Int Gene Research Articles

Horizontal gene transfer is a key determinant of antibiotic resistance genes profiles during chicken manure composting with the addition of biochar and zeolite

Livestock manure is an important reservoir of antibiotic resistance genes (ARGs). Biochar and zeolite are commonly used to improve the quality of compost, however, little is known about the impacts of these additives on the fate of ARGs during composting and the underlying mechanisms involved. In this study, zeolite (ZL), biochar (BC), or zeolite and biochar (ZB) simultaneously were added to chicken manure compost to evaluate their effects on the ARGs patterns. After composting, the abundance of ARGs reduced by 92.6% in control, while the reductions were 95.9%, 98.7% and 98.2% for ZL, BC, ZB, respectively. Co-occurrence network analysis indicated that the potential hosts for most ARGs were predominantly affiliated to Firmicutes such as Lactobacillus and Fastidiosipila. Furthermore, shifts in ARGs were significantly correlated with class 1 integrase gene (intI1), and structural equation models further revealed that intI1 gene contributed most (standardized total effect 0.92) to the ARGs-removal, which was trigged by horizontal gene transfer. Together these results suggest that the addition of zeolite and biochar mitigate the accumulation and spread of ARGs during composting, and the crucial role of horizontal gene transfer (HGT) on the behaviors of ARGs should pay more attention to in the future.

The first evaluation relationship of integron genes and the multidrug-resistance in class A ESBLs genes in enteropathogenic Escherichia coli strains isolated from children with diarrhea in Southwestern Iran.

Enteropathogenic Escherichia coli (EPEC) is one of the most important diarrheagenic agents among infants under 5years in developing countries. This study aimed to investigate the relationship of integron genes and class A extended-spectrum ESBLs genes in MDR E. coli strains isolated from children with diarrhea in Southwestern Iran. Totally, 321 fecal samples were collected from diarrheal children under 5years admitted to teaching hospitals of Abadan and Khorramshahr, southwest Iran. Routine bacteriological tests were performed for the identification of E. coli isolates. Multiplex PCR was used for the presence of eae, bfp, stx1, and stx2 genes to detected EPEC strains. Serogrouping was performed for EPEC strains. The EPEC isolates' antibiotic resistance pattern was determined by the disk-diffusion technique. All EPEC isolates were screened for integron and class A β-lactamase genes. Of the 14 EPEC isolates, 12 (85.7%) were found to be ESBL-positive by double disk synergy test (DDST) and PCR. In addition, blaCTX-M and blaTEM genes were detected in 83.3% (n = 10) and 58.3% (n = 7) of EPEC isolates, respectively. None of the isolates had the blaKPC gene. On the other hand, 64.2% (n = 9) and 7.1% (n = 1) were positive only for intlI and intlII genes, respectively. The results demonstrated that EPEC is one of the major causes of childhood diarrhea in our region and that the distribution of class 1 integrons and ESBLs in EPEC strains is highly prevalent. Moreover, the results revealed that continuous monitoring of the emergence and expansion of MDR in EPEC strains is necessary.

A Pilot Study Combining Ultrafiltration with Ozonation for the Treatment of Secondary Urban Wastewater: Organic Micropollutants, Microbial Load and Biological Effects

Ozonation followed by ultrafiltration (O3 + UF) was employed at pilot scale for the treatment of secondary urban wastewater, envisaging its safe reuse for crop irrigation. Chemical contaminants of emerging concern (CECs) and priority substances (PSs), microbial load, estrogenic activity, cell viability and cellular metabolic activity were measured before and immediately after O3 + UF treatment. The microbial load was also evaluated after one-week storage of the treated water to assess potential bacteria regrowth. Among the organic micropollutants detected, only citalopram and isoproturon were not removed below the limit of quantification. The treatment was also effective in the reduction in the bacterial loads considering current legislation in water quality for irrigation (i.e., in terms of enterobacteria and nematode eggs). However, after seven days of storage, total heterotrophs regrew to levels close to the initial, with the concomitant increase in the genes 16S rRNA and intI1. The assessment of biological effects revealed similar water quality before and after treatment, meaning that O3 + UF did not produce detectable toxic by-products. Thus, the findings of this study indicate that the wastewater treated with this technology comply with the water quality standards for irrigation, even when stored up to one week, although improvements must be made to minimise microbial overgrowth.

Fate of antibiotic resistant E. coli and antibiotic resistance genes during full scale conventional and advanced anaerobic digestion of sewage sludge.

Antibiotic resistant bacteria (ARB) and their genes (ARGs) have become recognised as significant emerging environmental pollutants. ARB and ARGs in sewage sludge can be transmitted back to humans via the food chain when sludge is recycled to agricultural land, making sludge treatment key to control the release of ARB and ARGs to the environment. This study investigated the fate of antibiotic resistant Escherichia coli and a large set of antibiotic resistance genes (ARGs) during full scale anaerobic digestion (AD) of sewage sludge at two U.K. wastewater treatment plants and evaluated the impact of thermal hydrolysis (TH) pre-treatment on their abundance and diversity. Absolute abundance of 13 ARGs and the Class I integron gene intI1 was calculated using single gene quantitative (q) PCR. High through-put qPCR analysis was also used to determine the relative abundance of 370 ARGs and mobile genetic elements (MGEs). Results revealed that TH reduced the absolute abundance of all ARGs tested and intI1 by 10-12,000 fold. After subsequent AD, a rebound effect was seen in many ARGs. The fate of ARGs during AD without pre-treatment was variable. Relative abundance of most ARGs and MGEs decreased or fluctuated, with the exception of macrolide resistance genes, which were enriched at both plants, and tetracyline and glycopeptide resistance genes which were enriched in the plant employing TH. Diversity of ARGs and MGEs decreased in both plants during sludge treatment. Principal coordinates analysis revealed that ARGs are clearly distinguished according to treatment step, whereas MGEs in digested sludge cluster according to site. This study provides a comprehensive within-digestor analysis of the fate of ARGs, MGEs and antibiotic resistant E. coli and highlights the effectiveness of AD, particularly when TH is used as a pre-treatment, at reducing the abundance of most ARGs and MGEs in sludgeand preventing their release into the environment.

Comparison of the elimination effectiveness of tetracycline and AmpC β-lactamase resistance genes in a municipal wastewater treatment plant using four parallel processes.

Municipal wastewater treatment plants (mWWTPs), considered reservoirs of antibiotic resistance genes (ARGs), are selected to compare the contributions of technology and process to ARG removal. Fifteen ARGs (tetA, tetB, tetC, tetE, tetG, tetL, tetM, tetO, tetQ, tetS, tetX, MOX, CIT, EBC, and FOX) and two integron genes (intI1, intI2) were tracked and detected in wastewater samples from a large-scale mWWTP with four parallel processes, including three biological technologies of AAO (anaerobic-anoxic-oxic), AB (adsorption-biodegradation), and UNITANK, two different disinfection technologies, and two primary sedimentation steps. The results showed that ARGs were widely detected, among which tetA and tetM had the highest detection rate at 100%. AAO was the most effective process in removing ARGs, followed by the AB and UNITANK processes, where the separation step was critical: 37.5% AmpC β-lactamase genes were reduced by the secondary clarifier. UV disinfection was more efficient than chlorination disinfection by 47.0% in ARG removal. Both disinfection and primary sedimentation processes could effectively remove integrons, and the swirling flow grit chamber was a more effective primary settling facility in total ARG removal than the aerated grit chamber. The tet genes and AmpC β-lactamase genes were significantly correlated with the water quality indexes of BOD5, CODCr, SS, TP, TOC, pH and NH4+-N (p < 0.05). In addition, the correlation between efflux pump genes and AmpC β-lactamase genes was strongly significant (r2 = 0.717, p < 0.01). This study provides a more powerful guide for selecting and designing treatment processes in mWWTPs with additional consideration of ARG removal.

A comparison of antibiotics, antibiotic resistance genes, and bacterial community in broiler and layer manure following composting.

Animal manure is an important source of antibiotics and antibiotic resistance genes (ARGs) in the environment. However, the difference of antibiotic residues and ARG profiles in layer and broiler manure as well as their compost remains unexplored. In this study, we investigated the profiles of twelve antibiotics, seventeen ARGs, and class 1 integrase gene (intI1) in layer and broiler manure, and the corresponding compost at large-scale. Compared with layer manure, broiler manure exhibited approximately six times more residual tetracyclines, especially chlortetracycline. The relative abundances of qnrS and ermA genes in broiler manure were significantly higher than those in layer manure. The concentration of tetracyclines not only had a significantly positive correlation with tetracycline resistance genes (tetA and tetC) but was also positively correlated with quinolone resistance (qepA, qnrB, and qnrS) and macrolide resistance (ermA and ermT). Most ARGs in manure were reduced after composting. However, the relative abundance of sulfonamide resistance gene sul1 increased up to 2.41% after composting, which was significantly higher than that of broiler (0.41%) and layer (0.62%) manure. The associated bacterial community was characterized by high-throughput 16S rRNA gene sequencing. The relative abundances of thermophilic bacteria had significant positive correlations with the abundance of sul1 in compost. The composting has a significant impact on the ARG-associated gut microbes in poultry manure. Variation partitioning analysis indicated that the change of bacterial community compositions and antibiotics contributed partially to the shift in ARG profiles. The results indicate that at industry-scale production broiler manure had more antibiotics and ARGs than layer manure did, and composting decreased most ARG abundances in poultry manure except for sulfonamide resistance genes.

Antimicrobial resistant gene prevalence in soils due to animal manure deposition and long-term pasture management.

The persistence of antimicrobial resistant (AMR) genes in the soil-environment is a concern, yet practices that mitigate AMR are poorly understood, especially in grasslands. Animal manures are widely deposited on grasslands, which are the largest agricultural land-use in the United States. These nutrient-rich manures may contain AMR genes. The aim of this study was to enumerate AMR genes in grassland soils following 14-years of poultry litter and cattle manure deposition and evaluate if best management practices (rotationally grazed with a riparian (RBR) area and a fenced riparian buffer strip (RBS), which excluded cattle grazing and poultry litter applications) relative to standard pasture management (continuously grazed (CG) and hayed (H)) minimize the presence and amount of AMR genes. Quantitative PCR (Q-PCR) was performed to enumerate four AMR genes (ermB, sulI, intlI, and blactx-m-32) in soil, cattle manure, and poultry litter environments. Six soil samples were additionally subjected to metagenomic sequencing and resistance genes were identified from assembled sequences. Following 14-years of continuous management, ermB, sulI, and intlI genes in soil were greatest (P < 0.05) in samples collected under long-term continuous grazing (relative to conservation best management practices), under suggesting overgrazing and continuous cattle manure deposition may increase AMR gene presence. In general, AMR gene prevalence increased downslope, suggesting potential lateral movement and accumulation based on landscape position. Poultry litter had lower abundance of AMR genes (ermB, sulI, and intlI) relative to cattle manure. Long-term applications of poultry litter increased the abundance of sulI and intlI genes in soil (P < 0.05). Similarly, metagenomic shotgun sequencing revealed a greater total number of AMR genes under long-term CG, while fewer AMR genes were found in H (no cattle manure) and RBS (no animal manure or poultry litter). Results indicate long-term conservation pasture management practices (e.g., RBS and RBR) and select animal manure (poultry litter inputs) may minimize the presence and abundance of AMR genes in grassland soils.

Antibiotic resistance genes in swine manure slurry as affected by pit additives and facility disinfectants

Manure storage facilities are critical control points to reduce antibiotic resistance genes (ARGs) in swine manure slurry before the slurry is land applied. However, little is known about how exogenous chemicals entering the manure storage facilities may affect the fate of ARGs. The objective of this study was to analyze the impact of six commonly used pit additives and four facility disinfectants on the concentration of ARGs in swine manure slurry. Bench scale reactors, each containing approximately 50 L of liquid swine manure, were dosed with additives or disinfectants and were sampled for 40 days. Seven antibiotic resistance genes along with the intI1 gene and the 16S rRNA gene were monitored. Out of the six additives tested, Sludge Away significantly reduced the time-averaged absolute abundance of erm(C), erm(F), tet(Q), and the 16S rRNA gene as compared to the no additive control. Out of the four disinfectants tested, Tek-Trol significantly reduced the time-averaged absolute abundance of erm(B), erm(C), erm(F), intI1, tet(Q), and tet(X) than did the no-disinfectant control. According to Spearman's rank correlation, three genes erm(F), tet(Q), and tet(X) showed a strong to perfectly positive correlation and the two genes erm(B) and tet(O) showed a moderate to strong correlation in both the additive and disinfectant tests. Overall, the disinfectants were more effective in controlling the absolute abundance of ARGs than were the pit additives.

Analysis of the Correlation Between Antibiotic Resistance Patterns and Virulence Determinants in Pathogenic Klebsiella pneumoniae Isolates from Egypt.

Klebsiella pneumoniae is responsible for a plethora of infections involving multiple body systems. This study investigated K. pneumoniae clinical isolates for virulence-associated characters and antibiotic resistance. First, antibiotic sensitivity was determined for 40 K. pneumoniae clinical isolates. Some virulence and resistance-associated factors were studied phenotypically and genotypically. Multiple resistance profiles were observed (multidrug resistant [MDR; 42.5%], extensive drug resistant [XDR; 35%], and pandrug resistant [PDR; 5%]). Moreover, CTX-M-1, TEM, qnrS, and qnrA genes were detected in 70%, 30%, 60%, and 30% of selected isolates, respectively, and 40% of tested isolates were extended-spectrum β-lactamases (ESBLs) producers. Interestingly, all ESBLs producers harbored class 1 integrase gene (IntI1), while 60% of ESBLs producers harbored both CTX-M-1 and TEM. All tested isolates were capsulated while 87.5% were biofilm producers. Fimbriae were detected in 90% of tested isolates (all were biofilm producers and type 3 fimbriae adhesion gene [mrkD] positive). Sequence analysis of OXA-48, qnrS, and IntI1 revealed 100% identity with published sequences, while sequencing of qnrA, OmpK-35, and iron regulatory protein gene (irp2) showed minor variations in the form of one or few single-nucleotide polymorphism. Altogether, the current study revealed that all MDR, XDR, and PDR K. pneumoniae isolates were multivirulent and all harbored 3-5 virulence genes and 2-9 antimicrobial resistance genes and exhibited 8 and 10 different virulence and antimicrobial resistance profiles, respectively. In this study, we also report a positive correlation between some virulence genes and antimicrobial resistance genes among K. pneumoniae tested isolates.

Treated wastewater irrigation promotes the spread of antibiotic resistance into subsoil pore-water

In the present study, we investigated the impact of treated wastewater (TWW) irrigation on the prevalence of antibiotic resistance genes (ARGs) in subsoil pore-water, a so-far under-appreciated matrix. We hypothesized that TWW irrigation increases ARG prevalence in subsoil pore-water. This hypothesis was tested using a multiphase approach, which consisted of sampling percolated subsoil pore-water from lysimeter-wells of a real-scale TWW-irrigated field, operated for commercial farming practices, and controlled, laboratory microcosms irrigated with freshwater or TWW. We monitored the abundance of six selected ARGs (sul1, blaOXA-58, tetM, qnrS, blaCTX-M-32 and blaTEM), the intI1 gene associated with mobile genetic elements and an indicator for anthropogenic pollution and bacterial abundance (16S rRNA gene) by qPCR. The bacterial load of subsoil pore water was independent of both, irrigation intensity in the field study and irrigation water type in the microcosms. Among the tested genes in the field study, sul1 and intI1 exhibited constantly higher relative abundances. Their abundance was further positively correlated with increasing irrigation intensity. Controlled microcosm experiments verified the observed field study results: the relative abundance of several genes, including sul1 and intI1, increased significantly when irrigating with TWW compared to freshwater irrigation. Overall, TWW irrigation promoted the spread of ARGs and intI1 in the subsoil pore-water, while the bacterial load was maintained. The combined results from the real-scale agricultural field and the controlled lab microcosms indicate that the dissemination of ARGs in various subsurface environments needs to be taken into account during TWW irrigation scenarios.

Investigation of reduction in risk from antibiotic resistance genes in laboratory wastewater by using O3 , ultrasound, and autoclaving.

Biological laboratory wastewater containing both antibiotic-resistant bacteria (ARB) and antibiotics is a potential source of antibiotic resistance genes (ARGs). Thus, we determined the efficacy of autoclaving, a common disinfection method, in eliminating 5 ARGs (sul1, sul2, tetW, tetM, amp) and the integrase-encoding gene intI1 from laboratory wastewater. Autoclaving (15min, 121°C) inactivated all bacteria including ARB, whereas ARGs persisted in the wastewater with limited reduction even after 60min of treatment. Ozonation (O3 ), ultrasound (US), O3 /US, and autoclaving followed by O3 were investigated for their ability to reduce ARGs in laboratory wastewater. With O3 and O3 /US, the reduction rate ranged from 5.44 to 7.13 log for all ARGs investigated. Wastewater treatment with US alone did not reduce ARGs under the present experimental conditions (150W, 53kHz). Among the four treatments, autoclaving followed by O3 treatment showed the highest reduction rates in the shortest time; however, further optimization and investigation are needed for the advanced treatment of bio-laboratory wastewater. Overall, this study provides novel insights into ARG sources and demonstrates that advanced oxidation methods can be useful to optimize laboratory wastewater treatment for ARG inactivation. PRACTITIONER POINTS: Bio-laboratory wastewater is potential reservoir of ARGs. Conventional autoclaving was not able to reduce ARGs to a low level. Autoclaving-O3 completely eliminate all the bacteria. Autoclaving-O3 reduced ARGs efficiently (6.12-7.86 logs removal in 60min).

Diverse Gene Cassette Arrays Prevail in Commensal Escherichia coli From Intensive Farming Swine in Four Provinces of China.

Multiple-drug resistance bacteria containing antimicrobial resistance genes (ARGs) are a concern for public health. Integrons are bacterial genetic elements that can capture, rearrange, and express mobile gene cassettes responsible for the spread of ARGs. Few studies link genotype and phenotype of swine-related ARGs in the context of mobile gene cassette arrays among commensal Escherichia coli (E. coli) in nonclinical livestock isolates from intensive farms. In the present study, a total of 264 isolates were obtained from 330 rectal swabs to determine the prevalence and characteristics of antibiotic-resistant gene being carried by commensal E. coli in the healthy swine from four intensive farms at Anhui, Hebei, Shanxi, and Shaanxi, in China. Antimicrobial resistance phenotypes of the recovered isolates were determined for 19 antimicrobials. The E. coli isolates were commonly nonsusceptible to doxycycline (75.8%), tetracycline (73.5%), sulfamethoxazole-trimethoprim (71.6%), amoxicillin (68.2%), sulfasalazine (67.1%), ampicillin (58.0%), florfenicol (56.1%), and streptomycin (53.0%), but all isolates were susceptible to imipenem (100%). Isolates [184 (69.7%)] exhibited multiple drug resistance with 11 patterns. Moreover, 197 isolates (74.6%) were detected carrying the integron-integrase gene (intI1) of class 1 integrons. A higher incidence of antimicrobial resistance was observed in the intI1-positive E. coli isolates than in the intI1-negative E. coli isolates. Furthermore, there were 17 kinds of gene cassette arrays in the 70 integrons as detected by sequencing amplicons of variable regions, with 66 isolates (94.3%) expressing their gene cassettes encoding for multiple drug resistance phenotypes for streptomycin, neomycin, gentamicin, kanamycin, amikacin, sulfamethoxazole-trimethoprim, sulfasalazine, and florfenicol. Notably, due to harboring multiple, hybrid, and recombination cassettes, complex cassette arrays were attributed to multiple drug resistance patterns than simple arrays. In conclusion, we demonstrated that the prevalence of multiple drug resistance and the incidence of class 1 integrons were 69.7 and 74.6% in commensal E. coli isolated from healthy swine, which were lower in frequency than that previously reported in China.

Prevalence of resistance and toxin genes in community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus clinical isolates.

Objective(s):Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major health hazards and became of greater public health concern since the emergence of community-acquired MRSA. This work aimed to study the prevalence of mecA, femA, femB, lukS-PV, lukF-PV (PVL), intI, and intII genes among community-acquired (CA) hospital-acquired (HA) MRSA to increase vigilance in the diagnosis and management of suspected infections. Materials and Methods: S. aureus isolates recovered from clinical samples were classified into community or hospital-acquired and tested for their antibiotic susceptibility against 19 antibiotics. All isolates were screened for mecA, femA, femB, lukS-PV, lukF-PV, intI, and intII genes. Statistical correlations were carried out.Results:Out of 338 S. aureus isolates, only 105 were MRSA and classified as 77 CA-MRSA and 28 HA-MRSA. mecA and femA genes were present in all HA-MRSA and CA-MRSA isolates. femB was found in all HA-MRSA and 93.5% of CA-MRSA isolates. PVL genes were detected in 28.6% HA-MRSA isolates and 92.2% CA-MRSA. intI gene was recovered from 60.7% HA-MRSA isolates and 37.7% CA-MRSA isolates while the intII gene recovered from only 10.7% HA-MRSA isolates and 6.5% CA-MRSA.Conclusion:The high prevalence of MRSA colonizing the groin, axilla, and nose may play a significant role in endogenous infection, re-infection, and also acts as a route for MRSA transmission. mecA and femA genes could be used as a sole and fast step for identification of MRSA, while PVL genes cannot be used as a sole stable marker for CA-MRSA identification.

Simultaneous removal of multidrug-resistant Salmonella enterica serotype typhimurium, antibiotics and antibiotic resistance genes from water by electrooxidation on a Magnéli phase Ti4O7 anode

Antibiotics and multidrug resistant bacteria are present extensively in different environmental matrices, and considered as contaminants of emerging concern. This study evaluated the feasibility and effectiveness of using electrooxidation (EO) with Magnéli phase Ti4O7 anode to simultaneously remove bacterial pathogens, antibiotics and antibacterial resistance genes (ARGs) from water. A model pathogen, multidrug-resistant Salmonella enterica serotype Typhimurium DT104, was completely deactivated with 6.2 log reduction in 15 min at the current density of 10.0 mA cm−2, while ARGs (i.e., TetG, floR and sul1), class 1 integron gene (intI1) and virulence genes (invA and spvC) that were contained in the pathogen were removed by as much as 99.65% to 99.94%. Model antibiotics, tetracycline (TC) and sulfadimethoxine (SDM), were degraded 97.95 % and 93.42 % in the same EO treatment system within 3 h. It was found that both direct electron transfer (DET) and OH-mediated oxidation played important roles in the removal of the ARGs during the EO process. EO may be a promising tool to control the spread of antibiotic resistence by removing pathogens, antibiotics and ARGs simultaneously.

Multidrug Resistance in Integron Bearing Klebsiella pneumoniae isolated from Alexandria University Hospitals, Egypt.

Klebsiella pneumoniae is by far one of the most common Enterobacteriaceae associated with hospital-acquired infections. The dissemination of multi drug resistant Klebsiella pneumoniae is causing difficulty to treat infections worldwide. Of additional concern, multi drug resistant Klebsiella pneumoniae acquires and transfers antibiotic resistance genes among other bacterial isolates. Integrons have the main role in the acquisition as well as dissemination of resistance genes. Accordingly we aimed to investigate the frequency of resistance genes sul1, sul2, tetA, tetB and aac (3) IIa, class one (int1 gene) and class two integrons(int2 gene) in Klebsiella pneumoniae clinical isolates from four major hospitals in Alexandria, Egypt using Polymerase Chain Reaction. In addition we aimed to evaluate the association between multidrug resistance and presence of integrons in hospital-acquired Klebsiella pneumoniae in our hospitals. To the best of our knowledge, there is little information about integrons and acquisition of multiple antibiotic resistance in Klebsiella pneumoniae in hospitals in Alexandria, Egypt. In this study 76 isolates were resistant to sulphamethoxazole /trimethoprim. Of these 38 isolates (50%) harbored both genes sul1 and sul2 genes. 42 isolates out of the 60 (70%) isolates that showed resistance to tetracycline were tetA or tetB positive. The prevalence of int1 gene among all isolates tested was 90%, while only one isolate harbored the int2 gene (1%). Our results were indicative of the high prevalence of multidrug resistant Klebsiella pneumoniae as well as integrons that were found to play an essential role in the dissemination of antibiotic resistance genes in our hospitals.

Antibiotic-Resistant Bacteria in Hydroponic Lettuce in Retail: A Comparative Survey.

Hydroponic produce is gaining popularity due to its suitability for urban agriculture. The general public also considers that hydroponic produce is free from microbiological contamination. In this study, we compared the frequency and abundance of tetracycline-resistant and sulphadiazine-resistant bacteria and the minimal inhibitory concentration (MIC) of these isolates in conventional, organic, and hydroponic lettuce sold in retail. We also determined the frequency of samples carrying tetB, tetX, sul1, sul2, and int1 genes by PCR and further quantified the copy number of tetX, sul1, and int1 genes in samples positive for these genes using qPCR. As expected, the number of resistant bacteria and the MICs of these isolates were lowest in hydroponic lettuce and highest in organic lettuce. All tested resistant genes, except int1, were detected in samples of all three production methods, but no significant difference was observed between the three groups in the frequency of samples carrying the resistance genes examined or in their copy number. To the best of our knowledge, it is the first study directly reporting the existence of antibiotic-resistant bacteria and resistance genes in hydroponic vegetables sold in retail. The result highlights that the risk of antibiotic-resistant bacteria contamination in hydroponic produce should be further investigated.

Genotyping and Virulence Analysis of Drug Resistant Clinical Klebsiella pneumoniae Isolates in Egypt

Klebsiella pneumoniae is a highly drug-resistant human pathogen responsible for a variety of serious infections. Integrons, mobile genetic elements capable of integrating antibiotic resistance genes, and the capsule are important virulence factors that increase bacteria resistance to phagocytosis and antimicrobial agents. Molecular typing is an effective tool for identifying the likely etiology of infection. This study aimed to investigate the presence of the rmpA, wcaG, intI1, intI2, and intI3 virulence genes in clinical Klebsiella pneumoniae isolates, and explore their molecular genotypes by using ERIC-PCR. Fifty Klebsiella pneumoniae strains were isolated from various specimens. Antimicrobial resistance was evaluated by using the disc diffusion method. Five genes were amplified by conventional PCR. Genotyping was performed molecularly by using ERIC-PCR. Forty-seven isolates were multi-drug resistant. In all, 18%, 36%, and 98% of the 50 K. pneumoniae isolates were positive for rmpA, wcaG, and intI1 genes, respectively; however, all isolates were negative for intI2 and intI3 genes. Dendogram analysis of the ERIC-PCR results showed 49 distinct patterns, arranged in five clusters. Our study demonstrates high levels of antibiotic resistance and virulence among clinical isolates of K. pneumoniae. Such resistance reflects a growing problem for public health. Further, the presence of integrons increases the horizontal spread of antibiotic resistance and virulence genes among bacterial isolates. The ERIC-PCR technique is an effective method for molecular typing and epidemiological studies of hospital-acquired infections.

Molecular Profiling of Class I Integron Gene in MDR Salmonella typhi Isolates

Typhoid fever is a paramount reason for horribleness that more mortal sin “around the sum ages aggregations clinched alongside iraq it initiated by salmonella typhi. Salmonella typhi is diagnosed serologically by the Widal test and confirmed by vitek and using polymerase chain reaction (PCR) based amplification of DNA from the bacterial samples of typhoid fever patients. The present study was designed to detect class I integron gene encoding antimicrobial of S. typhi using appropriate primers by PCR. These isolates of this study were collected from postgraduate laboratories (Prepared samples in vitro prepared diagnostics), they were a previous collected from carried out in Al Najaf provenance, throughout those period from July 2018 on March 2019 including 231 cases from blood, stool samples collected from patients suffering from typhoid fever were attended to Al-Sader Medical City and Al-Hakim General Hospital in Al-Najaf province. Biochemically tests and monovalent antisera gave 117 (50.64%) positive result S. typhi isolates and confirmed by Vitek system and PCR which showed positive result 59 (50.42%). Fifty nine isolates of S. typhi, were collected from patients with typhoid fever that distributed to 40/59 (34 %) from blood , 19/59 (15.1%) stool. Molecular detection revealed that most isolates of S. typhi were positive results to (intI) gene 43/59 isolate (the specific primer (intI) gene for S. typhi bacteria was designed in this study by using bioinformatics programs with NCBI website). According to the different diagnostic above, Vitek and PCR method were more sensitivity technique for S. typhi detection among typhoid patients. The results of virulence factors of S.typhi isolates were negative results for gelatinase, hemolysin, protease and capsulated. Multidrug resistance (MDR) of S. typhi isolates were represented by 18 antibiotics resistance to class and sub class of antibiotic. All S. typhi isolates appeared high resistance 100% to Aztreonam (AZM15), Nitrofurantion (F), Amoxicillin/clavulanicacid (AMC30), (PY25), Clarithromycin (CLR), Cefoxitin (FOX30), Penecillin(P10), Cefotaxime (CTX30), ampicillin (AMP), Meropenem (MEM), Tetracycline(TE30). Also resistance of isolates that revealed 91% to Impinem (IP ), 88% Ampicillin (AM10), 85%Amoxillin (AX), 81% Gentamicin (CN10), 80% Chloramphenicol (C30), 74% Cefpirome (CPR) and 68% Carbenicillin (CB).

Occurrence of emerging sulfonamide resistance (sul1 and sul2) associated with mobile integrons-integrase (intI1 and intI2) in riverine systems

Global use of antibiotics has exceedingly enhanced in agricultural, veterinary and prophylactic human use in recent days. Hence, these antibiotics can easily be found in the environment. This study revealed the occurrence of emerging MDR and ESBL producing strains, pollution profile, and factors integrons (intI1 and intI2) and environmental factors associated, in the riverine systems under different ecological and geo-climatic zones were investigated. The samples were collected based on anthropogenic intervention such as discharge of domestic wastes, industrial wastes, hospital, and municipal wastes. Among 160bacterial morphotypes, 121 (75.62%) exhibited MDR trait with maximum resistance towards lincosamide (CD = 71.3%), beta-lactams (P = 70.6%; AMX = 66.3%), cephalosporin (CZ = 60.6%; CXM = 34.4%), sulfonamide (COT = 50.6%; TR = 43.8%) followed by macrolide (E = 29.4%), tetracycline (TET = 18.8%), aminoglycosides (S = 18.8%; GEN = 6.3%), fluoroquinolones (NX = 18.1%; OF = 4.4%) and carbapenem (IPM = 5.0%). IntI1 gene was detected in 73 (60.3%) of isolates, whereas intI2 was found in 11 (9.09%) isolates. Eight (6.61%) isolates carried both integron genes (intI1 and intI2). sul1 and dfrA1 genes were detected in 53 (72.6%) and 63 (86.3%) isolates, respectively. A total of 103 (85.1%) were found ESBL positive with the presence of ESBL genes in 100 (97.08%) isolates. In riverine systems most prevalent ESBL gene blaTEM (93.0%) was detected alone as well as in combination with bla genes. The data can be utilized for public awareness and regulation of guidelines by local governing bodies as an alarming threat to look-out against the prevalent resistance in environment thereby assisting in risk management during epidemics. This study is a comprehensive investigation of emerging antibiotic pollutants and its resistance in bacteria associated with factors integrons-integrase responsible for its dissemination. It may also assist in global surveillance of antibiotic resistance and policies to curtail unnecessary antibiotic use.

Characterization of Antibiotic Resistance and Metal Homeostasis Genes in Midwest USA Agricultural Sediments

Microbial antibiotic resistance is a naturally occurring phenomenon that has grown in part with the use of antibiotics in agricultural operation. There are also known connections between genes for metal homeostasis and antibiotic resistance, and either antibiotics or metals may select for both kinds of genes. Antibiotics, metals, and their associated genes have the potential to enter agricultural drainage channels and migrate to downstream locations through receiving water bodies. A relatively new agricultural best management practice—the two-stage channel design—functions by sequestering sediments and dissolved constituents as they flow through agricultural ditches from surface runoff and tile drainage discharge. Sedimentation in agricultural watersheds may entrap aggregate pollutants including antibiotics, metals, and associated resistance genes before transport into the drainage system. Here, we characterized the abundance and diversity of 22 antibiotic resistance and metal homeostasis genes in three two-stage channels that had self-developed in an area dominated by agricultural land use. Additionally, we analyzed the sediments for 17 antibiotics and nine metals that could affect the selection of these genes. In these rural systems that drain into Lake Erie, the abundance of antibiotic resistance and metal homeostasis genes were on the lower end of ranges (e.g., &lt;106 gene copies g−1 of intI1) reported in other riverine and lake systems, with only five genes—intI1, aacA, mexB, cadA, and merA—differing significantly between sites. The diversity of antibiotic resistance and metal homeostasis genes for these sediment samples were largely similar to other human impacted environments. Few antibiotics were detected in two stage channel sediments, with concentrations below the quantifiable limits (&lt;0.02–34.5 µg kg−1 soil) in most cases. Likewise, metals were present at what could be considered background concentrations. Despite serving as important drainage channel features in a region dominated by agricultural land use, results serve as an important baseline reference against which other monitoring studies can be compared to assess the perturbation of antibiotics and metals on agricultural channel sediments.