Genome characteristics of a MDR Pseudomonas monteilii carrying a novel VIM-type ß-lactamase, blaVIM-84.

OBJECTIVES: This study aimed to determine the genetic environment and characterize plasmid carrying a novel VIM-type ß-lactamase (VIM-84) in a multidrug-resistant Pseudomonas monteilii isolate obtained from the human gut through whole-genome sequencing. METHODS: DNA extraction of P. monteilii L2757hy was performed using the Genomic DNA Isolation Kit (QIAGEN, Hilden, Germany). Whole-genome sequencing was performed by Illumina NovaSeq 6000 and Oxford Nanopore platforms. The transferability of resistance genes was screened single clonal on MHA plates containing rifampicin and meropenem. Verification was performed using MALDI/TOF-MS and PCR with Pseudomonas aeruginosa PAO1Ri as the recipient strain. RESULTS: L2757hy was identified as P. monteilii through sequencing and ANI analysis. The genome was assigned as ST147 and comprised a 6,130,057 bp chromosome with a GC content of 61.8% and a 49,704 bp plasmid. Several resistance genes, including blaIMP-1, aac(6')-IIa and tmexCD-toprJ, as well as virulence genes such as iroN, and wzaJ, were identified on the chromosome. A novel VIM-type blaVIM-84 was found on the plasmid, which was previously identified in Pseudomonas aeruginosa. Plasmid harboring blaVIM-84 was untypable, and it could be transferred to P. aeruginosa PAO1Ri and was associated with a class I integron with the genetic environment intI1-blaVIM-84-tniR-tniQ-tniB-tniA, likely derived from Tn402. CONCLUSIONS: Our study revealed that the novel blaVIM-84 gene was harbored by P. monteilii rather than P. aeruginosa. We suggested that P. monteilii may serve as a reservoir for resistance genes.

Distribution characteristics of integrons and correlation analysis of antibiotic resistance in urine isolated Enterobacter cloacae.

Objective: This study aims to understand the distribution of integrons among Enterobacter cloacae isolated from clinical urine specimens in our hospital, as well as the molecular characteristics of the variable region resistance gene cassette of integron-positive strains and its relationship with drug resistance. Methods: We collected a total of 80 strains of Enterobacter cloacae isolated from urine specimens of hospitalized patients in our hospital between August 2019 and July 2023, and conducted drug sensitivity testing on them. Polymerase Chain Reaction (PCR) technology was employed to screen these strains for Class 1, 2, and 3 integrons. Following this, the promoter and variable regions of integron-positive strains were amplified and sequenced. Additionally, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) was utilized for homology analysis of integron-positive strains. Results: Among the 80 clinical strains, Class 1 integrons were detected in 31 (38.8%) strains, and the following resistance gene cassettes were identified: aadA2, aadA1, aadB, aac(6'), and catB8. Three types of variable region promoters were observed: PcS (4 strains), PcW (7 strains), and PcH1 (17 strains), with consistently inactive downstream P2 promoters. Additionally, Class 2 integrons were detected in 5 (6.3%) strains, carrying the variable region resistance gene cassette dfrA1-sat2-aadA1. The promoters for Class 2 integrons were uniformly of the Pc2D-Pc2A-Pc2B-Pc2C type. No Class 3 integrons were detected. The strains containing integrons showed significantly higher resistance rates to ciprofloxacin, compound sulfamethoxazole, levofloxacin, gentamicin, amikacin, and tobramycin compared to those without integrons (P<0.05). 35 strains of Enterobacter cloacae carrying integrons are primarily classified into three genotypes: A, B, and C. These genotypes are mainly distributed in the urology department and Intensive Care Unit (ICU). The distribution of variable region gene boxes and promoter types is relatively concentrated in the same genotype. Conclusion: Our study confirmed that Enterobacter cloacae isolated from urine samples predominantly carries Class 1 integrons with an extended array of antibiotic-resistant genes. For future research, it is recommended to explore additional resistance mechanisms and evaluate the effectiveness of new therapeutic strategies. Clinicians should be vigilant about the possibility of clonal dissemination and implement enhanced infection control measures in hospital settings.

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.

Microalgae Enhances the Adaptability of Epiphytic Bacteria to Sulfamethoxazole Stress and Proliferation of Antibiotic Resistance Genes Mediated by Integron.

The transmission of ARGs in the microalgae-associated epiphytic bacteria remains unclear under antibiotic exposure, apart from altering the microbial community structure. In this study, Chlorella vulgaris cocultured with bacteria screened from surface water was examined to explore the spread of ARGs in the presence of sulfamethoxazole (SMX). The extracellular polymers released by Chlorella vulgaris could reduce antibiotic-induced collateral damage to bacteria, thus increasing the diversity of the microalgae-associated epiphytic bacteria. The abundances of sul1 and intI1 in the phycosphere at 1 mg/L SMX dose increased by 290 and 28 times, respectively. Metagenomic sequencing further confirmed that SMX bioaccumulation stimulated the horizontal transfer of sul1 mediated by intI1 in the microalgae-associated epiphytic bacteria, while reactive oxygen species (ROS)-mediated oxidative stress induced the SOS response and thus enhanced the transformation of sul1 in the J group. This is the first study to verify that microalgae protect bacteria from antibiotic damage and hinder the spread of ARGs mediated by SOS response, while the transfer of ARGs mediated by integron is promoted due to the bioaccumulation of SMX in the phycosphere. The results contribute to present comprehensive understanding of the risk of ARG proliferation by the presence of emerging contaminants residues in river.

A Comprehensive Study on the Distribution of Integrons and Their Gene Cassettes in Clinical Isolates.

Antibiotic resistance is a significant global health concern, leading to increased morbidity, mortality, and health care costs. Integrons are genetic elements that could acquire and express gene cassettes, including those that confer antibiotic resistance. This comprehensive study focused on the distribution of integrons and their gene cassettes in clinical isolates. This study explored the structure and classification of integrons with particular emphasis on Class I, II, III, and IV integrons. It also discussed the role of integrons in antibiotic resistance. The findings of this study contribute to a better understanding of the mechanisms underlying antibiotic resistance and provide valuable insights for developing strategies to combat this public health crisis.

Genomic Analysis of Antimicrobial Resistance in Pseudomonas aeruginosa from a "One Health" Perspective.

The "One Health" approach provides a comprehensive framework for understanding antimicrobial resistance. This perspective is of particular importance in the study of Pseudomonas aeruginosa, as it is not only a pathogen that affects humans but also persists in environmental reservoirs. To assess evolutionary selection for niche-specific traits, a genomic comparison of 749 P. aeruginosa strains from three environments (clinical, aquatic, and soil) was performed. The results showed that the environment does indeed exert selective pressure on specific traits. The high percentage of persistent genome, the lack of correlation between phylogeny and origin of the isolate, and the high intrinsic resistance indicate that the species has a high potential for pathogenicity and resistance, regardless of the reservoir. The flexible genome showed an enrichment of metal resistance genes, which could act as a co-selection of antibiotic resistance genes. In the plasmids, resistance genes were found in multigenic clusters, with the presence of a mobile integron being prominent. This integron was identified in several pathogenic strains belonging to distantly related taxa with a worldwide distribution, showing the risk of rapid evolution of resistance. These results provide a more complete understanding of the evolution of P. aeruginosa, which could help develop new prevention strategies.

Water chlorination increases the relative abundance of an antibiotic resistance marker in developing sourdough starters.

Multiple factors explain the proper development of sourdough starters. Although the role of raw ingredients and geography, among other things, have been widely studied recently, the possible effect of air quality and water chlorination on the overall bacterial communities associated with sourdough remains to be explored. Here, using 16S rRNA amplicon sequencing, we show that clean, filtered-air severely limited the presence of lactic acid bacteria in sourdough starters, suggesting that surrounding air is an important source of microorganisms necessary for the development of sourdough starters. We also show that water chlorination at levels commonly found in drinking water systems has a limited impact on the overall bacterial communities developing in sourdough starters. However, using targeted sequencing, which offers a higher resolution, we found that the abundance of integron 1, a genetic mechanism responsible for the horizontal exchange of antibiotic-resistance genes in spoilage and pathogenic bacteria, increased significantly with the level of water chlorination. Although our results suggest that water chlorination might not impact sourdough starters at a deep phylogenetic level, they indicate that it can favor the spread of genetic elements associated with spoilage bacteria. IMPORTANCE: Proper development of sourdough starters is critical for making tasty and healthy bread. Although many factors contributing to sourdough development have been studied, the effect of water chlorination on the bacterial communities in sourdough has been largely ignored. Researchers used sequencing techniques to investigate this effect and found that water chlorination at levels commonly found in drinking water systems has a limited impact on the overall bacterial communities developing in sourdough starters. However, they discovered that water chlorination could increase the abundance of integron 1, a genetic mechanism responsible for the horizontal exchange of antibiotic resistance genes in spoilage and pathogenic bacteria. This suggests that water chlorination could favor the growth of key spoilage bacteria and compromise the quality and safety of the bread. These findings emphasize the importance of considering water quality when developing sourdough starters for the best possible bread.

First detection of VEB-1 extended-spectrum ß-lactamase-producing Escherichia coli clinical isolate in Japan.

The extended-spectrum ß-lactamase (ESBL) gene, blaVEB-1, was identified for the first time in an Escherichia coli clinical isolate, JARB-RN-0061, from blood cultures in a Japanese general hospital in 2021. The isolate exhibited high resistance to broad-spectrum cephalosporins, including ceftazidime (MIC >128 mg/L) and cefepime (MIC = 16 mg/L). blaVEB-1 was identified during whole-genome sequencing and characterization of the isolate. JARB-RN-0061 belonged to the B2-O2:K1:H7-ST95-fimH41 lineage and was classified as presumptive extraintestinal pathogenic E. coli (ExPEC) and uropathogenic E. coli (UPEC). Moreover, the strain harbored multiple virulence genes on the chromosome. The Col156/IncFIB(AP001918)/IncFII(29)-type plasmid (114,216 bp), with clbB and tcpC genes involved in bacteremia, was unique to the fimH41 subclone. The blaVEB-1 gene was located on a non-typeable and non-conjugative plasmid, pJARB-RN-0061_VEB-1 (17,093 bp). It was embedded in the class 1 integron In1883-like, with multidrug resistance gene cassettes for aacA4, aadB, cmlA5, qnrVC4, and dfrA14. Notably, comparative analysis of the complete sequence of plasmid pJARB-RN-0061_VEB-1 revealed that it was highly homologous to the blaVEB-1-harboring plasmid, pMS2H7VEB-1 (100% coverage and 99.99% identity), except for the Tn3 family transposon (4,931 bp) and the plasmid pRHBSTW-00138_5 (97% coverage and 100% identity) harbored by Klebsiella quasipneumoniae subsp. similipneumoniae strains from hospital sewage in Japan and wastewater influent in the United Kingdom, respectively. The emergence of a human pathogenic E. coli clinical isolate with the blaVEB-1-carrying plasmid in the B2-ST95 worldwide pandemic lineage, characterized by the virulence potential of ExPEC or UPEC but a low prevalence of antimicrobial resistance, would raise public health concerns. IMPORTANCE: ESBLs are plasmid-mediated enzymes that confer resistance to clinically significant antimicrobial agents, such as broad-spectrum cephalosporins. Recently, the rapid spread of CTX-M-type ESBL-producing E. coli has become a global issue, including in Japan, where ESBL production in human pathogenic E. coli, such as the ExPEC and UPEC lineages, which typically harbor several virulence genes, is a severe public health concern. To date, VEB (Vietnamese extended-spectrum ß-lactamase) producers have been found only in hospital wastewater and rivers in Japan. Thus, we describe the first detection of a very rare human-derived blaVEB-1 gene in the E. coli B2-ST95 pandemic clonal lineage that is highly associated with ExPEC and UPEC in a Japanese clinical setting. Furthermore, we characterized the genomic features of plasmids harboring the class 1 integron-borne blaVEB-1. Our findings highlight the significance of closely monitoring ESBL-producing E. coli isolates to prevent the potential dissemination of this resistance determinant in Japan.

Differences in antimicrobial resistance-related genes of Trueperella pyogenes between isolates detected from cattle and pigs.

We investigated antimicrobial resistance-related genes in 109 isolates of Trueperella pyogenes that were isolated in cattle and pigs. All 89 tetracycline-resistant T. pyogenes isolates carried the resistance gene harbored either tetW, tetM, tetA(33), tetK, or tetL. The ermX or ermB were detected in 18 of 23 erythromycin-resistant isolates. Streptomycin-resistant aadA1, aadA9, aadA11, aadA24, strA, or strB were detected in 25 of 83 isolates. There were significant differences in the percentages of tetA(33), ermB, aadA1, aadA9, aadA11, or aadA24 carriage between cattle and pig isolates. In addition, the Class 1 gene cassette was detected only in 17 cattle isolates. This suggests that T. pyogenes isolates acquire resistance gene in each environment of cattle and pigs, and that the transmission of the bacteria between cattle and pigs is limited.

Comparing the activity of broad-spectrum beta-lactams in combination with aminoglycosides against VIM-producing Enterobacteriaceae.

Metallo-beta-lactamase (MBL)-producing carbapenem-resistant Enterobacteriaceae (CRE) infections continue to pose a serious threat to healthcare. Due to their unique active site, MBLs evade the activity of many novel beta-lactam/beta-lactamase inhibitor combinations, which have been specifically targeted toward those carbapenemases with serine active sites. Furthermore, resistance to most, if not all, other clinically relevant antimicrobial classes leaves few reliable therapeutic options. Combination therapy has thus played a vital role in the treatment of MBL-producing CRE infections. In this study, we utilized the static time-kill assay to investigate clinically relevant concentrations of cefepime, piperacillin-tazobactam, and meropenem alone and in combination with either amikacin or the novel plazomicin to determine if combinations of routinely used beta-lactam therapy with an aminoglycoside would achieve bactericidal activity against eight clinically isolated Verona integron-encoded MBL (VIM)-producing CRE. Furthermore, we compared this activity to the combination of aztreonam/avibactam, which has shown potent activity against MBL-producing CRE. Both aztreonam/avibactam and meropenem with either aminoglycoside were rapidly bactericidal within 4 hours and remained bactericidal through 24 hours against all isolates with few exceptions. Combinations including cefepime and piperacillin-tazobactam were also rapidly bactericidal, but activity after 24 hours was inconsistent depending upon the partner aminoglycoside and isolate. Further investigation is warranted to elucidate optimal antibiotic exposures against MBL-producing CRE, including novel agents in the pipeline.IMPORTANCECarbapenem-resistant Enterobacterales (CRE) are one of the most pressing antimicrobial-resistant threats at present. In addition to exhibiting resistance to many, if not all, commonly used antimicrobial agents, CRE achieves these resistant phenotypes through a variety of mechanisms, each of which can uniquely affect available treatment options. The present study is an in vitro investigation of several Verona integron-encoded metallo-beta-lactamase (VIM)-producing CRE isolated from patients at our academic medical center. Because metallo-beta-lactamases (MBLs) are inherently resistant to many of the novel treatments designed to treat CRE due to their different active site composition, we tested several antimicrobial combinations containing routinely utilized broad-spectrum beta-lactams and aminoglycosides. Our results further our understanding of combination therapy options against VIM-producing CRE, including with non-carbapenem-beta-lactams cefepime and piperacillin. By optimizing combinations of existing antimicrobial agents, we hope to expand the available armamentarium against these resistant pathogens.

Comparative genomics of IncQ1 plasmids carrying blaGES variants from clinical and environmental sources in Brazil.

IncQ-type plasmids have become important vectors in the dissemination of blaGES among different bacterial genera and species from different environments around the world, and studies estimating the occurrence of Guiana extended-spectrum (GES)-type ß-lactamases are gaining prominence. We analyzed the genetic aspects of two IncQ1 plasmids harboring different blaGES variants from human and environmental sources. The blaGES variants were identified using polymerase chain reaction (PCR) in Aeromonas veronii isolated from hospital effluent and Klebsiella variicola isolated from a rectal swab of a patient admitted to the cardiovascular intensive care unit in a different hospital. Antimicrobial-susceptibility testing and transformation experiments were performed for phenotypic analysis. Whole-genome sequencing was performed using Illumina and Oxford Nanopore platforms. The comparative analysis of plasmids was performed using BLASTn, and the IncQ1 plasmids showed a high identity and similar size. A. veronii harbored blaGES-7 in a class 1 integron (In2061), recently described by our group, and K. variicola carried blaGES-5 in the known class 1 integron. Both integrons showed a fused gene cassette that encodes resistance to aminoglycosides and fluoroquinolones, with an IS6100 truncating the 3'-conserved segment. The fused genes are transcribed together, although the attC site is disrupted. These gene cassettes can no longer be mobilized. This study revealed a mobilome that may contribute to the dissemination of GES-type ß-lactamases in Brazil. Class 1 integrons are hot spots for bacterial evolution, and their insertion into small IncQ-like plasmids displayed successful recombination, allowing the spread of blaGES variants in various environments. Therefore, they can become prevalent across clinically relevant pathogens.

PBP-3 directed therapy in VIM-producing Pseudomonas aeruginosa creates bacterial transformers, persisters in disguise.

OBJECTIVES: The proliferation of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa represents a significant public health threat. P. aeruginosa undergoes significant phenotypic changes that drastically impair antibiotic efficacy. The objectives of this study were (1) to quantify the time-course of killing of VIM-2-producing P. aeruginosa in response to aztreonam-based therapies (including avibactam for coverage of AmpC), and (2) to document the capacity of P. aeruginosa to undergo morphological transformations that facilitate persistence. METHODS: A well-characterised, clinical VIM-2-producing P. aeruginosa was studied in the hollow fibre infection model (HFIM) over 9 days (7 days of active antibiotic therapy, 2 days of treatment withdrawal) at a 107.5 CFU/mL starting inoculum. HFIM treatment arms included: growth control, aztreonam, ceftazidime/avibactam, aztreonam/ceftazidime/avibactam, polymyxin B, and aztreonam/ceftazidime/avibactam/polymyxin B. In addition, real-time imaging studies were conducted under static conditions to determine the time course of the reversion of persister cells. RESULTS: There was a pronounced discrepancy between OD620 and bacterial counts obtained from plating methods (hereafter referred to as 'OD-count discrepancy'). For aztreonam monotherapy, observed counts were 0 CFU/mL by 120 h. Despite this, there was a significant OD-count discrepancy compared with the pre-treatment 0 h. Between therapy withdrawal at 168 h and 216 h, all arms with suppressed counts had regrown to the system-carrying capacity. Real-time imaging of the P. aeruginosa filaments after drug removal showed rapid reversion from a long, filamentous phenotype to many individual rods within 2 h. CONCLUSION: Managing MBL-producing P. aeruginosa requires a multifaceted approach, focused on maximising killing and minimising proliferation of resistant and persistent subpopulations, which will involve eliminating drug-induced phenotypic transformers.

High prevalence of multidrug-resistant Pseudomonas aeruginosa carrying integron and exoA, exoS, and exoU genes isolated from burn patients in Ahvaz, southwest Iran: A retrospective study.

Background: Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. This study evaluated the relative frequency of exoenzymes (exo) A, U and S genes and integron classes (I, II, and III) among multi-drug-resistant clinical P. aeruginosa isolates from burn patients in Ahvaz, southwest of Iran. Methods: In this cross-sectional study P. aeruginosa isolates were recovered from 355 wound samples. The antimicrobial susceptibility test was done by disk agar diffusion method on Muller-Hinton agar according to the Clinical and Laboratory Standards Institute. MDR isolates were defined if they showed simultaneous resistance to 3 antibiotics. Extensively drug-resistant was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories. The presence of class I, II, and III integrons and virulence genes was determined using a PCR assay on extracted DNA. Results: Overall, 145 clinical P. aeruginosa isolates were confirmed with biochemical and PCR tests. Overall, 35% (52/145) of the isolates were taken from males and 64% (93/145) from female hospitalized burn patients. The highest resistance rates of P. aeruginosa isolates to antibiotics were related to piperacillin 59% (n = 86/145) and piperacillin-tazobactam 57% (n = 83/145). A total of 100% of isolates were resistant to at least one antibiotic. MDR and XDR P. aeruginosa had a frequency of 60% and 29%, respectively. The prevalence of integron classes I, II, and III in P. aeruginosa was 60%, 7.58%, and 3.44%, respectively. IntI was more common in MDR and XDR P. aeruginosa isolates. In addition, 70(48%) of P. aeruginosa isolates did not harbor integron genes. Besides, exoA, exoS, and exoU in P. aeruginosa had a frequency of 55%, 55%, and 56%, respectively. Conclusion: It was found that P. aeruginosa as a potent pathogen with strong virulence factors and high antibiotic resistance in the health community can cause refractory diseases in burn patients.

Molecular epidemiology, antibiotic resistance profile and frequency of integron 1 and 2 in adherent-invasive Escherichia coli isolates of colorectal cancer patients.

This study explores the prevalence of adherent-invasive Escherichia coli (AIEC) in colorectal cancer (CRC) patients and investigates the potential of effective intracellular antibiotics as a therapeutic strategy for CRC patients with AIEC infections. Considering the pivotal role of integrons in bacterial antibiotic resistance, the frequency of class 1 and 2 integrons in AIEC isolated from CRC patients, in one of the referenced 3 gastroenterology clinics in Isfahan, Iran was examined. AIEC strains were isolated from the colorectal biopsies and their antimicrobial sensitivity was assessed using the disc diffusion method. Polymerase chain reaction (PCR) was employed to detect intl1 and intl2. The multilocus sequence typing (MLST) method was utilized to type 10 selected isolates. Of the 150 samples, 24 were identified as AIEC, with the highest number isolated from CRC2 (33.4%) and CRC1 (29.16%), and the least from the FH group (8.3%) and control group (12.5%). int1 in 79.2% and int2 in 45.8% of AIEC strains were found and 41.6% of strains had both integrons. AIEC isolates with int1 exhibited the highest sensitivity to trimethoprim-sulfamethoxazole (57.9%), while those with int2 showed the highest sensitivity to ciprofloxacin (63.6%). A significant association between resistance to rifampin and integron 2 presence in AIEC isolates was observed. Furthermore, a significant correlation between integron 1 presence, invasion, survival, and replication within macrophages in AIEC strains was identified. MLST analysis revealed ST131 from CC131 with integron 1 as the most common sequence type (ST). The emergence of such strains in CRC populations poses a serious public health threat. The distribution pattern of STs varied among studied groups, with pandemic STs highlighting the importance of examining and treating patients infected with these isolates. Comprehensive prospective clinical investigations are warranted to assess the prognostic value of detecting this pathovar in CRC and to evaluate therapeutic techniques targeting drug-resistant AIECs, such as phage therapy, bacteriocins, and anti-adhesion compounds, for CRC prevention and treatment.

The relationship between integrons, antibiotic resistance genes and SXT resistance in Shigella flexneri strains.

OBJECTIVE: To investigate the correlation between sulfamethoxazole-trimethoprim (SXT) resistance in Shigella flexneri (S.flexneri) and the presence of integrons and relevant antibiotic resistance genes. METHODS: We collected 115 strains of Shigella flexneri isolated from feces of children with diarrhea in Jinan from 2012 to 2020 and determined the minimum inhibitory concentration (MIC) of SXT by Etest method. The presence of class 1, class 2, and class 3 integron genes, variable region antibiotic resistance gene cassettes, and sul1, sul2, sul3, and SXT elements were detected using polymerase chain reaction (PCR). Positive results were further analyzed by DNA sequencing and BLAST comparison. RESULTS: In total, the resistance rate to SXT was 60.9% among the 115 S.flexneri strains. The prevalence of class 1 and class 2 integrons were 88.7% and 87.0%, respectively, with no class 3 integrons detected. Among the strains, 13.0% carried typical class 1 integrons with variable region antibiotic resistance gene cassettes dfrA17-aadA5 and dfrV, while 85.2% carried atypical class 1 integrons with variable region antibiotic resistance gene cassette blaoxa-30-aadA1. The variable region antibiotic resistance gene cassettes of class 2 integrons were all dfrA1+sat1+aadA1. There was a statistical difference between the presence of class 1 integrons and class 2 integrons between the SXT-sensitive and resistant S.flexneri strains (χ2=22.800, χ2=16.365, P<0.01, P<0.01). Integrons carrying dfrV and dfrA1 by integrons also showed a statistical difference in SXT resistance (χ2=9.422, χ2=16.365, P<0.01, P<0.01). PCR revealed the presence of sul1 and sul2 in 13.0% and 47.0% of strains, respectively, with neither sul3 nor SXT elements detected. There was a significant difference between the presence of sul1, sul2 between the SXT-sensitive and resistant S.flexneri strains (χ2=9.588, χ2=65.445, P<0.01, P<0.01). CONCLUSION: In summary, integrons are involved in SXT resistance of S.flexneri, and dfrV, dfrA1, sul1, sul2 are closely related to SXT resistance of S.flexneri.

Clonal spread of trimethoprim-sulfamethoxazole-resistant Stenotrophomonas maltophilia isolates in a tertiary hospital.

Aim: The aims of this study were to: (i) determine antibiotic susceptibility of clinical Stenotrophomonas maltophilia isolates, (ii) investigate the presence of different classes of integrons and sul genes responsible for sulphonamide resistance, (iii) assess the molecular epidemiology of the isolates by determining their clonal relatedness, and (iv) investigate the potential sources of infection by collecting environmental samples when necessary. Methods: 99 S. maltophilia isolates from clinical specimens of hospitalized patients were screened by PCR for sul1, sul2, sul3 genes, and integron-associated integrase genes: intI1, intI2, and intI3. PFGE was used to determine the clonal relatedness of the isolates. Results: Susceptibility rates for trimethoprim-sulfamethoxazole, levofloxacin, and ceftazidime were 90.9%, 91.9%, and 53.5% respectively. All trimethoprim-sulfamethoxazole-resistant isolates were positive for intI1 and sul1. PFGE analysis revealed that 24 of the isolates were clonally related, clustering in seven different clones. Five of the nine trimethoprim-sulfamethoxazole-resistant isolates were clonally related. The first isolate in this clone was from a wound sample of a patient in the infectious diseases clinic, and the other four were isolated from the bronchoalveolar lavage samples of patients in the thoracic surgery unit. The patient with the first isolate neither underwent bronchoscopy nor stayed in the thoracic surgery unit. Although clustering was observed in bronchoalveolar lavage samples, no S. maltophilia growth was detected in environmental samples. Conclusion: The findings demonstrated that the sul1 gene carried by class 1 integrons plays an important role in trimethoprim-sulfamethoxazole resistance in S. maltophilia isolates. PFGE analysis revealed a high degree of genetic diversity. However, detection of clonally related isolates suggests the acquisition from a common source and/or cross-transmission of this microorganism between the patients.

Analysis of the Class 1 Integrons, Carbapenemase Genes and Biofilm Formation Genes Occurrence in Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant A. baumannii (CRAB) has spread rapidly in clinical settings and has become a key concern. The main objective of this study was to identify the distribution of integrons and biofilm-formation-related virulence genes in CRAB isolates. A total of 269 A. baumannii isolates (219 isolates of CRAB and 50 isolates of carbapenem-sensitive A. baumannii (CSAB)) were collected. Carbapenemase genes (bla KPC, bla VIM, bla IMP, bla NDM, and bla OXA-23-like) and biofilm-formation-related virulence genes (abal, bfms, bap, and cusE) were screened with PCR. Class 1 integron was screened with PCR, and common promoters and gene cassette arrays were determined with restriction pattern analysis combined with primer walking sequencing. Whole-genome sequencing was conducted, and data were analyzed for a bla OXA-23-like-negative isolate. All 219 CRAB isolates were negative for bla KPC, bla VIM, bla IMP, and bla NDM, while bla OXA-23-like was detected in 218 isolates. The detection rates for abal, bfms, bap, and cusE in 219 CRAB were 93.15%, 63.93%, 88.13%, and 77.63%, respectively. Class 1 integron was detected in 75 CRAB (34.25%) and in 3 CSAB. The single gene cassette array aacA4-catB8-aadA1 with relatively strong PcH2 promoter was detected in class 1 integrons. The bla OXA-23-like-negative CRAB isolate was revealed to be a new sequence type (Oxford 3272, Pasteur 2520) carrying bla OXA-72, bla OXA-259, and bla ADC-26. In conclusion, bla OXA-23-like was the main reason for CRAB's resistance to carbapenems. A new (Oxford 3272, Pasteur 2520) CRAB sequence type carrying the bla OXA-72, bla OXA-259, and bla ADC-26 was reported.

High-throughput microfluidic quantitative PCR system for the simultaneous detection of antibiotic resistance genes and bacterial and viral pathogens in wastewater.

Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan, collected three times a month between August 2022 and July 2023, were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R2 was ≥0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log10 copies/L. NoV-GII detection ratios and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1, int1, and blaCTX-M, irrespective of season. The less explored blaNDM-1 showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log10 copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1, and blaCTX-M varied seasonally. int1 was significantly correlated with blaCTX-M in autumn and spring, whereas it showed no correlation with blaNDM-1, questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE.

Genomic Insights into the First Emergence of blaNDM-5-Carrying Carbapenem-Resistant Salmonella enterica Serovar London Strain in China.

Carbapenem-resistant Salmonella enterica (S. enterica) pose a significant threat to public health, causing gastroenteritis and invasive infections. We report the first emergence of a carbapenem-resistant S. enterica serovar London strain, A132, carrying the blaNDM-5 gene in China. Whole-genome sequencing and bioinformatics analysis assigned A132 to be ST155, a multidrug-resistant clone frequently reported in China. The strain A132 exhibited resistance to multiple antibiotics, with 20 acquired antibiotic resistance genes (ARGs) identified, predominantly located on the IncFIB plasmid (pA132-1-NDM). Notably, the blaNDM-5 gene was located within an IS26 flanked-class 1 integron-ISCR1 complex, comprising two genetic cassettes. One cassette is the class 1 integron, which may facilitate the transmission of the entire complex, while the other is the blaNDM-5-containing ISCR1-IS26-flanked cassette, carrying multiple other ARGs. Genbank database search based on the blaNDM-5-carrying cassette identified a similar genetic context found in transmissible IncFIA plasmids from Escherichia coli (p91) and Enterobacter hormaechei (p388) with a shared host range, suggesting the potential for cross-species transmission of blaNDM-5. To our knowledge, this is the first reported case of Salmonella serovar London ST155 harboring blaNDM-5 gene. Phylogenetic analysis indicated a close relationship between A132 and eight S. London ST155 strains isolated from the same province. However, A132 differed by carrying the blaNDM-5 gene and four unique ARGs. Given the high transmissibility of the F-type plasmid harboring blaNDM-5 and 18 other ARGs, it is imperative to implement vigilant surveillance and adopt appropriate infection control measures to mitigate the threat to public health.

Molecular detection of Class 1, 2, and 3 integrons in hypervirulent and classic Klebsiella pneumoniae isolates: A cross-sectional study.

Background and Aims: The "hypervirulent" variant of Klebsiella pneumoniae (hvKp) is an emerging pathogen that cause life-threatening infection. The present study was conducted to identify the prevalence of hvKp and to investigate the presence class 1, 2, and 3 integrons in these isolates. Methods: A cross-sectional study was conducted at three teaching hospitals, Ahvaz, South-west of Iran, from January 1, 2019 to December 31, 2020. Samples were collected from inpatients and included only the first samples collected from each patient. K. pneumoniae strains were isolated from different specimens using biochemical test and confirmed by targeting 16S-23S rDNA internal transcribed spacer. HvKp isolates were recovered using string test and were further characterized by detection virulence-associated genes (rmpA, iucA, and magA). Antibiotic susceptibility patterns of isolates were determined using the disc diffusion method. Isolates were screened for presence the integron genes (intI, intII, and intIII) and repetitive element sequence-based polymerase chain reaction (PCR) performed to determine strain relatedness. SPSS version 22 was used for the data analysis. Results: Seventy-one (77%) of isolates showed multidrug-resistant (MDR) phenotype. HvKP accounted for 14% (13/92) of cKp isolated from blood (46%) and urinary tract infection (38%), and the great majority of them (61.5%; 8/13) exhibited MDR phenotype. Using the PCR assay, 29 of 92 isolates (31.5%) were found to have positive results for the presence of IntI. Three of the IntI-positive strains were hvKP. Class 2 integron was present in 8/92 cKp isolates. Integron Class 2 was found to coexist with Class 1 integron in 3/8 isolates. All integron-positive isolates (IntI and/or IntII) were resistant to at least three different classes of antibiotics and showed MDR phenotype. No Class 3 integrons were detected among the isolates. Conclusion: The results of our study revealed that considering the role of integrons in facilitating the acquisition and dissemination of resistance genes among bacteria, monitoring the emergence of hvKp, emphasizing on the mechanism of antimicrobial resistance, can prevent from the spread of carbapenemase-producing hvKp strains.