Int Gene Research Articles

Surfaces of gymnastic equipment as reservoirs of microbial pathogens with potential for transmission of bacterial infection and antimicrobial resistance.

Gymnastic equipment surfaces are shared by many people, and could mediate the transfer of bacterial pathogens. To better understand this detrimental potential, investigations on the reservoirs of bacterial pathogens and antimicrobial resistance on the surfaces of gymnastic equipment were performed by analyzing the bacterial community structures, prevalence of viable bacteria, and presence of antimicrobial resistance on both indoor and outdoor gymnastic facilities. The results of high-throughput 16S rDNA amplicon sequencing showed that Gram-positive bacteria on the surfaces of indoor gymnastic equipment significantly enriched, including the opportunistic pathogen Staphylococcus strains, while Enterobacteriaceae significantly enriched on surfaces of outdoor gymnastic equipment. The analysis of α-diversities showed a higher richness and diversity for bacterial communities on the surfaces of gymnastic equipment than the environment. Analysis of β-diversities showed that the bacterial communities on the surfaces of gymnastic equipment differ significantly from environmental bacterial communities, while the bacterial communities on indoor and outdoor equipment are also significantly different. Thirty-four bacterial isolates were obtained from the surfaces of gymnastic equipment, including three multidrug Staphylococcus and one multidrug resistant Pantoea. In particular, Staphylococcus hemolyticus 5-6, isolated from the dumbbell surface, is a multidrug resistant, hemolytic, high- risk pathogen. The results of quantitative PCR targeting antibiotic resistance related genes (intI1, sul1 and bla TEM) showed that the abundances of sul1 and bla TEM genes on the surfaces of gymnastic equipment are higher than the environment, while the abundances of sul1 gene on indoor equipment are higher than outdoor equipment. These results lead to the conclusion that the surfaces of gymnastic equipment are potential dissemination pathways for highly dangerous pathogens as well as antimicrobial resistance, and the risks of indoor equipment are higher than outdoor equipment.

Comparative study on genotypic properties of Pseudomonas aeruginosa isolated from subclinical mastitis in Türkiye

The results of antibacterial therapy in mastitis may be related to not only the antibiotic sensitivity of the etiological factors, but also to their abilities carrying integron and virulence genes. Integrons are mobile DNA element that can capture and carry genes, particularly those responsible for antibiotic resistance. Pseudomonas aeroginosa has numberless virulence factors which are contributed to bacterial invasion and toxicity. The aim of this study is to investigate antibiotic resistance, integron and virulence gene profiles of P. aeruginosa isolates obtained from cow milk with subclinical mastitis; further, was to evaluate the relationship between the antimicrobial resistance of bacteria with the of integron and virulence gene carrying. Material of the study is consisted with 32 (9.8%) of P. aeruginosa isolates obtained from 326 subclinical mastitic milk samples. After the bacterial identification was done by classical conventional methods, polymerase chain reaction (PCR) was used to verify the genus and species identifications of the isolates and to determine the integron and virulence gene profiles. Using disk diffusion method, antimicrobial resistance was determined to fifteen antibiotics against belonging to eleven antimicrobial families. The relationship the presence of integron and virulence genes associated with antibiotic resistance, further the relationship the presence of virulence genes and antimicrobial resistance was calculated with the Chi-Square (χ2) test. Ten of virulence genes (lasl, lasR, lasB, rhll, rhlR, rhlAB, plcH, plcN, ppyR, exoT) were found in all isolates whilst a virulence gene (aprA) was not present in any isolate. It was found that 34.4% of the isolates carried any integron gene. The results showed that the relationship is important between the presence of int genes and gentamicin, amikacin, tetracycline, cefoperazone, imipenem, aztreonam, ciprofloxacin, enrofloxacin resistance and exoU virulence gene presence. Also; there were also important associations between resistance to certain antibiotics and the presence of P. aeroginosa virulence genes: ciprofloxacin with lasA; netilmicin, cefoperazone and ciprofloxacin with pilB; netilmicin and ciprofloxacin with pelA; amikacin with algD, algU, algL; aztreonam with toxA; enrofloxacin with pvdA. All isolates obtained in this study showed multiple antibiotic resistance (MDR). In these cases, the presence of integron and some virulence genes could play a prominent role in the resistance of P. aeroginosa isolates to antimicrobial drugs. This study could be important in that it is the first study to show the presence of integrons and virulence genes in P. aeroginosa isolates originating from mastitic cow milk in Turkey. The presence of antibiotic-resistant P. aeroginosa strains in cattle farms may also pose a public health risk, as these bacteria can transmit their resistance genes to humans through food consumption.

Antimicrobial activity of cell free supernatants from probiotics inhibits against pathogenic bacteria isolated from fresh boar semen

The use of antibiotics with semen extender appears to be a practical solution to minimise bacterial growth in fresh boar semen preservation. Unfortunately, the excessive use of antibiotics promotes antimicrobial resistance (AMR). This becomes a worldwide concern due to the antimicrobial resistance genes transmitted to animals, environment, and humans. Probiotics are one of the alternative methods to reduce antibiotic use. They could inhibit pathogenic bacteria by producing antimicrobial substances in cell free supernatants (CFS). Nevertheless, there is no comprehensive study undertaken on inhibitory activity against pathogenic bacteria isolated from boar semen origin. Our study investigated the efficacy of CFS produced from selected probiotics: Bacillus spp., Enterococcus spp., Weissella spp., Lactobacillus spp., and Pediococcus spp. inhibiting pathogenic bacteria isolated from fresh boar semen. Besides, the semen-origin pathogenic bacteria are subjected to identification, antimicrobial resistance genes detection, and antibiotic susceptibility test (AST). Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis are the most common pathogens identified in boar semen with resistance to numerous antibiotics used in pig industry. The CFS with its antimicrobial peptides and/or bacteriocin constituent derived from selected probiotics could inhibit the growth of pathogenic bacteria carrying antimicrobial resistance genes (mcr-3 and int1 genes). The inhibition zones for Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis provided more efficient results in the CFS derived from Lactobacillus spp. and Pediococcus spp. than those of the CFS produced from Enterococcus spp., Weissella spp. and Bacillus spp., respectively. It is worth noted that as the incubation time increased, the antibacterial activity decreased conversely. Our results on CFS with its antimicrobial peptides and/or bacteriocin constituent inhibits semen-origin pathogenic bacteria guide the direction as a promising alternative method used in the semen extender preservation of the pig industry.

Microplastic biofilm, associated pathogen and antimicrobial resistance dynamics through a wastewater treatment process incorporating a constructed wetland

Microplastics in wastewater are colonized by biofilms containing pathogens and antimicrobial resistance (AMR) genes that can be exported into receiving water bodies. This study investigated establishment and changes in microplastic-associated biofilm and AMR during a conventional full-scale 2100 population equivalent wastewater treatment process combined with a free water surface polishing constructed wetland. Sequential microplastic colonization experiments were conducted at different stages of the wastewater treatment process, including in raw sewage, treated effluent and the constructed wetland. Two scenarios were tested in which the constructed wetland served as either (i) a polishing step or (ii) as primary recipient of sewage inoculated microplastics. Bacterial 16S rRNA gene sequencing was carried out for qualitative bacterial community analysis. qPCR was applied for quantitative analysis of AMR genes (sul1, ermB, tetW, intiI1), bacterial biomass (16S rRNA) and a human fecal marker (HF183). Microbial diversity on microplastics increased with incubation time. The initial sewage-derived biofilm composition changed more significantly in the wastewater effluent compared to the constructed wetland. Pathogen and AMR load decreased by up to two orders of magnitude after coupled conventional and constructed wetland treatment, while less impact was observed when sewage-inoculated microplastic material was directly transferred into the constructed wetland. Aeromonas, Klebsiella, and Streptococcus were key pathogenic genera correlated with AMR in microplastic-associated biofilms. Despite decreasing trends on human pathogens and AMR load along the treatment process, microplastic-associated biofilms were a considerable potential hotspot for AMR (intI1 gene) and accommodated Cyanobacteria and fish pathogens.

Distribution and dynamics of antibiotic resistance genes in a three-dimensional multifunctional biofilm during greywater treatment

Antibiotic resistance genes (ARGs) have been identified as serious threats to public health. Despite the widespread in various systems, dynamics of ARGs in three-dimensional multifunctional biofilm (3D-MFB) treating greywater are largely undefined. This work tracked the distributions and dynamics of eight target genes (intI1, korB, sul1, sul2, tetM, ermB, blaCTX-M and qnrS) in a 3D-MFB during greywater treatment. Results showed that hydraulic retention times at 9.0 h achieved the highest linear alkylbenzene sulfonate (LAS) and total nitrogen removal rates at 99.4% and 79.6%, respectively. ARGs presented significant liquid-solid distribution feature, but non-significant with biofilm position. Intracellular ARGs (predominant by intI1, korB, sul1 and sul2) at bottom biofilm were 210- to 4.2 × 104- fold higher than that in cell-free liquid. Extracellular polymeric substances (EPS)-attached LAS showed linear relationship with most of ARGs (R2 > 0.90, P < 0.05). Sphingobacteriales, Chlamydiales, Microthrixaceae, SB-1, Cryomorphaceae, Chitinophagaceae, Leadbetterella and Niabella were tightly bound up with target ARGs. Key is that EPS-attached LAS considerably determines the occurrence of ARGs, and microbial taxa play an important role in the dissemination of ARGs in the 3D-MFB.

Quantification of the mobility potential of antibiotic resistance genes through multiplexed ddPCR linkage analysis.

There is a clear need for global monitoring initiatives to evaluate the risks of antibiotic resistance genes (ARGs) towards human health. Therefore, not only ARG abundances within a given environment, but also their potential mobility, hence their ability to spread to human pathogenic bacteria needs to be quantified. We developed a novel, sequencing-independent method for assessing the linkage of an ARG to a mobile genetic element by statistical analysis of multiplexed droplet digital PCR (ddPCR) carried out on environmental DNA sheared into defined, short fragments. This allows quantifying the physical linkage between specific ARGs and mobile genetic elements, here demonstrated for the sulfonamide ARG sul1 and the Class 1 integron integrase gene intI1. The method's efficiency is demonstrated using mixtures of model DNA fragments with either linked and unlinked target genes: Linkage of the two target genes can be accurately quantified based on high correlation coefficients between observed and expected values (R2) as well as low mean absolute errors (MAE) for both target genes, sul1 (R2 = 0.9997, MAE = 0.71%, n = 24) and intI1 (R2 = 0.9991, MAE = 1.14%, n = 24). Furthermore, we demonstrate that adjusting the fragmentation length of DNA during shearing allows controlling rates of false positives and false negative detection of linkage. The presented method allows rapidly obtaining reliable results in a labor- and cost-efficient manner.

Dynamic Changes in Antibiotic Resistance Genes During Biological Fermentation of Chicken Manure and Pig Manure

Livestock manure is a major carrier that stores and transmits antibiotic resistance genes (ARGs). The aim of this study was to identify the crucial environmental factors that result in the change in relative abundance of ARGs and MGEs to explore effective methods to reduce the occurrence of ARGs. The abundance of ten ARGs and five MGEs were measured using real-time quantitative PCR with 0, 3, 7, 10, 14, 21, 28, 45, 60, and 75 d in CM and PM aerobic composting. The succession law of the bacterial community was analyzed using 16S rRNA high-throughput sequencing. Furthermore, the abundance of ARGs and MGEs was correlated with the properties (temperature, moisture content, pH, and DOC) of composting piles to identify the crucial factors affecting the variation in ARGs and MGEs. The results showed that the abundance of ARGs and MGEs in PM was higher than that in CM. Composting significantly reduced the relative abundance of nine ARGs and five MGEs. The removal ratios of three ARGs (tetM, tetT, and aacA) and four MGEs (ISEcp1, IS1216, IS613, and tnp614) in CM reached 99%. The removal ratios of nine ARGs[tetB(P), tetL, tetM, tetO, tetT, aacA, aadD,aphA3, and sat4] and four MGEs (ISEcp1, IS26, IS1216, and tnp614) reached 99% in PM. However, the abundance of the genes tetG, intI1, and IS6100 increased. Firmicutes and Actinobacteria were dominant in CM and PM during composting. Actinobacteria and Proteobacteria may have been the major host bacteria of tetG and intI1. The redundancy analysis showed that moisture and pH were the crucial factors that could influence the differences in the abundance of ARGs and MGEs. The composition and abundance of ARGs were driven by physicochemical properties, bacterial community, and MGEs. Composting could significantly reduce the abundance of ARGs in CM and PM, thus reducing the risk of ARGs being distributed via livestock manure application. However, some ARGs and MGEs remained in high abundance. Therefore, it is necessary to further optimize the composting process, improve the harmless treatment effect of livestock manure, and promote the safe agricultural use of organic fertilizers.

Insight into the spatiotemporal distribution of antibiotic resistance genes in estuarine sediments during long-term ecological restoration

In this study, we aimed to investigate the long-term spatiotemporal changes in hydrodynamics, antibiotics, nine typical subtypes of antibiotic resistance genes (ARGs), class 1 integron gene (intI1), and microbial communities in the sediments of a semi-enclosed estuary during ecological restoration with four treatment stages (influent (#1), effluent of the biological treatment area (#2), oxic area (#3), and plant treatment area (#4)). Ecological restoration of the estuary reduced common pollutants (nitrogen and phosphorus) in the water, whereas variations in ARGs showed noticeable seasonal and spatial features. The absolute abundance of ARGs at sampling site #2 considerably increased in autumn and winter, while it significantly increased at sampling site #3 in spring and summer. The strong intervention of biological treatment (from #1 to #2) and aerators (from #2 to #3) in the estuary substantially affected the distribution of ARGs and dominant antibiotic-resistant bacteria (ARB). The dominant ARB (Thiobacillus) in estuarine sediments may have low abundance but important dissemination roles. Meanwhile, redundancy and network analysis revealed that the microbial communities and intl1 were key factors related to ARG dissemination, which was affected by spatial and seasonal ecological restoration. A positive correlation between low flow velocity and certain ARGs (tetM, tetW, tetA, sul2, and ermC) was observed, implying that flow optimization should also be considered in future ecological restoration to remediate ARGs. Furthermore, the absolute abundance of ARGs can be utilized as an index to evaluate the removal capacity of ARGs by estuarine restoration.

Detection of Morganella morganii bound to a plastic substrate in surface water

ObjectivesAround the globe, escalation in rare opportunistic microbial infections is alarming as they are heading steadily towards 'superbug' status. In aquatic ecosystems, plastic fosters multidrug-resistant pathogenic bacteria and plays a significant role in trafficking antibiotic-resistant genes. In this study, we focused on a multidrug-resistant bacterial strain isolated from microbial communities found on plastic substrates of a volcanic lake in central Italy. MethodsExtended-spectrum beta-lactamase-producing strains were isolated from both raw water and plastic substrates for a comparative investigation using microbiological and molecular methods, and antibiotic susceptibility profiling was performed against a panel of ten antibiotics. ResultsMolecular identification and Basic Local Alignment Search Tool analysis confirmed an almost identical sequencing pattern of two isolated strains and their homology with Morganella morganii. Antibiotic susceptibility tests revealed their resistance to almost all tested antibiotics. Class 1 integron-associated gene (intI1) and seven antibiotic resistance genes were detected in both strains, confirming their superbug status. ConclusionTo our knowledge, this is the first study on the characterization of extended-spectrum beta-lactamase-producing M. morganii isolated from the biofilm of plastic substrates, depicting the potential toxicity of plastic in harbouring and dispersing virulent, multidrug-resistant, opportunistic human pathogens.

Microbial source tracking and land use associations for antibiotic resistance genes in private wells influenced by human and livestock fecal sources.

Antimicrobial resistance is a growing public health problem that requires an integrated approach among human, agricultural, and environmental sectors. However, few studies address all three components simultaneously. We investigated the occurrence of five antibiotic resistance genes (ARGs) and the class 1 integron gene (intI1) in private wells drawing water from a vulnerable aquifer influenced by residential septic systems and land-applied dairy manure. Samples (n=138) were collected across four seasons from a randomized sample of private wells in Kewaunee County, Wisconsin. Measurements of ARGs and intI1 were related to microbial source tracking (MST) markers specific to human and bovine feces; they were also related to 54 risk factors for contamination representing land use, rainfall, hydrogeology, and well construction. ARGs and intI1 occurred in 5%-40% of samples depending on target. Detection frequencies for ARGs and intI1 were lowest in the absence of human and bovine MST markers (1%-30%), highest when co-occurring with human and bovine markers together (11%-78%), and intermediate when co-occurring with just one type of MST marker (4%-46%). Gene targets were associated with septic system density more often than agricultural land, potentially because of the variable presence of manure on the landscape. Determining ARG prevalence in a rural setting with mixed land use allowed an assessment of the relative contribution of human and bovine fecal sources. Because fecal sources co-occurred with ARGs at similar rates, interventions intended to reduce ARG occurrence may be most effective if both sources are considered.

Chromate Removal by Enterobacter cloacae Strain UT25 from Tannery Effluent and Its Potential Role in Cr (VI) Remediation.

An indigenous chromate-resistant bacterial strain isolated from tannery effluent was identified based on morphological, biochemical, and 16S rRNA gene sequencing, as Enterobacter cloacae UT25. It was found to resist heavy metal ions such as Cr (VI), Pb (II), Cu (II), Co (II), Ni (II), Hg (II), and Zn (II) and antibiotics. The strain was able to remove 89 and 86% chromate, after 24h of incubation in a Luria-Bertani (LB) medium at an initial Cr (VI) concentration of 1000 and 1500µg/ml, respectively. Minimum inhibitory concentration (MIC) was observed for chromate to be 80,000 and 1850µg/ml, after 48h of incubation in LB and acetate minimal media (AMM), respectively. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) analysis showed discrete cells with intact and smooth cell walls and homogenous cytoplasm in the absence of metal stress, whereas chromate stress caused cell lysis and reduction in size, which was a characteristic response to Cr (VI) toxicity. Energy Dispersive X-Ray Spectroscopy (EDX) confirmed the adsorption of oxyanions to the cell wall which was one of the Cr (VI) removal mechanisms by the bacterium. Atomic Force Microscopy (AFM) micrographs of chromate-untreated and treated cells revealed Root Mean Square roughness (Rq) values of 16.25 and 11.26nm, respectively, indicating less roughness in the presence of stress. The partial gene sequence of class 1 integrons (intI1) of strain UT25 showed 94% homology with intI1 gene of strain Enterobacter hormaechei strain ECC59 plasmid pECC59-1. The present analysis highlighted the potential of E. cloacae UT25 as a promissory bacterium that could be applied in removing chromate from polluted environments.

The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2. Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ). Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication. We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics.

Fate of Horizontal-Gene-Transfer Markers and Beta-Lactamase Genes during Thermophilic Composting of Human Excreta

Thermophilic composting is a suitable treatment for the recycling of organic wastes for agriculture. However, using human excreta as feedstock for composting raises concerns about antibiotic resistances. We analyzed samples from the start and end of a thermophilic composting trial of human excreta, together with green cuttings and straw, with and without biochar. Beta-lactamase genes blaCTX-M, blaIMP, and blaTEM conferring resistance to broad-spectrum beta-lactam antibiotics, as well as horizontal gene transfer marker genes, intI1 and korB, were quantified using qPCR. We found low concentrations of the beta-lactamase genes in all samples, with non-significant mean decreases in blaCTX-M and blaTEM copy numbers and a mean increase in blaIMP copy numbers. The decrease in both intI1 and korB genes from start to end of composting indicated that thermophilic composting can decrease the horizontal spread of resistance genes. Thus, thermophilic composting can be a suitable treatment for the recycling of human excreta.

Impact of biochar amendment on antibiotic removal and ARGs accumulation in constructed wetlands for low C/N wastewater treatment

Constructed wetlands (CWs) amended with biochar to stimulate antibiotic and antibiotic resistance genes (ARGs) removal from carbon restricts wastewater holds particular application prospect due to high efficiency and good stability. In this study, we conducted biochar amended constructed wetland (BCW) and non-biochar amended constructed wetland (NCW) to explore the feasibility and mechanisms for sulfamethoxazole (SMX) and ARGs removal from low C/N wastewater. Results showed that biochar-amended considerably stimulated the biological oxidation and reduction process in CW, as evidenced by higher chemical oxygen demand (COD), nitrogen, and SMX removal performance (7.1 ± 2.6 %, 19.8 ± 3.6 %, 36.8 ± 6.2 %) than that in NCW. The accumulation of ARGs was negatively associated with SMX removal efficiency, where the int and sul genes performed lower concentration in BCW due to the higher SMX removal efficiency. The biochar’s dissolved organic carbon release in CWs indicated that water and alkaline media portray the optimum conditions for SMX and ARGs removal. Microbial community analysis exposed that more functional consortia capable of SMX metabolism (e.g., Arthrobacter, Ramlibacter, Flavobacterium, and norank_f_JG30-KF-CM45) were enriched in BCW. This study demonstrated the feasibility and mechanism of biochar-amended CWs for regulated SMX removal and ARGs accumulation, which provides new insights for enhancing the depuration ability of CWs.

Transcriptome analysis reveals adaptation mechanism of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) adults to benzoquinone stress

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is a cosmopolitan pest of stored grain. The secretions of the stink gland of T. castaneum contain benzoquinone, which acts as immune defense substances. However, high concentrations of secretions can be harmful to the individuals. The effects of benzoquinone produced by T. castaneum and the addition of benzoquinone to its living environment on T. castaneum were investigated, and the transcriptome of T. castaneum adults was analyzed to explore molecular mechanism of environmental adaptation of T. castaneum under benzoquinone stress by using Illumina sequencing technology. A total of 5361, 6923, and 83 differentially expressed genes were respectively identified in T. castaneum adults in different treated wheat flour (adults in T. castaneum infested wheat flour (referred to as T2) vs Control (adults in clear wheat flour, referred to as T1), adults in wheat flour with benzoquinone (referred to as T3) vs Control (T1), and T3 vs T2). We identified 2562 commonly up-regulated genes and 1931 commonly down-regulated genes in T2 vs T1 and T3 vs T1 treatments. Weighted gene co-expression network analysis (WGCNA) identified 6 modules composing of differentially co-expressed genes. The Turquoise module was extremely associated with the T2 sample. Turquoise module of genes was found to be highly correlated with the oxidation-reduction process (168 genes), macromolecular complex (154 genes), and oxidoreductase activity (169 genes). Three transcription factor genes, LOC664577, LOC661810, and LOC657046 were presented in Turquoise module as hub genes. The changes in gene expression indicated that T. castaneum may transfer toxic compounds into low-toxicity or non-toxic products through a variety of enzymes in response to benzoquinone stress. The current results provide the molecular mechanisms that may explain the adaptation of T. castaneum to benzoquinone stress.

High occurrence of antibiotic resistance genes in intensive aquaculture of hybrid snakehead fish

The increasing abundance of antibiotic resistance genes (ARGs), which are regarded as new pollutants, has raised public health concern. The use of antibiotics in aquaculture has promoted the evolution and spread of ARGs. The occurrence and abundance of ARGs in aquaculture has attracted extensive attention. However, the distribution and transmission of ARGs in aquaculture require further study. This study analyzed water and sediment from intensive culture of hybrid snakehead fish farm in Zhongshan, South China. Twenty-two types of ARGs were detected in all environmental samples. The relative abundance of sulfonamide resistance genes (sul1 and sul2) was the highest, ranging from 3.37×10-2 to 8.53×100 copies/16S rRNA gene. High occurrence of quinolones, phenicols, tetracycline resistance genes, and class 1 integrase gene (intI1) was also observed in pond water samples. This implies that pond water is one of the main reservoirs and origins of ARGs in the aquatic environment. Proteobacteria was the most abundant phylum in all the environmental samples, and its relative abundance ranged from 24.05% to 41.84%. Network and canonical correspondence analyses showed that a high abundance of ARGs (int1, sul1, sul2, qacEΔ1, aac6, and oqxA) was positively correlated with Proteobacteria, Cyanobacteria, and Bacteroidetes, and the abundance of Proteobacteria, Actinobacteria, and Patescibacteria was positively correlated with environmental factors (sulfide, nitrite nitrogen, pH, free chlorine, and ammonia nitrogen). These findings demonstrate the prevalence and persistence of ARGs in intensive fish farming in southern China. This suggests that ARG levels and microbiological community composition in aquaculture should be conventionally determined to assess potential risks to public health.

A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum β-Lactamases, Plasmid-Mediated AmpC β-Lactamases, and Integrons in Gram-Negative Bacteria.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, aac(6')-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

Investigation of antimicrobial susceptibility, class I, II, and III integrons among clinical isolates of Pseudomonas aeruginosa from hospitalized burn patients in Southern Iran

Abstract Introduction Burn infection due to Pseudomonas aeruginosa poses a considerable challenge in terms of graft loss, systemic sepsis, prolonged hospital stays, and even enhanced mortality. The aim of this study was to consider the susceptibility pattern to antibiotics and occurrence of class I, II, and III integrons in P. aeruginosa isolated from hospitalized burn patients. Materials and Methods A cross-sectional study was performed on 70 P. aeruginosa isolates obtained from blood, wounds, biopsy, and sputum samples from burn patients between January 2020 and March 2021 from different wards of a teaching hospital in Ahvaz, Iran. Results Antimicrobial susceptibility pattern was performed using the disk diffusion method. Also, PCR was carried out for the detection of intI1, intI2 and intI3 genes. Among the 70 P. aeruginosa isolates, 34 (48.6%) isolates were obtained from females and 36 (51.4%) isolates from males. PCR amplification revealed that 51.4% isolates (36/70) had intI1, 30% isolates (21/70) had intI2, and 12.9% isolates (9/70) had both genes. However, the intI3 gene was not detected in any isolates. Furthermore, there is no important association between resistance to antibiotics and the presence of intI genes. Conclusion According to our results, there were no statistically significant differences among resistance to antibiotics and the presence of intI and intI1 genes. However, the surveillance of patterns of antibiotic resistance and investigation into the role of integrons in controlling the scattering of antibiotic-resistant genes are recommended.

Preliminary Study on the Treatment Efficiency of Pasteurized Lime Thermal Alkaline Hydrolysis for Excess Activated Sludge and Reduction of Tetracycline Resistance Genes

Thermal alkaline hydrolysis is a common pretreatment method for the utilization of excess activated sludge (EAS). Owing to strict environment laws and need for better energy utilization, new methods were developed in this study to improve the efficiency of pretreatment method. Direct thermal hydrolysis (TH), pasteurized thermal hydrolysis (PTH), and alkaline pasteurized thermal hydrolysis (PTH + CaO and PTH + NaOH) methods were used to treat EAS. Each method was compared and analyzed in terms of dissolution in ammonium nitrogen (NH4 + -N) and soluble COD (SCOD) in EAS. Furthermore, the removal of tetracycline resistance genes (TRGs) and class 1 transposon gene intI1 from EAS was investigated. The NH4 + -N and SCOD concentrations in EAS treated by PTH were 1.24 and 2.58 times higher than those of TH. However, the removal efficiency of total TRGs and intI1 between the groups was comparable. The SCOD concentration of the PTH + NaOH group was 4.37 times higher than that of the PTH group, and the removal efficiency of total TRGs was increased by 9.52% compared with that by PTH. The NH4 + -N and SCOD concentrations of the PTH + CaO group could reach 85.04% and 92.14% of the PTH + NaOH group, but the removal efficiency of total TRGs by PTH + CaO was 19.78% lower than that by PTH + NaOH. Thus, to reduce the financial cost in actual operation, lime (CaO) can be used instead of a strong alkali (NaOH), and pasteurized steam at 70°C instead of conventional high-temperature heating to treat EAS. This study provides a reference for the development of alkaline hydrolysis under moderate temperatures along with the removal of TRGs in EAS.

Comparative genomics of trimethoprim-sulfamethoxazole-resistant Achromobacter xylosoxidans clinical isolates from Serbia reveals shortened variant of class 1 integron integrase gene.

Trimethoprim-sulfamethoxazole (SXT) is the preferable treatment option of the infections caused by Achromobacter spp. Our study aimed to analyze the SXT resistance of 98 Achromobacter spp. isolates from pediatric patients, among which 33 isolates were SXT-resistant. The presence of intI1 was screened by PCR and genome sequence analyses. The intI1 gene was detected in 10 of SXT-resistant isolates that had shorter intI1 PCR fragments named intI1S. Structural changes in intI1S were confirmed by genome sequencing and analyses which revealed 86 amino acids deletion in IntI1S protein compared to canonical IntI1 protein. All IntI1S isolates were of non-CF origin. Pan-genome analysis of intI1S bearing A. xylosoxidans isolates comprised 9052 genes, with the core genome consisting of 5455 protein-coding genes. Results in this study indicate that IntI1S isolates were derived from clinical settings and that cystic fibrosis (CF) patients were potential reservoirs for healthcare-associated infections that occurred in non-CF patients.