Resistance Gene Cassettes Research Articles

Identification and Functional Analysis of Git3 G Protein-Coupled Receptors in Ganoderma boninense PER71

G protein-coupled receptors (GPCRs) are integral components of eukaryotic heterotrimeric G proteins, playing crucial roles in detecting extracellular signals and initiating the activity of signaling proteins within cells to activate cellular responses to these signals. The objectives of this study are to identify and characterize the function of Git3, a Class III GPCR protein, in the oil palm pathogen Ganoderma boninense. To identify the potential genes encoded for GPCR in this fungus, intensive data mining on the genome and transcriptome data has been carried out. A total of six classes of GPCRs have been identified. These include Class II pheromone detectors, Class III carbon detectors, Class IV nitrogen detectors, Class VII proteins similar to glycosyltransferase, Class VIII proteins similar to hemolysin, and Class X protein receptors. Among these, the Class III protein Git3, postulated to be involved in glucose sensing and fungal pathogenicity, was selected for gene knockdown using RNA interference (RNAi). A plasmid, designated pUChph-GIT3, was constructed, to target git3 silencing by incorporating a hygromycin resistance gene cassette and antisense sequences of git3. Transformation of G. boninense PER71 with pUChph-GIT3 produced five potential Δgit3 gene-silenced mutants. PCR analysis confirmed the integration of the RNAi expression cassette into the fungal genome. Quantitative PCR (qPCR) analysis revealed significant reductions in git3 expression in three G. boninense mutants, M42, M66, and M5 by 47%, 23%, and 13%, respectively. The Disease Severity Index (DSI) indicated slower disease progression in oil palm plantlets infected with Δgit3 mutants compared to those infected with wild-type G. boninense PER71. In conclusion, this study successfully isolated and characterized the git3 GPCR from G. boninense and demonstrated that it might play a role during the early stages of infection, as the mutants were able to slow the progression of infection in oil palm plantlets.

Evaluation of antibiotics resistance and transmission risk of Escherichia coli in rice-frog coculture system

In order to assess the antibiotic resistance of Escherichia coli and its transmission risk in a rice-frog coculture system in Zhejiang Province, this study collected E. coli from isolated soil, field water, and frog feces from the rice-frog coculture systems in four different areas of Zhejiang Province. The collected isolates were identified by 16S rRNA sequencing, while their antibiotic-resistant phenotypes were determined by Kirby-Bauer (K-B) method. PCR was used to identify the antibiotic-resistant genotypes and integrons, while conjugative transfer experiments were used to assess resistance transmission characteristics. The results showed a high prevalence of antibiotic resistance in the 82 strains of E. coli tested, primarily against tetracycline, sulfisoxazole, amoxicillin, and erythromycin. Most of these strains exhibited multidrug resistance, with the Fuyang area demonstrating the highest resistance rate compared to the other three areas. Further PCR analysis identified the sul1 gene as the most frequently detected resistance gene (63.41%), followed by blaTEM, tetA, and tetB. Among the 16 antibiotic resistance genes (ARGs) detected, the Fuyang isolates consistencly exhibited higher detection rate of 9 ARGs in comparison to the other regions. Additionally, the integrase gene intI1 displayed the highest detection rate, with 14 strains (34.15%) of integrase-positive bacteria carrying gene cassettes. Four different gene cassette compositions were observed, with dfrA1-aadA1 and dfrA17-aadA5 being the most common combinations. Conjugative transfer experiments demonstrated successful transfer of gene cassettes in 4 out of 14 donor bacteria, with conjugation transfer frequencies ranging from 4.32×10-5 to 7.13×10-4. These findings revealed the severity of resistance in the Fuyang area among the four regions. Integrons play a significant role in mediating the resistance to multiple antibiotics in E. coli, facilitating the potential spread of resistance gene cassettes between different bacteria. Overall, this study provides valuable insights into the resistance status and transmission characteristics of E. coli in the rice-frog coculture system in Zhejiang Province, providing a theoretical basis for ensuring the food safety of rice crops.

Distribution characteristics of integrons and correlation analysis of antibiotic resistance in urine isolated Enterobacter cloacae.

This study aims to understand the distribution of integrons among Enterobacter cloacae isolated from clinical urine specimens in our hospital, as well as the molecular characteristics of the variable region resistance gene cassette of integron-positive strains and its relationship with drug resistance. We collected a total of 80 strains of Enterobacter cloacae isolated from urine specimens of hospitalized patients in our hospital between August 2019 and July 2023, and conducted drug sensitivity testing on them. Polymerase Chain Reaction (PCR) technology was employed to screen these strains for Class 1, 2, and 3 integrons. Following this, the promoter and variable regions of integron-positive strains were amplified and sequenced. Additionally, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) was utilized for homology analysis of integron-positive strains. Among the 80 clinical strains, Class 1 integrons were detected in 31 (38.8%) strains, and the following resistance gene cassettes were identified: aadA2, aadA1, aadB, aac(6'), and catB8. Three types of variable region promoters were observed: PcS (4 strains), PcW (7 strains), and PcH1 (17 strains), with consistently inactive downstream P2 promoters. Additionally, Class 2 integrons were detected in 5 (6.3%) strains, carrying the variable region resistance gene cassette dfrA1-sat2-aadA1. The promoters for Class 2 integrons were uniformly of the Pc2D-Pc2A-Pc2B-Pc2C type. No Class 3 integrons were detected. The strains containing integrons showed significantly higher resistance rates to ciprofloxacin, compound sulfamethoxazole, levofloxacin, gentamicin, amikacin, and tobramycin compared to those without integrons (P<0.05). 35 strains of Enterobacter cloacae carrying integrons are primarily classified into three genotypes: A, B, and C. These genotypes are mainly distributed in the urology department and Intensive Care Unit (ICU). The distribution of variable region gene boxes and promoter types is relatively concentrated in the same genotype. Our study confirmed that Enterobacter cloacae isolated from urine samples predominantly carries Class 1 integrons with an extended array of antibiotic-resistant genes. For future research, it is recommended to explore additional resistance mechanisms and evaluate the effectiveness of new therapeutic strategies. Clinicians should be vigilant about the possibility of clonal dissemination and implement enhanced infection control measures in hospital settings.

Functional genomic regions associated with blast disease resistance in rice predicted syntenic orthologs and potential resistance gene candidates from diverse cereal genomes

Blast disease is a major production constraint in cereal crops worldwide. Genome regions associated with blast resistance in rice were used in homology-based prediction of resistance gene orthologs across cereals. Seventy-four Resistance Gene Analogs (RGAs) that were collocated with rice QTLs (Quantitative Trait Loci) associated with blast resistance, predicted 89 orthologs from cereals,including 61 from finger millet. The RGA orthologs found hits in cereal transcriptomes derived from blast-infected tissues. Over 92% of the putative RGAs encoded NBS-LRR (Nucleotide Binding Site -Leucine Rich Repeat) proteins. Despite conserved primary protein structures, NBS-LRRs were variable in the encoding genes. Primers designed on conserved NBS-LRRs motifs amplified multiple fragments in PCR indicating the presence of highly similar paralogs in genomes. Rice chromosomes 1, 2, 4, 6, and 11 dock clusters of RGAs and syntenic clusters of orthologous RGAs were predicted from other cereal genomes identifying genome hotspots for resistance gene cassettes. RGAs in such cassettes were tightly linked with 50 %–90 % within-cluster similarity. Local finger millet germplasm identified multi-modes of resistance, and lack of correlation between the resistance traits points to independent expression of resistance mechanisms and associated genes in different tissues and development stages. The significant diversity, genomic abundance, and potential functional redundancy complicate use of RGAs for resistance breeding in given host genetic backgrounds, pathogen pools and agro-climates; therefore, robust technology pipelines that enable system-level analysis of expression and effectiveness can increase the practical use of RGAs.

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.

Screening Key Sites of Class 2 Integron Integrase that Impact Recombination Efficiency.

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.

Molecular Characterization of Resistance and Virulence Factors of Trueperella pyogenes Isolated from Clinical Bovine Mastitis Cases in China.

The present study was designed to investigate the resistance determinants and virulence factors of 45 Trueperella pyogenes isolates from clinical bovine mastitis in Hexi Corridor of Gansu, China. Minimum inhibitory concentrations (MICs) was tested by E-test method. Gene of antimicrobial resistance, virulence integrase and integron gene cassettes were determined by PCR and DNA sequencing. The T. pyogenes isolates exhibited high resistance to streptomycin (88.9%) and tetracycline (64.4%), followed by erythromycin (15.6%) and gentamicin (13.3%). Resistance to streptomycin was most commonly encoded by aadA9 (88.9%); and to tetracycline, by tetW (64.4%). Importantly, all streptomycin-resistant isolates carried aadA9 alone or in combination with aadA1, aadA11 and strA-strB. Similarly, all tetracycline-resistant isolates harbored tetW alone or in combination with tetA33. Meanwhile, ermX was detected in 13.3% isolates, only one erythromycin-resistant isolate was not identified for this gene. Moreover, all T. pyogenes isolates carried class 1 integrons, and 17.8% of them contained gene cassettes, including arrays aadA1-aadB (4.4%), aad A24-dfrA1-ORF1 (2.2%) and aadA1 (2.2%). Furthermore, all tested isolates harbored virulent genes plo and fimA, followed by fimC (88.9%), fimE (86.6%) nanP (75.6%), nanH (40.0%), cbpA (35.6%) and fimG (6.7%). To our knowledge, this is the first report of integron gene cassettes of T. pyogenes isolates from bovine mastitis cases in China. These findings are useful for developing the prevention and the virulence factors of T. pyogenes could be promising candidates for vaccine antigens for bovine mastitis caused by T. pyogenes in China.

Genomic and functional insights into antibiotic resistance genes floR and strA linked with the SXT element of Vibrio cholerae non-O1/non-O139.

The emergence and spread of antibiotic-resistant bacterial pathogens are a critical public health concern across the globe. Mobile genetic elements (MGEs) play an important role in the horizontal acquisition of antimicrobial resistance genes (ARGs) in bacteria. In this study, we have decoded the whole genome sequences of multidrug-resistant Vibrio cholerae clinical isolates carrying the ARG-linked SXT, an integrative and conjugative element, in their large chromosomes. As in others, the SXT element has been found integrated into the 5'-end of the prfC gene (which encodes peptide chain release factor 3 involved in translational regulation) on the large chromosome of V. cholerae non-O1/non-O139 strains. Further, we demonstrate the functionality of SXT-linked floR and strAB genes, which confer resistance to chloramphenicol and streptomycin, respectively. The floR gene-encoded protein FloR belongs to the major facilitator superfamily efflux transporter containing 12 transmembrane domains (TMDs). Deletion analysis confirmed that even a single TMD of FloR is critical for the export function of chloramphenicol. The floR gene has two putative promoters, P1 and P2. Sequential deletions reveal that P2 is responsible for the expression of the floR. Deletion analysis of the N- and/or C-terminal coding regions of strA established their importance for conferring resistance against streptomycin. Interestingly, qPCR analysis of the floR and strA genes indicated that both of the genes are constitutively expressed in V. cholerae cells. Further, whole genome-based global phylogeography confirmed the presence of the integrative and conjugative element SXT in non-O1/non-O139 strains despite being non-multidrug resistant by lacking antimicrobial resistance (AMR) gene cassettes, which needs monitoring.

Distribution diversity and expression regulation of class 1 integron promoters in clinical isolates of Morganella morganii.

Morganella morganii is an emerging nosocomial opportunistic pathogen with increasing multidrug resistance. Antibiotic resistance, driven primarily by the horizontal transfer of resistance genes, has become a global health crisis. Integrons, mobile genetic elements, are now understood to facilitate the transfer of these genes, contributing to the rapid proliferation of resistant strains. Understanding the regulatory role of integrons in drug resistance gene expression is crucial for developing novel strategies to combat this pressing public health issue. To investigate the distribution of promoter types in the variable regions of class 1 integrons isolated from clinical isolates of M. morganii and their regulatory role in the expression of downstream drug resistance gene cassettes. Ninety seven clinical isolates of M. morganii were screened for the presence of class 1 integrons (intI1) using polymerase chain reaction (PCR). Gene cassettes within the variable regions of positive isolates were characterized, and the gene cassette promoter Pc variants and downstream auxiliary promoter P2 were identified. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was employed for homology analysis. Recombinant plasmids containing different variable region promoters and gene cassettes were constructed to evaluate drug resistance genes and integrase (intI1) expression levels using reverse transcription-quantitative PCR (RT-qPCR) and antimicrobial susceptibility testing. Of the clinical isolates, 28.9% (n = 28/97) were positive for class 1 integrons. 24.7% (n = 24/97) of these isolates carried gene cassettes encoding resistance to aminoglycosides and trimethoprim. Three Pc promoter types (PcH1, PcS, and PcW) were identified, while all P2 promoters were inactive with a 14-base pair spacing between the -35 and -10 regions. ERIC-PCR analysis classified the integron-positive strains into 6 genotypes, with high consistency in promoter types and gene cassettes within each genotype. RT-qPCR and antimicrobial susceptibility testing demonstrated that strong promoters significantly enhanced the expression of downstream drug resistance gene cassettes compared to weak promoters. Additionally, RT-qPCR revealed a negative correlation between intI1 expression and Pc promoter strength. Class 1 integrons are prevalent in M. morganii. The promoter types within these integrons are diverse, and promoter strength is closely linked to downstream gene cassette expression. Integron-positive strains exhibit high homology, suggesting horizontal gene transfer and dissemination in clinical settings.

The characterisation of antimicrobial resistant Escherichia coli from dairy calves.

Introduction. Dairy calves, particularly pre-weaned calves have been identified as a common source of multidrug resistant (MDR) Escherichia coli.Gap statement. E. coli strains isolated from dairy calves and the location of their resistance genes (plasmid or chromosomal) have not been well characterised.Aim. To characterise the phenotypic and genotypic features as well as the population structure of antimicrobial-resistant E. coli isolated from calves located on dairy farms that feed waste-milk to their replacement calves.Methodology. Recto-anal swab enrichments from 40 dairy calves (≤ 14 days old) located on four dairy farms were examined for tetracycline, streptomycin, ciprofloxacin, and third-generation cephalosporin resistant E. coli. Whole genome sequencing was performed using both short- and long-read technologies on selected antimicrobial resistant E. coli.Results. Fifty-eight percent (23/40) of calves harboured antimicrobial resistant E. coli: 43 % (17/40) harboured tetracycline resistant, and 23 % (9/40) harboured chromosomal mediated AmpC producing E. coli. Whole genome sequencing of 27 isolates revealed five sequence types, with ST88 being the dominant ST (17/27, 63 % of the sequenced isolates) followed by ST1308 (3/27, 11 %), along with the extraintestinal pathogenic E. coli lineages ST69 (3/27, 11 %), ST10 (2/27, 7 %), and ST58 (2/27, 7 %). Additionally, 16 isolates were MDR, harbouring additional resistance genes that were not tested phenotypically. Oxford Nanopore long-read sequencing technologies enabled the location of multiple resistant gene cassettes in IncF plasmids to be determined.Conclusion. Our study identified a high incidence of tetracycline and streptomycin-resistant E. coli in dairy calves, and highlighted the presence of multidrug-resistant strains, emphasising the need for further investigation into potential associations with farm management practices.

Comparison of Antimicrobial Susceptibility of Escherichia Coli Isolated from Fecal Poultry and Bovine Housed in Tunisian Farms; Phylogroup Diversity and Detection of Tetracycline and Sulfonamides Resistant Genes With Integron Class1

Little detailed documentation researched the excessive use of antimicrobials such as tetracycline and sulfonamides in veterinary medicine in Tunisia and more studies are needed. A total of 58 of commensal Escherichia coli isolates recovered from fecal samples of healthy poultry (n=31) and bovin (n=27) recovered from farms in Tunisia were examinated for 20 antimicrobial as well as, researched the presence of integron, variable regions (VRs), phylogroupes, tetracycline (tetA, tetB et tetC) and sulfonamides(sul1, sul2, sul3) resistance genes. The most frequently resistance in poultry origin were to tetracycline (94.3%), sulfonamide (70.69%), nalidixic acid (61.29), amoxicillin (58%), to trimthetoprim-sulfamethoxazole and streptomycin with the same rate (64.51%), ticarcilline (58%). Whereas, the bovine isolates were most resistant to streptomycin (55.5%), to amoxicillin (18.5), to tetracycline (37%), and have a moderate same rates to kanamycine, to trimthetoprim-sulfamethoxazole 11.11, to nalidixic acid and to sulfonamide 7.4%, For poultry and bovine class 1 integron were detected in 20, 6 isolates, respectively as well as class 2 integron were found in 2 and 1 isolates, respectively. Class 1 integrons were significantly associated with poultry origin (p=0.001). For poultry sul1, sul2, and sul3 genes were detected in 14 (46.2 %), 7 (23.8 %), and 4 (8.9 %) resistant isolates, respectively. Whereas, for bovine 5 isolates were resistant to sulfonamide and sul1 and sul2 genes were detected in 4 and 1 isolates with absence of sul3 genes. and tetracycline genes tetA, tetB genes were observed in 27 (84.37 %) and 8 (25%) resistant isolates, respectively while, TetC was not detected amongst our isolates. Seven arrangement gene cassette were detected; dfrA1-satA1-aadA1 in one identical DNA fragments with approximate size of 2000 bp and six arrangements of resistance gene cassettes of class 1 integron were detected; dfrA1+aadA1 (5isolates); for dfrA17+aadA1, dfrA12+ orfF+aadA2 each one two 2isolates; one isolate for aadA1 and dfrA5, respectively. In poultry, 16 isolates were found to belong to phylogroup A (sub- groupA1: 12, sub- groupA0: 4); 9 to B1, 1 to B2 and 5 to phylogroup D. However, in bovine 9 isolates have the phylogroups A1, 7 isolates B1, 4 isolates B2, and 3 isolates found to phylogroup D. Our results showed that the prevalence of resistance in E. coli isolates from poultry was much higher than that in bovin.

Analysis of antibiotic resistance gene cassettes in a newly identified Salmonella enterica serovar Gallinarum strain in Korea

Antimicrobial resistant pathogens are a global health threat driven by the indiscriminate use of antimicrobials. Antimicrobial resistance can be acquired by resistance genes encoded by mobile genetic elements. In this study, we identified a strain of Salmonella enterica serovar Gallinarum (SG4021) from an infected chicken in Korea and characterized the presence of resistance genes in its plasmid by whole genome sequencing. The sequence was then compared with that of a plasmid (P2) from strain SG_07Q015, the only other strain of S. Gallinarum isolated in Korea for which a genome sequence is available. The results revealed that both strains harbored nearly identical DNA carrying antibiotic resistance gene cassettes inserted into integron In2 of the transposable element Tn21, namely an aadA1 resistance gene conferring resistance to aminoglycosides and a sul1 resistance gene conferring resistance to sulfonamide. Interestingly, despite the presence of sul1 in SG4021, an antibiotic sensitivity test revealed that it was sensitive to sulfonamides. Further analysis revealed that this disparity was due to the insertion of a ~ 5 kb ISCR16 sequence downstream of the promoter driving sul1 expression in SG4021. Using various mutants, we showed that the insertion of ISCR16 blocked the expression of the sul1 gene from the upstream promoter. Therefore, the functionality of antimicrobial resistance genes determines phenotypic antimicrobial resistance.

Molecular Characterization of Antimicrobial Resistance of Vibrio Species Isolated from Fish in Egypt

Vibrio sp. are the most widely recognized and serious pathogen in fish and shellfish marine aquaculture around the world generated disease, not only to fish but also to human causing gastroenteritis. Vibriosis is a common disease caused by a number of Vibrio spp.; V. harveyi, V. parahaemolyticus, V. alginolyticus, V. anguillarum, V. vulnificus etc. Vibrio sp. are Gram negative, straight or curved short rods, non-sporulating, non-capsulated, arranged singly or in chains, motile, oxidase positive, catalase positive, citrate positive, string test positive, indol positive, urease negative, H2S negative. The current study was conducted to spot light on identification, molecular characterization and antimicrobial resistance of Vibrio sp. isolated from fish in Egypt and to detect the presence of some of β- lactams resistance genes and class 1 integron. About 52 Vibrio isolates were isolated from 150 fish (75 Oreochromus niloticus, 50 Mugil cephalus, 25 Clarias gariepinus) in Kafr El-Sheikh Governorate in Egypt. Twenty isolates of Vibrio species were tested against 11 antimicrobials for antimicrobial resistance. All the isolates were highly sensitive (100%) to ciprofloxacin and norfloxacin. V. alginolyticus showed high resistance to Ampicillin and intermediate resistance to streptomycin, cefotaxime and erythromycin. V. cholerae showed high resistance to Ampicillin and intermediate resistance to erythromycin. V. parahaemolyticus showed high resistance to gentamycin and intermediate resistance to Ampicillin. V. fluvialis showed high and intermediate resistance to Ampicillin. V. splendidus showed high resistance to Ampicillin and intermediate resistance to streptomycin and cefotaxime. Five isolates showing multidrug resistance were tested for blaTEM , blaCMY2 ,blaCTX genes and class 1 integron. The genes blaTEM was detected in 100% of the isolates, while blaCMY2 and blaCTX genes were detected in 0% of the isolates. Class 1 integron was detected with a percentage of (100%) in the 5 examined isolates. A class 1 integrons bearing streptomycin/ spectinomycin resistant gene cassette of aadA2 were discovered on V. parahaemolyticus isolate. As there is a little reports about antibiotic resistance in Vibrio, this study highlight on the incidence of some of β-lactams resistance genes and class 1 integrons in Vibrio species.

Phenotypic and genotypic discrepancies for carbapenemase-producing Citrobacter freundii in multiple isolates from a single patient

BackgroundCarbapenemase-producing gram-negative organisms continue to be a significant healthcare concern and a therapeutic challenge. Members of the genus Citrobacter have emerged as increasingly multidrug resistant and versatile healthcare-associated pathogens. In this study we investigated five KPC-producing Citrobacter freundii isolates, from the same patient, that presented unusual phenotypic characteristics including false susceptibility to carbapenems detection by culture-based methods.MethodsThe isolates were tested for antimicrobial susceptibility using broth microdilution and disk diffusion. Production of serine carbapenemase was confirmed with the mCIM (modified carbapenem inactivation method) test. Genotypes were determined by PCR and whole genome sequencing analysis.ResultsThe five isolates were susceptible to meropenem by broth microdilution and presented varying colonial morphologies and levels of susceptibility to carbapenems by multiple phenotypic methods, despite being positive for carbapenemase production by mCIM and positive for blaKPC by PCR. Whole genome sequence analysis showed that three of the five highly related isolates harbor an additional gene cassette, including blaCARB-2, ant(2''), aadA2, dfrA19, catB3, cmlA1, mph(E), msr(E), and qnrA1. The presence of these genes explains the difference in phenotypes observed.ConclusionFailure to detect and completely eradicate the carbapenemase-producing C. freundii in the urine with ertapenem therapy, likely due to the presence of a heterogeneous population, resulted in the phenotypic and genotypic adaptations of the organism as it disseminated to the bloodstream and kidneys. The fact that carbapenemase-producing C. freundii can elude detection by phenotypic methods and can so easily acquire and transfer resistance gene cassettes is of concern.

Class 1 integrons and multiple mobile genetic elements in clinical isolates of the Klebsiella pneumoniae complex from a tertiary hospital in eastern China.

The emergence of highly drug-resistant K. pneumoniae, has become a major public health challenge. In this work, we aim to investigate the diversity of species and sequence types (STs) of clinical Klebsiella isolates and to characterize the prevalence and structure of class 1 integrons. Based on the whole genome sequencing, species identification was performed by 16S rRNA gene homology and average nucleotide identity (ANI) analysis. STs were determined in accordance with the international MLST schemes for K. pneumoniae and K. variicola. Integron characterization and comparative genomic analysis were performed using various bioinformatic tools. Species identification showed that the 167 isolates belonged to four species: K. pneumoniae, K. variicola subsp. variicola, K. quasipneumoniae and K. aerogenes. Thirty-six known and 5 novel STs were identified in K. pneumoniae, and 10 novel STs were identified in K. variicola subsp. variicola. Class 1 integrons were found in 57.49% (96/167) of the isolates, and a total of 169 resistance gene cassettes encoding 19 types of resistance genes, including carbapenem resistance gene (bla IPM-4) and class D β-lactamases gene (bla OXA-1 and bla OXA-10), were identified. Among the 17 complete genomes, 29 class 1 integrons from 12 groups were found, only 1 group was encoded on chromosomes. Interestingly, one plasmid (pKP167-261) carrying two copies of approximately 19-kb IS26-Int1 complex resistance region that contains an integron and a multidrug resistance gene fragment. The results of this work demonstrated that the species and STs of the clinical Klebsiella isolates were more complex by the whole genome sequence analysis than by the traditional laboratory methods. Finding of the new structure of MGEs related to the resistance genes indicates the great importance of deeply exploring the molecular mechanisms of bacterial multidrug resistance.

Influence of class 2 integron integrase concentration on gene cassette insertion and excision in vivo.

Integron can capture and express antimicrobial resistance gene cassettes and plays important roles in horizontal gene transfer. The establishment of a complete in vitro reaction system will help to reveal integron integrase mediated site-specific recombination process and regulation mechanism. As an enzymatic reaction, the concentration of integrase is assumed to have a great influence on the reaction rate. To determine the influence of different concentrations of integrase on the reaction rate and to find the best range of enzyme concentration were essential to optimizing the in vitro reaction system. In this study, plasmids with gradient transcription levels of class 2 integron integrase gene intI2 under different promoters were constructed. Among plasmids pI2W16, pINTI2N, pI2W, and pI2NW, intI2 transcription levels ranged from about 0.61-fold to 49.65-fold of that in pINTI2N. And the frequencies of gene cassette sat2 integration and excision catalyzed by IntI2 were positively correlated with the transcription levels of intI2 within this range. Western blotting results indicated high expression of IntI2 partly existed in the form of an inclusion body. When compared with Pc of class 1 integron, the spacer sequence of PintI2 can increase the strength of PcW but decrease the strength of PcS. In conclusion, the frequencies of gene cassette integration and excision were positively correlated with the concentration of IntI2. intI2 driving by PcW with PintI2 spacer sequence can obtain the optimum IntI2 concentration required to achieve the maximum recombination efficiency in vivo in this study.

Uncovering the diversity and contents of gene cassettes in class 1 integrons from the endophytes of raw vegetables

Rapid spread of antibiotic resistance genes (ARGs) in pathogens is threatening human health. Integrons allow bacteria to integrate and express foreign genes, facilitating horizontal transfer of ARGs in environments. Consumption of raw vegetables represents a pathway for human exposure to environmental ARGs. However, few studies have focused on integron-associated ARGs in the endophytes of raw vegetables. Here, based on the approach of qPCR and clone library, we quantified the abundance of integrase genes and analyzed the diversity and contents of resistance gene cassettes in class 1 integrons from the endophytes of six common raw vegetables. The results revealed that integrase genes for class 1 integron were most prevalent compared with class 2 and class 3 integron integrase genes (1–2 order magnitude, P < 0.05). The cucumber endophytes harbored a higher absolute abundance of integrase genes than other vegetables, while the highest bacterial abundance was detected in cabbage and cucumber endophytes. Thirty-two unique resistance gene cassettes were detected, the majority of which were associated with the genes encoding resistance to beta-lactam and aminoglycoside. Antibiotic resistance gene cassettes accounted for 52.5 % of the functionally annotated gene cassettes, and blaTEM-157 and aadA2 were the most frequently detected resistance cassettes. Additionally, carrot endophytes harbored the highest proportion of antibiotic resistance gene cassettes in the class 1 integrons. Collectively, these results provide an in-depth view of acquired resistance genes by integrons in the raw vegetable endophytes and highlight the potential health risk of the transmission of ARGs via the food chain.

Antimicrobial resistance and molecular characterization of gene cassettes from class 1 integrons in Salmonella strains.

Introduction. The emergence of antibiotic-resistant Salmonella isolates is a global concern and has been attributed to the indiscriminate use of antibiotics in humans and animals. Integrons are mobile gene elements closely related to bacterial drug resistance. Among them, class 1 integrons containing various resistance gene cassettes could play an important role in disseminating and maintaining antibiotic resistance in Salmonella isolates.Hypothesis. Salmonella class 1 integrons have a relationship with Salmonella drug resistance.Aim. This study aims to investigate the distribution of class 1 integrons and their variable regional molecular characteristics, as well as the diversity of the promoters and drug sensitivity among Salmonella strains.Methodology. A total of 111 Salmonella strains, collected between 2018 and 2020, underwent fully automated bacterial identification using the VITEK 2 Compact system and an antibiotic sensitivity test. PCR was employed to screen class 1 integrase genes (IntI1) and integron variable regions, while promoter type and variable region gene cassette characteristics were determined using sequencing analysis.Results. A total of 24 IntI1-positive strains were detected in 111 Salmonella strains. Moreover, IntI1-positive strains exhibited statistically significant resistance to ceftazidime, ciprofloxacin, levofloxacin, ceftriaxone, trimethoprim/sulfamethoxazole and azithromycin compared to integron-negative strains (P<0.05). The multidrug resistance rate of IntI1-positive strains was significantly higher than that of negative strains. Variable regions were observed in 6 of the 24 IntI1-positive strains. Four gene cassettes were detected, namely dfrA17-aadA5, dfrA12-aadA2, aadA22 and aar-3-dfrA27. Finally, 3 types of class 1 integron variable region promoters were identified in 24 strains, including PcW, PcH1 and PcWTGN-10; they are all relatively weak promoters.Conclusion. The integron and the drug resistance genes carried by integron have a certain relationship with drug resistance.

Detection of multidrug resistant pathogenic bacteria and novel complex class 1 integrons in campus atmospheric particulate matters

Recent advances provided overwhelming evidence that atmospheric particulate matters carry a substantial amount of antibiotic resistance genes (ARGs). It has also been documented that polluted air facilitates transmission of bacterial pathogenesis and antimicrobial resistance (AMR). These investigations generally used culture-independent approaches which reveal sophisticated microbiomic and resistomic compositions in particulate matters, while culture-dependent methods directly demonstrating presence of live, functional bacteria has not been fully applied. In recent years, efforts undertaken worldwide managed to reduce air particulate matter pollution, leading to cleaner air in many parts of world, including China. Whether atmospheric particulate matters may still function as vehicles for pathogenic bacteria and AMR in improving air conditions is turning into an interesting question to address. In attempt to answer this question, a culture-dependent approach is used to find out the putative role of atmospheric particulate matters in relatively ‘clean’ air to transmit pathogenic bacteria and AMR in this work. By harvesting particulate matters in an unindustrialized and less-polluted university campus, culturing and identifying bacteria in particulate matters, and characterizing pathogenesis and AMR properties of these bacteria, interesting findings were made that even in relatively ‘clean’ air, antibiotic-resistant pathogenic bacteria are prevalent; and that mobile genetic elements including integrons are widespread. In particular, in air samples collected, multidrug-resistant hemolytic Bacillus strains that may pose significant health threat could be identified. Complex class 1 integrons, two of which carry novel antibiotic resistant gene cassette arrays, were also found for the first time in airborne bacteria, suggesting the danger of horizontal transfer of AMR in air. In conclusion, using culture-dependent methods, this work shows that atmospheric particulate matters are viable vehicles for the transmission of bacterial pathogenesis and AMR, and that even in relatively ‘clean’ air, the threat of airborne antibiotic-resistant pathogens is significant.

Multi‑locus sequence and drug resistance analysis of Salmonella infection in children with diarrhea in Guangdong to identify the dominant ST and cause of antibiotic‑resistance.

Multi-locus sequence typing (MLST) can be used to analyze the homology among the drug resistance gene cassettes in Salmonella and determine the prevalence. Information extracted using this technique can provide a theoretical basis for hospitals to devise protocols to control Salmonella infections. The aim of the present study was to investigate the possible association between drug resistance and integrons in clinical isolates of Salmonella from human fecal samples. Therefore, in the present study, 52 clinical fecal isolates of non-duplicate (i.e., not genome contamination) Salmonella were harvested from children with diarrhea and used for bacterial identification using biochemical tests, drug susceptibility analysis by antibiotic susceptibility testing and serotype identification using an agglutination assay. In total, seven Salmonella housekeeping genes (chorismate synthase, β sliding clamp of DNA polymerase III, uroporphyrinogen-III synthase, histidinol dehydrogenase, phosphoribosylaminoimidazole carboxylase catalytic subunit, 2-oxoglutarate dehydrogenase E1 component and homoserine dehydrogenase) were amplified and sequenced using MLST, before sequence alignment was performed against the Pub MLST database to determine the sequence-typed (ST) strains and construct genotypic evolutionary diagrams. Subsequently, the 52 Salmonella strains were subdivided into 11 serotypes and 11 sequence types. The dominant subtypes were found to be Salmonella typhimurium ST34 and ST19, which were diversely distributed. However, no new subtypes were found. Although the serotypes, including ST19, ST29, ST34, ST40, ST11, ST27, ST469, ST365, ST1499, ST413 and ST588, were closely associated with the MLST subtype, they did not correspond entirely. The detection rate of class I integrons was 38.46% (20/52), but no class II and III integrons were detected. The variable regions of three of 20 class I integrons were found to be amplified, whereas nine gene cassettes, including dihydrofolate reductase A12, open reading frame F, aminoglycoside-adenylyltransferase (aad)A2, aadA22, aadA23, aadA1, cadmium-translocating P-type ATPase 2, lincosamide and linF, were associated with drug resistance. These data suggest that Class I integrons are important factors underlying drug resistance in Salmonella, which may serve a role in the spread of drug resistance and warrant specific focus. In addition, MLST typing and serotyping should be applied cooperatively in epidemiological research.