Int Gene Research Articles

Faecal microbiota and antibiotic resistance genes in migratory waterbirds with contrasting habitat use

Migratory birds may have a vital role in the spread of antimicrobial resistance across habitats and regions, but empirical data remain scarce. We investigated differences in the gut microbiome composition and the abundance of antibiotic resistance genes (ARGs) in faeces from four migratory waterbirds wintering in South-West Spain that differ in their habitat use. The white stork Ciconia ciconia and lesser black-backed gull Larus fuscus are omnivorous and opportunistic birds that use highly anthropogenic habitats such as landfills and urban areas. The greylag goose Anser anser and common crane Grus grus are herbivores and use more natural habitats. Fresh faeces from 15 individuals of each species were analysed to assess the composition of bacterial communities using 16S rRNA amplicon-targeted sequencing, and to quantify the abundance of the Class I integron integrase gene (intI1) as well as genes encoding resistance to sulfonamides (sul1), beta-lactams (blaTEM, blaKPC and blaNDM), tetracyclines (tetW), fluoroquinolones (qnrS), and colistin (mcr-1) using qPCR. Bacterial communities in gull faeces were the richest and most diverse. Beta diversity analysis showed segregation in faecal communities between bird species, but those from storks and gulls were the most similar, these being the species that regularly feed in landfills. Potential bacterial pathogens identified in faeces differed significantly between bird species, with higher relative abundance in gulls. Faeces from birds that feed in landfills (stork and gull) contained a significantly higher abundance of ARGs (sul1, blaTEM, and tetW). Genes conferring resistance to last resort antibiotics such as carbapenems (blaKPC) and colistin (mcr-1) were only observed in faeces from gulls. These results show that these bird species are reservoirs of antimicrobial resistant bacteria and suggest that waterbirds may disseminate antibiotic resistance across environments (e.g., from landfills to ricefields or water supplies), and thus constitute a risk for their further spread to wildlife and humans.

The behavior of antibiotic-resistance genes and their relationships with the bacterial community and heavy metals during sewage sludge composting

The main contributors to antibiotic resistance genes (ARGs) profiles during the composting process under the addition of biochar (BC) and peat (PT) were not fully explored. This study investigated the influence of BC and PT amendment on ARGs fate, the bacterial community and heavy metals in sewage sludge compost. Compared to control, BC and PT declined the total abundances of ARGs by 17.6% and 43.0% after composting. Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were the dominant phyla across the composting process, among which, members of Firmicutes (mainly Bacillaceae) were the potential hosts for multiple ARGs. BC and PT addition declined the abundance of bacterial pathogens such as Bacteroides and Pseudomonas. Besides, the concentrations of copper (Cu), zinc (Zn) and lead (Pb) were less in BC and PT treatments than control on day 40, and these metals displayed significant positive correlations to sul1 and intI1. Furthermore, variation partitioning analysis (VPA) revealed that the bacterial community exhibited the most contribution to the ARG patterns, as much as 34.0%, followed by heavy metals (10.8%) and intI1 gene (1.5%). These results suggested that biochar and peat can reduce the risks of ARGs in sewage sludge mainly by sharping the bacterial composition.

Unraveling the riverine antibiotic resistome: The downstream fate of anthropogenic inputs

River networks are one of the main routes by which the public could be exposed to environmental sources of antibiotic resistance, that may be introduced e.g. via treated wastewater. In this study, we applied a comprehensive integrated analysis encompassing mass-flow concepts, chemistry, bacterial plate counts, resistance gene quantification and shotgun metagenomics to track the fate of the resistome (collective antibiotic resistance genes (ARGs) in a microbial community) of treated wastewater in two Swiss rivers at the kilometer scale. The levels of certain ARGs and the class 1 integron integrase gene (intI1) commonly associated with anthropogenic sources of ARGs decreased quickly over short distances (2-2.5 km) downstream of wastewater discharge points. Mass-flow analysis based on conservative tracers suggested this decrease was attributable mainly to dilution but ARG loadings frequently also decreased (e.g., 55.0-98.5 % for ermB and tetW) over the longest studied distances (6.8 and 13.7 km downstream). Metagenomic analysis confirmed that ARG of wastewater-origin did not persist in rivers after 5 ~ 6.8 km downstream distance. sul1 and intI1 levels and loadings were more variable and even increased sharply at 5 ~ 6.8 km downstream distance on one occasion. While input from agriculture and in-situ positive selection pressure for organisms carrying ARGs cannot be excluded, in-system growth of biomass is a more probable explanation. The potential for direct human exposure to the resistome of wastewater-origin thus appeared to typically abate rapidly in the studied rivers. However, the riverine aquatic resistome was also dynamic, as evidenced by the increase of certain gene markers downstream, without obvious sources of anthropogenic contamination. This study provides new insight into drivers of riverine resistomes and pinpoints key monitoring targets indicative of where human sources and exposures are likely to be most acute.

Detection of integron genes in the plasmid DNA of multidrug resistant Pseudomonas aeruginosa isolated from surgical wounds of some patients in Benin City, Nigeria

Pseudomonas aeruginosa is an opportunistic pathogen with the capability to cause serious surgical wound infections and remains a major healthcare problem. Plasmid is an extra chromosomal material in bacterial cells and confers resistance to the cell against many antibiotics. Genetic elements such as integron are implicated in conferring multidrug resistance (MDR) to P. aeruginosa . This study aims at investigating the occurrence of integron genes (int1, int2, int3) in the plasmid DNA and their ability to cause MDR in P. aeruginosa . In total, 284 different wound swabs were collected, P. aeruginosa isolated and screened using standard laboratory methods. Antibiotics susceptibility tests were carried out using Kirby-Bauer disk diffusion method. Polymerase chain reaction (PCR) was also carried out using P. aeruginosa plasmid DNA as a template to detect the presence/absence of the integron genes using different pairs of specific primers. The results reveal that 34 (54.8%) of the microbes isolated were P. aeruginosa . Most of the isolates showed notable resistance to antibiotics, most notably against Ceftazidime, Augmentin, Cefixime and Gentamicin . Eleven isolates harbors the plasmid DNA . PCR amplification showed that 6 (54.5%) of the P. aeruginosa isolates harbor integron class 1 genes, non harbors the integron class 2 genes while 3 (27.3%) possess the integron class 3 genes. The isolates with these genes were highly resistant to most of the antibiotics used. int1 gene was prevalent then int3.
 Keywords: Antimicrobial, Wound infection, Integron, Polymerase chain reaction, Plasmid DNA

Prevalence of Antibiotic Resistance Genes, Heavy Metal Resistance Genes and the Class 1 Integron Gene in Well Water and Tap Water Samples from Four Cities in Henan Province, China.

The presence of multi-resistance to both antibiotics and heavy metals in drinking water poses a significant risk to human health. Herein, we utilized qPCR to assess patterns of antibiotic resistance genes (ARGs), heavy metal resistance genes (HMRGs), and class 1 integron (intI1) gene expression levels in well and tap water samples from four cities in Henan Province, China. The relative abundance of most index values was higher in well water relative to tap water, or was highest in Shangqiu City and lowest in Puyang City on average. The expression of ARG was closely correlated with that of intI1 and HMRG in both well and tap water. Overall, our data highlighted the health threat posed by ARGs in the drinking water supply and underscore the potential for the transfer of these genes between bacteria with the aid of intI1 under selective pressure associated with human activity and heavy metal stress.

Responses of aerobic granular sludge to fluoroquinolones: Microbial community variations, and antibiotic resistance genes

In this study, aerobic granular sludge (AGS) was operated under high levels of ammonium for removing three fluoroquinolones (FQs), i.e., ciprofloxacin (CFX), ofloxacin (OFX), and norfloxacin (NFX) at 3, 300, and 900 µg/L, respectively. Two key objectives were to investigate the differential distribution of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in sludge fractions and to evaluate correlations between ARGs and MGEs to nitrifying and denitrifying bacteria. AGS showed excellent stability under the exposure of FQs, with nitrite-oxidizing bacteria (NOB) more sensitive to FQs than ammonium-oxidizing bacteria (AOB). Specific oxygen utilization rates (SOUR) showed a reduction of 26.9% for NOB but only 4.0% of the reduced activity of AOB by 3 μg/L FQs. AGS performed better removal efficiencies for CFX and NFX than OFX, and the efficiencies increased with their elevated concentrations, except at 900 μg/L FQs. The elevated FQ concentrations led to a significant enrichment of intI1 and genus Thauera, while qnrD and qnrS showed no accumulation. Compared to nitrifiers, FQs relevant ARGs and the intI1 gene preferred to exist in denitrifiers, and the abundance of denitrifiers behaved a decreasing trend with the sludge size. Two quinoline-degrading bacteria were found in the AGS system, i.e., Alicycliphilus and Brevundimonas, possibly carrying qnrS and qnrD, respectively. Their relative abundance increased with the sludge size, which was 2.18% in sludge <0.5 mm and increased to 3.70% in sludge >2.0 mm, suggesting that the AGS may be a good choice in treating FQs-containing wastewater.

Fate of antibiotic resistance genes and metal resistance genes during the thermophilic fermentation of solid and liquid swine manures in an ectopic fermentation system

Environmental pollution due to resistance genes from livestock manure has become a serious issue that needs to be resolved. However, little studies focused on the removal of resistance genes in simultaneous processing of livestock feces and urine. This study investigated the fate of antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and class 1 integron-integrase gene (intI1) during thermophilic fermentation of swine manure in an ectopic fermentation system (EFS), which has been regarded as a novel system for efficiently treating both feces and urine. The abundances of MRGs and tetracycline resistance genes were 34.44–97.71% lower in the EFS. The supplementation of heavy metals significantly increased the abundance of intI1, with the enhancement effect of copper being more prominent than that of zinc. The highest abundances of resistance genes and intI1 were observed at high Cu levels (A2), indicating that Cu can increase the spreading of resistance genes through integrons. Network analysis revealed the co-occurrence of ARGs, MRGs, and intI1, and these genes potentially shared the same host bacteria. Redundancy analysis showed that the bacterial community explained most of the variations in ARGs, and environmental factors had influences on ARGs abundances by modulating the bacterial community composition. The decreased Sphingomonas, Comamonas, Acinetobacter, Lactobacillus, Bartonella, Rhizobium, and Bacteroides were mainly responsible for the reduced resistance genes. These results demonstrate that EFS can reduce resistance genes in simultaneous processing of livestock feces and urine.

Genetic characterization, mechanisms and dissemination risk of antibiotic resistance of multidrug-resistant Rothia nasimurium

Rothia nasimurium is part of the commensal flora of humans and other animals and has recently received increasing attention for its multidrug-resistance (MDR) and pathogenicity. Currently, no systematic reports characterize the genetics, mechanisms, and dissemination risks of antibiotic resistance in MDR R. nasimurium. Here, we present the first report outlining a MDR strain of R. nasimurium, E1706032a, isolated from ducks exhibiting clinical sickness. Phylogenetic analysis indicates that E1706032a mostly likely originated in the commensal bacteria of Amazona aestiva in Florida. E1706032a is resistant to beta-lactams, aminoglycosides, macrolides, sulfonamides, fluoroquinolones, rifamycins, tetracyclines, lincosamides and chloramphenicol. Genetic sequences related to drug resistance were detected, including resistance genes (aac(6')-Ib, ant(3″)-Ia, sul1, dfrA7, erm(X)), efflux pumps (tetZ, qacEΔ1, cmx, phosphate ABC transporter ATP-binding protein), and resistance-related proteins (hydrolase of the metallo-beta-lactamase (MBLs), mycinamicin resistance protein (myrA), DNA-directed RNA polymerase subunit beta (rpoB) variants, etc). E1706032a carries an IS481-like element, IS5564 and IS6-like elements, and IS6100 along with several novel transposases of the IS3 family. E1706032a also carries the class 1 integron gene IntI1, which is downstream adjacent to the gene cassettes aac(6')-Ib, tetZ, dfrA27, ant(3″)-Ia, qacEΔ1, sul1, cmx and upstream adjacent to gene tnpA of IS6100. Genetic analysis suggests that E1706032a carries wide antibiotic resistance and dissemination potential through movable elements and thus has the potential to cause difficult-to-treat infections in animals and humans. The dissemination of E1706032a from parrots in Florida to ducks in eastern China indicates a cross-regional public health infection risk that should be evaluated for risk of global spreading.

High Prevalence of Drug Resistance and Class 1 Integrons in Escherichia coli Isolated From River Yamuna, India: A Serious Public Health Risk.

Globally, urban water bodies have emerged as an environmental reservoir of antimicrobial resistance (AMR) genes because resistant bacteria residing here might easily disseminate these traits to other waterborne pathogens. In the present study, we have investigated the AMR phenotypes, prevalent plasmid-mediated AMR genes, and integrons in commensal strains of Escherichia coli, the predominant fecal indicator bacteria isolated from a major urban river of northern India Yamuna. The genetic environment of blaCTX–M–15 was also investigated. Our results indicated that 57.5% of the E. coli strains were resistant to at least two antibiotic classes and 20% strains were multidrug resistant, i.e., resistant to three or more antibiotic classes. The multiple antibiotic resistance index of about one-third of the E. coli strains was quite high (>0.2), reflecting high contamination of river Yamuna with antibiotics. With regard to plasmid-mediated AMR genes, blaTEM–1 was present in 95% of the strains, followed by qnrS1 and armA (17% each), blaCTX–M–15 (15%), strA-strB (12%), and tetA (7%). Contrary to the earlier reports where blaCTX–M–15 was mostly associated with pathogenic phylogroup B2, our study revealed that the CTX-M-15 type extended-spectrum β-lactamases (ESBLs) were present in the commensal phylogroups A and B1, also. The genetic organization of blaCTX–M–15 was similar to that reported for E. coli, isolated from other parts of the world; and ISEcp1 was present upstream of blaCTX–M–15. The integrons of classes 2 and 3 were absent, but class 1 integron gene intI1 was present in 75% of the isolates, denoting its high prevalence in E. coli of river Yamuna. These evidences indicate that due to high prevalence of plasmid-mediated AMR genes and intI1, commensal E. coli can become vehicles for widespread dissemination of AMR in the environment. Thus, regular surveillance and management of urban rivers is necessary to curtail the spread of AMR and associated health risks.

Comparison of Class 2 Integron Integrase Activities.

Integrons play important roles in the dissemination of antimicrobial resistant genes among bacteria. Class 2 integrons usually has an internal stop codon, TAA, in integrase genes (intI2), leading to a truncated integrase, IntI2*. However, a few class 2 integrons with a natural full-length integrase have been reported. In this study, the sequences of natural full-length intI2 were extracted from INTEGRALL database and analyzed. A total of 236 sequences of intI2 were retrieved from INTEGRALL database, only seven of which were natural full-length intI2 genes and could be divided into five types according to their coding amino acid sequence. Quantitative real-time PCR was used to detect gene cassette sat2 integration and excision efficiency catalyzed by different natural full-length IntI2s. The results showed that all five IntI2s could catalyze attI2 × attCsat2 integration and attCdfrA1/sat2 × attCsat2/aadA1 excision in Escherichia coli. Integration and excision frequency catalyzed by IntI2A176 was highest and was about twofold as high as those catalyzed by IntI2S175_A176. The secondary structure of the IntI2 was predicted by online software. Polymorphisms of these five IntI2s were limited within residues 172, 174, 175, 176 and 256, and these residues were all far away from the predicted DNA binding regions or catalyzed sites. Influence of amino acid sequence polymorphisms of these natural full-length IntI2s on their catalyzed activities is limited.

Integron and its role in antimicrobial resistance: A literature review on some bacterial pathogens.

In recent years, different acquired resistance mechanisms, including transposons, bacteriophages, plasmids, and integrons have been identified as involved in the spread of resistance genes in bacteria. The role of integrons as mobile genetic elements playing a central role in antibiotic resistance has been well studied and documented. Integrons are the ancient structures that mediate the evolution of bacteria by acquiring, storing, disposing, and resorting to the reading frameworks in gene cassettes. The term integron describes a large family of genetic elements, all of which are able to capture gene cassettes. Integrons were classified into three important classes based on integrase intI gene sequence. Integrons can carry and spread the antibiotic resistance genes among bacteria and are among the most significant routes of distribution of resistance genes via horizontal transfer. All integrons have three essential core features. The first feature is intI, the second one is an integron-associated recombination site, attI, and an integron-associated promoter, Pc, is the last feature. Among them, the class 1 integron is a major player in the dissemination of antibiotic resistance genes across pathogens and commensals. Various classes of integrons possessing a wide variety of gene cassettes are distributed in bacteria throughout the world. This review thus focuses on the distribution of integrons among important bacteria.

IntI1 Type Mobile Genetic Elements Co-selected Antibiotic-Resistant Genes in Untreated Hospital Wastewaters.

Dissemination of antibiotic-resistant genes (ARGs) from hospital wastewaters (HWWs) is facilitated by the horizontal gene transfer (HGT) and involves association of ARGs with mobile genetic elements (MGEs). In our previous study, HWWs were found to have relatively high copy numbers of ARGs aadA, tetA, cmlA, sul1, and qnrS. In this study, therefore, the same HWWs were also monitored for 3 MGEs class 1 integron (intI1), insertion sequence common region 1 (ISCR1) and conjugative transposon Tn916/Tn1545 by using quantitative polymerase chain reaction. The gene intI1 with 7.4 × 102 average copy number/mL was found to be the most prevalent MGE and was up to two orders of magnitude higher than ISCR1 (5.5 × 100 average copy number/mL, p < 0.05) and Tn916/Tn1545 (2.3 × 100 average copy number/mL, p < 0.05) in all HWWs tested. Positive correlation between intI1 and the aadA, tetA, cmlA and sul1 genes indicated that the MGEs harbouring class1 integron most likely played major role in co-selecting all these ARGs together.

Antimicrobial resistance in Escherichia coli isolates from frugivorous (Eidolon helvum) and insectivorous (Nycteris hispida) bats in Southeast Nigeria, with detection of CTX-M-15 producing isolates

Thirty-five Escherichia coli isolates obtained from the liver, spleen and intestines of 180 frugivorous and insectivorous bats were investigated for antimicrobial resistance phenotypes/genotypes, prevalence of Extended-Spectrum beta-lactamase (ESBL) production, virulence gene detection and molecular typing. Eight (22.9 %) of the isolates were multidrug resistant (MDR). Two isolates were cefotaxime-resistant, ESBL-producers and harbored the blaCTX-M-15 gene; they belonged to ST10184-D and ST2178-B1 lineages. tet(A) gene was detected in all tetracycline-resistant isolates while int1 (n = 8) and blaTEM (n = 7) genes were also found. Thirty-three of the E. coli isolates were assigned to seven phylogenetic groups, with B1 (45.7 %) being predominant. Three isolates were enteropathogenic E. coli (EPEC) pathovars, containing the eae gene (with the variants gamma and iota), and lacking stx1/stx2 genes. Bats in Nigeria are possible reservoirs of potentially pathogenic MDR E. coli isolates which may be important in the ecology of antimicrobial resistance at the human-livestock-wildlife-environment interfaces. The study reinforces the importance of including wildlife in national antimicrobial resistance monitoring programmes.

Molecular Study of Class1 and 2 Integrons in Methicillin-resistant Staphylococcus Aureus

Background and Aim: Methicillin-Resistant Staphylococcus aureus (MRSA) had appeared in the last few decades and became a significant health problem. The study aimed to evaluate the prevalence of class 1 and class 2 integrons among MRSA clinical isolates from different healthcare-associated infections (HAIs) and the prevalence of multi-drug resistance (MDR) in these isolates. Materials and Methods: Two hundred Staphylococcus aureus (S. aureus) isolates from different HAIs were evaluated for the presence of the mecA gene, class 1 (intI1) and class 2 (intI2) integrons via the polymerase chain reaction (PCR) beside the determination of antibiotics sensitivity. Results: MRSA was determined by resistance to cefoxitin in 152 isolates (76%) of isolated S. aureus. The molecular study showed the prevalence of mecA, intI1, and intI2 genes among the isolated MRSA strains as follows: 100%, 70.4%, 13.1, respectively, and there were 12 isolates (7.9%) that had both intI1 and intI2 genes. There was a significant high incidence of resistance to gentamicin (P=0.01), amikacin (P=0.005), tobramycin (P=0.035), erythromycin (P=0.035), clindamycin (P=0.08), levofloxacin (P=0.0001) and ciprofloxacin (P=0.0001) in MRSA strains positive for intI1 and/or intI2 genes compared to MRSA strains negative for intI genes and non-methicillin resistant S. aureus (Non- MRSA) strains. There was a significantly high prevalence of MDR among MRSA strains positive for intI1 and/or intI2 genes (62.8%) compared to MRSA strains negative for intI genes (28.8%) and Non- MRSA strains (8.3%), (P=0.0001). Conclusion: The present study revealed a high prevalence of MRSA strains among S. aureus clinical isolates with a high prevalence of the mecA gene in those isolates. There was also a high prevalence of intI1gene but not intI2 gene. The mecA and intI genes were associated with the development of MDR in S. aureus.

Antibiotic resistance gene load and irrigation intensity determine the impact of wastewater irrigation on antimicrobial resistance in the soil microbiome

Treated wastewater (TWW) irrigation is a useful counter-measure against the depletion of freshwater (FW) resources. However, TWW contains several contaminants of emerging concern, such as antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs). Thus, TWW irrigation might promote the spread of antimicrobial resistance in soil environments. In the present work, we hypothesized that the ARG load and irrigation intensity define the effect of TWW irrigation on ARG spread dynamics in soil. This hypothesis was tested using a multiphase approach: a) comparing soil from a full-scale, commercially operated, TWW irrigated field with non-irrigated soil, b) long-term sampling of the TWW irrigated field over one year with different irrigation intensities and intercepted by irrigation breaks and c) laboratory-scale soil microcosms irrigated with TWW compared to FW. Six ARGs, the integrase gene intI1 and the 16S rRNA were quantified using qPCR. In addition, effects of TWW irrigation on bacterial community composition of microcosm-samples were analysed with 16S rRNA amplicon sequencing. The genes sul1, qnrS, blaOXA-58, tet(M) and intI1 were significantly more abundant in the TWW irrigated field soil, whereas blaCTX−-M-32 and blaTEM, the least abundant genes in the TWW irrigation, showed higher abundance in the non-irrigated soil. The relative abundance of sul1, qnrS, blaOXA-58, tet(M) and intI1 correlated with TWW irrigation intensity and decreased during irrigation breaks. Despite the decrease, the levels of these genes remained consistently higher than the non-irrigated soil indicating persistence upon their introduction into the soil. Microcosm experiments verified observations from the field study: TWW irrigation promoted the spread of ARGs and intI1 into soil at far elevated levels compared to FW irrigation. However, the impact of TWW irrigation on 16S rRNA absolute abundance and the soil microbial community composition was negligible. In conclusion, the impact of TWW irrigation depends mainly on the introduced ARG load and the irrigation intensity.

Clonal Lineage Analysis of Shigella flexneri Isolates Circulating in Ahvaz, Iran.

Shigellosis is a significant public health challenge particularly in developing countries, and the spread of antibiotic resistance genes through integron structures has become an important problem in the treatment of Shigellosis. The aim of this study was to investigate the genetic association of Shigella flexneri antibiotic resistant clones collected from Ahvaz between 2013 and 2015 by pulse field gel electrophoresis (PFGE) method. A total of 45 S. flexneri isolates, which were resistant to ampicillin and cotrimoxazole, were obtained from patients with Shigellosis referred to Ahvaz hospitals during 2013 - 2015. PCR was performed to evaluate the frequency of Sul1, int1, blaOXA, and int2 genes. In addition, pulse field gel electrophoresis method was used to investigate the genetic relationship between 40 S. flexneri isolates. PCR results showed that the highest frequency was related to the sul1 gene with 80% (36 isolates) and the lowest frequency was related to class 2 integron with 15.5% (7 isolates); 31.11% (14 isolates) of the isolates were sul1 and int1. Also, 13.33% (6 isolates) had blaOXA and int1 genes, simultaneously. But none of the isolates had class 1 integrons and class 2 integrons at the same time. PFGE results showed 25 different pulsotype patterns, of which 16 isolates had their own unique pattern and were divided into 16 pulsotypes, and 27 isolates were divided into 9 pulsotypes. int2 and sul1 resistance genes had an upward trend from 2013 to 2015 and the results of PFGE indicated a different origin of S. flexneri clones.

On-farm screw press and rotary drum treatment of dairy manure-associated antibiotic residues and resistance.

An on-farm solid-liquid separator (SLS) and rotary drum composter (RD) manure treatment system was monitored for its impact on antibiotic residues and antibiotic resistance genes (ARGs). Administered antibiotics were tracked, and treatment system mass flows were quantified. Total amounts of antibiotic residues and ARGs were calculated from measured concentrations and mass flows. Only oxytetracycline (OTC) and sulfadimethoxine (SDM) were detected in the manure treatment system influent. No β-lactams were measured despite comprising ∼25% of the antibiotics administered. Nearly 80% of OTC and>90% of SDM partitioned into SLS liquid effluent (SL). The RD reduced the mass of OTC remaining in the SLS solid effluent (SS) significantly by 50%, whereas the mass of SDM appeared to increase after RD treatment. All four ARGs tested were detected in influent, with>70% of the sul1, blaOXA-1 , and intI1 genes (normalized by the 16S ribosomal RNA gene) partitioning into the SL. In contrast, about eight times more normalized tetO gene copies partitioned into the SS than in the SL. All ARGs remaining in the SS were significantly reduced by the RD treatment, with a noteworthy 98% reduction in normalized tetO gene copies. This study provides insight into on-farm levels of antibiotic residues and ARGs in dairy manure, their partitioning during SLS treatment, and their fate after a high-temperature RD treatment reaching 72.2 ± 0.18 °C near the outlet. It also notes the importance of mass-flow standardization of data, and the need to work towards standardization of manure system sampling protocols for antibiotic residues and ARGs.

Detection of &amp;lt;i&amp;gt;Acinetobacter baumannii&amp;lt;/i&amp;gt; in the Oropharynx of Long-Term Hospitalized Patients and the Expression of &amp;lt;i&amp;gt;IntI&amp;lt;/i&amp;gt; Gene Induced by Different Antibiotics

Acinetobacter baumannii is the main drug resistant bacteria in clinic at present, and its drug resistance is still rising rapidly. Integrons play an important role in bacterial acquisition of exogenous drug resistance genes. This study investigated A. baumannii colonization in oropharynx and the integron gene carrying, and the expressions of integrase gene were determined when exposed to the different concentrations of antibiotics. 64 hospitalized patients were collected during January 2019 to June in respiratory department of our hospital (the hospitalized time more than 14 days) in our experiment. All throat swab collections were used for DNA extraction, and A. baumannii identification and integron gene detection were done by PCR assay. A. baumannii strains isolated from the oropharynx were identified by MALD-TOF-MS technology and the drug resistance was also analyzed. When expose to a series of ceftazidime and imipenem (10 μg/mL, 5 μg/mL, 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL), the expressions of integron gene in the strains originated from oropharynx were determined by qRT-PCR assay. The results of the PCR showed that 60 patients had detected the A. baumannii Ab-ITS gene and rA gene in throat swabs, wherein 43 patients with symptoms of infection and 17 without symptoms of infection. There was no significant difference in Ab-ITS gene and rA gene detection rates in the symptoms of infection and no symptoms of infection (P > 0.05). In total 60 cases of samples, 29 cases had detected the IntI genes. 6 strains of A. baumannii isolated from the throat swabs of hospitalized patients were multi-drug resistant bacteria with IntI gene and variable region genes. Gene sequencing analysis revealed that the variable region gene cassettes were aacA4-catB8-aadA1-qacEdelta1. When exposed to the concentration of 1 μg/mL and 5 μg/mL ceftazidime, the mRNA expression of IntI had significantly increase compared to the negative control (P IntI had significantly increase compared to the negative control (P A. baumannii and carries ingegron genes in long-term hospitalized patients. The improper use of ceftazidime and imipenem might also contribute to the up-regulation of the integrase genes expression and the enhancement of bacterial resistance, which requires more attention in clinical work.

Dynamic changes of plasma extracellular vesicle long RNAs during perioperative period of colorectal cancer

ABSTRACT Extracellular vesicles (EVs) long RNAs (exLRs) have been shown to be indicators for the diagnosis and prognosis of colorectal cancer (CRC); however, the dynamic changes of exLRs during perioperative period and their cellular sources in CRC remains largely unknown. In this study, exLR sequencing (exLR-seq) was performed on plasma samples from three CRC patients at four time points (before surgery [T0], after extubation [T1], 1 day after surgery [T2], and 3 days after surgery [T3]). Bioinformatics approaches were used to investigate the profile and biofunctions of exLRs and their cellular sources. Greater than 12,000 mRNAs and 2,000 lncRNAs were reliably detected in each exLR-seq sample. Compared with T0, there were 110 differentially expressed genes (DEGs) in T1, 60 DEGs in T2, and 50 DEGs in T3. A total of 11 DEGs were found at all three time points and were related to membrane potential. In addition, compared to T0, 22 differentially expressed lncRNAs (DELRs) were found in T1, 19 DELRs in T2, and 38 DELRs in T3. Moreover, only three DELRs were detected at all three time points. Interestingly, EVs from CD8 + T cells, CD4+ memory T cells and NK cells decreased after surgery and the absolute quantity of EVs from immune cells were reduced as well. In summary, this study was the first to characterize the dynamic changes of exLRs during perioperative period and the cellular sources. These findings established the foundation for further studies involving the effects of these dynamically changed exLRs on CRC.

Analyses of the relationships among biofilm activity, antibiotic resistance and expressions of SHV, TEM, CTX and IntI in Klebsiella pneumoniae

Aim: Klebsiella pneumoniae as an opportunistic pathogen is responsible for nosocomial, urinary tract infections, pneumonia, bacteremia, liver abscesses, and respiratory and blood infections in patients. The aim of our study is to examine the antibiotic sensitivity, expression levels of IntI, TEM, SHV and CTX genes, which play a role in multidrug resistance, biofilm formation and even the relationships among them. Materials and Methods: Identification was implemented by carrying out the VITEK2 system and phenotypic confirmation of the ESBL producing isolates by using a combined disc test. Antibiotic sensitivity was implemented by disc diffusion method and microdilution methods toward various antibiotics. Furthermore, biofilm formation was examined through microtitration plate method. Results: The expressions of IntI, TEM, SHV and CTX were implemented in qRT-PCR. 19 were susceptible to all antibiotics, 17 were ESBL (+) and 16 were carbapenem-resistant among 52. The expression of these genes was upregulated in ESBL (+) strains and produced biofilm and the expression of these genes was upregulated, too. Conclusion: The results of the study revealed the potential role of the mentioned genes in biofilm formation and antibiotic resistance, enabling the development of new drugs. Further studies will be essential so as to determine when or how these systems are included in antibiotic resistance and biofilm formation.