Integron Gene Research Articles

Industrial-scale aerobic composting of livestock manures with the addition of biochar: Variation of bacterial community and antibiotic resistance genes caused by various composting stages

The presence of large amounts of antibiotic resistance genes (ARGs) in livestock manures poses an impending, tough safety risk to ecosystems. To investigate more comprehensively the mechanisms of ARGs removal from industrial-scale composting of livestock manure based on biochar addition, we tracked the dynamics of bacterial community and ARGs at various stages of aerobic composting of livestock manures with 10% biochar. There were no significant effects of biochar on the bacterial community and the profiles of ARGs. During aerobic composting, the relative abundance of ARGs and mobile genetic elements (MGEs) showed overall trends of decreasing and then increasing. The key factor driving the dynamics of ARGs was bacterial community composition, and the potential hosts of ARGs were Caldicoprobacter, Tepidimicrobium, Ignatzschineria, Pseudogracilibacillus, Actinomadura, Flavobacterium and Planifilum. The retention of the thermophilic bacteria and the repopulation of the initial bacteria were the dominant reasons for the increase in ARGs at maturation stage. Additionally, among the MGEs, the relative abundance of transposon gene was substantially removed, while the integron genes remained at high relative abundance. Our results highlighted that the suitability of biochar addition to industrial-scale aerobic composting needs to be further explored and that effective measures are needed to prevent the increase of ARGs content on maturation stage.

Sources and Drivers of ARGs in Urban Streams in Atlanta, Georgia, USA.

The spread of antibiotic resistance genes (ARGs) in the aquatic environment is an emerging concern in the interest of protecting public health. Stemming the environmental dissemination of ARGs will require a better understanding of the sources and drivers of ARGs in the water environment. In this study, we used direct measurement of sewage-associated molecular markers, the class 1 integron gene, standard water quality parameters, and watershed characteristics to evaluate the sources and drivers of ARGs in an urban watershed impacted by a gradient of human activities. Quantitative polymerase chain reaction (qPCR) was used to quantify the abundance of the sewage-associated HF183, the E. coli fecal indicator, class 1 integron gene (int1), and the ARGs sulI, sulII, tetW, tetM, ampC, and blaSHV in stream water samples collected from the Proctor Creek watershed in Atlanta, Georgia. Our findings show that ARGs were widely distributed, with detection frequencies of 96% (sulI and sulII), 82% (tetW and tetM), and 49% (ampC and blaSHV). All the ARGs were positively and significantly correlated (r > 0.5) with the HF183 and E. coli markers. Non-linear machine learning models developed using generalized boosting show that more than 70% of the variation in ARG loads in the watershed could be explained by fecal source loading, with other factors such as class 1 integron, which is associated with acquired antibiotic resistance, and environmental factors contributing < 30% to ARG variation. These results suggest that input from fecal sources is a more critical driver of ARG dissemination than environmental stressors or horizontal gene transfer in aquatic environments highly impacted by anthropogenic pollution. Finally, our results provide local watershed managers and stakeholders with information to mitigate the burden of ARGs and fecal bacteria in urban streams.

Characterization of Escherichia coli strains isolated from geese by detection of integron-mediated antimicrobial resistance

Current research shows that the resistance of Escherichia coli (E. coli) is mainly related to integron gene cassettes. To assess the resistance of E. coli of goose origin and the carriage of its integron genes in four farms in Heilongjiang Province, antibiotic resistance phenotypes and the presence of various types of integrons were investigated. In this study, test strains were sampled and isolated from the farms, and 109 test strains were tested for drug sensitivity of 15 different antimicrobial drugs by the Kb disc diffusion method. Polymerase chain reaction was used to detect E. coli in three types of integrase genes (intI1, intI2, and intI3) and for sequencing analysis of the class I integron gene cassette. Susceptibility test results show that more than 70% of tested strains exhibit resistant phenotypes to ampicillin, amoxicillin, imipenem, tetracycline, and doxycycline. The detection rate of class I integrons was 68.91%, while class II integrons and class III integrons were not detected. The detection rate of class I integrin gene cassette was 7.42%. Sequence analysis showed that strains carried different integron gene cassettes: dfrA17-aadA5, dfrA1-aadA1, dfrA27-arr-3, and aminoglycoside 3''-nucleotidyltransferase. Results suggest that the detection rate of class I integrons is highly correlated with their drug resistance. Class I integrons provide a valuable guide to studying the spread and the expression of resistance genes and thus finding effective measures to prevent bacterial resistance.

Molecular Characterization and Antimicrobial Resistance Profiling of Extended Spectrum Beta-lactamase (ESBL) Producing Escherichia coli in Bovines from J and K, India

Background: Higher prevalence of ESBL producers is alarming in dairy sector. So limiting antimicrobial use may curtail the selection and persistence of predominant ESBL and conjugative plasmids among strains. The study was aimed to determine the occurrence of extended-spectrum beta-lactamase (ESBL) producing E. coli as well as their genetic diversity, antimicrobial resistance and integrons in bovines. Methods: A total of 180 faecal samples were screened for the presumptive ESBL producing E. coli isolates. All ESBL producing isolates were subjected to the screening test and were confirmed as ESBL producers by double disc. Further, these ESBL producers were tested for the presence of bla genes and those found positive were tested for multidrug resistance (MDR) by disk diffsion. MDR positive isolates were further tested for the presence of intI 1, intI 2 and intI 3 genes. Result: A total of 360 presumptive ESBL producing E. coli isolates were obtained. Out of which, 154 (42.77%) isolates were found to be resistant and were confirmed as ESBL producers. Of 154 ESBL isolates, 120 (77.92%) isolates carried the gene/s screened for blaTEM, blaCTX-M and blaSHV genes are were declared as multi drug resistant. Out of 120 MDR isolates, 59 tested positive for the integron gene.

Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis.

The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and the transconjugant strains. Sequence analysis revealed a conjugative multidrug resistance plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a β-lactamase gene. The 7.1 kb element and the previously reported element Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that these elements generate a circular copy of one specific strand without leaving an empty site on the donor molecule, in contrast to the movement of integron gene cassettes or ICE/IMEs discovered to date. These elements are termed SEs (strand-biased circularizing integrative elements): SE-6945 (the 7.1 kb element) and SE-6283 (Tn6283). The copy number and location of SE-6945 in the chromosome affected the antibiotic resistance levels of the transconjugants. SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that SEs are involved in the spread of AMR genes among marine bacteria.

Emergence of hypermucoviscous colistin-resistant high-risk convergent Klebsiella pneumoniae ST-2096 clone from Pakistan.

Klebsiella pneumoniae convergent clones are considered a threat to healthcare settings. Here we report a comprehensive genomic profiling of an emerging colistin-resistant K. pneumoniae ST-2096 convergent clone from Pakistan. Methods: Whole-genome sequencing was performed and raw reads were assembled antimicrobial resistance and virulence genes were predicted using various online tools. Results & conclusion: The phenotypically multidrug-resistant (MDR) and hypermucoviscous (hv) colistin-resistant K. pneumoniae (hvCRKP-10718), which, intriguingly, possessed a wide range of antimicrobial resistance (blaTEM-1A, blaOXA-1, blaOXA-232, blaCTX-M-15, blaSHV-106, oqxA, oqxB, aac(6')-Ib-cr, aadA2, aac(6')-Ib-cr, armA, tetD, mphE, msrE, fosA, dfrA1, dfrA12, dfrA14, catB3, sul1) and virulence determinants (RmpA/RmpA2, yersiniabactin [ybt], aerobactin [iuc/iut], enterobactin). Furthermore, the acquisition of various mobile genetic elements (MDR/virulent plasmids, type II integron gene cassette, insertional sequences, transposases) and associated hv capsular type made this MDR/hv isolate a convergent clone belonging to a high-risk lineage (ST-2096). Based on core-genome multilocus sequence typing and single-nucleotide polymorphism analysis, this isolate showed ≥99% nucleotide identity with MDR K. pneumoniae isolates from India, depicting its evolutionary background. This study provides a comprehensive genomic profiling of this high-risk convergent K. pneumoniae ST-2096 clone from Pakistan. Comparative genomics of MDR/hv colistin-resistant K. pneumoniae isolates with other MDR convergent strains from the Indian subcontinent indicated the emergence of this evolving superbug.

Pseudomonas aeruginosa from pet Chinese stripe-necked turtles (Ocadia sinensis) demonstrating antimicrobial and heavy metal resistance

Leading nosocomial pathogen Pseudomonas aeruginosa has increasingly been reported to be an opportunistic pathogen. In this study, a total of twenty P. aeruginosa isolates were isolated from 40 pet Chinese stripe-necked turtles and examined for their antimicrobial and heavy metal resistance properties. All isolates were multidrug resistance by scoring multiple antimicrobial resistance indices ≥0.2. In the disc distribution test, 100% resistance to ampicillin and oxacillin were detected. In addition to that, 14 (70%) isolate demonstrated amoxicillin resistance. Imipenem, fosfomycin, gentamycin, tobramycin and piperacillin resistance were detected in 40%, 15%, 20%, 10% and 5% of the isolates, respectively. The ESBLs gene that predominated in this study was blaSHV (55%), followed by blaTEM (50%), blaCTX (10%) and blaOXA (5%). The most frequent aminoglycoside resistance gene in this study was aac(6´)-Ib (40%). Class1 integron integrase gene intI1 and class 1 integron gene cassette gene aadA1 were detected in 45% and 35% of the isolates, respectively. All P. aeruginosa isolates demonstrated Cu and Cd resistance. CzcA and CopA genes were detected in 65% and 30% of the isolates, respectively. These findings reveal the presence of pet turtle-born P. aeruginosa can be a potential risk to public health and cannot be excluded as a non-nosocomial source of infections.

Inverse PCR-based detection reveal novel mobile genetic elements and their associated genes in the human oral metagenome

The human oral cavity is one of the hotspots harboring multiple mobile genetic elements (MGEs), which are segments of DNA that can move either within bacterial genomes or between bacterial cells that can facilitate the spreading of genetic materials, including antimicrobial resistance genes. It is, therefore, important to investigate genes associated with the MGEs as they have a high probability of dissemination within the bacterial population under selective pressure from human activities. As one-third of oral bacteria are not yet culturable in the laboratory condition, therefore, in this work, it is aimed to detect and identify the genetic contexts of MGEs in the oral cavity through an inverse PCR (IPCR)-based approach on the oral metagenomic. The human oral metagenome was extracted from saliva samples collected from healthy individuals in Tromsø, Norway. The extracted DNA was partially digested with the HindIII restriction enzyme and self-circularized by ligation. DNA primers targeting each MGE were designed to amplify outwards from the MGEs and used for the IPCR on the circularized DNA products. The IPCR amplicons were cloned into a pCR-XL-2-TOP vector, screened, and sequenced. Out of 40 IPCR amplicons, we confirmed and verified the genetic contexts of 11 samples amplified with primers targeting integron gene cassettes (GCs), IS431 composite transposons, and Tn916 conjugative transposons (tet(M) and xis-int). Novel integron GCs, MGEs, and variants of Tn916 conjugative transposons were identified, which is the first report using the IPCR technique to detect the genetic contexts of MGEs in the oral metagenomic DNA.

Novel Mobile Integrons and Strain-Specific Integrase Genes within Shewanella spp. Unveil Multiple Lateral Genetic Transfer Events within The Genus.

Shewanella spp. are Gram-negative bacteria that thrive in aquatic niches and also can cause infectious diseases as opportunistic pathogens. Chromosomal (CI) and mobile integrons (MI) were previously described in some Shewanella isolates. Here, we evaluated the occurrence of integrase genes, the integron systems and their genetic surroundings in the genus. We identified 22 integrase gene types, 17 of which were newly described, showing traits of multiple events of lateral genetic transfer (LGT). Phylogenetic analysis showed that most of them were strain-specific, except for Shewanella algae, where SonIntIA-like may have co-evolved within the host as typical CIs. It is noteworthy that co-existence of up to five different integrase genes within a strain, as well as their wide dissemination to Alteromonadales, Vibrionales, Chromatiales, Oceanospirillales and Enterobacterales was observed. In addition, identification of two novel MIs suggests that continuous LGT events may have occurred resembling the behavior of class 1 integrons. The constant emergence of determinants associated to antimicrobial resistance worldwide, concomitantly with novel MIs in strains capable to harbor several types of integrons, may be an alarming threat for the recruitment of novel antimicrobial resistance gene cassettes in the genus Shewanella, with its consequent contribution towards multidrug resistance in clinical isolates.

Monitoring and Evaluation of Antibiotic Resistance Pattern of Escherichia coli Isolated from Drinking Water Sources in Ardabil Province of Iran

Background and Objective: Antibiotic resistance is rapidly spreading among bacteria, such as Escherichia coli, which have been discharged into water sources by humans and animals, in part due to genes carried by integrons. The goal of this study was to track and assess the pattern of antibiotic resistance among Escherichia coli strains isolated from drinking water sources in Iran’s Ardabil province. Methods: Escherichia coli strains were isolated and identified using standard biochemical and microbiological techniques from Ardabil province’s water supply sources in 2019 and 2020. The class 1 integron gene was detected using the polymerase chain reaction (PCR). The disk diffusion method was used to determine antibiotic resistance and sensitivity. Results: The Int I gene was found in 47 out of 200 isolates (23.5%). The antibiotic streptomycin had the highest resistance (100%), whereas gentamycin, ciprofloxacin, tobramycin, tetracycline, cefipime, imipenem, meropenem, chloramphenicol, and trimethoprim-sulfamethoxamide had the lowest resistance. Gentamycin, ciprofloxacin, tobramycin, tetracycline, cefipime, imipenem, meropenem, and trimethoprim-sulfamethoxazole, on the other hand, had the highest sensitivity of 100%, while streptomycin had the lowest (0%). Also, the highest level of pollution of Escherichia coli in drinking water supply sources in the province is in Parsabad city (42.86%), and the lowest level of pollution is in Kosar city (6.67%). Conclusion: According to the findings of this study, the high prevalence of Escherichia coli strains resistant to the antibiotic streptomycin circulating in water sources should be considered a major problem in terms of antibiotic resistance spreading among bacteria in water.

Flaviflexus equikiangi sp. nov. isolated from faeces of Equus kiang (Tibetan wild ass) and carrying a class 1 integron gene cassette in its genome

Two Gram-stain-positive, catalase-negative, non-spore-forming, cocci-shaped strains (dk850T and JY899) were isolated from the feces of Equus kiang in the Qinghai-Tibet Plateau of China. 16S rRNA gene sequence-based phylogenetic analyses showed that strains dk850T and JY899 belong to the genus Flaviflexus, closest to F. salsibiostraticola KCTC 33148T, F. ciconiae KCTC 49253T and F. huanghaiensis H5T. The DNA G + C content of strain dk850T was 62.9%. The digital DNA-DNA hybridization values of strain dk850T with the closely related species were below the 70% threshold for species demarcation. The two strains grew best at 28°C on brain heart infusion (BHI) agar with 5% sheep blood. All strains had C18:1ω9c and C16:0 as the major cellular fatty acids. MK-9(H4) was the major menaquinone in strain dk850T. The major polar lipids included diphosphatidylglycerol and an unidentified phospholipid. Strains dk850T and JY899 were identified as carrying a class 1 integron containing the aminoglycoside resistance gene aadA11, both strains were resistant to spectinomycin and streptomycin. Based on several lines of evidence from phenotypic and phylogenetic analyses, strains dk850T and JY899 represent a novel species of the genus Flaviflexus, for which the name Flaviflexus equikiangi sp. nov. is proposed. The type strain is dk850T(= CGMCC 1.16593T = JCM 33598T).

Water quality and antimicrobial resistant profile of Escherichia coli isolated from dug wells used for domestic purposes

The rapid spread of antimicrobial resistance (AMR) poses a significant threat to public safety. The use of water containing resistant bacteria could increase the risk of spreading AMR. This study assessed the quality of 143 dug wells used for domestic purposes in some communities in Nigeria and determined the resistance profile of isolated &lt;em&gt;Escherichia coli (E. coli)&lt;/em&gt;. The Microbact&lt;sup&gt;TM&lt;/sup&gt; identification kit was used to identify the isolates, and the susceptibility profile was evaluated using the Kirby-Bauer disc diffusion method. The combination disc technique was used to test all isolates for extended spectrum beta lactamase (ESBL) production. Polymerase chain reaction was used to identify ESBL genes, Integrons, and plasmid-mediated quinolone resistance genes. A total of 110 (76.9%) wells were contaminated by coliform bacteria. Of these, 94 (84.45%) wells yielded 202 &lt;em&gt;E. coli&lt;/em&gt; strains. The isolates were commonly resistant to ampicillin (60.9%) but were all susceptible to meropenem. Seventy-seven (38.1%) isolates were multi-drug resistant. Two isolates harbored blaCTX-M and blaTEM separately while four (19%) ciprofloxacin-resistant isolates carried the oqxAB/aac-lb-cr gene. All isolates with resistance genes harbored class 1 and/2 Integrons. Most wells had coliform counts far above the World Health Organization’s recommended limit, indicating that they are unsafe to drink. The presence of multidrug-resistant isolates in well water poses a serious risk to consumers since it might lead to outbreaks of untreatable water-borne diseases.

Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities.

Integrons are microbial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the performance of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class one integrons are collecting and concentrating resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

Exploring the abundance and influencing factors of antimicrobial resistance genes in manure plasmidome from swine farms

Plasmids play a critical role in the dissemination of antimicrobial resistance genes (ARGs), however, a systematical understanding of ARGs originated from plasmids in swine production is currently lacking. Herein, quantitative polymerase chain reaction was applied to determine the prevalence of ten ARGs and the class1 integron gene intI1 of plasmid source in swine manure from 44 farms in Sichuan, Hubei and Hebei provinces, China. All assayed ARGs were observed in plasmid DNA samples, and the average absolute abundance of aac(6’)-Ib-cr, blaNDM, blaCTX-M, optrA, ermB, floR, mcr-1, qnrS, tetM, sul1 and intI1 were 7.09, 2.90, 4.67, 6.62, 7.55, 7.14, 4.08, 4.85, 7.16, 7.11 and 8.07 of 10 log copies/gram, respectively. IntI1 showed a high correlation (r > 0.8, P < 0.01) with the abundance of aac(6’)-Ib-cr and sul1 in swine manure. Moreover, the farm scale (i.e., herd population) and geographical location were not found to be critical factors influencing the absolute abundance of ARGs of plasmid DNA in swine farms. However, the concentrations of florfenicol, Cu, Zn, Fe, total phosphorus (TP) and total potassium (TK) demonstrated a significant correlation with the abundance of several ARGs. Particularly, Cu and Zn had high correlations with optrA and blaCTX-M, respectively. Our results demonstrated that antibiotics, heavy metals and environmental nutrients are likely jointly contributing to the long-term persistence of ARGs in swine production. This study provides insights into the abundance and influencing factors of ARGs from swine manure, which is of significance for assessing and reducing the public health risks in livestock production.

Co-selective Pressure of Cadmium and Doxycycline on the Antibiotic and Heavy Metal Resistance Genes in Ditch Wetlands.

Abuse of heavy metals and antibiotics results in the dissemination of metal resistance genes (MRGs) and antibiotic resistance genes (ARGs). Ditch wetlands are important sinks for heavy metals and antibiotics. The relationships between bacterial communities and MRG/ARG dissemination under dual stresses of heavy metals and antibiotics remain unclear. The responses of MRGs and ARGs to the co-selective pressure of cadmium (Cd) and doxycycline (DC) in ditch wetlands were investigated after 7-day and 84-day exposures. In ecological ditches, residual rates of Cd and DC varied from 0.4 to –5.73% and 0 to –0.61%, respectively. The greatest total relative abundance of ARGs was observed in the Cd 5 mg L–1 + DC 50 mg L–1 group. A significant level of DC (50 mg L–1) significantly reduced the total relative abundances of MRGs at a concentration of 5 mg L–1 Cd stress. Redundancy analysis indicated that Cd and DC had strong positive effects on most ARGs and MRGs after a 7-day exposure. Meanwhile, the class 1 integron gene (intI1) exhibited strong positive correlations with most ARGs and cadmium resistance genes (czcA) after an 84-day exposure. Network analysis showed that Acinetobacter and Pseudomonas were the potential dominant host genera for ARGs and MRGs, and tetracycline resistance genes (tetA), czcA, and intI1 shared the same potential host bacteria Trichococcus after an 84-day exposure.

Plants inhibit the relative abundance of sulfonamide resistance genes and class 1 integron by influencing bacterial community in rhizosphere of constructed wetlands

Antibiotic resistance genes (ARGs) commonly detected in wastewater can potentially lead to a health crisis. Constructed wetlands (CWs) remove ARGs and sulfonamides (SAs) from wastewater, but the importance of plants in the process is seldom reported. We compared the effect of three wetland plant species (Cyperus alternifolius, Juncus effuses, and Cyperus papyrus), sample distance from the root, and SA presence on the environmental abundance of class 1 integron (intI1) and SA resistance genes (sul) using specially designed CW rhizoboxes. Quantitative polymerase chain reaction revealed that the relative abundance of the target genes in planted CWs, especially in C. alternifolius planted CWs, was significantly lower than that in unplanted CWs (P < 0.05). The substrate in the rhizosphere or near-/moderate-rhizosphere (closest to the root) showed the lowest average relative abundance of the target genes, while the bulk substrate (without the root) showed the highest abundance of these genes, irrespective of the planted species. Further, the influence of plants was more evident after 8 weeks of wastewater treatment. The trend was the same in SA-treated and untreated groups, although the relative abundance of the target genes was significantly higher in the former (P < 0.05). The weaker correlation between the intI1 and sul genes in the rhizosphere and near-/moderate-rhizosphere in comparison to the bulk substrate in the SA group suggested that the risk of horizontal gene transfer was probably higher in the bulk substrate and unplanted CW. A partial least-squares path model revealed that dissolved organic carbon and oxygen content significantly influenced SA concentration, microbial community, and intI1 genes, and then shaping the sul genes together. Finally, redundancy analysis suggested that abundance of sul genes was influenced by bacteria enriched in the bulk substrate and unplanted CWs. The findings provide new insights into the importance for controlling risk of ARGs by wetland plants.

High-Throughput Screening of Antimicrobial Resistance Genes and Their Association With Class 1 Integrons in Urban Rivers in Japan

Antimicrobial resistance (AMR) is a serious public health concern. Many countries have implemented AMR surveillance programs for humans and animals, but a scheme for monitoring AMR in the environment has not yet been established. Class 1 integrons, which can acquire antimicrobial resistance genes (ARGs) to gene cassettes, were proposed as a candidate to evaluate the anthropogenic impacts on AMR. However, the association between class 1 integrons and ARGs in aquatic environments is less studied and requires further elucidation. This study used high-throughput quantitative polymerase chain reaction (HT-qPCR) to characterize the pollution profiles of ARGs and mobile gene elements (MGEs) in 24 urban rivers in Tokyo and its surrounding area. The abundance of class 1 integron-integrase gene (intI1) and the array of class 1 integron gene cassettes were also determined. In total, 9–53 target genes were detected per sample, and their abundances increased following effluent discharge from wastewater treatment plants. The river and wastewater samples were categorized based on their HT-qPCR profiles, indicating that this method was useful for characterizing the pollution status in aquatic environments. The prevalence ofintI1in the rivers was observed. Some ARGs and MGEs were positively correlated withintI1, indicating thatintI1could be used as a proxy for monitoring these ARGs and MGEs in urban rivers. Long-read sequencing of class 1 integron gene cassettes revealed that one to three ARGs were present in the gene cassettes. Regardless of the sample type,blaGES-24,aadA2, andqacHwere dominant in the gene cassettes. The source and spread of class 1 integrons carrying these ARGs in aquatic environments should be further monitored.

High Prevalence of Antimicrobial Resistance and Integron Gene Cassettes in Multi-Drug-Resistant Klebsiella pneumoniae Isolates From Captive Giant Pandas (Ailuropoda melanoleuca).

Multi-drug-resistant Klebsiella pneumoniae (MDR K. pneumonia) is increasingly being reported with corresponding increase in morbidity and mortality all over the world. However, limited information is available concerning MDR K. pneumonia in giant pandas. The objective of this study was to grasp the drug resistance profile of MDR K. pneumonia isolated from giant pandas. A total of 182 K. pneumoniae isolates were collected from fresh feces of 94 captive giant pandas of different ages and sex and separated by season. We performed a standard disk diffusion antimicrobial susceptibility test with the isolates and further evaluated the antibiotic resistance genes (ARGs) of multi-drug-resistant strains by high-throughput quantitative PCR. In addition, we then analyzed mobile genetic elements (MGEs), integron gene cassettes, and the multi-locus sequence typing of multi-drug-resistant strains by PCR. Antimicrobial susceptibility testing results demonstrated that a total of 30 (16.5%) K. pneumoniae isolates showed multiple drug resistances. The thirty MDR K. pneumonia isolates were mainly resistant to amoxicillin (100.0%), doxycycline (86.7%), chloramphenicol (60.0%), compound trimethoprim (60.0%) and trimethoprim (56.7%). Fifty different types of antibiotic resistance genes were found, which included a total of 671 antibiotic resistance genes, in the 30 multi-drug-resistant isolates. The top ten resistance genes were: vanTC-02, aacC, blaCTX-M-04, blaSHV-01, blaSHV-02, ampC-04, blaOXY, tetD, blaTEM and tetA-02. Thirteen mobile genetic elements were detected, of which IS26 (96.67%) and intI1 (96.67%) had the highest frequency. The thirty MDR K. pneumonia isolates were negative for the traA, traF, tnsA, IS1133, ISpa7, ISkpn6, intI2 and intI3 genes. Moreover, a further investigation of integrons revealed that two types of specific gene cassettes (dfrA12 + orfF + aadA2 and dfrA12 + orfF) were identified in class 1 integrons. Multi-locus sequence typing results showed that 22 STs in the thirty MDR K. pneumonia isolates were identified, the main type was ST37 (5/30). Our results illustrate that effective surveillance and strict biosecurity strategies should be taken to prevent the spread of multi-drug-resistant bacteria, and monitor the emergence of mobile genetic elements and integrons.

Distribution and Molecular Characterization of Functional Class 2 Integrons in Clinical Proteus mirabilis Isolates.

BackgroundIntegrons are the main mode of horizontal transmission of drug-resistance genes and are closely related to drug resistance in clinical bacteria. In this study, the distributions of class 1, 2, and 3 integron gene cassettes were investigated in 150 Proteus mirabilis (P. mirabilis) isolates from patients, and molecular characterization of functional class 2 integrons was further analyzed.MethodsClass 1, 2, and 3 integrons were screened by polymerase chain reaction (PCR) in 150 clinical P. mirabilis isolates. The variable regions of the integrons were determined by restriction analysis and sequencing. Internal stop codons mutations in class 2 integrons and their common promoters were also determined by sequencing. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was used to analyze the phylogenetic relations of class 2 integron-positive isolates.ResultsClass 1 integrons were detected in 69 (46%) of 150 P. mirabilis isolates, and six different gene cassette arrays were detected, with the most prevalent being dfrA32-aadA2. Class 2 integrons were detected in 61 (40.7%) of 150 P. mirabilis isolates, and three different gene cassette arrays were detected, including sat2-aadA1, which was detected for the first time in a class 2 integron. Nearly similar ERIC-PCR fingerprinting patterns were detected in 45 (73.8%) of 61 class 2 integron-positive isolates. The functional class 2 integron was detected in three P. mirabilis isolates having the same gene cassette, dfrA1-sat2-aadA1, in the variable region and four novel open reading frames with unknown functions. Same PintI2 and Pc promoters were detected in these three functional class 2 integron isolates, as was found in other class 2 integron isolates. However, these three strains did not totally show identical homology and drug sensitivity.ConclusionAlthough functional class 2 integrons have low distribution and relatively conserved molecular characteristics, they can still form clinical dissemination and drug resistance expression.

Molecular Detection of Integrons, Colistin and β-lactamase Resistant Genes in Salmonella enterica Serovars Enteritidis and Typhimurium Isolated from Chickens and Rats Inhabiting Poultry Farms.

The rapid growth of multidrug-resistant Salmonella is a global public health concern. The aim of this study was to detect integrons, colistin and β-lactamase resistance genes in Salmonella enteritidis and typhimurium. A total of 63 isolates of S. enteritidis (n = 18) and S. typhimurium (n = 45) from fecal samples of layers and rats at chicken farms were screened for antibiotic resistant genes. Conventional PCR was performed for the detection of integrons (classes 1, 2, and 3), colistin (mcr-1-5) and β-lactamase (blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaCTX-M-15, blaTEM, blaSHV, and blaOXA) resistant genes. Of these isolates, 77% and 27% of S. typhimurium and S. enteritidis harboured the mcr-4 encoded gene for colistin, respectively. The prevalence of class 1 integrons for S. typhimurium and S. enteritidis was 100% for each serovar, while for class 2 integrons of S. typhimurium and S. enteritidis it was 49% and 33% respectively, while class 3 integron genes was not detected. Our study also detected high levels of β-lactamase encoding genes (bla gene), namely blaCTX-M, blaCTX-M-1, blaCTX-M-9 and blaTEM from both S. typhimurium and S. enteritidis. This, to our knowledge, is the first report of mcr-4 resistance gene detection in Salmonella serovars in South Africa. This study also highlights the importance of controlling rats at poultry farms in order to reduce the risk of transmission of antibiotic resistance to chickens and eventually to humans.