Int Gene Research Articles

Molecular Detection of Integrons I and II in Some Clinical Isolates from Niger Delta University Teaching Hospital, Okolobiri, Nigeria

Aim: This study aimed at detecting the integron-integrase I and II gene in Gram negative bacteria. Study Design: Cross-sectional and descriptive study design were utilized in this study. Place and Duration of Study: Niger Delta University Teaching Hospital Okolobiri and Niger Delta University Molecular Laboratory CHS, Wilberforce Island, Amassoma, between February 2023 and April 2023. Methodology: This study was specifically carried out on clinical isolates with a population figure obtained from 6 different samples collected from Niger Delta University Teaching Hospital, okolobiri in Bayelsa state. Of 500 different clinical samples, 100 clinical isolates were isolated and identified using Standard Bacteriological methods. Genomic DNA was extracted by boiling method. The Integrons I and II genes were detected using Polymerase Chain Reaction. Results: Of the 100 isolates used for molecular analysis, the Int I1 gene was detected in 54 isolates of which 8(44.4%) were Escherichia coli,14(63.63%) were Pseudomonas aeruginosa and 14(70%) were Klebsiella oxytoca. IntI2 gene was detected in 78 isolates of which16(88.88%) were Escherichia coli,20(90.90%) was Pseudomonas aeruginosa and 10(50%) were Klebsiella oxytoca. IntI1 and IntI2 genes were detected in 38 isolates of which9(50%) were Escherichia coli,12(54.54%) was Pseudomonas aeruginosa and 6(30%) were Klebsiella oxytoca. Conclusion: This study showed that there was a high prevalence of class I and II integrons in multi-drug-resistant Gram-negative bacteria, therefore justifying the extent of resistance observed.

Bacterial communities and tetracycline resistance genes in the soil-rice continuum upon tetracycline exposure

Edible plants are known as key vectors for transmission of antibiotic resistance genes (ARGs) to humans. However, little is known about how antibiotic exposure affects the abundance of ARGs in the soil-rice continuum. This study assessed tetracycline (TC) content, tetracycline resistance genes (TRGs), class 1 integron-integrase gene (intI1), and bacterial communities in the mature soil-rice continuum exposed to varying concentrations of TC (0, 50, 100, and 300 ppm). As TC exposure levels increased, the TC content in rhizosphere soils and roots increased accordingly. However, TC residue was not detected in the grains. Additionally, sample type (rhizosphere soils, roots, and grains) was the key determinant of bacterial communities and TRGs profiles in the soil-rice continuum. The bacterial communities and TRGs profiles in the rhizosphere soils and roots exposed to TC >50 ppm were more dissimilar to those without TC than to those exposed to TC <50 ppm. The abundance of TRGs in the soil-rice continuum increased with the TC concentrations but declined when TC > 50 ppm. This decline was primarily due to changes in the abundance of tetX and tetZ. Variation partitioning analysis revealed that the TRGs profiles in rhizosphere soils is strongly affected by bacterial communities. In contrast, the variation in TRGs profiles in roots was predominantly explained by bacterial communities-TC interactions. However, the changes in TRGs profiles of grains could not be attributed to the bacterial communities, TC, or intI1. This study provides valuable insights into the response of TRGs profiles in the soil-rice continuum to TC exposure.

The influence of urbanization and water reclamation plants on fecal indicator bacteria and antibiotic resistance in the Los Angeles River watershed: A case study with complementary monitoring methods.

Urban land use and water reclamation plants (WRPs) can impact fecal indicator bacteria (FIB) and antimicrobial resistance (AMR) in coastal watersheds. However, there is a lack of studies exploring these effects on the US West Coast. Additionally, there is limited research using a complementary approach across culture-, qPCR-, and metagenomics-based techniques for characterizing environmental AMR. In this study, sixteen locations were sampled in the Los Angeles River, encompassing both upstream and downstream of three WRPs discharging into the river. Culture-dependent methods quantified Enterococcus, total coliforms, E. coli, and extended spectrum beta-lactamase-producing E. coli as a low-cost screening tool for AMR, while qPCR measured selected antibiotic resistance genes (ARGs): sul1, ermF, tetW, blaSHV, along with intI1 and 16S rRNA genes. Bacteroides HF183 and crAssphage markers were quantified via ddPCR. All samples underwent shotgun sequencing to investigate gene abundance and mobility and an overall risk score for AMR. Results reveal downstream sites contain ARGs at least two orders of magnitude greater than upstream locations. Developed areas had the highest ARG sequence abundances and the most ARG classes as indicated by metagenomic analysis. WRP effluent exhibited elevated ARGs and co-location of ARGs, mobile genetic elements, and pathogens. A culture-based assessment of AR in E. coli and Pseudomonas aeruginosa revealed increased resistance ratios for most antibiotics from upstream to downstream a WRP discharge point. This study highlights the impacts of land use and WRPs on ARGs and FIB, offering a multi-pronged analysis of AMR.

Effects of sulfamethoxazole and copper on the natural microbial community from a fertilized soil

Cattle manure or its digestate, which often contains antibiotic residues, can be used as an organic fertilizer and copper (Cu) as a fungicide in agriculture. Consequently, both antibiotics and Cu are considered soil contaminants. In this work, microcosms were performed with soil amended with either manure or digestate with Cu and an antibiotic (sulfamethoxazole, SMX) co-presence and the planting of Lactuca sativa. After the addition of the organic amendments, a prompt increase in the microbial activity and at the same time of the sul1 and intI1 genes was observed, although ARGs generally decreased over time. In the amended and spiked microcosms, the microbial community was able to remove more than 99% of SMX in 36 days and the antibiotic did not bioaccumulate in the lettuce. Interestingly, where Cu and SMX were co-present, ARGs (particularly sul2) increased, showing how copper had a strong effect on resistance persistence in the soil. Copper also had a detrimental effect on the plant-microbiome system, affecting plant biomass and microbial activity in all conditions except in a digestate presence. When adding digestate microbial activity, biodiversity and lettuce biomass increased, with or without copper present. Not only did the microbial community favour plant growth, but lettuce also positively influenced its composition by increasing bacterial diversity and classes (e.g., Alphaproteobacteria) and genera (e.g., Bacillus), thus indicating a good-quality soil.Key points• Cattle digestate promoted the highest microbial activity, diversity, and plant growth• Cattle digestate counteracted detrimental contaminant effects• Cu presence promoted antibiotic cross-resistance in soilGraphical

A single-center analysis of clonal transmission of carbapenem-resistant Acinetobacter baumannii among intensive care unit patients during the COVID-19 pandemic

Carbapenem-Resistant Acinetobacter baumannii (CRAB) outbreak in intensive care units (ICUs) is a significant problem for healthcare facilities. In this study, we aimed to investigate the occurrence of CRAB isolates among ICU-admitted patients during the three waves of the COVID-19 pandemic in Iran using Multiple-Locus Variable Number Tandem-Repeat Analysis (MLVA). We obtained 50 (A) baumannii isolates from tracheal aspirate and blood culture samples. In the disc diffusion method, all isolates were cefotaxime, ceftriaxone, and cefepime-resistant, while 98% (49/50) of isolates were resistant to piperacillin, piperacillin-tazobactam, ceftazidime, and ciprofloxacin. Levofloxacin and tobramycin resistance was found in 76% (38/50) of isolates. In the microbroth dilution test all isolates were resistant to imipenem, 98% (49/50) to meropenem, 68% (34/50) to colistin, and 20% (10/50) to polymyxin (B) Based on the PCR findings, all isolates harbored blaOXA−40, ISAba-1, and int-2 genes. There were no isolates found that have the blaOXA−58, blaOXA−143, blaVEB−1, blaVIM, and int-3 genes. Among Extended-spectrum beta-lactamases (ESBL) genes, blaCTX−M, blaTEM, blaSHV, blaGES, and blaPER−1 have a prevalence of 42% (21/50), 84% (42/50), 58% (29/50), 78% (39/50), and 54% (27/50), respectively. 74% (37/50) of the isolates had the blaOXA−23 gene, while all of the isolates carried the blaOXA−40 gene. Among MBL genes, blaIPM, blaGIM, blaSIM, and blaNDM−1 have a prevalence of 20% (10/50), 8% (4/50), 22% (11/50), and 60% (30/50), respectively. The prevalence of int-1 was documented as 74% (37/50). Accordingly, all isolates were identified as CRAB. The co-existence of blaOXA−23/int-2 and blaOXA−23/isaba-1 was 74% (37/50). The co-existence of blaNDM−1/ISAba-1 was observed in 30 (60%) isolates. Using an 80% similarity threshold on the dendrogram constructed through MLVA typing, all isolates were grouped into two clusters: cluster A with 9 isolates from wave 3, and cluster B with 41 isolates from waves 3, 4, and 5. Our study confirms a clonal transmission of CRAB during the study period and suggests using molecular typing methods like MLVA in healthcare settings to identify dominant clones, antibiotic resistance patterns, and transmission routes. This will help to better manage the emergence and spread of antibiotic-resistant strains in future outbreaks.

Removing high strength lincomycin in pharmaceutical wastewater by a bacteria microalgae consortium co-immobilized filter

Lincomycin (LIN) in pharmaceutical wastewater would enter municipal wastewater treatment plants and decrease their performance, leading to residual LIN enter the natural environment and pose serious eco-risk. In this study, a bacterium-microalgae consortium co-immobilized filter (BMCCF) was established and used to remove LIN in artificial pharmaceutical wastewater treatment plants effluents (PWWTPE). LIN removal mechanisms and degradation products’ eco-toxicity was studied, and the abundance change of class 1 integrase gene (intI1) and antibiotic resistance genes (ARGs) was monitored. As a result, 98.54% of 82 mg L-1 LIN was removed within 7 days, LIN removal was mainly attributed to bio-degradation by the Bacillus subtilis strain, and LIN degradation products were less toxic than their substrate. Therefore, the BMCCF established in this study provides a promising alternative for the bio-treatment of pharmaceutical wastewater containing high concentration of LIN.

Evaluation of antibiotics resistance and transmission risk of Escherichia coli in rice-frog coculture system

In order to assess the antibiotic resistance of Escherichia coli and its transmission risk in a rice-frog coculture system in Zhejiang Province, this study collected E. coli from isolated soil, field water, and frog feces from the rice-frog coculture systems in four different areas of Zhejiang Province. The collected isolates were identified by 16S rRNA sequencing, while their antibiotic-resistant phenotypes were determined by Kirby-Bauer (K-B) method. PCR was used to identify the antibiotic-resistant genotypes and integrons, while conjugative transfer experiments were used to assess resistance transmission characteristics. The results showed a high prevalence of antibiotic resistance in the 82 strains of E. coli tested, primarily against tetracycline, sulfisoxazole, amoxicillin, and erythromycin. Most of these strains exhibited multidrug resistance, with the Fuyang area demonstrating the highest resistance rate compared to the other three areas. Further PCR analysis identified the sul1 gene as the most frequently detected resistance gene (63.41%), followed by blaTEM, tetA, and tetB. Among the 16 antibiotic resistance genes (ARGs) detected, the Fuyang isolates consistencly exhibited higher detection rate of 9 ARGs in comparison to the other regions. Additionally, the integrase gene intI1 displayed the highest detection rate, with 14 strains (34.15%) of integrase-positive bacteria carrying gene cassettes. Four different gene cassette compositions were observed, with dfrA1-aadA1 and dfrA17-aadA5 being the most common combinations. Conjugative transfer experiments demonstrated successful transfer of gene cassettes in 4 out of 14 donor bacteria, with conjugation transfer frequencies ranging from 4.32×10-5 to 7.13×10-4. These findings revealed the severity of resistance in the Fuyang area among the four regions. Integrons play a significant role in mediating the resistance to multiple antibiotics in E. coli, facilitating the potential spread of resistance gene cassettes between different bacteria. Overall, this study provides valuable insights into the resistance status and transmission characteristics of E. coli in the rice-frog coculture system in Zhejiang Province, providing a theoretical basis for ensuring the food safety of rice crops.

Prevalence of Resistance Genes Among Multidrug-Resistant Gram-Negative Bacteria Isolated from Waters of Rivers Swat and Kabul, Pakistan.

The waters of rivers Swat and Kabul are the main water source for domestic and irrigation purposes in the northwestern part of Pakistan. However, this water has been contaminated due to human activities. This study aimed to analyze the water of these rivers for occurrence of antibiotic resistance genes among Gram-negative bacteria. Samples were collected from 10 different locations of these rivers. The samples were processed for the isolation of Gram-negative bacteria. Isolated bacteria were checked against 12 different antibiotics for susceptibility. The isolates were also analyzed for the presence of seven antibiotic resistance genes. A total of 50 bacterial isolates were recovered that belonged to five different bacterial genera, that is, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Raoultella terrigena (Klebsiella terrigena), and Pseudomonas fluorescens. Antibiotic resistance pattern was cefixime 72%, cephalothin 72%, ampicillin 68%, nalidixic acid 68%, kanamycin 54%, streptomycin 42%, sulfamethoxazole-trimethoprim 28%, chloramphenicol 28%, meropenem 8%, gentamicin 8%, amikacin 2%, and tobramycin 2%. The prevalence of bla-TEM gene was 72% (n = 36), aadA gene 34% (n = 17), sul gene 32% (n = 16), bla-CTXM gene 12% (n = 6), int gene 66% (n = 33), and int1 gene 6% (n = 3). This information highlights the need for controlling and monitoring the release of domestic wastes to rivers.

Quinolone and Tetracycline-Resistant Biofilm-Forming Escherichia coli Isolates from Slovak Broiler Chicken Farms and Chicken Meat

Escherichia coli isolates from intensive poultry production are associated with antimicrobial resistance and worldwide health problems. The aim of the study was to detect and evaluate the phenotypic and genotypic antimicrobial resistance, biofilm formation, phylogenetic typing, and virulence factors in E. coli isolates from the rectal swabs of chickens from two farms and swabs of chicken meat purchased from Slovakian food markets. Interpretative readings of minimal inhibitory concentration (MIC) revealed dominant resistance to ampicillin (&gt;50%) in both groups. We also detected higher resistance to ciprofloxacin (45%), tetracycline, ampicillin + sulbactam, and trimethoprim + sulfonamide (each &gt;30%). Here, 28.57% of the strains studied were multidrug-resistant (MDR). The formation of weak biofilms was confirmed in 8.8% of E. coli, while one of the strains obtained from chicken cloacal swabs was classified as a strong biofilm producer. The most frequently confirmed phylogenetic groups in E. coli were B1 and A1 in all groups. PCR detection revealed the presence of genes encoding tetracycline resistance (tetAB) and plasmid-mediated quinolone resistance (qnrABS), and Int1 (52.9%), Tn3 (76.5%), kpsMT II (8.8%), fimA (97.1%), cvaC (38.2%), and iutA (76.5%) genes in the strains studied. Our results demonstrate that chickens and chicken meat were the source of antibiotic-resistant, biofilm-forming, and virulent E. coli, representing a potential risk from the point of view of the One Health concept.

Fate of fluoroquinolones associated with antimicrobial resistance in circular periurban agriculture

Animal antibiotic use contributes to antimicrobial resistance (AMR) in humans. While animal manure benefits soil fertility, it also acts as hotspot for antibiotic residues, antibiotic-resistant bacteria, and their genes. Amending soils with poultry litter is recognized as “magic” among horticulture farmers and it remains a common practice globally. However, this poses a risk especially in countries where prophylactic use of antibiotics is allowed. In Argentina, fluoroquinolones are used in this way besides being listed as essential medicines and classified as “watch” by the World Health Organization. Antibiotic selective pressure can favour AMR in the environment but the fate of antibiotic residues and AMR dissemination from these practices remains poorly understood. Our research addresses this gap with a biological model tracing fluoroquinolones from poultry to soil to lettuce and tracking anthropogenic AMR with the proposed biomarker genes sul1 and intI1. Fresh poultry litter was stored for six months before application in a horticulture field experiment. The experiment included control and manured plots where lettuce was cultivated till harvest. Enrofloxacin concentration was 7.3 μg/kg in fresh poultry litter, while its metabolite ciprofloxacin was 39.22 μg/kg after storage. Although no fluoroquinolones were detected in soils, lettuce from manured plots contained enrofloxacin and ciprofloxacin at 14.97 and 9.77 μg/kg, respectively, providing evidence of fluoroquinolone bioaccumulation in plants. Abundance of sul1 and intI1 in poultry litter was not affected by storage. Manured soils showed better soil quality than controls, but sul1 gene abundance was 1.6 times higher, reaching 7.61 Log sul1/g soil. A less sensitive, but significant effect was registered for intI1. These findings show that static storage is insufficient to stop the transmission of antibiotics and AMR biomarkers from poultry to horticulture. Amending soil with industrial poultry litter contributes to pollution with these emergent contaminants and risks human antibiotic exposure through fresh vegetables.

Metagenomic insights into plasmid-mediated antimicrobial resistance in poultry slaughterhouse wastewater: antibiotics occurrence and genetic markers.

Slaughterhouse wastewater represents important convergence and concentration points for antimicrobial residues, bacteria, and antibiotic resistance genes (ARG), which can promote antimicrobial resistance propagation in different environmental compartments. This study reports the assessment of the metaplasmidome-associated resistome in poultry slaughterhouse wastewater treated by biological processes, employing metagenomic sequencing. Antimicrobial residues from a wastewater treatment plant (WWTP) that treats poultry slaughterhouse influents and effluents were investigated through high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Residues from the macrolide, sulfonamide, and fluoroquinolone classes were detected, the latter two persisting after the wastewater treatment. The genetic markers 16S rRNA rrs (bacterial community) and uidA (Escherichia coli) were investigated by RT-qPCR and the sul1 and int1 genes by qPCR. After treatment, the 16S rRNA rrs, uidA, sul1, and int1 markers exhibited reductions of 0.67, 1.07, 1.28, and 0.79 genes copies, respectively, with no statistical significance (p > 0.05). The plasmidome-focused metagenomics sequences (MiSeq platform (Illumina®)) revealed more than 100 ARG in the WWTP influent, which can potentially confer resistance to 14 pharmacological classes relevant in the human and veterinary clinical contexts, in which the qnr gene (resistance to fluoroquinolones) was the most prevalent. Only 7.8% of ARG were reduced after wastewater treatment, and the remaining 92.2% were associated with an increase in the prevalence of ARG linked to multidrug efflux pumps, substrate-specific for certain classes of antibiotics, or broad resistance to multiple medications. These data demonstrate that wastewater from poultry slaughterhouses plays a crucial role as an ARG reservoir and in the spread of AMR into the environment.

Genomic analysis of Enterobacteriaceae from colorectal cancer patients at a tertiary hospital in Ghana: a case-control study.

Colorectal cancer (CRC) is a severe gastrointestinal cancer and a leading cause of cancer-related deaths in Ghana. The potential role of gut Enterobacteriaceae in the increasing incidence of CRC in Ghana is yet to be thoroughly investigated. In this study, Enterobacteriaceae from CRC patients and healthy control participants were analyzed by whole genome sequencing to identify genomic features that are associated with CRC. Socio-demographic data showed a significant association between age and alcohol consumption and CRC. Escherichia coli was the most abundant Enterobacteriaceae isolated from the study participants and they were predominantly intestinal commensals. Escherichia coli isolates belonging to phylogroup D encoded the highest number of virulence genes. The agn43 and int genes were widespread in Escherichia coli isolates from the CRC patients. Multilocus sequence types of potentially pathogenic Escherichia coli from the CRC patients also encoded genes involved in aggregation, adherence and biofilm formation. The ampC2 and ampH antimicrobial resistance genes were also widespread in the genome of the Escherichia coli isolates. This study highlights the virulence tendencies of Escherichia coli from CRC patients and their ability to transfer virulence determinants to other Enterobacteriaceae residing in the gut.

The simultaneous addition of chitosan and peat enhanced the removals of antibiotics resistance genes during biogas residues composting

Direct reuse of biogas residue (BR) has the potential to contribute to the dissemination of antibiotic resistance genes (ARGs). Although high-temperature composting has been demonstrated as an effective method for the harmless treatment of organic waste, there is few researches on the fate of ARGs in high-temperature composting of BR. This research examined the impact of adding 5% chitosan and 15% peat on physicochemical characteristics, microbial communities, and removal of ARGs during BR-straw composting in 12 Biolan 220L composters for 48 days. Our results showed that the simultaneous addition of chitosan and peat extended the high-temperature period, and increased the highest temperature to 74 °C and germination index. These effects could be attributed to the presence of thermophilic cellulose-decomposing genera (Thermomyces and Thermobifida). Although the microbial communities differed compositionally among temperature stages, their dissimilarity drastically reduced at final stage, indicating that the impact of different treatments on microbial community composition decreases at the end of composting. Peat had a greater impact on aerobic genera capable of cellulose degradation at thermophilic stage than chitosan. Surprisingly, despite the total copy number of ARGs significantly decreased during composting, especially in the treatment with both chitosan and peat, intl1 gene abundance significantly increased 2 logs at thermophilic stage and maintained high level in the final compost, suggesting there is still a potential risk of transmission and proliferation of ARGs. Our work shed some lights on the development of waste resource utilization and emerging contaminants removal technology.

Identification Of Metallo?-Lactamses and Integron Genes In Pseudomonas Aeruginosa Isolated From Burn Injury Patients: Phenotype And Genotype

Pseudomonas aeruginosa, which generates metallo-?-lactamase (MBL), is the cause of infections linked to burn patients, and this is a growing global problem. The objectives of the recent study were to determine antibiotic susceptibility of P. aeruginosa isolates, the presence of MBLs genes, and Integron gene class-1 . One hundred burn patients at the Al-Hussein Teaching Hospital in Al Smawah, Iraq, were isolated clinically. Twenty (20%) of these were determined to be P. aeruginosa by 16sRNA and biochemical testing. To further identify the antibiotic sensitivity for each isolate was employed by Disc Diffusion Method . The phenotypic MBL production For detected isolates was evaluated by Combined Disc Diffusion Method (CDD). Integron gene class -1 (Int-1) and Genes encoding MBLs were identified by Polymerase Chain Reaction (PCR). P. aeruginosa was shown to be totally resistant to ceftriaxone, ampicillin, piperacillin, gentamicin, and ciprofloxacin in this investigation. 90% of the isolates were determined to be multidrug resistant, and different levels of moderate to lowest resistance were observed in Amikacin, Meropenem, Aztreonam, Cefixime, and Levofloxacin. While 90% of the isolates possessed the Int-1 gene, our recorded the absence of All MBLs genes (Bla IMP, Bla VIM, Bla GIM ) From All isolates under investigation. Illustrating for the first time how crucial it is to put in place appropriate infection control measures in order to give patients the best care possible and stop the development of these resistant microbes among burn patients.

Dietary Artichoke (Cynara scolymus) Extract Ameliorated the Growth Performance, Humoral Immune Parameters and Resistance Against Aeromonas hydrophila in Goldfish (Carassius auratus)

Abstract This trial investigated the efficacy of artichoke (Cynara scolymus) extract (AE) on the growth performance, immunity, antioxidant parameters, and resistance against Aeromonas hydrophila of goldfish (Carassius auratus). For this purpose, a total number of 470 goldfish with initial weight 5.70±0.2 g were fed with four experimental diets including 0 (T0), 100 (T1), 150 (T2), and 200 (T3) mg kg−1 diet AE for 8 weeks. At the end of feeding trial, growth performances, serum immune parameters, and mucus antioxidant enzymes were measured. Fish were challenged with A. hydrophila, and the antioxidant and immunity-related gene expression were investigated. Based on the results, the highest final weight (FW) and weight gain (WG) were attained in T2 and T3 (P&lt;0.05). Immune factors including ACH50, lysozyme, and total immunoglobulin in T2 and T3 showed the highest values (P&lt;0.05). The expression of GR, IL1 β, TNF α, HSC70, HSP70, and HSP90 β genes in T1, T2, and T3 were higher than the control (P&lt;0.05). The GST expression was significantly enhanced in T2 (P&lt;0.05). The present study demonstrated that the administration of AE, especially at doses of 150 mg kg−1, could improve the growth, immunity, and antioxidant parameters, as well as enhance disease resistance against A. hydrophila in goldfish.

Occurrence of chlorine-resistant Pseudomonas aeruginosa in hospital water systems: threat of waterborne infections for patients

BackgroundSeveral healthcare-associated infection outbreaks have been caused by waterborne Pseudomonas aeruginosa exhibiting its ability to colonize water systems and resist conventional chlorine treatment. This study aims to investigate the occurrence of Pseudomonas aeruginosa in hospital drinking water systems and the antimicrobial resistance profiles (antibiotic and chlorine resistance) of isolated strains.MethodsWe investigated the presence of Pseudomonas aeruginosa in water and biofilms developed in nine hospital water systems (n = 192) using culture-based and molecular methods. We further assessed the survival of isolated strains after exposure to 0.5 and 1.5 ppm concentrations of chlorine. The profile of antibiotic resistance and presence of antibiotic resistance genes in isolated strains were also investigated.ResultsUsing direct PCR method, Pseudomonas aeruginosa was detected in 22% (21/96) of water and 28% (27/96) of biofilm samples. However, culturable Pseudomonas aeruginosa was isolated from 14 samples. Most of P. aeruginosa isolates (86%) were resistant to at least one antibiotic (mainly β-lactams), with 50% demonstrating multidrug resistance. Moreover, three isolates harbored intI1 gene and two isolates contained blaOXA−24,blaOXA−48, and blaOXA−58‌ genes. Experiments with chlorine disinfection revealed that all tested Pseudomonas aeruginosa strains were resistant to a 0.5 ppm concentration. However, when exposed to a 1.5 ppm concentration of chlorine for 30 min, 60% of the strains were eliminated. Interestingly, all chlorine-resistant bacteria that survived at 30-minute exposure to 1.5 ppm chlorine were found to harbor the intI1 gene.ConclusionsThe detection of antimicrobial resistant Pseudomonas aeruginosa in hospital water systems raises concerns about the potential for infections among hospitalized patients. The implementation of advanced mitigation measures and targeted disinfection methods should be considered to tackle the evolving challenges within hospital water systems.

Pneumococcal transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of serotype 19F in Indonesia

Genetic evolution of resistance due to mutations and transposon insertions is the primary cause of antimicrobial resistance in Streptococcus pneumoniae. Resistance to macrolide, tetracycline, and chloramphenicol is caused by the insertion of specific genes that carried by transposon (Tn). This study aims to analyze transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of S. pneumoniae serotype 19F in Indonesia. S. pneumoniae serotype 19F isolates were collected from nasopharyngeal swab specimens from different regions in Indonesia. Genomic DNA was extracted from sixteen isolates and whole genome sequencing was performed on Illumina platform. Raw sequence data were analyzed using de novo assembly by ASA3P and Microscope server. The presence of transposons was identified with detection of int and xis genes and visualized by pyGenomeViz. The genome size of S. pneumoniae ranges from 2,040,117 bp to 2,437,939 bp, with a GC content of around 39 %. ST1464 (4/16) and ST271 (3/16) were found as the predominant sequence type among isolates. Tn2010 was the most common transposon among S. pneumoniae serotype 19F isolates (7/16) followed by Tn2009 (4/16), and Tn5253 (3/16). We identified two deletion sites within the tetM gene (2 bp and 58 bp) that confer tetracycline susceptibility from one isolate. This study suggests that genomic analysis can be employed for the detection and surveillance of antimicrobial resistance genes among S. pneumoniae strains isolated from various regions in Indonesia.

Vegetable phylloplane microbiomes harbour class 1 integrons in novel bacterial hosts and drive the spread of chlorite resistance

Bacterial hosts in vegetable phylloplanes carry mobile genetic elements, such as plasmids and transposons that are associated with integrons. These mobile genetic elements and their cargo genes can enter human microbiomes via consumption of fresh agricultural produce, including uncooked vegetables. This presents a risk of acquiring antimicrobial resistance genes from uncooked vegetables. To better understand horizontal gene transfer of class 1 integrons in these compartments, we applied epicPCR, a single-cell fusion-PCR surveillance technique, to link the class 1 integron integrase (intI1) gene with phylogenetic markers of their bacterial hosts. Ready-to-eat salads carried class 1 integrons from the phyla Bacteroidota and Pseudomonadota, including four novel genera that were previously not known to be associated with intI1. We whole-genome sequenced Pseudomonas and Erwinia hosts of pre-clinical class 1 integrons that are embedded in Tn402-like transposons. The proximal gene cassette in these integrons was identified as a chlorite dismutase gene cassette, which we showed experimentally to confer chlorite resistance. Chlorine-derived compounds such as acidified sodium chlorite and chloride dioxide are used to disinfectant raw vegetables in food processing facilities, suggesting selection for chlorite resistance in phylloplane integrons. The spread of integrons conferring chlorite resistance has the potential to exacerbate integron-mediated antimicrobial resistance (AMR) via co-selection of chlorite resistance and AMR, thus highlighting the importance of monitoring chlorite residues in agricultural produce. These results demonstrate the strength of combining epicPCR and culture-based isolation approaches for identifying hosts and dissecting the molecular ecology of class 1 integrons.

Comparative performance of electronegative membrane filtration and automated concentrating pipette for detection of antibiotic resistance genes and microbial markers in river water samples

The target viral and bacterial concentrations in river water are essential for environmental monitoring and public health studies. Filtration-based methods are commonly employed, yet challenges arise due to recoverability and filter pore size. This study aimed to compare the performance of electronegative membrane filtration (EMF) and automated Concentrating Pipette (CP) Select (InnovaPrep) methods for quantifying antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial and viral markers in river water samples. Fifty-four river water samples were collected from upstream and downstream locations in a river in Japan. The CP Select method was modified by adding MgCl2 and using different tips. The recovery efficiencies for total coliforms and Escherichia coli were assessed, and class 1 integron-integrase gene (intI1), 16S rRNA, gene encoding sulfonamide resistance (sul1), cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), and Escherichia coli gene (sfmD) were detected. CP Select showed recovery efficiencies of 45 %–63 % for total coliforms and 17 %–35 % for E. coli. The intI1, 16S rRNA, sul1, crAssphage, PMMoV, and sfmD concentrations using the modified CP Select method were 10.1 ± 0.5, 8.7 ± 0.2, 7.7 ± 0.2, 6.7 ± 0.2, 5.4 ± 0.2, and 3.5 ± 0.5 log10 copies/L, respectively. Higher intI1 and sul1 concentrations were observed downstream, with the highest contribution percentage (22 % and 21 %) using CP Select or EMF. The modified CP Select method with 0.05 μm tips yielded more quantifiable results for all target genes and greater PMMoV concentrations (p < 0.05). Positive correlations were found among bacterial, ARG/MGE, and viral markers (Spearman's ρ = 0.71 for 16S rRNA and sfmD, 0.88 for intI1 and sul1, and 0.64 for PMMoV and crAssphage). The modified CP Select method demonstrated effective recovery of bacteria and quantification of ARGs, MGEs, and microbial markers in river water. Further studies are required to validate these methods and confirm their applicability in diverse environmental contexts.

Effects of polypropylene microplastics on digestion performance, microbial community, and antibiotic resistance during microbial anaerobic digestion

As an emerging pollutant, microplastics (MPs) have attracted increasing attention worldwide. The effects of polypropylene (PP) MPs on digestion performance, behaviors of dominant microbial communities, antibiotic resistance genes (ARGs) and mobile genetic elements in microbial anaerobic digesters were investigated. The results showed that the addition of PP-MPs to digesters led to an increase in methane production of 10.8% when 300 particles/g TSS of PP-MPs was introduced compared with that in digester not treated with PP-MPs. This increase was attributed to the enrichment of acetogens such as Syntrophobacter (42.0%), Syntrophorhabdus (27.0%), and Syntrophomonas (10.6%), and methanogens including Methanobacterium and Methanosaeta. tetX was highly enriched due to PP-MP exposure, whereas parC exhibited the greatest increase (35.5% – 222.7%). Horizontal gene transfer via ISCR1 and intI1 genes might play an important role in the spread of ARGs. Overall, these findings provide comprehensive insight into the ecological dynamics of PP-MPs during microbial anaerobic digestion.