5, and aadA6) and/or trimethoprim (dhfrIb, dhfrXII, and dhfrXVII) resistance genes. The ExPEC strains belonged to different phylogenetic groups, depending on their host origin. Strains isolated from animal tissues belonged to either a commensal group (group A or B1) or a virulent group (group B2 or D), while the majority of the human isolates belonged to a virulent group (group B2 or D). Although the limited number of isolates evaluated in the present study prevents firm epidemiological conclusions from being made, on a more global scale, these data demonstrate that extraintestinal isolates of E. coli can possess relatively distinct intra- and intergroup resistance gene profiles, with animal isolates presenting a more heterogeneous group than human isolates.

Antimicrobial resistance genes in enterotoxigenic Escherichia coli O149:K91 isolates obtained over a 23-year period from pigs.

A total of 112 Escherichia coli O149:K91 strains isolated from pigs with diarrhea in Quebec, Canada, between 1978 and 2000 were characterized for their genotypic antimicrobial resistance profiles. Tests for resistance to 10 antimicrobial agents were conducted. Resistance to tetracycline and sulfonamides was found to be the most frequent, but resistance to cefotaxime and ceftiofur was absent. An increase in the number of isolates resistant to at least three antimicrobials was observed over time. The distribution of 28 resistance genes covering six antimicrobial families (beta-lactams, aminoglycosides, phenicols, tetracycline, trimethoprim, and sulfonamides) was assessed by colony hybridization. Significant differences in the distributions of tetracycline [tet(A), tet(B), tet(C)], trimethoprim (dhfrI, dhfrV, dhfrXIII), and sulfonamide (sulI, sulII) resistance genes were observed during the study period (1978 to 2000). Sixty percent of the isolates possessed a class 1 integron, illustrating the importance of integrons in the epidemiology of antibiotic resistance in E. coli strains from pigs. Amplification of the integron's variable region resulted in four distinct fragments of 1, 1.3, 1.6, and 1.8 kb, with the 1.6- and 1.8-kb fragments appearing only during the last half of the study period. Examination of linkages among the different resistance genes showed a variety of positive and negative associations. Association analysis of isolates divided into two groups, those isolated between 1978 and 1989 and those isolated between 1990 and 2000, revealed the appearance of new positive resistance gene associations. Our genotypic resistance analyses of ETEC isolates from pigs indicate that many of the antibiotic resistance genes behind phenotypic resistance are not static but, rather, are in a state of flux driven by various selection forces such as the use of specific antimicrobials.