INSTIs and NNRTIs Potently Inhibit HIV-1 Polypurine Tract Mutants in a Single Round Infection Assay

Integrase strand transfer inhibitors (INSTIs) are a class of antiretroviral compounds that prevent the insertion of a DNA copy of the viral genome into the host genome by targeting the viral enzyme integrase (IN). Dolutegravir (DTG) is a leading INSTI that is given, usually in combination with nucleoside reverse transcriptase inhibitors (NRTIs), to treat HIV-1 infections. The emergence of resistance to DTG and other leading INSTIs is rare. However, there are recent reports suggesting that drug resistance mutations can occur at positions outside the integrase gene either in the HIV-1 polypurine tract (PPT) or in the envelope gene (env). Here, we used single round infectivity assays to measure the antiviral potencies of several FDA-approved INSTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) against a panel of HIV-1 PPT mutants. We also tested several of our promising INSTIs and NNRTIs in these assays. No measurable loss in potency was observed for either INSTIs or NNRTIs against the HIV-1 PPT mutants. This suggests that HIV-1 PPT mutants are not able, by themselves, to confer resistance to INSTIs or NNRTIs.

Multiple and High-Risk Clones of Extended-Spectrum Cephalosporin-Resistant and blaNDM-5-Harbouring Uropathogenic Escherichia coli from Cats and Dogs in Thailand

Resistance to extended-spectrum cephalosporins (ESC) and carbapenems in Escherichia coli (E. coli), increasingly identified in small animals, indicates a crisis of an antimicrobial resistance situation in veterinary medicine and public health. This study aimed to characterise the genetic features of ESC-resistant E. coli isolated from cats and dogs with urinary tract infections in Thailand. Of 72 ESC-resistant E. coli isolated from diagnostic samples (2016–2018), blaCTX-M including group 1 (CTX-M-55, -15 and -173) and group 9 (CTX-M-14, -27, -65 and -90) variants were detected in 47 isolates (65.28%) using PCR and DNA sequencing. Additional antimicrobial resistance genes, including plasmid-mediated AmpC (CIT and DHA), blaNDM-5, mcr-3, mph(A) and aac(6′)-Ib-cr, were detected in these isolates. Using a broth microdilution assay, all the strains exhibited multidrug-resistant phenotypes. The phylogroups were F (36.11%), A (20.83%), B1 (19.44%), B2 (19.44%) and D (4.17%), with several virulence genes, plasmid replicons and an integrase gene. The DNA fingerprinting using a repetitive extragenic palindromic sequence-PCR presented clonal relationships within phylogroups. Multiple human-associated, high-risk ExPEC clones associated with multidrug resistance, including sequence type (ST) 38, ST131, ST224, ST167, ST354, ST410, ST617 and ST648, were identified, suggesting clonal dissemination. Dogs and cats are a potential reservoir of ESC-resistant E. coli and significant antimicrobial resistance genes.

Complete genome analysis of enterotoxigenic Escherichia coli K88 phage vB_EcoP_E212, a novel genus Lederbergvirus

The features and genome annotation of a newly bacteriophage v B_EcoP_E212 (referred to as E212) which isolated from farm sewage collected in Jilin, China was describes in this study. Bacteriophage E212 belongs to the family Podoviridae, order Caudovirales through transmission electron microscopy. This phage specifically infects enterotoxigenic Escherichia coli K88. The dsDNA molecule of phage E212 was 38252 bp in length and contained 46.98% G + C content. It has been predicted to contain 53 ORFs, and no tRNAs. Phage E212 carried the integrase gene, and no homologues of virulence factors or antimicrobial resistance genes were found in this phage. Phage E212 was assigned to the genus Lederbergvirus in accordance with nucleotide sequence alignment and phylogenetic analysis.

Biochar and Hyperthermophiles as Additives Accelerate the Removal of Antibiotic Resistance Genes and Mobile Genetic Elements during Composting

Sewage treatment plants are known as repositories of antibiotic resistance genes (ARGs). Adding biochar and inoculating with exogenous microbial agents are common ways to improve the quality of compost. However, little is known about the effects of these exogenous additives on the fate of ARGs during composting and the related mechanisms. In this study, municipal sludge was taken as the research object to study the ARG-removal effects of four composting methods: ordinary compost (CT), compost with hyperthermophiles (HT), compost with hyperthermophiles and 2.0% biochar (HT2C) and compost with hyperthermophiles and 5.0% biochar (HT5C). Real-time quantitative PCR (qPCR) and 16S rRNA high-throughput sequencing were conducted to analyze the ARGs, MGEs and bacterial community. After composting, the abundance of ARGs in CT was reduced by 72.7%, while HT, HT2C and HT5C were reduced by 80.7%, 84.3% and 84.8%, respectively. Treatments with different proportions of biochar added (HT2C, HT5C) had no significant effect on the abundance of ARGs. Network analysis showed that Firmicutes and Nitrospirae were positively associated with most ARGs and may be potential hosts for them. In addition, redundancy analysis further showed that the class 1 integrase gene (intI1), pH and organic carbon had a greater effect on ARGs. Our findings suggested that the combination of hyperthermophiles and biochar during the composting process was an effective way to control ARGs and mobile genetic elements (MGEs), thus inhibiting the spread and diffusion of ARGs in the environment and improving the efficiency of treating human and animal diseases.

Functional characterization of a gene cluster responsible for inositol catabolism associated with hospital-adapted isolates of Enterococcus faecium

Enterococcus faecium is a nosocomial, multidrug-resistant pathogen. Whole genome sequence studies revealed that hospital-associated E. faecium isolates are clustered in a separate clade A1. Here, we investigated the distribution, integration site and function of a putative iol gene cluster that encodes for myo-inositol (MI) catabolism. This iol gene cluster was found as part of an ~20 kbp genetic element (iol element), integrated in ICEEfm1 close to its integrase gene in E. faecium isolate E1679. Among 1644 E. faecium isolates, ICEEfm1 was found in 789/1227 (64.3 %) clade A1 and 3/417 (0.7 %) non-clade A1 isolates. The iol element was present at a similar integration site in 180/792 (22.7 %) ICEEfm1-containing isolates. Examination of the phylogenetic tree revealed genetically closely related isolates that differed in presence/absence of ICEEfm1 and/or iol element, suggesting either independent acquisition or loss of both elements. E. faecium iol gene cluster containing isolates E1679 and E1504 were able to grow in minimal medium with only myo-inositol as carbon source, while the iolD-deficient mutant in E1504 (E1504∆iolD) lost this ability and an iol gene cluster negative recipient strain gained this ability after acquisition of ICEEfm1 by conjugation from donor strain E1679. Gene expression profiling revealed that the iol gene cluster is only expressed in the absence of other carbon sources. In an intestinal colonization mouse model the colonization ability of E1504∆iolD mutant was not affected relative to the wild-type E1504 strain. In conclusion, we describe and functionally characterise a gene cluster involved in MI catabolism that is associated with the ICEEfm1 island in hospital-associated E. faecium isolates. We were unable to show that this gene cluster provides a competitive advantage during gut colonisation in a mouse model. Therefore, to what extent this gene cluster contributes to the spread and ecological specialisation of ICEEfm1-carrying hospital-associated isolates remains to be investigated.

Arms race in a cell: genomic, transcriptomic, and proteomic insights into intracellular phage–bacteria interplay in deep-sea snail holobionts

Background Deep-sea animals in hydrothermal vents often form endosymbioses with chemosynthetic bacteria. Endosymbionts serve essential biochemical and ecological functions, but the prokaryotic viruses (phages) that determine their fate are unknown. Results We conducted metagenomic analysis of a deep-sea vent snail. We assembled four genome bins for Caudovirales phages that had developed dual endosymbiosis with sulphur-oxidising bacteria (SOB) and methane-oxidising bacteria (MOB). Clustered regularly interspaced short palindromic repeat (CRISPR) spacer mapping, genome comparison, and transcriptomic profiling revealed that phages Bin1, Bin2, and Bin4 infected SOB and MOB. The observation of prophages in the snail endosymbionts and expression of the phage integrase gene suggested the presence of lysogenic infection, and the expression of phage structural protein and lysozyme genes indicated active lytic infection. Furthermore, SOB and MOB appear to employ adaptive CRISPR–Cas systems to target phage DNA. Additional expressed defence systems, such as innate restriction–modification systems and dormancy-inducing toxin–antitoxin systems, may co-function and form multiple lines for anti-viral defence. To counter host defence, phages Bin1, Bin2, and Bin3 appear to have evolved anti-restriction mechanisms and expressed methyltransferase genes that potentially counterbalance host restriction activity. In addition, the high-level expression of the auxiliary metabolic genes narGH, which encode nitrate reductase subunits, may promote ATP production, thereby benefiting phage DNA packaging for replication. Conclusions This study provides new insights into phage–bacteria interplay in intracellular environments of a deep-sea vent snail.

Degradation of antibiotic resistance genes and mobile gene elements in dairy manure anerobic digestion

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene (sulII), tetracycline resistance genes (tetW), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes (ermF), class 1 integrase gene (intI1), and transposase gene (tnpA). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced (p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins–Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

Epidemiological Analysis of Multidrug-Resistant Acinetobacter baumannii Isolates in a Tertiary Hospital Over a 12-Year Period in China

Acinetobacter baumannii is an important nosocomial pathogen, which is multidrug resistant (MDR). Acinetobacter baumannii has become a major threat to public health worldwide due to its ability to easily acquire resistant genes. In order to analyze its epidemiology characteristics and the genetic evolution, A. baumannii isolates obtained from a Chinese tertiary hospital in the past 12 years (2008–2019), 295 isolates of non-repetitive A. baumannii, were recovered from patients and wards environments. The resistance genes were analyzed using antimicrobial susceptibility testing. The genetic relatedness of 295 isolates was identified by multilocus sequence typing (MLST) and eBURST analysis. It was found that the antibiotic-resistant and carbapenemase-resistant genes of all the 295 MDR A. baumannii in the hospital have not changed significantly over the past 12 years; all of them were resistant to multiple antibiotics except the polymyxin E and tigecycline. The results of drug-resistant genes showed that the detection rates of carbapenemase-resistant genes blaOXA−23, blaTEM−1, and blaOXA−66 were 97.6, 75.3, and 71.9%, respectively, which were detected almost every year from 2008 to 2019. Additionally, 16s rRNA methylation enzyme gene armA, aminoglycoside-resistant gene ant(3")-I, and class I integrase gene could also have a high positive rate. By MLST, these isolates were assigned to 12 sequence types (STs), including ST369, ST208, ST195, ST191, ST368, ST530, ST469, ST451, ST229, ST381, ST543, and ST1176. eBURST analysis showed that 9 STs with ST208 as the founder genotype belonged to Group 1 except for ST229, ST530, and ST1176. Therefore, most MDR A. baumannii isolates had a relatively close genetic relationship. Notably, the predominant ST208 and ST369 at the early stage changed to ST451 in 2019, indicating that the complex and diverse genetic background of the prevalence of A. baumannii isolates in the hospital. Overall, further epidemiological surveillance and genetic evolution analysis of A. baumannii are required, which can provide new strategies for the prevention and control of A. baumannii infections.

Method for Determining the Profile of Antibiotic Resistance Genes in the Vibrio cholera Strains by RT-PCR

The aim of the work was to design and carry out experimental studies of a set of reagents to identify the spectrum of genes that determine the resistance of the Vibrio cholerae strains to antibacterial drugs.Materials and methods. V. cholerae strains isolated from humans and environmental objects during epidemiological complications and the cholera-free period were included in the study. Sensitivity to antimicrobial drugs was evaluated by the disk diffusion method. Whole genome sequencing was performed on an Illumina MiSeq. The profile of resistance genes was determined based on a comparison with the ResFinder database. The temperature regime, the composition of the reaction mixtures, and the reaction parameters were optimized; the specificity, sensitivity and reproducibility of the constructed prototype test system were measured.Results and discussion. The spectrum of antibiotic resistance and the profile of resistance genes were determined for the studied strains. To develop multiplex PCR, we selected the most common in the V. cholerae populations genes, which are responsible for resistance to tetracycline (tetA), streptomycin (strA), florfenicol/ chloramphenicol (floR) and trimethoprim/sulfamethoxazole (two variants of the dihydrofolate reductase gene: dfrA1 and dhfR), as well as SXT element integrase gene (int). In the reaction, markers were specifically detected in accordance with the genomic resistance profile, which correlates with the phenotypic manifestation of resistance determined by the disco-diffusion method. The sensitivity of the developed panel of primers and probes for V. cholerae strains was 103 –104 CFU/ml. Therefore, taking into account the specificity, rapidity and simplicity of the reaction, the developed system of primers and probes can be successfully applied for a preliminary assessment of the resistance of the V. cholerae strains to antimicrobial agents.