Int Gene Research Articles

Seasonal Variation and Influencing Factor Analysis of Antibiotic Resistance Genes in Water Supply Reservoirs of Central China

This study quantified an integron gene intI1 and 19 antibiotic resistance genes(ARGs) to identify the ARGs pollution characteristics in 11 drinking water reservoirs of central China. The results indicated that the ARGs abundance did not change significantly over time in the studied reservoir waterbodies. Tetracycline, sulfonamide, and β-lactam ARGs were dominant. The high abundance and detection rate of two sulfonamide ARGs(sul1 and sul2) suggested that they were the predominant ARGs. No polymyxin resistance genes(mcr-1) were detected, which indicated that the antibiotic restriction policy of China has achieved positive outcomes. Compared with that in other environmental media, the ARGs abundance in the reservoir environment was low. The correlation analysis showed relevance between the water quality indicators and the ARGs, which suggested that the water quality indexes can be used as ARGs pollution indicators in the reservoir environment. The abundance and detection rate of carbapenem ARGs were low owing to their dosage restriction and high degradability. Tetracycline ARGs were closely related to the other resistance gene types, which might have been due to horizontal gene transfer. Although the overall correlation between intI1 and ARGs was modest, it might be the main reason for the spread of several individual ARGs in the reservoir environment.

Influence of soil characteristics and metal(loid)s on antibiotic resistance genes in green stormwater infrastructure in Southern California

The synergetic effects of metal(loid)s and soil characteristics on bacterial antibiotic resistance genes (ARGs) in green stormwater infrastructure (GSI) has been relatively understudied. Surface soil samples from six GSIs in Southern California over three time periods were assessed for selected ARGs, class 1 integron-integrase genes (intI1), 16S rRNA genes, and bioavailable and total concentrations of nine metal(loid)s, to investigate the relationships among ARGs, soil characteristics, and co-occurring metal(loid)s. Significant correlations existed among relative gene abundances (sul1, sul2, tetW, and intI1), total metal(loid)s (arsenic, copper, lead, vanadium, and zinc), and bioavailable metal(loid) (arsenic) (r = 0.29–0.61, padj < 0.05). Additionally, soil texture, organic matter, and nutrients within GSI appeared to be significantly correlated with relative gene abundances of sul1, sul2, and tetW (r = −0.57 to 0.59, padj < 0.05). Multiple regression models significantly improved the estimation of ARGs in GSI when considering multiple effects of soil characteristics and metal(loid)s (r = 0.74, padj < 0.001) compared to correlation results. Total arsenic was a significant (positive) correlate in all the regression models of relative gene abundances. This work provides new insights into co-dependencies between GSI ARGs and co-occurring metal(loid)s, indicating the need for risk assessment of metal(loid)-influenced ARG proliferation.

Strict relationship between class 1 integrons and resistance to sulfamethoxazole in Escherichia coli

Antibiotic resistance is a health concern. Class 1 integrons (Int1) are genetic elements that contribute to the problem, as they carry different antibiotic resistance genes in their variable region, frequently dfrA (resistance to trimethoprim) and, in their conserved region, the sul1 gene (resistance to sulfonamides, e.g. sulfamethoxazole). These are synthetic antibiotics that work by blocking two enzymes in the folic acid synthesis pathway. In the clinic, the combination of trimethoprim (TMP) and sulfamethoxazole (SMX), called co-trimoxazole (SXT), is widely used. A collection of 230 uropathogenic Escherichia coli strains was studied with three objectives: i) to analyze their phenotype of susceptibility to antifolate antibiotics, ii) to determine the genetic basis of their resistance to SMX, and iii) to correlate the phenotypic and genotypic data with the presence of Int1. The prevalence of resistance to SMX, TMP, and SXT was 54%, 45%, and 41%, respectively. The mobile genes sul1, sul2 and sul3, which confer resistance to sulfonamides, were PCR-surveyed: all sulfa-resistant strains were found to contain at least one of these genes, with the exception of three strains. For these latter, the possibility of being target folP mutants could be ruled out, pointing to the existence of a still unknown mechanism of resistance to SMX in E. coli. All 50 strains previously cataloged as positive for Int1 (because they had an intI1 gene for the integrase) were resistant to SMX: most had sul1, alone or with sul2 or sul3, others only had sul2, and one lacked every sul gene. In addition, 16 sul1+intI1- strains were found to contain other typical integron sequences. That is, in no case was the sul1 gene detected independently of other Int1 sequences. Therefore, we propose that the sul1 gene would be a good marker for the presence of Int1, as well as the intI1 gene. Following this criterion, the prevalence of Int1 in our collection increased from 22% (50 intI1+) to 29% (66 intI1+ and/or sul1+). Of these 66 Int1+ strains, 63 were resistant to TMP. The main conclusion in this work is that the presence of a class 1 integron would always require a sulfamethoxazole resistant cellular context. In more general terms, these integrons appear to be closely related to resistance to antifolate compounds.

Antibiotic-resistant Gram-negative Bacteria in Urban Flies, and the Increased Risk Posed by Open-air Markets in Mexico City

Aims: Flies are known to spread antibiotic resistant bacteria (ARB), especially from farms to cities; but they may also play a role in the intra-urban dispersion of ARB, in conjunction with poor sanitary conditions. Here, we characterized gram-negative ARB isolated from urban flies (Lucilia and Sarcophaga spp.), and the co-relation with the periodic installation of two open-air markets in Mexico City.&#x0D; Methodology: Forty-two flies were individually captured, and 116 gram-negatives (68 of them Escherichia coli) were isolated from them. Resistance prevalence, and the presence of class 1 integrons was assessed.&#x0D; Results: The isolates were resistant to an average of 2.26 antibiotics (2.6 for E. coli), and 33% of E. coli isolates carried the intI1 gene. Thirteen percent of E. coli isolates produced extended-spectrum beta-lactamases (ESBL), all of them CTX-M, alone or, mostly, along TEM enzymes. Comparing data from market-free days vs. days when open-air markets were installed, the average number of resistance phenotypes per E. coli isolate went from 2.14 to 3.09; the number of resistance phenotypes per fly from 4.62 to 8.88; the average number of resistances per isolate per fly from 1.25 to 2.43; and the ESBL-producing carriage rate per fly from 0.08 to 0.38, respectively (P &lt;.05). Other resistance parameters, were consistently higher among flies captured on market days, but differences were not significant.&#x0D; Conclusion: Urban flies in Mexico City carry a high number of gram-negative ARB; the presence of open-air markets significantly increase the risk of fly-mediated ARB spreading to the neighboring areas.

Release of Antibiotic-Resistance Genes from Hospitals and a Wastewater Treatment Plant in the Kathmandu Valley, Nepal

Hospitals and wastewater treatment plants (WWTPs) are high-risk point sources of antibiotic-resistance genes (ARGs) and antibiotic-resistant bacteria. This study investigates the occurrence of clinically relevant ARGs (sul1, tet(B), blaCTX-M, blaNDM-1, qnrS) and a class one integron (intI1) gene in urban rivers, hospitals, and municipal wastewater in the Kathmandu Valley, Nepal. Twenty-five water samples were collected from three rivers, six hospitals, and a wastewater treatment plant to determine the concentrations of ARGs and intI1 using quantitative polymerase chain reactions. From the results, all tested ARGs were detected in the river water; also, concentrations of ARGs in WWTP and hospital effluents varied from 6.2 to 12.5 log10 copies/L, highlighting the role of a WWTP and hospitals in the dissemination of ARGs. Except for blaNDM-1, significant positive correlations were found between intI1 and other individual ARGs (r = 0.71–0.96, p &lt; 0.05), indicating the probable implications of intI1 in the transfer of ARGs. Furthermore, this study supports the statement that the blaNDM-1 gene is most likely to be spread in the environment through untreated hospital wastewater. Due to the interaction of surface water and groundwater, future research should focus on ARGs and factors associated with the increase/decrease in their concentration levels in drinking water sources of the Kathmandu Valley.

Effect of Gene Editing on Alzheimer's Disease

Abstract Background Alzheimer’s disease is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, and, eventually, the ability to carry out the simplest tasks. In most people with Alzheimer’s, symptoms first appear in their mid-60s. Current estimates suggest that 44 million people live with dementia worldwide at present. This is predicted to more than triple by 2050 as the population ages. Alzheimer’s disease is currently ranked as the sixth leading cause of death in the United States, but recent estimates indicate that the disorder may rank third, just behind heart disease and cancer, as a cause of death for older people Treatment is currently targeted toward symptomatic therapy, although trials are underway that aim to reduce the production and overall burden of pathology within the brain. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has shown promise in certain neurological disorders, it provides a precise editing to human genome and reflect an efficient curative therapy. We aimed to investigate the efficacy of knock-out of the APP gene “Amyloid precursor protein gene(APBA2)that consequently modify the expression of Amyloid protein in leucocytes cell line using CRISPR-cas9 technology. Methods The gene expression profile of Alzheimer's disease was downloaded from biological bioinformatics databases,and based on bioinformatics analysis, we figured out that APP gene was overexpressed in Alzheimer's disease in both brain and peripheral tissues such as plasma, fibroblast and PMNLs. We used PMNLs as the source of gene for edition in our study .We knocked out the APP gene in leucocytes cell lines using CRISPR-cas9 technology. Finally, the gene editing efficacy was evaluated by cell viability assay, the gene expression was measured by qPCR and the Amyloid protein expression was proved by Immunofluorescence. Results knockout of APP gene int Leucocytes Cell line resulted in reduction in cell viability that was associated with marked reduction in the amyloid protein and gene expressions. Conclusion knockout of APP(APBA2) gene represents a promising therapeutic strategy in Alzheimer's disease.

High frequency of multidrug-resistant (MDR) Klebsiella pneumoniae harboring several \u03b2-lactamase and integron genes collected from several hospitals in the north of Iran

BackgroundKlebsiella pneumoniae is one of the leading causes of hospital outbreaks worldwide. Also, antibiotic-resistant K. pneumoniae is progressively being involved in invasive infections with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the incidence of resistance genes (integron types and β-lactamase-encoded genes) among clinical isolates of K. pneumoniae.MethodsIn this cross-sectional study, a total of 100 clinical samples were obtained from hospitalized patients in three teaching hospitals in the north of Iran, from November 2018 and October 2019. Antimicrobial susceptibility testing was performed using disk agar diffusion test in line with CLSI recommendations. For colistin, minimum inhibitory concentration (MIC) was determined using broth microdilution. Based on antibiogram, multi-drug resistant (MDR) and extensive-drug resistant (XDR) strains were detected. Finally, integron types and β-lactamase resistance genes were identified using polymerase chain reaction technique.ResultsThe most and least clinical samples were related to the urine and bronchoalveolar lavage, respectively. Based on the antibiogram results, amikacin and gentamicin exhibited good activity against K. pneumoniae strains in vitro. The high resistance rate (93%) to ampicillin/sulbactam predicts the limited efficacy of this antibiotic, in the hospitals studied. Among all the 100 isolates, the frequency of MDR and XDR phenotypes were 58% and 13%, respectively, while no pan-drug resistant (PDR) strains were found. In the MDR K. pneumoniae strains, the prevalence of blaSHV, blaTEM, blaCTX-M-15, blaKPC, blaOXA-48, blaNDM β-lactamase genes were 91.4%, 82.7%, 79.3%, 29.3%, 36.2% and 6.9%, respectively, however 91.4% of the isolates were carrying intI gene. Class II and III integrons were not detected in any isolates.ConclusionThe MDR K. pneumoniae is becoming a serious problem in hospitals, with many strains developing resistance to most available antimicrobials. Our results indicate co-presence of a series of β-lactamase and integron types on the MDR strains recovered from hospitalized patients. The increasing rate of these isolates emphasizes the importance of choosing an appropriate antimicrobial regimen based on antibiotic susceptibility pattern.

Distribution of quinolone and macrolide resistance genes and their co-occurrence with heavy metal resistance genes in vegetable soils with long-term application of manure.

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) has become an increasingly serious global public health issue. This study investigated the distribution characteristics and influencing factors of ARB and ARGs in greenhouse vegetable soils with long-term application of manure. Five typical ARGs, four heavy metal resistance genes (MRGs), and two mobile genetic elements (MGEs) were quantified by real-time quantitative polymerase chain reaction (qPCR). The amount of ARB in manure-improved soil greatly exceeded that in control soil, and the bacterial resistance rate decreased significantly with increases in antibiotic concentrations. In addition, the resistance rate of ARB to enrofloxacin (ENR) was lower than that of tylosin (TYL). Real-time qPCR results showed that long-term application of manure enhanced the relative abundance of ARGs in vegetable soils, and the content and proportion of quinolone resistance genes were higher than those of macrolide resistance genes. Redundancy analysis (RDA) showed that qepA and qnrS significantly correlated with total and available amounts of Cu and Zn, highlighting that certain heavy metals can influence persistence of ARGs. Integrase gene intI1 correlated significantly with the relative abundance of qepA, qnrS, and ermF, suggesting that intI1 played an important role in the horizontal transfer of ARGs. Furthermore, there was a weakly but not significantly positive correlation between specific detected MRGs and ARGs and MGEs. The results of this study enhance understanding the potential for increasing ARGs in manure-applied soil, assessing ecological risk and reducing the spread of ARGs.

New Estimation of Antibiotic Resistance Genes in Sediment Along the Haihe River and Bohai Bay in China: A Comparison Between Single and Successive DNA Extraction Methods.

Sediment is thought to be a vital reservoir for antibiotic resistance genes (ARGs). Often, studies describing and comparing ARGs and their potential hosts in sediment are based on single DNA extractions. To date, however, no study has been conducted to assess the influence of DNA extraction efficiency on ARGs in sediment. To determine whether the abundance of ARGs is underestimated, we performed five successive extraction cycles with a widely used commercial kit in 10 sediment samples collected from the Haihe River and Bohai Bay. Our results showed that accumulated DNA yields after five extractions were 1.8–3.1 times higher than that by single DNA extractions. High-throughput sequencing showed that insufficient DNA extraction could generate PCR bias and skew community structure characterization in sediment. The relative abundances of some pathogenic bacteria, such as Enterobacteriales, Lactobacillales, and Streptomycetales, were significantly different between single and successive DNA extraction samples. In addition, real-time fluorescent quantitative PCR (qPCR) showed that ARGs, intI1, and 16S rRNA gene abundance strongly increased with increasing extraction cycles. Among the measured ARGs, sulfonamide resistance genes and multidrug resistance genes were dominant subtypes in the study region. Nevertheless, different subtypes of ARGs did not respond equally to the additional extraction cycles; some continued to have linear growth trends, and some tended to level off. Additionally, more correlations between ARGs and bacterial communities were observed in the successive DNA extraction samples than in the single DNA extraction samples. It is suggested that 3–4 additional extraction cycles are required in future studies when extracting DNA from sediment samples. Taken together, our results highlight that performing successive DNA extractions on sediment samples optimizes the extractable DNA yield and can lead to a better picture of the abundance of ARGs and their potential hosts in sediments.

Genotyping, drug resistance and virulence factors of Candida species isolated from patients using long-term inhaled steroids.

In this study, it was aimed to determine the isolation frequency and species distribution of Candida species isolated from asthmatic patients using long-term inhaled steroids. It was also aimed to determine the drug resistance patterns and the frequency of erg11, HWP1, ALS1, INT1, SAP1 PLB1 genes in isolates. Genotyping of Candida strains isolated from patients and healthy control group was performed by PCR-RFLP method. Drug resistance was investigated phenotypically, and the presence of erg11 resistance genes and HWP1, ALS1, INT1, SAP1 PLB1 virulence genes were investigated by PCR method. C albicans was the most isolated species in steroid-using patients and healthy control groups (patients: 44.2%; control group: 30.8%). C tropicalis and C glabrata were found to have the highest rates of non-albicans Candida in patients with 17.4% and 13.77%, respectively. Azole resistance was found to be significantly higher in isolates isolated from patients compared to the control group. Similarly, the presence of erg11 resistance gene was highest in C albicans (17.65%), C glabrata (12.5%) and C tropicalis (8.3%) strains in the control group, while C parapsilosis was highest in patients. (57.1%) and C glabrata (54.2%) strains. Compared to the control group, the virulence of Candida strains isolated from the patients was found to be higher. Presence of HWP1, ALS1, INT1, SAP1 and PLB1 genes in patients were determined as 72.1%, 63.9%, 68.9%, 57.38% and 54.5%, respectively. These rates were 29.4%, 35.3%, 25.5%, 17.7% and 23.5% in the healthy control group, respectively. In asthma patients using long-term inhaled steroids, both Isolation rates of Candida species, drug resistance rates, presence of virulence genes were found to be significantly higher in patients than in the control group. We think that this may be due to the suppression of cellular immunity by long-term steroid use.

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.

Metagenomic strategies identify diverse integron-integrase and antibiotic resistance genes in the Antarctic environment.

The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high‐throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non‐Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside‐modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D β‐lactamase (blaOXA‐205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase (aadA6), and the other is a qacG2 gene.

Elevated levels of antibiotic resistance in groundwater during treated wastewater irrigation associated with infiltration and accumulation of antibiotic residues

Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

Persisting antibiotic resistance gene pollution and its association with human sewage sources in tropical marine beach waters.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are pollutants of worldwide concern that threaten human health and ecosystems. Anthropogenic activities and wastewater could be ARB and ARG pollution sources; however, research on ARG abundance and microbial source tracking (MST) of contamination in tropical marine waters is limited. This study examined spatiotemporal variations of six ARGs (blaNDM, blaTEM, blaVIM, mcr-1, sul1, and tetQ) against the widely used antibiotic groups and a class 1 integron-integrase gene (intI1) at two Thai tropical recreational beaches (n=41). Correlations between ARGs and sewage-specific MST markers (i.e., crAssphage and human polyomaviruses [HPyVs]) and fecal indicator bacteria (i.e., total coliforms, fecal coliforms, and enterococci) were also investigated. BlaTEM, intI1, sul1, and tetQ were ubiquitous at both beaches (85.4-100% detection rate); intI1 was the most abundant (3-6 orders in log10 copies/100mL), followed by blaTEM (2-4 orders), sul1 (2-3 orders), and tetQ (2-4 orders). BlaNDM was found in 7.3% (up to 4 orders), and no mcr-1 was detected. Interestingly, blaVIM was prevalent at one beach (2-5 orders; n=17), but found in only one sample at the other (4 orders). Temporal, but not spatial, differences were noticed; blaTEM was at higher levels in the wet season. IntI1 correlated with sul1 and tetQ (Spearman's rho=0.47-0.97), suggesting potential horizontal gene transfer. CrAssphage, but not HPyVs, correlated with intI1, sul1, and tetQ (Spearman's rho=0.50-0.74). Higher numbers of ARGs tended to co-occur in samples with higher crAssphage concentrations, implying sewage contribution to the marine water, with a persisting ARG background. This study provides insight into the ARG pollution status of tropical coastal waters and suggests crAssphage as a proxy for ARG pollution, which could facilitate effective management policies to minimize ARG dissemination in marine environments.

Assessing Class 1 Integron Presence in Poultry Litter Amended with Wood Biochar and Wood Vinegar.

Class 1 integrons are mobile genetic elements that facilitate the spread of antibiotic resistance genes among bacteria. The use of prophylactic antibiotics has resulted in the rise of antibiotic resistance genes accumulating in a wide range of settings, including poultry houses and the agricultural fields where poultry litter is applied as a fertilizer. Biochar and wood vinegar are forest products wastes that have generated increasing attention as additives to agricultural soils. The objectives of this study were to observe the prevalence of class 1 integrons in poultry litter blended with biochar and wood vinegar over time and to verify a modified class 1 integron screening assay. Poultry litter blends were sampled and screened for class 1 integrons using polymerase chain reaction, and 80 products, 79 of which showed positive, were sent for DNA sequencing. The GenBank® BLAST database was used to verify the presence of the class 1 integron-integrase gene (intI1). There was no change in prevalence over time in poultry litter blends. Out of 79 PCR products that were intI1 positive, 78 showed at least 95% sequence identity to intI1 encoding bacteria and 64 showed at least 97% sequence identity. This indicates that this method was effective for conducting baseline surveillance of class 1 integrons in poultry litter and poultry litter-blended biochar and/or wood vinegar. Most significantly, class 1 integron prevalence did not decrease over time, further supporting the recalcitrance of these elements and the need for improved monitoring systems.

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.

The Occurrence and Characterization of Class I, II, and III Integrons Among Carbapenemase-Producing Clinical Strains of Acinetobacter baumannii in Tehran, Iran

Background: Acinetobacter baumannii has emerged as a critical pathogen with high morbidity and mortality in long-term hospitalized patients who stay in intensive care units. Carbapenemases and integrons are two critical DNA elements that contribute to the emergence of multidrug-resistant (MDR) A. baumannii. Objectives: The current study aimed at characterization and molecular detection of class 1, 2, and 3 integrons among carbapenemase-producing A. baumannii strains recovered from a clinical setting in Tehran, Iran. Methods: A total of 65 non-replicated clinical strains were considered in this study. Class 1, 2, and 3 carbapenemase genes and clonal relatedness of the isolates were investigated by PCR assay. Results: The prevalence of carbapenemases was as follows: blaOXA23 (92.31%), blaVIM (69.23%), and blaNDM (1.54%). In addition, PCR sequencing confirmed the presence of gene cassette arrays consisting of aacA4-catB8-aadA1 (12/46, 26.09%), aadB-aadA1 (26.09%, 12/46), arr2-cm1A5 (30.43%, 14/46), and dfrA1-aadA1 (7.39%, 8/46) in class 1 integron and dfrA1-sat2 (52.94%, 9/17), and sat2-aadA1 (47.06%, 8/17) in class 2 integron. Sequence-based typing of both blaOXA-51-like and ampC revealed the following distribution of three different clone types among isolates: clonal complex (CC) 10 (46.15%, 30/65), CC2 (40%, 26/65), and CC3 (13.85%, 9/65). Statistical analysis showed that the presence of the intI1, blaOXA23, blaVIM, or blaNDM genes can significantly increase the acquiring MDR phenotypes in A. baumannii isolates. Conclusions: High prevalence of carbapenemase-producing A. baumannii harboring integrons is alarming public health. It seems that class 1 integron can be served as a predictive biomarker for the presence of MDR phenotypes in the clinical setting. However, integrons do not carry carbapenemases in these strains.

Nanoscale zero-valent iron inhibits the horizontal gene transfer of antibiotic resistance genes in chicken manure compost

Livestock manure has been identified as a significant hotspot for antibiotic resistance genes (ARGs). However, the impact of nanoscale zero-valent iron (nZVI) on the fate of ARGs during livestock manure composting remains poorly understood. Here, we investigated the evolution of ARGs in chicken manure compost exposed to 100 and 600 mg kg−1 nZVI. The results showed that nZVI addition reduced the concentration of some antibiotics such as doxycycline and sulfamethoxazole. Furthermore, nZVI addition decreased the abundances of most ARGs at the end of composting, but nZVI dosage did not have any significant effect. The abundances of the dominant ARGs (sul1 and sul2) were significantly correlated to the class 1 integron-integrase gene (intI1). A network analysis revealed that the genera Bacteroides, Bacillus, Corynebacterium, Thiopseudomonas and Pseudomonas were the main potential hosts for multiple ARGs, and the decreased abundance of these bacteria contributed to the removal of ARGs. Structural equation models demonstrated that the reduction in intI1 played a predominant role in ARG removal. The nZVI also had direct effects on the intI1 abundance. These findings suggest that the addition of nZVI is a promising strategy to minimize ARG release in chicken manure compost.

Detection of carbapenemase-producing, hypervirulent Klebsiella spp. in wastewater and their potential transmission to river water and WWTP employees

Wastewater treatment plants (WWTPs) release drug-resistant microorganisms to water bodies (with effluents), and WWTP employees are exposed to bioaerosol emissions from the processed wastewater. Bacteria of the genus Klebsiella, in particular carbapenemase-producing (CP), hyper-virulent (Hvr) strains of Klebsiella pneumoniae, play a special role in this process. Klebsiella spp. strains isolated from wastewater, river water and the upper respiratory tract of WWTP employees were analyzed in this study. The isolated strains were identified as K. pneumoniae (K. pn) or K. non-pneumoniae (K. npn). The prevalence of nine types of genes encoding resistance to beta-lactams, nine genes encoding virulence factors and K1/K2 capsular serotypes, three genes encoding multi drug effluent pump systems, and the class 1 integron-integrase gene was determined by PCR. A total of 284 Klebsiella spp. isolates were obtained in the study: 270 environmental strains and 14 strains from the upper respiratory tract. Among environmental isolates 90.7% (245/270) harbored beta-lactam resistance genes, 17.4% (47/270) were classified as CP strains, 11.1% (30/270) were classified as Hvr strains, and 1.9% (5/270) were classified as CP-Hvr strains. CP-Hvr strains were also isolated from WWTP employees. Genes encoding β-lactamases (including carbapenemases), complete efflux pump systems and the K1 serotype were identified more frequently in K. pn strains. In turn, K. npn strains were characterized by a higher prevalence of blaSHV and intI1 genes and K2 serotype gene. The strains isolated from wastewater and river water also differed in the abundance of drug resistance and virulence genes. The results of the study indicate that CP-Hvr K. pn strains are possibly transmitted from wastewater via bioareosol to the upper respiratory tract of WWTP employees. blaGES-type carbapenemases significantly contributed to the spread of drug resistance in the environment.

Microplastics deteriorate the removal efficiency of antibiotic resistance genes during aerobic sludge digestion

Sludge from wastewater treatment plants (WWTPs) is considered to be reservoirs of antibiotic resistance genes (ARGs), which can be efficiently removed by sludge treatment processes, e.g., aerobic sludge digestion. However, recent studies report microplastics, which also accumulate in sludge, may serve as carriers for ARGs. In the presence of microplastics, whether ARGs can still be efficiently destroyed by aerobic sludge digestion remains to be urgently investigated. In this study, the fate of ARGs during aerobic digestion was investigated with and without the addition of three prevalent categories of (i.e., polyvinyl chloride (PVC), polyethylene (PE), and polyethylene terephthalate (PET)). Nine ARGs and class 1 integron-integrase gene (intI1) that represents the horizontal transfer potential of ARGs were tested in this study. Compared with the control, the ARGs removal efficiency decreased by 129.6%, 137.0%, and 227.6% with the presence of PVC, PE, and PET, respectively, although a negligible difference was observed with their solids reduction efficiencies. The abundance of potential bacterial hosts of ARGs and intI1 increased in the reactors with the addition of microplastics, suggesting that microplastics potentially selectively enriched bacterial hosts and promoted the horizontal transfer of ARGs during aerobic sludge digestion. These may have contributed to the deteriorated ARGs removal efficiency. This study demonstrated that microplastics in sludge would decrease the ARGs removal efficiency in aerobic digestion process, potentially leading to more ARGs entering the local environment during sludge disposal or utilization.