Microniches in biofilm depth are hot-spots for antibiotic resistance acquisition in response to in situ stress

Class 1 integrons play a major role in antibiotic resistance dissemination among Gram-negative bacteria. They are genetic platforms able to capture, exchange and express antibiotic resistance gene cassettes. The integron integrase, whose expression is regulated by the bacterial SOS response, is the key element of the integron catalyzing insertion/excision/shuffling of gene cassettes. We previously demonstrated that the basal level of integrase expression and in consequence, its activity, is increased via the starvation-induced stringent response in the biofilm population. However, biofilms are heterogeneous environments where bacteria are under various physiological states. Here we thus analyzed at the bacterial level, the SOS response and integrase expression within the biofilm, using confocal microscopy and flow cytometry. We showed that in the absence of exogenous stress, only a small number of bacteria (~ 1%) located in the depth of the biofilm induce the SOS-response leading to a high level of integrase expression, through both a stringent response-dependent and -independent manner. Our results thus indicate that few bacteria located in microniches of the biofilm depth undergo sufficient endogenous stress to promote the acquisition of antibiotic resistance, forming a reservoir of bacteria ready to rapidly resist antibiotic treatments.

Characterization and Antimicrobial Resistance of Environmental and Clinical Aeromonas Species Isolated from Fresh Water Ornamental Fish and Associated Farming Environment in Sri Lanka

The aims of this study were to characterize and investigate antimicrobial susceptibility and presence of integrons in 161 Aeromonas spp. isolated from ornamental freshwater fish farming environment, apparently healthy and diseased fish. Phylogenetic analyses of the gyrB gene sequences identified Aeromonas veronii as the most abundant species (75.8%) followed by Aeromonas hydrophila (9.3%), Aeromonas caviae (5%), Aeromonas jandaei (4.3%), Aeromonas dhakensis (3.7%), Aeromonas sobria (0.6%), Aeromonas media (0.6%), and Aeromonas popoffii (0.6%). Susceptibility to thirteen antimicrobials was determined and antimicrobial resistance frequencies were: amoxicillin (92.5%), enrofloxacin (67.1%), nalidixic acid (63.4%), erythromycin (26.1%), tetracycline (23.6%), imipenem (18%), trimethoprim-sulfamethoxazole (16.8%), and gentamicin (16.8%). Multi-drug resistance (MDR) was widespread among the isolates (51.6%, 83/161) with 51.6% (63/122) A. veronii isolates being MDR. In addition, 68.3% of isolates had multiple antibiotic resistance (MAR) indexes higher than 0.2, suggesting that they originated from a high-risk source of contamination where antimicrobials are often used. In all, 21.7% isolates carried class 1 integrons, with 97.1% having gene cassettes, while there were 12 isolates carrying class 2 integron gene cassettes. Our findings highlight that the aquatic environment and ornamental fish act as reservoirs of multidrug resistant Aeromonas spp. and underline the need for a judicious use of antimicrobials and timely surveillance of antimicrobial resistance (AMR) in aquaculture.

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

Integrons are bacterial 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 PCR 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 use 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 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

Molecular study of metallo-β-lactamases and integrons in Acinetobacter baumannii isolates from burn patients

Background Productions of metallo-β-lactamases enzymes are the most common mechanism of antibiotic resistance to all beta-lactam classes (except monobactams) in Acinetobacter baumannii. MBLs are usually associated with gene cassettes of integrons and spread easily among bacteria. The current study was performed to detect the genes encoding MBLs and integron structures in A. baumannii isolates from burn patients. Methods This study was performed on 106 non-duplicate A. baumannii isolates from burn patients referred to Shahid Motahari Hospital in Tehran. Antibiotic susceptibility of A. baumannii isolates was performed using disk diffusion and broth microdilution method in accordance with the CLSI guidelines. The presence of class 1 integron and associated gene cassettes as well as MBLs-encoding genes including blaVIM, and blaIMP were investigated using PCR and sequencing techniques. Results In this cross-sectional study all (100%) of the A. baumannii isolates examined were multidrug resistant. All isolates were sensitive to colistin and simultaneously all were resistant to imipenem. PCR assays showed the presence of blaVIM and blaIMP genes in 102 (96.2%) and 62 (58.5%) isolates of A. baumannii respectively. In addition, 62 (58.5%) of the A. baumannii isolates carried integron class 1, of which 49 (79.0%) were identified with at least one gene cassette. Three types of integron class 1 gene cassettes were identified including: arr2, cmlA5, qacE1 (2300 bp); arr-2, ereC, aadA1, cmlA7, qacE1 (4800 bp); and aac(3)-Ic, cmlA5 (2250 bp). Conclusion A high prevalence of MBLs genes, especially blaVIM, was identified in the studied MDR A. baumannii isolates. In addition, most of the strains carried class 1 integrons. Furthermore, the gene cassettes arrays of integrons including cmlA5 and cmlA7 were detected, for the first time, in A. baumannii strains in Iran.

Predicting the taxonomic and environmental sources of integron gene cassettes using structural and sequence homology of attC sites

Integrons are bacterial genetic elements that can capture mobile gene cassettes. They are mostly known for their role in the spread of antibiotic resistance cassettes, contributing significantly to the global resistance crisis. These resistance cassettes likely originated from sedentary chromosomal integrons, having subsequently been acquired and disseminated by mobilised integrons. However, their taxonomic and environmental origins are unknown. Here, we use cassette recombination sites (attCs) to predict the origins of those resistance cassettes now spread by mobile integrons. We modelled the structure and sequence homology of 1,978 chromosomal attCs from 11 different taxa. Using these models, we show that at least 27% of resistance cassettes have attCs that are structurally conserved among one of three taxa (Xanthomonadales, Spirochaetes and Vibrionales). Indeed, we found some resistance cassettes still residing in sedentary chromosomal integrons of the predicted taxa. Further, we show that attCs cluster according to host environment rather than host phylogeny, allowing us to assign their likely environmental sources. For example, the majority of β-lactamases and aminoglycoside acetyltransferases, the two most prevalent resistance cassettes, appear to have originated from marine environments. Together, our data represent the first evidence of the taxonomic and environmental origins of resistance cassettes spread by mobile integrons.

Horizontal transfer and evolution of wall teichoic acid gene cassettes in Bacillus subtilis

Background: Wall teichoic acid (WTA) genes are essential for production of cell walls in gram-positive bacteria and necessary for survival and variability in the cassette has led to recent antibiotic resistance acquisition in pathogenic bacteria. Methods: Using a pan-genome approach, we examined the evolutionary history of WTA genes in Bacillus subtilis ssp. subtilis. Results: Our analysis reveals an interesting pattern of evolution from the type-strain WTA gene cassette possibly resulting from horizontal acquisition from organisms with similar gene sequences. The WTA cassettes have a high level of variation which may be due to one or more independent horizontal transfer events during the evolution of Bacillus subtilis ssp. subtilis. This swapping of entire WTA cassettes and smaller regions within the WTA cassettes is an unusual feature in the evolution of the Bacillus subtilis genome and highlights the importance of horizontal transfer of gene cassettes through homologous recombination within B. subtilis or other bacterial species. Conclusions: Reduced sequence conservation of these WTA cassettes may indicate a modified function like the previously documented WTA ribitol/glycerol variation. An improved understanding of high-frequency recombination of gene cassettes has ramifications for synthetic biology and the use of B. subtilis in industry.

Carbapenem-Resistant Klebsiella Pneumoniae in Southwest China: Molecular Characterizations and Risk Factors Caused by NDM and KPC Producers

Background: Klebsiella pneumoniae is one of the most common Enterobacteriaceae. In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) has become one of the most important carbapenem-resistant Enterobacteriaceae. CRKP are usually resistant to antibiotics. Up to this day, the emergence of carbapenemase-producing K. pneumoniae has been a challenge for treatment of clinical infection.Methods: (i) 66 non-repetitive clinical CRKP isolates were identified by matrix-assisted laser analytical ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and drug sensitivity analysis was performed by Vitek2 Compact. EDTA-synergy test and mCIM / eCIM test were used to detect drug-resistant phenotypes. (ii) Carbapenemase genes, extended-spectrum β-lactamase genes (ESBLs), cephalosporinase gene (AmpC), virulence genes, integron and resistance gene cassettes were amplified by PCR. (iii) Plasmid typing was performed by plasmid conjugation assay and PCR-based replicon typing (PBRT) method. (iv) The genetic environments of KPC-2 and NDM-1 were analyzed by using overlapping PCR. (v) MLST was used to analyze the molecular epidemiological characteristics of CRKP. (vi) Risk factors of CRKP infection by logistic regression model.Results: Our study revealed that 42 of the 66 CRKP isolates obtained from patients were identified as blaKPC-2, 24 blaNDM-1-positive strains were identified (20 blaNDM-1 and 4 blaNDM-5), of which 18 were from the neonatal departments. And CRKP strains were ESBL (extended-spectrum β-lactamases) and AmpC enzymes producer, Notably, we found two CR-hvKp (carbapenem-resistant hypervirulent klebsiella pneumoniae) strains, which contains blaKPC-2 gene and other resistant genes. Two of the 42 KPC-2-producing CRKP strains were positive for transconjugants, and the plasmid typing was the IncFII type. And two NDM-producing CRKP strains tested positive for transconjugants, which belonged to the lncX3 plasmid. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (tnpA-tnpR-ISkpn8-blaKPC-2) and conserved regions (blaNDM−1-bleMBL-trpF-tat) are associated with the dissemination of KPC-2 and NDM-1. Intl1 carrying drug resistance gene cassettes were widely distributed in CRKP. According to the MLST results, a total of 13 ST types were measured in 66 CRKP strains, ST11 and ST4495 were the main ST types, and the latter was the newly discovered ST type. Hematological disease, tracheal cannula and prior use of β-lactams and β-lactamase inhibitor combination were identified as independent risk factors for CRKP infections.Conclusion: These findings manifested the need for intensive surveillance and precautions to monitor the further spread of KPC and NDM in southwest China.

Construction of an Agrobacterium Mediated RNAI Genetic Transformation Vector Targeting the Replicase Gene of Indian cassava mosaic virus and Evaluation of Their Transformation Ability in Cassava Immature Leaf Lobes

Cassava mosaic virus is one of the major problems affecting cassava industry in India. Currently there are no effective strategy to completely protect cassava from cassava mosaic viruses. In order to attain cassava mosaic virus resistance RNAi vectors targeting the replicase gene of Indian cassava mosaic virus is constructed in this study. Their efficiency to transform cassava immature leaf lobes were also studied here. Replicase gene of Indian cassava mosaic virus in Tamilnadu are cloned and sequenced. Conserved domains are identified and sub cloned to CSIRO RNAi vector system and transformation studies are done in immature cassava leaves. Two different RNAi vectors were constructed, utilizing a conserved 440bp of 5’ end of ICMV Rep (AC1) gene which also corresponds to a part of AC4 gene, and functions as a viral RNAi suppressor protein. The partial Rep gene of ICMV was cloned in sense and anti-sense orientations in the RNAi intermediate vector, pHANNIBAL. After cloning into pHANNIBAL, the cloned RNAi gene cassettes of ICMV is released and cloned into the binary vector, pART27, which contains the kanamycin-resistance gene as a plant selectable marker. In order to use hygromycin as a selection agent in cassava genetic transformation, RNAi–Rep gene cassettes of ICMV were cloned into pCAMBIA1305.2. These constructs were named pICR1 and pICR2 respectively. The Genetic transformation studies in cassava leaves done using pICR2 vector could generate PCR positive plants. An agrobacterium mediated replicase RNAi vector is developed and that can be transformed into cassava immature leaf lobes. Their efficiency to silence the Indian cassava mosaic virus should be studied further.