Int Gene Research Articles

Prevalence of antibiotic resistance genes in drinking water of the Kathmandu Valley, Nepal

In resource-limited settings, fecal indicator bacterial analysis is the only microbiological water quality test performed, and emerging contaminants, such as antibiotic resistance genes (ARGs), are often neglected. To address this knowledge gap, water samples were collected from shallow wells (n = 24), deep wells (n = 16), stone spouts (n = 14), springs (n = 8), tanker filling stations (TFS; n = 12), water tankers (n = 12), and drinking water treatment plants (DWTPs; n = 6 each of raw and treated water) of the Kathmandu Valley, Nepal, between December 2015 and August 2016 to investigate the presence of five ARGs (blaNDM-1, blaCTX−M, tetB, qnrS, and sul1) and class one integrase gene (intI1). All ARGs were detected in water samples from shallow wells and stone spouts, whereas blaNDM-1 and blaCTX−M were not detected in deep wells and springs. tetB and intI1 were detected at a greater number of water tanker samples than TFS, indicating that water tankers were not disinfected regularly. Only sul1 and intI1 were detected in treated water of DWTPs. Persistence of intI1 throughout the water treatment process and significantly strong correlation (ρ > 0.5, p < 0.05) with the majority of ARGs included in our study suggested the suitability of intI1 to assess the contamination and fate of ARGs in drinking water of the valley. Low free chlorine levels in treated water before pipeline distribution urge water quality managers to evaluate tap water for pathogens and ARGs. Further studies are recommended on emerging contaminants, such as antibiotics, in drinking water sources that are not routinely assessed but can provide selective pressure for the spread of antibiotic resistance in the environment.

The overlap of soil and vegetable microbes drives the transfer of antibiotic resistance genes from manure-amended soil to vegetables

Livestock manure, as a major source of antibiotic resistance genes (ARGs), could further transfer ARGs from soil to vegetables when it's used as fertilizer in field and then pose threat to human health. Meanwhile, manure inputs and vegetable planting also affect soil bacterial communities, but these effects on the transmission of ARGs from soil to vegetable is still lacking. Here, lettuce and endive were cultivated in manure-amended soils using pot experiment. The distribution of bacterial community, ARGs and intI1 gene were studied in manure-amended soil and vegetable roots and leaves at harvest. High-throughput sequencing analysis demonstrated that planting vegetables exerted significant effect on soil bacterial communities, which partly explained the decrease of certain ARGs and the intI1 gene in planted soil than in control soil. ARGs in vegetable and soil were interconnected. The bacterial community compositions among root endophyte, leaf endophyte, and phyllosphere were varied by Hierarchical clustering analysis. Higher abundance of shared bacterial taxa was found between root endophytes and soil microbes, which could lead to a relative higher detection frequency of ARGs in root endophyte. Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes were dominant in the plant endophyte and phyllosphere microbes and had intensive correlations with ARGs. Taken together, our findings provided valuable insights into the role of bacterial community structure in the dissemination of ARGs from manure-amended soil to vegetables.

Abundance of integrons in halophilic bacteria.

Integrons are genetic platforms used for expressing open reading frames (ORFs) arranged in gene cassettes. Excision and integration of gene cassettes are controlled by their associated integron integrase (IntI). Using IntegronFinder software, we analyzed all complete halophilic genomes available in the HaloDom database, along with selected partial halophilic genomes. We identified 18 new complete bacterial integrons and 46 clusters of attC sites lacking a neighboring integron integrase (CALINs). Different classes of insertion sequences (ISs) were also identified within and near integrons and CALINs, with an abundance of IS1182 elements and different ISs that can presumably mobilize adjacent genomic structures. Different promoters of intI genes (PintI) showed nearby binding sites for arginine repressor (ArgR), raising the possibility that IntI expression and recombination activity are regulated by these proteins. Our findings revealed the existence of new integrons in halophilic bacteria with possible adaptive roles.

Prevalence of trimethoprim/sulfamethoxazole resistance genes among Stenotrophomonas maltophilia clinical isolates in Egypt.

Stenotrophomonas maltophilia is an important multidrug resistant nosocomial pathogen. Trimethoprim/sulfamethoxazole (TMP/SMX) is considered the drug of choice for treatment of S. maltophilia infections, thus emerging resistance to TMP/SMX poses a serious threat. In the present study we aimed to investigate the frequency of TMP/SMX resistance genes (sul1, sul2, dfrA), and to evaluate their relatedness with integron 1 (int1), and insertion sequence common regions (ISCR) among 100 S. maltophilia from different clinical isolates in Egypt. Isolates were identified biochemically and confirmed by VITEK2. Detection of sul1, sul2, and dfrA genes, int1 and ISCR elements was performed by PCR. Among the 16 TMP/SMX resistant isolates, sul1 gene was detected in all of them, and it was associated with int1 gene presence in all resistant isolates. The sul2 gene was detected in 6 out of 16 resistant isolates (37.5%), and only 2 of the 16 resistant isolates (12.5%) harboured dfrA gene. ISCR was detected in 10 of the resistant isolates (62.5%) and in 4 of them it was associated with the presence of sul2 gene. Among the 84 TMP/SMX sensitive isolates, sul1 gene was detected in 15 (17.8%), int1 in 16 (19%) and ISCR in 6 (7.1%). None of the susceptible isolates had sul2 or dfrA genes. These findings point out an increasing frequency of TMP/SMX resistance genes among S. maltophilia clinical isolates in our region, so the adoption of prudent use of S. maltophilia antimicrobial agents and the establishment of a surveillance system are desperately needed.

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

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

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

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

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

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

Molecular Characterization of Antibiotic Resistant Genes among Gram Negative Clinical Isolates

Increasing antibiotic resistance is a threat worldwide. Deep studies have to be done for correct and appropriate usage of antibiotics for treating infections efficiently and to curtail the spread of resistance. Hence, this study is aimed at understanding the prevalence of antibiotic resistance genes among 35 gram-negative bacterial isolates. By performing biochemical tests, the isolates were identified to be Escherichia coli (n = 15), Klebsiella oxytoca (n = 3), Klebsiella pneumoniae (n = 5), Proteus mirabilis (n = 4), Proteus vulgaris (n = 1) and Pseudomonas aeruginosa (n = 7). Antibiotic susceptibility tests showed that most E. coli isolates were resistant to ampicillin, levofloxacin and cefazolin. Ampicillin resistance was found in all of Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates. All Proteus mirabilis isolates were resistant to ciprofloxacin and co-trimoxazole. Pseudomonas aeruginosa showed maximum resistance to gentamycin and tobramycin. All 35 isolates were sensitive to amikacin, imipenem and meropenem. Polymerase Chain Reaction showed that AmpC and SHV genes were predominant in E. coli and Klebsiella species respectively. Among Proteus mirabilis and Pseudomonas aeruginosa, IntI gene was more prevailing. NDM1 gene was not detected in any of the isolates. This study also showed the prevalence of isolates resistant to multiple drugs due to the co-existence of resistant genes. Therefore, control of spread of infections, routine study in this field and proper guidelines for prescribing antibiotics is very necessary.&#x0D; HIGHLIGHTS&#x0D; &#x0D; This study is aimed at understanding the prevalence of antibiotic resistance genes among 35 gram-negative bacterial isolates&#x0D; A considerable number of isolates had antibiotic resistance genes and some isolates showed resistance to multiple drugs due to the co-existence of resistant genes&#x0D; Infections caused by bacteria that are resistant to antibiotics are difficult to treat and may sometimes lead to death&#x0D; Regular study and proper use of antibiotics is essential to curtail the spread of antibiotic resistant bacteria. Regular&#x0D; &#x0D; GRAPHICAL ABSTRACT

Genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria (MDRB) isolated from the gut microbiota of highly antibiotic treated Salmo salar

The main objective of this study was the genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria isolated from the intestinal microbiota of aquaculture salmon treated with high concentrations of antibiotics. In 82 multidrug resistant bacterial isolates, the prevalence of both the conserved elements of the integrons, qacEΔ1 and sul1 genes, and the variable region (VR) was determined. Further, whole genome sequencing and complete genetic analysis was performed in VR-positive isolates. Despite the fact that 100% of the bacterial isolates presented the intI1 gene, only 12.3% carried the qacEΔ1 and sul1 genes and only two (2.4%) presented a VR with gene cassettes. In the Pseudomonas baetica 25P2F9 isolate, a VR carrying aac(6')31, qacH, and blaOXA-2 gene cassettes was described, whereas the VR of Aeromonas salmonicida 30PB8 isolate showed a dfrA14 gene cassette. The array of gene cassettes found in the Pseudomonas isolate appears with high frequency in clinically relevant pathogens such as Pseudomonas aeruginosa or Escherichia coli. Additionally, it was possible to determine that these integrons are contained in plasmids and coul be easily transferred. Resistome analysis demonstrated that both isolates carried a great diversity of antibiotic resistance genes, including many β-lactamases. Even in the Aeromonas isolate a new oxacillin-hydrolyzing beta-lactamase gene was described (blaOXA-956). The presence of multidrug resistant bacteria and clinically relevant genetic elements in the salmon intestinal microbiota make the aquaculture a hotspot in the phenomenon of antibiotic resistance; therefore, the control of antibiotics used in this activity is a key point to avoid its escalation.

Occurrence and distribution of Carbapenem-resistant Enterobacterales and carbapenemase genes along a highly polluted hydrographic basin

We determined the distribution and temporal variation of Carbapenem Resistant Enterobacterales (CRE), carbapenemase-encoding genes and other antibiotic resistance genes (ARGs) in a highly polluted river (Lis River; Portugal), also assessing the potential influence of water quality to this distribution.Water samples were collected in two sampling campaigns performed one year apart (2018/2019) from fifteen sites and water quality was analyzed. CRE were isolated and characterized. The abundance of four ARGs (blaNDM, blaKPC, tetA, blaCTX-M), two Microbial Source Tracking (MST) indicators (HF183 and Pig-2-Bac) and the class 1 integrase gene (IntI1) was measured by qPCR. Resultsconfirmed the poor quality of the Lis River water, particularly in sites near pig farms. A collection of 23 CRE was obtained: Klebsiella (n = 19), Enterobacter (n = 2) and Raoultella (n = 2). PFGE analysis revealed a clonal relationship between isolates obtained in different sampling years and sites. All CRE isolates exhibited multidrug resistance profiles. Klebsiella and Raoultella isolates carried blaKPC while Enterobacter harbored blaNDM. Conjugation experiments were successful for only four Klebsiella isolates. All ARGs were detected by qPCR on both sampling campaigns. An increase in ARGs and IntI1 abundances was detected in sites located downstream of wastewater treatment plants. Strong correlations were observed between blaCTX-M, IntI1 and the human-pollution marker HF183, and also between tetA and the pig-pollution marker Pig-2-bac, suggesting that both human- and animal-derived pollution in the Lis River are a potential source of ARGs. Plus, water quality parameters related to eutrophication and land use were significantly correlated with ARGs abundances. Our findings demonstrated that the Lis River encloses high levels of antibiotic resistant bacteria and ARGs, including CRE and carbapenemase-encoding genes. Overall, this study provides a better understanding on the impacts of water pollution resulting from human and animal activities on the resistome of natural aquatic systems.

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

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

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

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

Tracking antibiotic resistance through the environment near a biosolid spreading ground: Resistome changes, distribution, and metal(loid) co-selection

The application of urban wastewater treatment plants (WWTPs) products to agricultural lands has contributed to the rising level of antibiotic resistance and drawn a critical public health concern. It has not been thoroughly investigated at which spatial scales a biosolid applied area as a potentially predominant source affects surrounding soil resistomes. This study investigated distribution and impact of WWTP biosolids treated with anaerobic digestion on an agricultural area. Heterotrophic plate counts (HPCs) and quantitative polymerase chain reaction (qPCR) were performed for detection of selected antibiotic-resistant bacteria (ARB), selected antibiotic resistance genes (ARGs), intI1 genes, and 16S rRNA genes. Biosolid samples contained significantly higher levels of selected ARGs than the raw agricultural soils (p < 0.05). The average relative abundances of intI1, sul1, blaSHV, and ermB genes were significantly higher in biosolid-amended soils than nearby agricultural soils (p < 0.05). Spatial interpolation analysis of relative gene abundances of intI1, sul1, sul2, and tetW across the studied area further indicated directional trends towards the northwest and southeast directions, highlighting possible airborne spread. Concentrations of Co, Cu, Ni, and Fe were found to be significantly and positively correlated with relative abundances of intI1, sul1, and tetW genes (p < 0.05). The resistance ratios of culturable antibiotic-resistant bacteria in agricultural soils with biosolid amendments were generally identical to those without biosolid amendments. This study will advance the understanding of the antibiotic resistome in agricultural soils impacted by long-term waste reuse and inform the evaluation strategies for future biosolids application and management.

Accumulation of antibiotic resistance genes in pakchoi (Brassica chinensis L.) grown in chicken manure-fertilized soil amended with fresh and aged biochars.

Biochar has been used to alleviate the contamination of antibiotic resistance genes (ARGs) in soil and to inhibit ARGs transfer from soil to plants. However, the effect of aged biochar on ARGs abundance in soil and ARGs enrichment in plants are scarcely investigated. In this study, a pot experiment was conducted to compare the effects of fresh and aged biochars on the accumulation of five typical ARGs including tetX, tetW, sul2, ermB, and intI1 in a chicken manure-fertilized soil and in pakchoi (Brassica chinensis L.). Results showed that both biochars significantly decreased the abundance of tetW, sul2, and ermB and increased the abundance of tetX and intI1 in soil. However, the accumulation of all tested ARGs in pakchoi were significantly decreased by both biochars. At the lower addition rate (1%), the fresh biochar was superior to the aged biochar in decreasing the accumulation of some genes (tetW, tetX, and sul2) in pakchoi, whereas an opposite tendency was observed for other genes (ermB and intI1). As the addition rate increased to 2%, the difference between the two biochars diminished, and a similar capacity of decreasing ARGs transfer was observed. The reduction in ARGs accumulation in pakchoi was highly related to the type of ARGs, the biochar addition level, and the aging of biochar. Our results provide insights into the naturally aged biochar on the fate of ARGs in a soil-plant system.

Microplastics can selectively enrich intracellular and extracellular antibiotic resistant genes and shape different microbial communities in aquatic systems

Microplastics (MPs), as emerging contaminants, are posing potential risks to environment, and animal and human health. The ubiquitous presence of MPs in natural ecosystems provides favorable platform to selectively adsorb antibiotic resistant genes (ARGs) and bacteria (ARB) and bacterial assemblages, especially in wastewater which is hotspot for MPs, ARGs and ARB. In this study, the selective capture of intracellular ARGs (iARGs), extracellular ARGs (eARGs), and bacterial assemblages by MPs with different materials (i.e. polyethylene, polyvinylchloride, and polyethylene terephthalate) and sizes (200 μm and 100 μm) was investigated. The results showed that iARGs (i.e. i-TetA, i-TetC, i-TetO, i-sul1), integron-integrase gene (intI1), and eARGs (i.e. e-TetA and e-blaTEM) were selectively enriched on MPs. Relative abundances of i-sul1, i-TetA, and intI1 were generally higher than that of i-TetC and i-TetO on all MPs. Moreover, MPs also have strong effects on the formation of microflora in wastewater, which resulted in different bacterial communities and functions in the wastewater and on the MPs. These findings suggested that MPs could affect the selective enrichment of ARB and ARGs in water environment.

Antimicrobial Resistance and Molecular Characterization of Gene Cassettes from Class 1 Integrons in Escherichia coli Strains.

To investigate the antimicrobial resistance and molecular characterization of gene cassettes from class 1 integrons in Escherichia coli strains isolated from hospitalized patients. Bacterial identification was conducted using the Vitek-2 Compact system, and antimicrobial susceptibility analysis was performed using the Kirby-Bauer method. Class 1 integrons, integrase genes, the variable regions of integrons and promoters from the isolated E. coli were screened by polymerase chain reaction, and subjected to DNA sequencing. In total, 138 E. coli strains were collected from the hospitalized patients, most from urine specimens (41.30%, 57/138). Antimicrobial resistance to ampicillin (89.86%) was most prevalent, with 79.99% of strains being multidrug-resistant (MDR). The class 1 integron integrase intI1 gene was detected in 67.39% of the isolates (93/138). Three gene cassette arrays and 5 antimicrobial resistance gene cassettes were detected in 69 of the class 1 integron-positive strains. The most common gene cassette array was dfrA17-aadA5. Of the 93 intI1-positive strains, 5 different common promoters were detected. The most prevalent common promoter was PcH1, and most isolates contained the dfrA17-aadA5 gene cassette array. In summary, antimicrobial resistance and MDR were prevalent among E. coli isolates in our city Weifang in Shandong Provence China. Gene cassettes of the class 1 integron variable region mostly conferred resistance to traditional antimicrobials. Weak promoters in the variable regions were predominant in this study. Integrons pose a great threat to the treatment of MDR bacterial infections and further investigations are needed.

Effects of activated sludge and UV disinfection processes on the bacterial community and antibiotic resistance profile in a municipal wastewater treatment plant.

Wastewater tertiary treatment has been pointed out as an effective alternative for reducing the concentration of antibiotic resistant bacteria and genes (ARB and ARGs) in wastewaters. The present work aimed to build on the current knowledge about the effects of activated sludge and UV irradiation on antibiotic resistance determinants in biologically treated wastewaters. For that, the microbial community and ARGs' composition of samples collected after preliminary (APT), secondary (AST), and tertiary (ATT) treatments in a full-scale wastewater treatment plant using a modified activated sludge (MAS) system followed by an UV stage (16mJ/cm2) were investigated through culture-dependent and independent approaches (including metagenomics). A total of 24 phyla and 460 genera were identified, with predominance of Gammaproteobacteria in all samples. Pathogenic genera corresponded to 8.6% of all sequences on average, mainly Acinetobacter and Streptococcus. Significant differences (p < 0.05) in the proportion of pathogens were observed between APT and the other samples, suggesting that the secondary treatment reduced its abundance. The MAS achieved 64.0-99.7% average removal efficiency for total (THB) and resistant heterotrophic bacteria, although the proportions of ARB/THB have increased for sulfamethoxazole, cephalexin, ciprofloxacin, and tetracycline. A total of 107 copies/mL of intI1 gene remained in the final effluent, suggesting that the treatment did not significantly remove this gene and possibly other ARGs. In accordance, metagenomic results suggested that number of reads recruited to plasmid-associated ARGs became more abundant in the pool throughout the treatment, suggesting that it affected more the bacteria without these ARGs than those with it. In conclusion, disinfected effluents are still a potential source for ARB and ARGs, which highlights the importance to investigate ways to mitigate their release into the environment.

High Frequency of Class I and II Integrons and the Presence of aadA2 and dfrA12 Gene Cassettes in the Clinical Isolates of Acinetobacter baumannii from Shiraz, Southwest of Iran

Background: Acinetobacter baumannii is a global concern that causes healthcare-associated infections due to multidrug resistance against commercially available antimicrobial agents. Objectives: The present study was conducted to determine the antimicrobial susceptibility of A. baumannii isolates from clinical specimens in Shiraz, Iran. In addition, the possible relationship of susceptibility patterns with the presence of integrons and related gene cassettes is investigated. Methods: Acinetobacter baumannii isolates were collected, and their susceptibility to various antibiotics was tested using the Kirby-Bauer disk diffusion method. Moreover, molecular analysis were performed to detect the presence of the OXA-51-like gene, as well as class I, II, and III integrons, and associated gene cassettes. Results: The majority of isolates were resistant to imipenem (99.4%), piperacillin (98.2%), gentamycin (98.2%), meropenem (97.7%), ceftazidime (95.4%), amikacin (95.4%), and trimethoprim-sulfamethoxazole (90.8%). All strains showed multidrug resistance to the tested antibiotics. The distribution analysis of integrons genes revealed that 90.2, 72.4, and 12.1% of the isolates carried intI1, intI2, and intI3 genes, respectively. Moreover, two types of prevalent gene cassettes, including aad and dfr, were detected in class 1 integron-carrying strains. Conclusions: The current study showed the high prevalence of A. baumannii isolates harboring integrons in our investigated medical center, which may indicate the distribution of multidrug resistance events. The different gene cassette arrays highlight the remarkable role of geographical issues in disseminating multidrug-resistant (MDR) isolates. This could be attributed to distinct therapeutic interventions in different areas. The results demonstrate the necessity of continuous surveillance to prevent the distribution of multidrug resistance among A. baumannii strains in Iran.

Metagenomic community composition and resistome analysis in a full-scale cold climate wastewater treatment plant

BackgroundWastewater treatment plants are an essential part of maintaining the health and safety of the general public. However, they are also an anthropogenic source of antibiotic resistance genes. In this study, we characterized the resistome, the distribution of classes 1–3 integron-integrase genes (intI1, intI2, and intI3) as mobile genetic element biomarkers, and the bacterial and phage community compositions in the North End Sewage Treatment Plant in Winnipeg, Manitoba. Samples were collected from raw sewage, returned activated sludge, final effluent, and dewatered sludge. A total of 28 bacterial and viral metagenomes were sequenced over two seasons, fall and winter. Integron-integrase genes, the 16S rRNA gene, and the coliform beta-glucuronidase gene were also quantified during this time period.ResultsBacterial classes observed above 1% relative abundance in all treatments were Actinobacteria (39.24% ± 0.25%), Beta-proteobacteria (23.99% ± 0.16%), Gamma-proteobacteria (11.06% ± 0.09%), and Alpha-proteobacteria (9.18 ± 0.04%). Families within the Caudovirales order: Siphoviridae (48.69% ± 0.10%), Podoviridae (23.99% ± 0.07%), and Myoviridae (19.94% ± 0.09%) were the dominant phage observed throughout the NESTP. The most abundant bacterial genera (in terms of average percent relative abundance) in influent, returned activated sludge, final effluent, and sludge, respectively, includes Mycobacterium (37.4%, 18.3%, 46.1%, and 7.7%), Acidovorax (8.9%, 10.8%, 5.4%, and 1.3%), and Polaromonas (2.5%, 3.3%, 1.4%, and 0.4%). The most abundant class of antibiotic resistance in bacterial samples was tetracycline resistance (17.86% ± 0.03%) followed by peptide antibiotics (14.24% ± 0.03%), and macrolides (10.63% ± 0.02%). Similarly, the phage samples contained a higher prevalence of macrolide (30.12% ± 0.30%), peptide antibiotic (10.78% ± 0.13%), and tetracycline (8.69% ± 0.11%) resistance. In addition, intI1 was the most abundant integron-integrase gene throughout treatment (1.14 × 104 gene copies/mL) followed by intI3 (4.97 × 103 gene copies/mL) while intI2 abundance remained low (6.4 × 101 gene copies/mL).ConclusionsWastewater treatment successfully reduced the abundance of bacteria, DNA phage and antibiotic resistance genes although many antibiotic resistance genes remained in effluent and biosolids. The presence of integron-integrase genes throughout treatment and in effluent suggests that antibiotic resistance genes could be actively disseminating resistance between both environmental and pathogenic bacteria.

Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats.

Wild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates (13,7%). Based on the analysis of the coexistence of 4 genes encoding ESBLs/AmpC (blaCTX-M, blaTEM, blaSHV, blaAmpC) and class 1 and 2 integrons genes (intI1, intI2) a subset of two resistance profiles was observed among the investigated E. coli isolates carrying blaAmpC, blaSHV, and blaCTX-M, blaTEM, class 1 and 2 integrons, respectively. The E. coli isolates were categorized into 4 phylogenetic groups A (39.4%), B2 (24.25%), D (24.25%) and B1 (12.1%). The pathogenic B2 and D groups were mainly typical for the Laridae family. Among the 28 virulence factors (Vfs) detected in pathogenic phylogenetic groups B2 and D, 7 were exclusively found in those groups (sfa, vat, tosA, tosB, hly, usp, cnf), while 4 VFs (fecA, fyuA, irp2, kspMTII) showed a statistically significant association (P≤0.05) with phylogroups A and B1. Our results indicated that strains belonging to commensal phylogroups A/B1 possess extensive iron acquisition systems (93,9%) and autotransporters (60,6%), typical for pathogens, hence we suggest that these strains evolve towards higher levels of virulence. This study, which is a point assessment of the virulence and drug resistance potential of wild birds, confirms the importance of taking wild birds as a reservoir of strains that pose a growing threat to humans. The E. coli analyzed in our study derive from different phylogenetic groups and possess an arsenal of antibiotic resistance genes and virulence factors that contribute to their ability to cause diseases.