In vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli in the mammalian gut.

The widespread sulfonamide resistance genes sul1, sul2, and sul3 in food and gut bacteria have attracted considerable attention. In this study, we assessed the in vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli, using a murine model. High fitness costs were incurred for sul1 and sul3 gene-dependent E. coli strains in vivo. A fitness advantage was found in three of the eight mice after intragastric administration of sul2 gene-dependent E. coli strains. We isolated three compensatory mutant strains (CMSs) independently from three mice that outcompeted the parent strain P2 in vivo. Whole-genome sequencing revealed seven identical single nucleotide polymorphism (SNP) mutations in the three CMSs compared with strain P2, an additional SNP mutation in strain S2-2, and two additional SNP mutations in strain S2-3. Furthermore, tandem mass tag-based quantitative proteomic analysis revealed abundant differentially expressed proteins (DEPs) in the CMSs compared with P2. Of these, seven key fitness-related DEPs distributed in two-component systems, galactose and tryptophan metabolism pathways, were verified using parallel reaction monitoring analysis. The DEPs in the CMSs influenced bacterial motility, environmental stress tolerance, colonization ability, carbohydrate utilization, cell morphology maintenance, and chemotaxis to restore fitness costs and adapt to the mammalian gut environment.IMPORTANCESulfonamides are traditional synthetic antimicrobial agents used in clinical and veterinary medical settings. Their long-term excessive overuse has resulted in widespread microbial resistance, limiting their application for medical interventions. Resistance to sulfonamides is primarily conferred by the alternative genes sul1, sul2, and sul3 encoding dihydropteroate synthase in bacteria. Studying the potential fitness cost of these sul genes is crucial for understanding the evolution and transmission of sulfonamide-resistant bacteria. In vitro studies have been conducted on the fitness cost of sul genes in bacteria. In this study, we provide critical insights into bacterial adaptation and transmission using an in vivo approach.

Marine bacteria harbor the sulfonamide resistance gene sul4 without mobile genetic elements.

Marine bacteria are possible reservoirs of antibiotic-resistance genes (ARGs) originating not only from clinical and terrestrial hot spots but also from the marine environment. We report here for the first time a higher rate of the sulfonamide-resistance gene sul4 in marine bacterial isolates compared with other sul genes. Among four sulfonamide-resistance genes (sul1, sul2, sul3, and sul4), sul4 was most abundant (45%) in 74 sulfonamide-resistant marine isolates by PCR screening. The order of abundance was sul4 (33 isolates) >sul2 (6 isolates) >sul3 (5 isolates) >sul1 (1 isolate). Whole-genome sequencing of 23 isolates of sul4-expressing α- and γ-proteobacteria and bacilli revealed that sul4 was not accompanied by known mobile genetic elements. This suggests that sul4 in these marine isolates is clonally transferred and not horizontally transferable. Folate metabolism genes formed a cluster with sul4, suggesting that the cluster area plays a role in folate metabolism, at which sul4 functions as a dihydropteroate synthase. Thus, sul4 might be expressed in marine species and function in folate synthesis, but it is not a transferable ARG.

Emergence of the fourth mobile sulfonamide resistance gene sul4 in clinical Salmonella enterica.

The fourth mobile sulfonamide resistance gene sul4 has been discovered in many metagenomic datasets. However, there is no reports of it in cultured bacteria. In this study, a sul4 positive clinical Salmonella enterica SC2020597 was obtained by conventional Salmonella isolation methods and characterized by species identification and antimicrobial susceptibility testing. Meanwhile, the genomic DNA was sequenced using both long-read and short-read methods. Following that, the complete genome was analyzed by bioinformatic methods. The sul4 gene in S. enterica SC2020597 differed from the sul4 identified in metagenomic data by one amino acid and could confer full resistance to sulfamethoxazole. Genetic location analysis showed that the sul4 in SC2020597 was carried by a complex chromosomally integrated hybrid plasmid. ISCR20-like was strongly associated with the mobilization of sul4 by core genetic context analysis. To the best of our knowledge, this is the first report of the emergence of sul4 in clinically cultured S. enterica. More important, the sul4 has the potential to spread to other bacteria with the help of mobile elements.

Corrigendum: The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

[This corrects the article DOI: 10.3389/fmicb.2023.1058350.].

The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

Introduction: Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2. Methods: Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ). Results: Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication. Discussion: We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics.

Impacts of Chemical and Organic Fertilizers on the Bacterial Communities, Sulfonamides and Sulfonamide Resistance Genes in Paddy Soil Under Rice-Wheat Rotation.

The responses of sulfonamides, sulfonamide-resistance genes (sul) and soil bacterial communities to different fertilization regimes were investigated by performing a field experiment using paddy soil with no fertilizer applied, chemical fertilizer applied, organic fertilizer applied, and combination of chemical and organic fertilizer applied. Applying organic fertilizer increased the bacterial community diversity and affected the bacterial community composition. Eutrophic bacteria (Bacteroidetes, Gemmatimonadetes, and Proteobacteria) were significantly enriched by applying organic fertilizer. It was also found organic fertilizer application increased sulfamethazine content and the relative abundances of sul1 and sul2 in the soil. In contrast, applying chemical fertilizer significantly increased the abundance of Nitrospirae, Parcubacteria, and Verrucomicrobia and caused no obvious changes on sul. Correlation analysis indicated that sul enrichment was associated with the increases in sulfamethazine content and potential hosts (e.g., Novosphingobium and Rhodoplanes) population. The potential ecological risks of antibiotics in paddy soil with organic fertilizer applied cannot be ignored.

Enhanced control of sulfonamide resistance genes and host bacteria during thermophilic aerobic composting of cow manure.

Traditional composting has already shown a certain effect in eliminating antibiotic residues, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs). It is worth noting that the rebounding of ARGs and the succession of the bacterial community during conventional aerobic composting are still serious threats. Considering the probable risk, improved and adaptable technologies are urgently needed to control antibiotic resistance efficiently. This study monitored how thermophilic aerobic composting affected the ARGs, as well as the bacterial diversity during the composting of cow manure spiked with sulfamethoxazole (SMX) at different concentrations. Results showed that the degradation of SMX was enhanced during thermophilic aerobic composting (control > SMX25 > SMX50 > SMX100) and was no longer detected after 20 days of composting. High temperature or heat significantly stimulated the rebounding of certain genes. After 35 days, the abundance of detected genes (sul2, sulA, dfrA7, and dfrA1) significantly decreased (p < 0.05) in control and antibiotic-spiked treatments, except for sul1. The addition of three concentrations of SMX elicited a sharp effect on bacterial diversity, and microbial structure in SMX25 led to significant differences with others (p < 0.05). The network analysis revealed more rigorous interactions among ARGs and abundant genera, suggesting that the host of ARGs potentially increased at low concentrations of SMX. Especially, genera g_norank_f__Beggiatoaceae, Ruminiclostridium, Caldicoprobacter, g_norank_o_MBA03, Hydrogenispora, and Ruminiclostridium_1 were major potential hosts for sul1. In conclusion, the rebounding of ARGs could be intermitted partially, and more efficient control of antibiotic resistance could be achieved in the thermophilic composting compared to conventional methods.

Large-scale pattern of resistance genes and bacterial community in the tap water along the middle and low reaches of the Yangtze River.

Antibiotic and metal resistance genes (ARGs and MRGs) in tap water are of great public health concern. However, very fewer studies focused on the relationship between resistance genes and opportunistic pathogens in tap water. In this study, the diversity and abundance of resistance genes and bacterial community from tap water at a large-scale along the middle and lower reaches of the Yangtze River were investigated. The total relative abundances of ARGs and MRGs were 2.95 × 10-3-1.22 × 10-1 and 1.93 × 10-3-1.20 × 10-1 copies/16S rRNA, respectively. The blaTEM and merP detected were major ARG and MRG subtypes, respectively. Mobile genetic elements (Intl1 and tnpA) showed significant correlations with the abundance of ARGs. Heavy metals also played a vital role in the co-selection of ARGs. Surprisingly, there were still eight opportunistic pathogens in tap water, among which Escherichia coli, Helicobacter pylori, Mycoplasma pneumoniae, and Porphyromonas gingivalis were the potential host of ARGs and MRGs. Escherichia coli had the highest abundance, while Bacillus anthracis had the highest detected frequency (100%), a widespread opportunistic pathogen in tap water.

Corrigendum: Diverse Mobile Genetic Elements and Conjugal Transferability of Sulfonamide Resistance Genes (sul1, sul2, and sul3) in Escherichia coli Isolates From Penaeus vannamei and Pork From Large Markets in Zhejiang, China.

[This corrects the article DOI: 10.3389/fmicb.2019.01787.].

Development and evaluation of a Luminex xTAG assay for sulfonamide resistance genes in Escherichia coli and Salmonella isolates.

Clinically occurring sulfonamide resistance in gram-negative bacteria is codified by several sul genes, mostly associated with the mobilized genetic elements named integrons, and integrons are frequently found in plasmids. There are four sul genes (sul1, sul2, sul3 and sul4) that encode resistance to sulfonamides. The aim of the present study was to develop a bead-based xTAG assay for the simultaneous detection of all four sul genes and related Class 1 integrons (int1) in Escherichia coli and Salmonella isolates. The limits of detection ranged from 10 to 1000 copies/µL of input purified plasmid DNA. Forty-one bacterial isolates from clinical samples were examined using the newly developed xTAG assay and also by conventional PCR to determine the relative performance of each. The results obtained by xTAG assay showed higher detection rates and accuracy for sul genes than conventional PCR. It indicated that the xTAG-multiplex PCR is a convenient method for rapid identification of sul genes.

Diverse Mobile Genetic Elements and Conjugal Transferability of Sulfonamide Resistance Genes (sul1, sul2, and sul3) in Escherichia coli Isolates From Penaeus vannamei and Pork From Large Markets in Zhejiang, China.

High prevalence rates of sulfonamide resistance genes sul1, sul2, and sul3 have been observed in Gram-negative bacteria isolated from humans, domestic animals, and aquaculture species worldwide. We investigated the distribution characteristics, location, conjugative transferability, and genetic environments of sul genes from Escherichia coli isolates collected from Penaeus vannamei and pork samples from three large markets in Zhejiang, China. The prevalence rates of sul genes in sulfonamide-resistant E. coli isolates from P. vannamei and pork samples were 90.0 and 88.6%, respectively, and the prevalence of sul1 and sul2 was significantly higher than that of sul3 (p < 0.05). Twenty-four representative sul-positive E. coli isolates were analyzed in detail. Southern blot hybridization confirmed that sul genes of E. coli isolates were located on plasmids and/or chromosomes. Transfer of resistance through conjugation was observed in all 18 E. coli isolates harboring sul genes on plasmids. Replicon typing identified seven different incompatibility groups and IncF was the dominant replicon type among sul gene-containing plasmids from both sources. PCR walking analysis indicated that 87.5% (35/40) of sul gene-related fragments carried insertion sequences (ISs) belonging to a variety of families in diverse sites, with IS26 occurring most frequently. In addition, the sul1 gene was detected mainly in fragments carrying class 1 integrons. Co-location on the same fragment with resistance genes that may contribute to the persistence and dissemination of sul1 and/or sul2 genes. The diversity of mobile genetic elements and resistance genes adjacent to sul3 was much lower than those adjacent to sul1 and sul2, especially those located in chromosomes, which reduced the transmission potential of the sul3 gene. In conclusion, combined with the results of clonal relatedness analysis by PFGE and MLST of 24 representative E. coli isolates from P. vannamei and pork samples, it showed that a small number of sul genes were vertically transmitted among E. coli from P. vannamei and that horizontal gene transfer was likely the main transmission mechanism of sul genes from both sources. Our results provide important information to better understand the risk of transmission of sul genes from seafood and meat to humans.

Sulfadiazine degradation in soils: Dynamics, functional gene, antibiotic resistance genes and microbial community.

Sulfonamides and their corresponding antibiotic resistance genes (ARGs) are widespread in the environment, which leads to a major threat to global health crisis. Biodegradation plays a major role in sulfonamides removal in soil ecosystem, but the degradation dynamics and the associated functional bacteria in situ remain unclear. In this study, aerobic degradation of sulfadiazine (SDZ) at two dosages (1 and 10 mg/kg) was explored for up to 70 days in two different agricultural soils. The removal of SDZ in all treatments followed first-order multi-compartment model with half-life times of 0.96-2.57 days, and DT50 prolonged with the increase of initial dosage. A total of seven bacterial genera, namely Gaiella, Clostrium_sensu_stricto_1, Tumebacillus, Roseiflexus, Variocorax, Nocardioide and Bacillus, were proposed as the potential SDZ-degraders. sadA gene was for the first time detected in soil samples, but other functional genes might also participate in SDZ degradation. The enrichment of sulfonamide resistance genes was found after 70 days' incubation, which might result in the spread of ARGs in soil. This study can add some new insights towards SDZ degradation in soil ecosystem and provide a potential resource for the bioremediation of SDZ-contaminated soil.

Fate of sulfonamide resistance genes during sludge anaerobic fermentation: Roles of sludge components and fermentation pHs.

This study assessed potential effects of two neglected factors (sludge components and pH values) on the fate of sulfonamide (sul) resistance genes during sludge anaerobic fermentation. It was found that sludge with different contents of protein, carbohydrate and humic acid caused no significant changes in the abundances of sul genes. Nevertheless, sul genes were sensitive to pHs (4-10), and the maximum attenuations (0.8-1.1 log unit) were obtained at pH 10. Mechanism exploration indicated that pHs drove the community evolution of sulfonamide resistant bacteria (SRB), most of which were affiliated to the pH-enriched phyla but not the pH-enriched dominant genera. In addition, the relative abundances of SRB were decreased under both acidic and alkaline conditions. Furthermore, the abundances of intI 1 as well as the sul-carrying abilities of plasmid and extracellular DNA were all reduced at test pHs, indicating that the potential of horizontal gene transfer among bacteria was restricted.

On sulfonamide resistance, sul genes, class 1 integrons and their horizontal transfer in Escherichia coli.

Class 1 integrons (Int1) contribute to antibiotic multiresistance in Gram-negative bacteria. Being frequently carried by conjugative plasmids, their spread would depend to some extent on their horizontal transfer to other bacteria. This was the main issue that was addressed in this work: the analysis of Int1 lateral transfer in the presence of different antibiotic pressures. Strains from a previously obtained collection of Escherichia coli K12 carrying natural Int1+ conjugative plasmids were employed as Int1 donors in conjugation experiments. Two recipient strains were used: an E. coli K12 and an uropathogenic E. coli isolate. The four antibiotics employed to select transconjugants in LB solid medium were ampicillin, trimethoprim, sulfamethoxazole, and co-trimoxazole. For this purpose, adequate final concentrations of the three last antibiotics had to be determined. Abundant transconjugants resulted from the mating experiments and appeared in most -but not all-selective plates. In those supplemented with sulfamethoxazole or co-trimoxazole, transconjugants grew or not depending on the genetic context of the recipient strain and on the type of gene conferring sulfonamide resistance (sul1 or sul2) carried by the Int1+ plasmid. The horizontal transfer of a recombinant plasmid bearing an Int1 was also assayed by transformation and these experiments provided further information on the viability of the Int1+ clones. Overall, results point to the existence of constraints for the lateral transfer of Int1 among E. coli bacteria, which are particularly evidenced under the antibiotic pressure of sulfamethoxazole or of its combined formula co-trimoxazole.

A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria.

Sulfonamide-degrading bacteria have been discovered in various environments, suggesting the presence of novel resistance mechanisms via drug inactivation. In this study, Microbacterium sp. CJ77 capable of utilizing various sulfonamides as a sole carbon source was isolated from a composting facility. Genome and proteome analyses revealed that a gene cluster containing a flavin-dependent monooxygenase and a flavin reductase was highly up-regulated in response to sulfonamides. Biochemical analysis showed that the two-component monooxygenase system was key enzymes for the initial cleavage of sulfonamides. Co-expression of the two-component system in Escherichia coli conferred decreased susceptibility to sulfamethoxazole, indicating that the genes encoding drug-inactivating enzymes are potential resistance determinants. Comparative genomic analysis revealed that the gene cluster containing sulfonamide monooxygenase (renamed as sulX) and flavin reductase (sulR) was highly conserved in a genomic island shared among sulfonamide-degrading actinobacteria, all of which also contained sul1-carrying class 1 integrons. These results suggest that the sulfonamide metabolism may have evolved in sulfonamide-resistant bacteria which had already acquired the class 1 integron under sulfonamide selection pressures. Furthermore, the presence of multiple insertion sequence elements and putative composite transposon structures containing the sulX gene cluster indicated potential mobilization. This is the first study to report that sulX responsible for both sulfonamide degradation and resistance is prevalent in sulfonamide-degrading actinobacteria and its genetic signatures indicate horizontal gene transfer of the novel resistance gene.

Occurrence of Transferable Integrons and sul and dfr Genes Among Sulfonamide-and/or Trimethoprim-Resistant Bacteria Isolated From Chilean Salmonid Farms.

Salmon farming industry in Chile currently uses a significant quantity of antimicrobials to control bacterial pathologies. The main aims of this study were to investigate the presence of transferable sulfonamide- and trimethoprim-resistance genes, sul and dfr, and their association with integrons among bacteria associated to Chilean salmon farming. For this purpose, 91 Gram-negative strains resistant to sulfisoxazole and/or trimethoprim recovered from various sources of seven Chilean salmonid farms and mainly identified as belonging to the Pseudomonas genus (81.0%) were studied. Patterns of antimicrobial resistance of strains showed a high incidence of resistance to florfenicol (98.9%), erythromycin (95.6%), furazolidone (90.1%) and amoxicillin (98.0%), whereas strains exhibited minimum inhibitory concentrations (MIC90) values of sulfisoxazole and trimethoprim of >4,096 and >2,048 µg mL-1, respectively. Strains were studied for their carriage of these genes by polymerase chain reaction, using specific primers, and 28 strains (30.8%) were found to carry at least one type of sul gene, mainly associated to a class 1 integron (17 strains), and identified by 16S rRNA gene sequencing as mainly belonging to the Pseudomonas genus (21 strains). Of these, 22 strains carried the sul1 gene, 3 strains carried the sul2 gene, and 3 strains carried both the sul1 and sul2 genes. Among these, 19 strains also carried the class 1 integron-integrase gene intI1, whereas the dfrA1, dfrA12 and dfrA14 genes were detected, mostly not inserted in the class 1 integron. Otherwise, the sul3 and intI2 genes were not found. In addition, the capability to transfer by conjugation these resistance determinants was evaluated in 22 selected strains, and sul and dfr genes were successfully transferred by 10 assayed strains, mainly mediated by a 10 kb plasmid, with a frequency of transfer of 1.4 × 10-5 to 8.4 × 10-3 transconjugant per recipient cell, and exhibiting a co-transference of resistance to florfenicol and oxytetracycline, currently the most used in Chilean salmon industry, suggesting an antibacterial co-selection phenomenon. This is the first report of the characterization and transferability of integrons as well as sul and dfr genes among bacteria associated to Chilean salmon farms, evidencing a relevant role of this environment as a reservoir of these genes.

Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes.

Soil represents a significant reservoir of antibiotic resistance genes (ARGs), which can potentially spread across distinct ecosystems and be acquired by pathogens threatening human as well as animal health. Currently, information on the identity and diversity of these genes, enabling anticipation of possible future resistance development in clinical environments and the livestock sector, is lacking. In this study, we applied functional metagenomics to discover novel sulfonamide as well as tetracycline resistance genes in soils derived from forest and grassland. Screening of soil metagenomic libraries revealed a total of eight so far unknown ARGs. The recovered genes originate from phylogenetically diverse soil bacteria (e.g., Actinobacteria, Chloroflexi, or Proteobacteria) and encode proteins with a minimum identity of 46% to other antibiotic resistance determinants. In particular forest soil ecosystems have so far been neglected in studies focusing on antibiotic resistance. Here, we detected for the first time non-mobile dihydropteroate synthase (DHPS) genes conferring resistance to sulfonamides in forest soil with no history of exposure to these synthetic drugs. In total, three sulfonamide resistant DHPSs, differing in taxonomic origin, were discovered in beech or pine forest soil. This indicates that sulfonamide resistance naturally occurs in forest-resident soil bacterial communities. Besides forest soil-derived sulfonamide resistance proteins, we also identified a DHPS affiliated to Chloroflexi in grassland soil. This enzyme and the other recovered DHPSs confer reduced susceptibility toward sulfamethazine, which is widely used in food animal production. With respect to tetracycline resistance, four efflux proteins affiliated to the major facilitator superfamily (MFS) were identified. Noteworthy, one of these proteins also conferred reduced susceptibility toward lincomycin.

High-Throughput Screen Identifying the Thiosemicarbazone NSC319726 Compound as a Potent Antimicrobial Lead Against Resistant Strains of Escherichia coli.

Antibiotic discovery is vital when considering the increasing antimicrobial resistance threat. The aim of this work was to provide a high-throughput screen (HTS) assay using multidrug-resistant Escherichia coli strains to enable further research into antimicrobial lead discovery and identify novel antimicrobials. This study describes a primary HTS of a diverse library of 7884 small molecules against a susceptible E. coli strain. A secondary screening of 112 molecules against four E. coli strains with different susceptibility profiles revealed NSC319726 as a potential antimicrobial lead serving as a novel template. NSC319726 is a good candidate for an analoguing program.

Characterization of Bacterial Communities and Their Antibiotic Resistance Profiles in Wastewaters Obtained from Pharmaceutical Facilities in Lagos and Ogun States, Nigeria.

In Nigeria, pharmaceutical wastewaters are routinely disseminated in river waters; this could be associated with public health risk to humans and animals. In this study, we characterized antibiotic resistant bacteria (ARB) and their antibiotic resistance profile as well as screening for sul1 and sul2 genes in pharmaceutical wastewater effluents. Bacterial composition of the wastewater sources was isolated on non-selective media and characterized by the polymerase chain reaction (PCR) amplification of the 16S rRNA genes, with subsequent grouping using restriction fragment length polymorphism (RFLP) and sequencing. The antibiotics sensitivity profiles were investigated using the standard disk diffusion plate method and the minimum inhibitory concentrations (MICs) of selected antibiotics on the bacterial isolates. A total of 254 bacterial strains were isolated, and majority of the isolates were identified as Acinetobacter sp., Klebsiella pneumonia, Proteus mirabilis, Enterobacter sp. and Bacillus sp. A total of 218 (85.8%) of the bacterial isolates were multidrug resistant. High MICs values were observed for all antibiotics used in the study. The result showed that 31.7%, 21.7% and 43.3% of the bacterial isolates harbored sul1, sul2, and Intl1 genes, respectively. Pharmaceuticals wastewaters are potential reservoirs of ARBs which may harbor resistance genes with possible risk to public health.

Establishment of a Multiplex Loop-Mediated Isothermal Amplification Method for Rapid Detection of Sulfonamide Resistance Genes (sul1, sul2, sul3) in Clinical Enterobacteriaceae Isolates from Poultry.

Antimicrobial resistance genes play an important role in mediating resistance to sulfonamide in Gram-negative bacteria. While PCR is the current method to detect sulfonamide resistance genes (sul1, sul2, sul3), it is time-consuming and costly and there is an urgent need to develop a more convenient, simpler and rapid test for the sul. In this study, we describe a multiplex loop-mediated isothermal amplification (m-LAMP) assay we developed for the rapid and simultaneous detection of three sul. This m-LAMP assay successfully detected seven reference strains with different sul genotypes, but was negative for nine sul-negative reference strains. The m-LAMP products were verified by HinfI restriction enzyme digestion and the detection limit of the test was 0.5 pg genomic DNA per reaction. Testing 307 sulfonamide-resistant Enterobacteriaceae clinical isolates with the m-LAMP revealed all were positive for the sul with sul2 (79.5%) and sul1 (64.5%) being most prevalent, and sul3 the least (12.1%). Of the Enterobacteriaceae isolates tested, the Salmonella Indiana, a newly emerging serovar resistant to numerous antimicrobials, were most commonly positive with 33% having sul3.