Cassette Arrays Research Articles

Analysis of the Class 1 Integrons, Carbapenemase Genes and Biofilm Formation Genes Occurrence in Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant A. baumannii (CRAB) has spread rapidly in clinical settings and has become a key concern. The main objective of this study was to identify the distribution of integrons and biofilm-formation-related virulence genes in CRAB isolates. A total of 269 A. baumannii isolates (219 isolates of CRAB and 50 isolates of carbapenem-sensitive A. baumannii (CSAB)) were collected. Carbapenemase genes (bla KPC, bla VIM, bla IMP, bla NDM, and bla OXA-23-like) and biofilm-formation-related virulence genes (abal, bfms, bap, and cusE) were screened with PCR. Class 1 integron was screened with PCR, and common promoters and gene cassette arrays were determined with restriction pattern analysis combined with primer walking sequencing. Whole-genome sequencing was conducted, and data were analyzed for a bla OXA-23-like-negative isolate. All 219 CRAB isolates were negative for bla KPC, bla VIM, bla IMP, and bla NDM, while bla OXA-23-like was detected in 218 isolates. The detection rates for abal, bfms, bap, and cusE in 219 CRAB were 93.15%, 63.93%, 88.13%, and 77.63%, respectively. Class 1 integron was detected in 75 CRAB (34.25%) and in 3 CSAB. The single gene cassette array aacA4-catB8-aadA1 with relatively strong PcH2 promoter was detected in class 1 integrons. The bla OXA-23-like-negative CRAB isolate was revealed to be a new sequence type (Oxford 3272, Pasteur 2520) carrying bla OXA-72, bla OXA-259, and bla ADC-26. In conclusion, bla OXA-23-like was the main reason for CRAB's resistance to carbapenems. A new (Oxford 3272, Pasteur 2520) CRAB sequence type carrying the bla OXA-72, bla OXA-259, and bla ADC-26 was reported.

Emergence of extensive drug resistance and high prevalence of multidrug resistance among clinical Proteus mirabilis isolates in Egypt

BackgroundProteus mirabilis is an opportunistic pathogen that has been held responsible for numerous nosocomial and community-acquired infections which are difficult to be controlled because of its diverse antimicrobial resistance mechanisms.MethodsAntimicrobial susceptibility patterns of P. mirabilis isolates collected from different clinical sources in Mansoura University Hospitals, Egypt was determined. Moreover, the underlying resistance mechanisms and genetic relatedness between isolates were investigated.ResultsAntimicrobial susceptibility testing indicated elevated levels of resistance to different classes of antimicrobials among the tested P. mirabilis clinical isolates (n = 66). ERIC-PCR showed great diversity among the tested isolates. Six isolates (9.1%) were XDR while all the remaining isolates were MDR. ESBLs and AmpCs were detected in 57.6% and 21.2% of the isolates, respectively, where blaTEM, blaSHV, blaCTX−M, blaCIT−M and blaAmpC were detected. Carbapenemases and MBLs were detected in 10.6 and 9.1% of the isolates, respectively, where blaOXA−48 and blaNDM−1 genes were detected. Quinolone resistant isolates (75.8%) harbored acc(6')-Ib-cr, qnrD, qnrA, and qnrS genes. Resistance to aminoglycosides, trimethoprim-sulfamethoxazole and chloramphenicol exceeded 80%. Fosfomycin was the most active drug against the tested isolates as only 22.7% were resistant. Class I or II integrons were detected in 86.4% of the isolates. Among class I integron positive isolates, four different gene cassette arrays (dfrA17- aadA5, aadB-aadA2, aadA2-lnuF, and dfrA14-arr-3-blaOXA−10-aadA15) and two gene cassettes (dfrA7 and aadA1) were detected. While class II integron positive isolates carried four different gene cassette arrays (dfrA1-sat1-aadA1, estXVr-sat2-aadA1, lnuF- dfrA1-aadA1, and dfrA1-sat2).ConclusionP. Mirabilis ability to acquire resistance determinants via integrons may be held responsible for the elevated rates of antimicrobial resistance and emergence of XDR or even PDR strains limiting the available therapeutic options for management of infections caused by those strains.

Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene.

The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. To identify sources for the aac(6')-Im fragment found in A. baumannii. MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different. The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

Molecular Characterization of Class 1 Integrons and Carbapenem-Resistant Genes in Enterobacter cloacae Complex Isolates.

Enterobacter cloacae complex (ECC) widely exists in the hospital environment and is one of the important conditional pathogens of hospital-acquired infection. To investigate the distribution of integrons and carbapenem-resistant genes in clinical ECC, 70 isolates of ECC from non-sputum specimens were collected. Class 1 and class 2 integron integrase gene intI1 and intI2, as well as common carbapenem-resistant genes, blaKPC, blaVIM, blaIMP, blaNDM, blaGES, and blaOXA-23, were screened. Gene cassette arrays and common promoters of class 1 integron together with subtypes of carbapenem-resistant genes were determined by sequencing. Resistant rates to commonly used antimicrobial agents between class 1 integron-positive and integron-negative ECC isolates were analyzed. The whole-genome of blaNDM-7 harboring Enterobacter hormaechei was sequenced and the sequence around blaNDM-7 was analyzed. Twenty isolates were positive for intI1. Nineteen different antimicrobial-resistant gene cassettes and 11 different gene cassette arrays, including aadA22-lnuF, were detected in this study. Common promoters of class 1 integron PcH1, PcW, PcW-P2, and PcH2 were detected in 12, 4, 3, and 1 isolates, respectively. The rates of antimicrobial resistance of intI1-positive isolates were higher than those of intI1-negative isolates to clinical commonly used antimicrobial agents. Carbapenem-resistant genes blaKPC-2, blaNDM-1, blaNDM-2, and blaNDM-7 were detected in 2, 1, 1, and 1 isolates, respectively. blaNDM-7 was located between bleMBL and IS5. To the best of our knowledge, this study reported for the first time of blaNDM-7 in ECC isolate in China.

Identification of promoter activity in gene-less cassettes fromVibrionaceae superintegrons.

Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions. ICs generally encode promoterless genes, whose expression relies on the platform-associated Pc promoter, with the cassette array functioning as an operon-like structure regulated by the distance to the Pc. This is relevant in large sedentary chromosomal integrons (SCIs) carrying hundreds of ICs, like those in Vibrio species. We selected 29 gene-less cassettes in four Vibrio SCIs, and explored whether their function could be related to the transcription regulation of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Additionally, we identified the transcription start sites of gene-less ICs through 5'-RACE. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 cassette downstream, increasing trimethoprim resistance>512-fold in V. cholerae and Escherichia coli. Furthermore, one cassette with an antisense promoter can reduce trimethoprim resistance when cloned downstream. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in SCIs and their clinical importance if captured by mobile integrons.

Cassette recombination dynamics within chromosomal integrons are regulated by toxin-antitoxin systems.

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here, we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.

Characterization of an NDM-1-Producing Citrobacter koseri Isolate from China.

The continuous rise in carbapenemase-producing Enterobacteriaceae infections is a major public health concern. However, there is limited information available on New Delhi metallo-β-lactamase-1 (NDM-1) producing Citrobacter koseri. In this study, we isolated a blaNDM-1-carrying C. koseri from a stool sample of an inpatient. Our aim was to investigate the phenotypic and genomic features of this clinically derived carbapenem-resistant C. koseri isolate and to characterize the transmission pattern of the IncFII/IncN plasmid that carries the blaNDM-1 gene. S1-PFGE, Southern blot and conjugation assay confirmed the presence of blaNDM-1 gene in a conjugative plasmid. C. koseri L2395 and transconjugant L2395-EC600 strains showed similar resistance spectrum. Whole-genome analysis revealed that pL2395_NDM is an IncFII/IncN plasmid with a length of 67,839 bp. Moreover, blaNDM-1 gene was found encoded in the ISKpn19-blaNDM-1-ble-tnpF-dsbD-cutA-ISKpn19 cassette array. Phylogenetic analysis revealed that strain L2395 was close to an IMP-4-bearing C. koseri from Australia. Ongoing surveillance will be essential to control and prevent the spread of carbapenem-resistant Citrobacter spp. in the future.

Meta-analysis of the Vmp-like sequences of Lyme disease Borrelia: evidence for the evolution of an elaborate antigenic variation system.

VMP-like sequence (vls) antigenic variation systems are present in every Lyme disease Borrelia strain with complete genome sequences. The linear plasmid-encoded vls system consists of a single expression site (vlsE) and contiguous array(s) of silent cassettes that have ~90% identity with the central cassette region of the cognate vlsE gene; antigenic variation occurs through random, segmental, and unidirectional recombination of vls silent cassette sequences into the vlsE expression site. Automated annotation programs do not accurately recognize vls silent cassette sequences, so these regions are not correctly annotated in most genomic sequences. In this study, the vls sequences were re-analyzed in the genomic sequences of 31 available Lyme disease Borrelia and one relapsing fever Borrelia organisms, and this information was utilized to systematically compare the vls systems in different species and strains. In general, the results confirm the conservation of the overall architecture of the vls system, such as the head-to-head arrangement of vlsE and a contiguous series of vlsS silent cassette sequences and presence of inverted repeat sequences between the two regions. However, the data also provide evidence for the divergence of the vls silent cassette arrays through point mutations, short indels, duplication events, and rearrangements. The probable occurrence of convergent evolution toward a vls system-like locus is exemplified by Borrelia turcica, a variable large protein (Vlp) expressing organism that is a member of the relapsing fever Borrelia group.

Dissecting molecular evolution of class 1 integron gene cassettes and identifying their bacterial hosts in suburban creeks via epicPCR.

Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution. We applied a single-cell fusion PCR-based technique-emulsion, paired isolation and concatenation PCR (epicPCR)-to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host. Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5'-untranslated region (5'-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5'-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5'-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli. Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes.

A Wohlfahrtiimonas chitiniclastica with a novel type of blaVEB-1-carrying plasmid isolated from a zebra in China.

Wohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which causes infections in immunocompromised patients and some animals. Herein, we reported a W. chitiniclastica BM-Y from a dead zebra in China. The complete genome sequencing of BM-Y showed that this isolate carried one chromosome and one novel type of blaVEB-1-carrying plasmid. Detailed genetic dissection was applied to this plasmid to display the genetic environment of blaVEB-1. Three novel insertion sequence (IS) elements, namely ISWoch1, ISWoch2, and ISWoch3, were found in this plasmid. aadB, aacA1, and gcuG were located downstream of blaVEB-1, composing a gene cassette array blaVEB-1-aadB-aacA1-gcuG bracketed by an intact ISWoch1 and a truncated one, which was named the blaVEB-1 region. The 5'-RACE experiments revealed that the transcription start site of the blaVEB-1 region was located in the intact ISWoch1 and this IS provided a strong promoter for the blaVEB-1 region. The spread of the blaVEB-1-carrying plasmid might enhance the ability of W. chitiniclastica to survive under drug selection pressure and aggravate the difficulty in treating infections caused by blaVEB-1-carrying W. chitiniclastica. To the best of our knowledge, this is the first report of the genetic characterization of a novel blaVEB-1-carrying plasmid with new ISs from W. chitiniclastica.

An expanded CRISPR-Cas9-assisted recombineering toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates.

Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa. This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.

High incidence of multiple intI1 genomic gene cassettes in Aeromonas strains

Antimicrobial resistance profiles of Aeromonas have been used as an effective indicator for monitoring resistances in aquatic environments. Integron gene cassettes are a major reservoir of antibiotic resistance genes in bacteria. To date, little has been reported on the antimicrobial resistance or integrons of Aeromonas in bullfrog aquaculture. In this study, 27 isolates belonging to four Aeromonas species (A. hydrophila, A. veronii, A. caviae, and A. jandaei) were isolated from farmed diseased American bullfrogs (Rana catesbeiana). all of them were multidrug-resistant, exhibiting resistance to at least seven drugs from five antibiotic classes. Class 1 integrons (intI1s), containing nine types of gene cassette arrays encoding resistance to trimethoprim (dfrA1, dfrA12, dfrA15, dfrA17), streptomycin/spectinomycin (aadA2), fluoroquinolones/aminoglycosides (aac(6′)-Ib-cr), rifampin (arr3), proline-rich antimicrobial peptides (ptrB), and multiple antimicrobial agents involved in biofilm development (dgs), were detected in 26 (26/27, 96.3%) of the isolates. Seven patterns were identified, and six of them contained two or three gene cassettes, in 27 isolates. This work highlights unusually high frequency (21/27, 77.8%) of multiple intI1 gene cassette arrays and three novel intI1 gene cassettes or arrays (dgc, ptrB, and aac(6′)-Ib-cr-arr3) in the isolates of Aeromonas spp. from American bullfrogs.

Class 1 integron carrying qacEΔ1 gene confers resistance to disinfectant and antibiotics in Salmonella

Salmonella has presented increasingly alarming rates of antimicrobial resistance believed to be a result of a high prevalence of integrons. It is speculated that disinfectant-resistant isolates are due to the expression of qacEΔ1, an efflux pump located in the 3′ conserved sequence (3′CS) of class 1 integrons. With this concern, we tested the antibiotic and disinfectant resistance of 581 Salmonella strains collected from different sources, and characterized their integron structures. Gene expression and induction experiments were also performed. Results showed that Salmonella have high resistance to antimicrobials, especially to sulfonamides (SAs, 78.83 %), tetracyclines (TCs, 75.04 %) and benzalkonium chloride (BC, 87.26 %). The multi-drug resistance (MDR) frequency reached up to 63.17 %, and the prevalence of intI1 was 45.78 %. Molecular characterization of class 1 integrons exhibited nine different gene cassette arrays, of these, dfrA12-orf-aadA2 (n = 75), EstX (n = 25) and aadA2 (n = 14) were the most frequent. Importantly, 74.06 % of intI1-positive isolates were carrying qacEΔ1-sul1 genes in the 3′CS. This study also demonstrated that phenotypic resistance to both antibiotics and disinfectants was significantly correlated with the emergence of intI1 (p < 0.05). 91.37 % of qacEΔ1-sul1 positive Salmonella were found with disinfectant resistance. Additionally, expression of qacEΔ1 gene in Escherichia coli confirmed qacEΔ1 is predominantly involved in conferring disinfectant resistance. Disinfectant induction experiments further implicated qacEΔ1 in disinfectant resistance. RT-qPCR revealed a disinfectant-mediated increase in the relative expression of antibiotic-resistant genes (ARGs), aadA2 and dfrA12 on the integron, and efflux pump genes (mdtH and acrD) indicating that disinfectant could trigger co or cross-resistance. Therefore, our study confirmed that using disinfectant could provide selection pressure for strains with acquired resistance to antibiotics, providing new insights into the public health impact of Salmonella and guide continued efforts in antimicrobial stewardship and prevention of antibiotic resistance.

Tracing the Sources and Prevalence of Class 1 Integrons, Antimicrobial Resistance, and Trace Elements Using European Honey Bees.

Surveillance of antimicrobial resistance is essential for an effective One Health response. This study explores the efficacy of European honey bees (Apis mellifera) for biomonitoring antimicrobial resistance (AMR) in urban areas. Class 1 integrons (intI1) are investigated as a universal AMR indicator, as well as associated cassette arrays and trace element contaminants at a city-wide scale. Class 1 integrons were found to be pervasive across the urban environment, occurring in 52% (75/144) of the honey bees assessed. The area of waterbodies within the honey bee's foraging radius was associated with intI1 prevalence, indicating an exposure pathway for future investigation to address. Trace element concentrations in honey bees reflected urban sources, supporting the application of this biomonitoring approach. As the first study of intI1 in honey bees, we provide insights into the environmental transfer of bacterial DNA to a keystone species and demonstrate how intI1 biomonitoring can support the surveillance of AMR.

Class 1 integrons in clinical and swine industry isolates of Salmonella Typhimurium from Colombia, dating 1997 to 2017.

Background. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) has been linked to outbreaks of foodborne gastroenteritis disease, and the emergence of antimicrobial-resistant clones. In Colombia, laboratory surveillance of Salmonella spp. between 1997-2018 revealed that S. Typhimurium was the most ubiquitous serovar (27.6 % of all Salmonella isolates), with increasing levels of resistance to several families of antibiotics.Hypothesis. Resistant isolates of S. Typhimurium recovered from human clinical, food and swine samples carry class 1 integrons that are linked to antimicrobial resistance genes.Aim. Identify class 1 integrons, and investigate their association with other mobile genetic elements, and their relationship to the antimicrobial resistance of Colombian S. Typhimurium isolates.Methods. In this study, 442 isolates of S. Typhimurium were analysed, of which 237 were obtained from blood culture, 151 from other clinical sources, 4 from non-clinical sources and 50 from swine samples. Class 1 integrons and plasmid incompatibility groups were analysed by PCR and whole-genome sequencing (WGS), and regions flanking integrons were identified by WGS. The phylogenetic relationship was established by multilocus sequence typing (MLST) and single-nucleotide polymorphism (SNP) distances for 30 clinical isolates.Results . Overall, 39 % (153/392) of the human clinical isolates and 22 % (11/50) of the swine S. Typhimurium isolates carried complete class 1 integrons. Twelve types of gene cassette arrays were identified, including dfr7-aac-bla OXA-2 (Int1-Col1), which was the most common one in human clinical isolates (75.2 %, 115/153). Human clinical and swine isolates that carried class 1 integrons were resistant to up to five and up to three antimicrobial families, respectively. The Int1-Col1 integron was most prevalent in stool isolates and was associated with Tn21. The most common plasmid incompatibility group was IncA/C.Conclusions. The widespread presence of the IntI1-Col1 integron in Colombia since 1997 was striking. A possible relationship between integrons, source and mobile elements that favour the spread of antimicrobial resistance determinants in Colombian S. Typhimurium was identified.

Identifying Suitable Three-Dimensional Bio-Printed Scaffold Architectures to Incubate in a Perfusion Bioreactor: Simulation and Experimental Approaches

Abstract Traditional cell culturing methods are limited in their ability to supply growth medium to cells within scaffolds. To address this, we developed a custom perfusion bioreactor that allows for dynamic medium supply to encapsulated or seeded cells. Our custom-designed bioreactor improves the in vivo stimuli and conditions, which may enhance cell viability and proliferation performance. Some of the efforts include using dual medium tanks to replace the medium without stopping perfusion and a newly designed perfusion chamber that can accommodate an array of cassettes allowing for a wide assortment of scaffold shapes and sizes. In this paper, we explored the response of fluid flow to certain types of scaffold pore geometries and porosities using simulation and experimental approaches. Various pore geometries were considered, such as uniform triangular, square, diamond, circular, and honeycomb having uniform and variable sizes. Finally, bone tissue architecture was mimicked and simulated to identify the impact of fluid flow. Based on the results, optimum pore geometry for scaffolds were determined. We explored real-time fluid flow response on scaffolds fabricated with 8% Alginate, 4% Alginate-4% Carboxymethyl Cellulose (CMC), and 2% Alginate-6% CMC incubated, allowing a constant fluid flow for various periods such as 1, 2, 4, and 8 h. The change of fabricated scaffolds was determined in terms of swelling rate, i.e., change of filament width and material diffusion, i.e., comparison of dry material weight before and after incubation. This comparative study can assist in application-based materials selection suitable for incubating in a perfusion bioreactor.

Co-proliferation of antimicrobial resistance genes in tilapia farming ponds associated with use of antimicrobials

The prevalence of antimicrobial resistance genes (ARGs) in aquaculture has raised serious public concerns for food safety and human health, but its relationships to the use of antimicrobials in aquacultural ponds and even to their residues in the whole aquatic environment remain unclear. In this study, a better coverage of 323 target ARGs and 40 mobile genetic elements (MGEs) was analyzed in sediment using a smart chip-based high-throughput quantitative PCR approach (HT-qPCR) in random 20 ponds of a tilapia farming base in southern China, whose antimicrobial residues were reported previously. In total, 159 ARGs and 29 MGEs were quantified in 58 surface sediment samples across the ponds. Absolute abundance of ARGs ranged from 0.2 to 13.5 × 106 copies g−1, dominated by the categories of multidrug and sulfonamides. The quantified ARGs abundance and the antimicrobial compound residues were significantly correlated instead with the antimicrobial categories, mainly compounds in categories of fluoroquinolones and sulfonamides and trimethoprim (TMP). Antimicrobial residues alone explained 30.6 % of the ARGs' variation quantified in sediment across the ponds, indicating the clear link between antimicrobials and the proliferation of ARGs in aquaculture. Co-proliferation of the ARGs with non-related antimicrobial compounds quantified in sediment was also observed, especially for aminoglycosides' ARGs, which were highly associated with integrons (intI 1) as argued being carried by the intI 1 gene cassette arrays. Physicochemical properties of sediment (pH, electric conductivity, and total sulfur content) highly contributed the variations of the quantified ARGs abundance (21 %) across all the sediment samples equaling to the MGEs (20 %), suggesting co-selection for ARGs' proliferation in the aquaculture environment. This study provides insights into the interactions between residual antimicrobials and ARGs, which would enhance the understandings on the use and management of antimicrobials in aquaculture worldwide to strategize mitigation of antimicrobial resistance in aquaculture.

Spring of Warta River as a Source of Antibiotic-Resistant Coliform Bacteria

The aim of the study was to evaluate the occurrence of integron-carrying coliform bacteria in the spring water of Warta river, a major river in Poland. We isolated 18 strains (12 Escherichia coli, three Pantoea sp., 2 Citrobacter freundii, and 1 Klebsiella oxytoca) harbouring class 1 and/or class 2 integrons. Class 1 integrons contained aadA1, dfrA7, dfrA17, dfrA1-aadA1, and dfrA17-aadA5 gene cassette arrays, whereas the genetic content of class 2 integrons was stable and consisted of sat2-aad1 gene cassette array. The strains carrying integrons were resistant to 4–11 antimicrobials, most frequently to sulfamethoxazole, ampicillin, piperacillin, trimethoprim, and trimethoprim/sulfamethoxazole.

Contribution of different class 2 integron elements to fitness costs in multi-drug resistant Escherichia coli and evaluation of their adaptability in “farm-to-table” environments

Integrons play a pivotal role in the dissemination of antimicrobial resistance, because they can capture and express exogenous antimicrobial resistance genes. This study aimed to elucidate the structure and contribution of different elements of class 2 integrons to fitness costs in their host bacteria and evaluate their adaptability to the “farm-to-table” process. We mapped 27 typical class 2 integrons of Escherichia coli isolated from aquatic foods and pork products, each harboring an inactive truncated class 2 integrase gene and the gene cassette (GC) array dfrA1-sat2-aadA1 with strong Pc2A/Pc2B promoters. Notably, the fitness costs associated with class 2 integrons depended on the Pc promoter strength and quantity and content of GCs in the array. Additionally, the costs of integrases were activity-dependent, and a balance was identified between GC capture ability and integron stability, which could explain the inactive truncated integrase identified. Although typical class 2 integrons exhibited low-cost structures in E. coli, the bacteria incurred biological costs, including decreasing growth rates and biofilm formation, in farm-to-table environments, especially under low-nutrient conditions. Nevertheless, sub-inhibitory antibiotic concentrations led to the selection of class 2 integron-carrying bacteria. This study provides important insights into how integrons may travel from preharvest to consumer goods.

Uncovering Bacterial Hosts of Class 1 Integrons in an Urban Coastal Aquatic Environment with a Single-Cell Fusion-Polymerase Chain Reaction Technology.

Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbor class 1 integrons. We developed a modified version of epicPCR (emulsion, paired isolation, and concatenation polymerase chain reaction (PCR)) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first application of epicPCR for targeting variable, multigene loci of interest. We also identified the Rhizobacter genus as novel hosts of class 1 integrons. These findings establish epicPCR as a powerful tool for linking taxa to class 1 integrons in environmental bacterial communities and offer the potential to direct mitigation efforts toward hotspots of class 1 integron-mediated dissemination of AMR.