Rapid discrimination methods for clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using the N-terminal sequence of the flaA gene and investigation of antimicrobial resistance.

Although the genus Aeromonas inhabits the natural environment, it has also been isolated from hospital patient specimens as a causative agent of Aeromonas infections. However, it is not known whether clinical strains live in the natural environment, and if these strains have acquired antimicrobial resistance. In this study, we performed the typing of flagellin A gene (flaA) of clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using Polymerase Chain Reaction (PCR) assay with newly designed primers. Detection rates of the clinical and environmental flaA types of A. hydrophila were 66.7% and 88.2%, and the corresponding rates for A. veronii biovar sobria were 66.7% and 90.9%. The PCR assays could significantly discriminate between clinical and environmental strains of both species in approximately 4 h. Also, among the 63 clinical Aeromonas strains used, only one extended-spectrum ß-lactamase-producing bacteria, no plasmid-mediated quinolone resistance bacteria, and only four multidrug-resistant bacteria were detected. Therefore, the PCR assays could be useful for the rapid diagnosis of these Aeromonas infections and the monitoring of clinical strain invasion into water-related facilities and environments. Also, the frequency of drug-resistant Aeromonas in clinical isolates from Okinawa Prefecture, Japan, appeared to be low.

Antimicrobial resistance and genome characteristics of Salmonella enteritidis from Huzhou, China.

Salmonella enteritidis is a main pathogen responsible for sporadic outbreaks of gastroenteritis, and therefore is an important public health problem. This study investigated the drug resistance and genomic characteristics of S. enteritidis isolated from clinical and food sources in Huzhou, Zhejiang Province, China, from February 1, 2021, to December 30, 2023. In total, 43 S. enteritidis strains isolated during the study period were subjected to virulence gene, drug resistance gene, genetic correlation, antibiotic resistance, and multilocus sequence typing analyses. All 43 isolates were identified as ST11, and contained 108 virulence-related genes. Drug sensitivity analysis of the 43 isolates showed resistance rates of 100% to nalidixic acid and 90.70% to ampicillin and ampicillin/sulbactam. Multidrug resistance is a serious issue, with 81.40% of strains resistant to three or more antibacterial drugs. Genome sequencing indicated that S. enteritidis possessed 23 drug resistance genes, of which 14 were common to all 43 isolates. Phylogenetic analysis based on core genome single-nucleotide polymorphisms divided the 43 S. enteritidis strains into three clusters, with the 10 samples from an outbreak forming an independent branch located in cluster 3.

Assessment of the pathogen genomics landscape highlights disparities and challenges for effective AMR Surveillance and outbreak response in the East African community.

The East African Community (EAC) grapples with many challenges in tackling infectious disease threats and antimicrobial resistance (AMR), underscoring the importance of regional and robust pathogen genomics capacities. However, a significant disparity exists among EAC Partner States in harnessing bacterial pathogen sequencing and data analysis capabilities for effective AMR surveillance and outbreak response. This study assesses the current landscape and challenges associated with pathogen next-generation sequencing (NGS) within EAC, explicitly focusing on World Health Organization (WHO) AMR-priority pathogens. The assessment adopts a comprehensive approach, integrating a questionnaire-based survey amongst National Public Health Laboratories (NPHLs) with an analysis of publicly available metadata on bacterial pathogens isolated in the EAC countries. In addition to the heavy reliance on third-party organizations for bacterial NGS, the findings reveal a significant disparity among EAC member States in leveraging bacterial pathogen sequencing and data analysis. Approximately 97% (n = 4,462) of publicly available high-quality bacterial genome assemblies of samples collected in the EAC were processed and analyzed by external organizations, mainly in Europe and North America. Tanzania led in-country sequencing efforts, followed by Kenya and Uganda. The other EAC countries had no publicly available samples or had all their samples sequenced and analyzed outside the region. Insufficient local NGS sequencing facilities, limited bioinformatics expertise, lack of adequate computing resources, and inadequate data-sharing mechanisms are among the most pressing challenges that hinder the EAC's NPHLs from effectively leveraging pathogen genomics data. These insights emphasized the need to strengthen microbial pathogen sequencing and data analysis capabilities within the EAC to empower these laboratories to conduct pathogen sequencing and data analysis independently. Substantial investments in equipment, technology, and capacity-building initiatives are crucial for supporting regional preparedness against infectious disease outbreaks and mitigating the impact of AMR burden. In addition, collaborative efforts should be developed to narrow the gap, remedy regional imbalances, and harmonize NGS data standards. Supporting regional collaboration, strengthening in-country genomics capabilities, and investing in long-term training programs will ultimately improve pathogen data generation and foster a robust NGS-driven AMR surveillance and outbreak response in the EAC, thereby supporting global health initiatives.

Whole genome sequencing analysis on antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia.

IMPORTANCE: The emergence and rapid increase in the incidence of multidrug-resistant (MDR) bacteria in pig farms has become a serious concern and reduced the choice of effective antibiotics. OBJECTIVE: This study analyzed the phylogenetics and diversity of antibiotic resistance genes (ARGs) and molecularly identified the source of ARGs in antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. METHODS: Forty-four antibiotic-resistant E. coli isolates from fecal samples from 44 pig farms in Banten Province, Indonesia, were used as samples. The samples were categorized into 14 clusters. Sequencing was performed using the Oxford Nanopore Technologies MinION platform, with barcoding before sequencing with Nanopore Rapid sequencing gDNA-barcoding (SQK-RBK110.96) according to manufacturing procedures. ARG detection was conducted using ResFinder, and the plasmid replicon was determined using PlasmidFinder. RESULTS: Three phylogenetic leaves of E. coli were identified in the pig farming cluster in Banten Province. The E. coli isolates exhibited potential resistance to nine classes of antibiotics. Fifty-one ARGs were identified across all isolates, with each cluster carrying a minimum of 10 ARGs. The ant(3'')-Ia and qnrS1 genes were present in all isolates. ARGs in the E. coli pig farming cluster originated mainly from plasmids, accounting for an average of 89.4%. CONCLUSIONS AND RELEVANCE: The elevated potential for MDR events, coupled with the dominance of ARGs originating from plasmids, increases the risk of ARG spread among bacterial populations in animals, humans, and the environment.

Effect of erythromycin residuals in food on the development of resistance in Streptococcus pneumoniae: an in vivo study in Galleria mellonella.

Background: The use of antimicrobials to treat food animals may result in antimicrobial residues in foodstuffs of animal origin. The European Medicines Association (EMA) and World Health Organization (WHO) define safe antimicrobial concentrations in food based on acceptable daily intakes (ADIs). It is unknown if ADI doses of antimicrobials in food could influence the antimicrobial susceptibility of human-associated bacteria. Objectives: This aim of this study was to evaluate if the consumption of ADI doses of erythromycin could select for erythromycin resistance in a Galleria mellonella model of Streptococcus pneumoniae infection. Methods: A chronic model of S. pneumoniae infection in G. mellonella larvae was used for the experiment. Inoculation of larvae with S. pneumoniae was followed by injections of erythromycin ADI doses (0.0875 and 0.012 µg/ml according to EMA and WHO, respectively). Isolation of S. pneumoniae colonies was then performed on selective agar plates. Minimum inhibitory concentrations (MICs) of resistant colonies were measured, and whole genome sequencing (WGS) was performed followed by variant calling to determine the genetic modifications. Results: Exposure to single doses of both EMA and WHO ADI doses of erythromycin resulted in the emergence of erythromycin resistance in S. pneumoniae. Emergent resistance to erythromycin was associated with a mutation in rplA, which codes for the L1 ribosomal protein and has been linked to macrolide resistance in previous studies. Conclusion: In our in vivo model, even single doses of erythromycin that are classified as acceptable by the WHO and EMA induced significant increases in erythromycin MICs in S. pneumoniae. These results suggest the need to include the induction of antimicrobial resistance (AMR) as a significant criterion for determining ADIs.

Characterizing the gut phageome and phage-borne antimicrobial resistance genes in pigs.

BACKGROUND: Mammalian intestine harbors a mass of phages that play important roles in maintaining gut microbial ecosystem and host health. Pig has become a common model for biomedical research and provides a large amount of meat for human consumption. However, the knowledge of gut phages in pigs is still limited. RESULTS: Here, we investigated the gut phageome in 112 pigs from seven pig breeds using PhaBOX strategy based on the metagenomic data. A total of 174,897 non-redundant gut phage genomes were assembled from 112 metagenomes. A total of 33,487 gut phage genomes were classified and these phages mainly belonged to phage families such as Ackermannviridae, Straboviridae, Peduoviridae, Zierdtviridae, Drexlerviridae, and Herelleviridae. The gut phages in seven pig breeds exhibited distinct communities and the gut phage communities changed with the age of pig. These gut phages were predicted to infect a broad range of 212 genera of prokaryotes, such as Candidatus Hamiltonella, Mycoplasma, Colwellia, and Lactobacillus. The data indicated that broad KEGG and CAZy functions were also enriched in gut phages of pigs. The gut phages also carried the antimicrobial resistance genes (ARGs) and the most abundant antimicrobial resistance genotype was diaminopyrimidine resistance. CONCLUSIONS: Our research delineates a landscape for gut phages in seven pig breeds and reveals that gut phages serve as a key reservoir of ARGs in pigs. Video Abstract.

Occurrence, genetic characterization, and antibiotic susceptibility of Cronobacter spp. isolated from low water activity functional foods in Brazil.

Cronobacter spp. are bacterial pathogens isolated from a wide variety of foods. This study aims at evaluating the occurrence of Cronobacter spp. in low water activity functional food samples, detect the presence of virulence genes, and determine the antibiotic susceptibility of strains. From 105 samples, 38 (36.2%) were contaminated with Cronobacter spp. The species identified by polymerase chain reaction (PCR) and sequencing analyses (rpoB and fusA genes, respectively) were C. sakazakii (60.3%), C. dublinensis (25.4%), C. turincensis (9.5%), and C. malonaticus (4.8%). Nineteen fusA alleles were identified, including four new alleles. The virulence genes were identified by PCR and all isolates were positive for ompX and sodA genes, 60.3% to cpa gene, and 58.7% to hly gene. Using the disk diffusion method, antibiotic susceptibility to twelve antibiotics was assessed twice, separated by a 19-month period. In the first test, the isolates showed diverse antibiotic susceptibility profiles, with nineteen isolates (30.2%) being multi-drug resistant (resistant to three or more antibiotic classes), in the second, the isolates were susceptible to all antibiotics. Cronobacter spp. in functional foods demonstrates the need for continued investigation of this pathogen in foods, and further research is needed to clarify the loss of resistance of Cronobacter strains.

AcrAB-TolC efflux pump overexpression and tet(A) gene mutation increase tigecycline resistance in Klebsiella pneumoniae.

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-ß-naphthylamide dihydrochloride (PaßN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

Prevalence of the CRISPR-cas system and its association with antibiotic resistance in clinical Klebsiella pneumoniae isolates.

BACKGROUND AND OBJECTIVE(S): CRISPR-Cas is a prokaryotic adaptive immune system that protects bacteria and archaea against mobile genetic elements (MGEs) such as bacteriophages plasmids, and transposons. In this study, we aimed to assess the prevalence of the CRISPR-Cas systems and their association with antibiotic resistance in one of the most challenging bacterial pathogens, Klebsiella pneumoniae. MATERIALS AND METHODS: A total of 105 K. pneumoniae isolates were collected from various clinical infections. Extended-spectrum ß-lactamases (ESBLs) phenotypically were detected and the presence of ESBL, aminoglycoside-modifying enzymes (AME), and CRISPR-Cas system subtype genes were identified using PCR. Moreover, the diversity of the isolates was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS: Phenotypically, 41.9% (44/105) of the isolates were found to be ESBL producers. A significant inverse correlation existed between the subtype I-E CRISPR-Cas system's presence and ESBL production in K. pneumoniae isolates. Additionally, the frequency of the ESBL genes blaCTX-M1 (3%), blaCTX-M9 (12.1%), blaSHV (51.5%), and blaTEM (33.3%), as well as some AME genes such as aac(3)-Iva (21.2%) and ant(2'')-Ia (3%) was significantly lower in the isolates with the subtype I-E CRISPR-Cas system in comparison to CRISPR-negative isolates. There was a significant inverse correlation between the presence of ESBL and some AME genes with subtype I-E CRISPR-Cas system. CONCLUSION: The presence of the subtype I-E CRISPR-Cas system was correlated with the antibiotic-resistant gene (ARGs). The isolates with subtype I-E CRISPR-Cas system had a lower frequency of ESBL genes and some AME genes than CRISPR-negative isolates.

Global distribution and genetic characterization of blaOXA-positive plasmids in Escherichia coli.

In recent years, the emergence of blaOXA-encoding Escherichia coli (E. coli) poses a significant threat to human health. Here, we systematically analyzed the global geographic distribution and genetic characteristics of 328 blaOXA-positive E. coli plasmids based on NCBI database. Twelve blaOXA variants have been discovered, with blaOXA-1 (57.93%) being the most common, followed by blaOXA-10 (11.28%) and blaOXA-48 (10.67%). Our results suggested that blaOXA-positive E. coli plasmids were widespread in 40 countries, mainly in China, the United States, and Spain. MLST analysis showed that ST2, ST43, and ST471 were the top three host STs for blaOXA-positive plasmids, deserving continuing attention in future surveillance program. Network analysis revealed a correlation between different blaOXA variants and specific antibiotic resistance genes, such as blaOXA-1 and aac (6')-Ib-cr (95.79%), blaOXA-181 and qnrS1 (87.88%). The frequent detection of aminoglycosides-, carbapenems- and even colistin-related resistance genes in blaOXA-positive plasmids highlights their multidrug-resistant potential. Additionally, blaOXA-positive plasmids were further divided into eight clades, clade I-VIII. Each clade displayed specificity in replicon types and conjugative transfer elements. Different blaOXA variants were associated with specific plasmid lineages, such as blaOXA-1 and IncFII plasmids in clade II, and blaOXA-48 and IncL plasmids in clade I. Overall, our findings provide a comprehensive insight into blaOXA-positive plasmids in E. coli, highlighting the role of plasmids in blaOXA dissemination in E. coli.

Colistin-resistance genes in Escherichia coli isolated from patients with urinary tract infections.

BACKGROUND: The incidence of antimicrobial resistance is alarmingly high because it occurs in humans, environment, and animal sectors from a "One Health" viewpoint. The emergence of plasmid-carried mobile colistin-resistance (MCR) genes limits the efficacy of colistin, which is the last-line treatment for multidrug resistance (MDR) against gram-negative infections. OBJECTIVES: The current study aimed to investigate emergence of colistin-resistance (MCR 1-5) genes in E. coli isolated from patients with urinary tract infections (UTIs) in Jordan. METHODS: E. coli (n = 132) were collected from urine specimens. The E. coli isolated from human UTI patients were examined the resistance to colistin based on the presence of MCR (1-5). All isolates were tested against 20 antimicrobials using the standard disk diffusion method. The broth microdilution technique was used to analyze colistin resistance. In addition, the MCR (1-5) genes were detected using multiplex PCR. RESULTS: Out of the 132 isolates, 1 isolate was colistin-resistant, having a minimum inhibitory concentration of 8 µg/mL and possessing MCR-1. All the E. coli isolates showed high resistance to penicillin (100%), amoxicillin (79.55%), cephalexin (75.76%), nalidixic acid (62.88%), tetracycline (58.33%), or cefepime (53.79). CONCLUSION: To our knowledge, this is the first report on the presence of plasmid-coded MCR-1 in E. coli from a patient with UTIs in Jordan. This is a problematic finding because colistin is the last-line drug for the treatment of infections caused by MDR gram-negative bacteria. There is a crucial need to robustly utilize antibiotics to control and prevent the emergence and prevalence of colistin-resistance genes.

Metagenomic analysis of the bacterial microbiome, resistome and virulome distinguishes Portuguese Serra da Estrela PDO cheeses from similar non-PDO cheeses: An exploratory approach.

This study aimed to evaluate the microbiome, resistome and virulome of two types of Portuguese cheese using high throughput sequencing (HTS). Culture-dependent chromogenic methods were also used for certain groups/microorganisms. Eight samples of raw ewe's milk cheese were obtained from four producers: two producers with cheeses with a PDO (Protected Designation of Origin) label and the other two producers with cheeses without a PDO label. Agar-based culture methods were used to quantify total mesophiles, Enterobacteriaceae, Escherichia coli, Staphylococcus, Enterococcus and lactic acid bacteria. The presence of Listeria monocytogenes and Salmonella was also investigated. The selected isolates were identified by 16S rRNA gene sequencing and evaluated to determine antibiotic resistance and the presence of virulence genes. The eight cheese samples analyzed broadly complied with EC regulations in terms of the microbiological safety criteria. The HTS results demonstrated that Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus plantarum, Lacticaseibacillus rhamnosus, Enterococcus durans and Lactobacillus coryniformis were the most prevalent bacterial species in cheeses. The composition of the bacterial community varied, not only between PDO and non-PDO cheeses, but also between producers, particularly between the two non-PDO cheeses. Alpha-diversity analyses showed that PDO cheeses had greater bacterial diversity than non-PDO cheeses, demonstrating that the diversity of spontaneously fermented foods is significantly higher in cheeses produced without the addition of food preservatives and dairy ferments. Despite complying with microbiological regulations, both PDO and non-PDO cheeses harbored potential virulence genes as well as antibiotic resistance genes. However, PDO cheeses exhibited fewer of these virulence and antibiotic resistance genes compared to non-PDO cheeses. Therefore, the combination of conventional microbiological methods and the metagenomic approach could contribute to improving the attribution of the PDO label to this type of cheese.

Genomic Characterization and Phylogenetic Analysis of Linezolid-Resistant Enterococcus from the Nostrils of Healthy Hosts Identifies Zoonotic Transmission.

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZDR) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZDR enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZDR-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZDR enterococci in all ecological niches and body parts to direct appropriate control strategies.

Prevalence and molecular detection of Staphylococcus aureus resistance to antibiotics.

In Algeria, the issue of antibiotic resistance is on the rise, being the Staphylococcus aureus infection as a significant concern of hospital-acquired infections. The emergence of antibiotic resistance in this bacterium poses a worldwide challenge. The aim of this study aims to establish the incidence of S aureus strains in Algeria as well as identify phenotypic and genotypic resistance based on the "mecA" and "nuc" genes. From 2014 to 2017, a total of 185 S aureus strains were isolated from patients at a hospital in the city of Rouïba, Algiers the number of isolates was slightly higher in males at 58.06% compared to females at 41.94%, resulting in a sex ratio of 1.38. the Oxacillin and Cefoxitin DD test (1 µg oxacillin disk and 30 µg cefoxitin disk) identified 42 strains as resistant. The results indicated high resistance to lactam antibiotics, with penicillin having a 100% resistance rate. There was also significant resistance to oxacillin (51.25%) and cefoxitin (50%). This resistance was frequently associated with resistance to other antibiotic classes, such as aminoglycosides (50%) and Macrolides (28.29%). To confirm methicillin-resistant characteristics, a polymerase chain reaction (PCR) multiplex was conducted on 10 isolates (6 SARM; 4 MSSA) on a phenotypic level. Three isolates tested positive for "mecA," while 7 were negative. All strains carry the nuc gene, which is specific to S aureus. In Algeria, the incidence of S aureus resistance is slightly lower compared to other countries, but it is increasing over time. It is now more crucial than ever to restrict the proliferation of multidrug-resistant strains and reduce undue antibiotic prescriptions. To achieve this, it is vital to keep updated on the epidemiology of this bacterium and its antibiotic susceptibility. This will enable the formulation of appropriate preventive control measures to manage its progression.

Metagenomic approach revealed the mobility and co-occurrence of antibiotic resistomes between non-intensive aquaculture environment and human.

BACKGROUND: Aquaculture is an important food source worldwide. The extensive use of antibiotics in intensive large-scale farms has resulted in resistance development. Non-intensive aquaculture is another aquatic feeding model that is conducive to ecological protection and closely related to the natural environment. However, the transmission of resistomes in non-intensive aquaculture has not been well characterized. Moreover, the influence of aquaculture resistomes on human health needs to be further understood. Here, metagenomic approach was employed to identify the mobility of aquaculture resistomes and estimate the potential risks to human health. RESULTS: The results demonstrated that antibiotic resistance genes (ARGs) were widely present in non-intensive aquaculture systems and the multidrug type was most abundant accounting for 34%. ARGs of non-intensive aquaculture environments were mainly shaped by microbial communities accounting for 51%. Seventy-seven genera and 36 mobile genetic elements (MGEs) were significantly associated with 23 ARG types (p < 0.05) according to network analysis. Six ARGs were defined as core ARGs (top 3% most abundant with occurrence frequency > 80%) which occupied 40% of ARG abundance in fish gut samples. Seventy-one ARG-carrying contigs were identified and 75% of them carried MGEs simultaneously. The qacEdelta1 and sul1 formed a stable combination and were detected simultaneously in aquaculture environments and humans. Additionally, 475 high-quality metagenomic-assembled genomes (MAGs) were recovered and 81 MAGs carried ARGs. The multidrug and bacitracin resistance genes were the most abundant ARG types carried by MAGs. Strikingly, Fusobacterium_A (opportunistic human pathogen) carrying ARGs and MGEs were identified in both the aquaculture system and human guts, which indicated the potential risks of ARG transfer. CONCLUSIONS: The mobility and pathogenicity of aquaculture resistomes were explored by a metagenomic approach. Given the observed co-occurrence of resistomes between the aquaculture environment and human, more stringent regulation of resistomes in non-intensive aquaculture systems may be required. Video Abstract.

Enhanced removal of ciprofloxacin and associated antibiotic-resistant genes from wastewater using a biological aeration filters in combination with Fe3O4-modified zeolite.

Antibiotics release into the water environment through sewage discharge is a significant environmental concern. In the present study, we investigated the removal of ciprofloxacin (CIP) in simulated sewage by biological aeration filter (BAF) equipped with Fe3O4-modified zeolite (Fe3O4@ZF). Fe3O4@ZF were prepared with impregnation method, and the Fe3O4 particles were successfully deposited on the surface of ZF in an amorphous form according to the results of XPS and XRD analysis. The modification also increased the specific surface area (from 16.22 m²/g to 22 m²/g) and pore volume (from 0.0047 cm³/g to 0.0063 cm³/g), improving the adsorption efficiency of antibiotics. Fe3O4 modified ZF improved the treatment performance significantly, and the removal efficiency of CIP in BAF-Fe3O4@ZF was 79%±2.4%. At 10ml/L CIP, the BAF-Fe3O4@ZF reduced the relative abundances of antibiotics resistance genes (ARGs) int, mexA, qnrB and qnrS in the effluent by 57.16%, 39.59%, 60.22%, and 20.25%, respectively, which effectively mitigate the dissemination risk of ARGs. The modification of ZF increased CIP-degrading bacteria abundance, such as Rhizobium and Deinococcus-Thermus, and doubled bacterial ATP activity, promoting CIP degradation. This study offers a viable, efficient method to enhance antibiotic treatment and prevent leakage via sewage discharge.

Molecular Prevalence and Geographical Variations of Carbapenem-Resistant Klebsiella pneumoniae ST15 Isolates in a Tertiary Hospital in Ningbo, China.

BACKGROUND In China, the most prevalent type of CRKP is ST11, but the high-risk clone ST15 has grown in popularity in recent years, posing a serious public health risk. Therefore, we investigated the molecular prevalence characteristics of ST15 CRKP detected in a tertiary hospital in Ningbo to understand the current potential regional risk of ST15 CRKP outbreak. MATERIAL AND METHODS We collected and evaluated 18 non-duplicated CRKP strains of ST15 type for antibiotic resistance. Their integrons, virulence genes, and resistance genes were identified using polymerase chain reaction (PCR), and their homology was determined using MALDI-TOF MS. RESULTS The predominant serotype of 18 ST15 CRKP strains was K5. ST15 CRKP exhibited the lowest antimicrobial resistance to Cefoperazone/sulbactam (11.1%), followed by trimethoprim/sulfamethoxazole (22.2%). Resistance gene testing revealed that 14 out of 18 ST15 CRKP strains (77.8%) carried Klebsiella pneumoniae carbapenemase 2 (KPC-2), whereas all ST15 CRKP integrons were of the intI1 type. Furthermore, virulence gene testing revealed that all 18 ST15 CRKP strains carried ybtS, kfu, irp-1, and fyuA genes, followed by the irp-2 gene (17 strains) and entB (16 strains). The homology analysis report showed that 2 clusters had closer affinity, which was mainly concentrated in classes C and D. CONCLUSIONS The ST15 CRKP antibiotic resistance rates demonstrate clear geographical differences in Ningbo. Additionally, some strains carried highly virulent genes, indicating a possible evolution towards carbapenem-resistant highly virulent strains. To reduce the spread of ST15 CRKP, we must rationalize the clinical use of antibiotics and strengthen resistance monitoring to control nosocomial infections.

Keeping up with the pathogens: improved antimicrobial resistance detection and prediction from Pseudomonas aeruginosa genomes.

BACKGROUND: Antimicrobial resistance (AMR) is an intensifying threat that requires urgent mitigation to avoid a post-antibiotic era. Pseudomonas aeruginosa represents one of the greatest AMR concerns due to increasing multi- and pan-drug resistance rates. Shotgun sequencing is gaining traction for in silico AMR profiling due to its unambiguity and transferability; however, accurate and comprehensive AMR prediction from P. aeruginosa genomes remains an unsolved problem. METHODS: We first curated the most comprehensive database yet of known P. aeruginosa AMR variants. Next, we performed comparative genomics and microbial genome-wide association study analysis across a Global isolate Dataset (n = 1877) with paired antimicrobial phenotype and genomic data to identify novel AMR variants. Finally, the performance of our P. aeruginosa AMR database, implemented in our AMR detection and prediction tool, ARDaP, was compared with three previously published in silico AMR gene detection or phenotype prediction tools-abritAMR, AMRFinderPlus, ResFinder-across both the Global Dataset and an analysis-naïve Validation Dataset (n = 102). RESULTS: Our AMR database comprises 3639 mobile AMR genes and 728 chromosomal variants, including 75 previously unreported chromosomal AMR variants, 10 variants associated with unusual antimicrobial susceptibility, and 281 chromosomal variants that we show are unlikely to confer AMR. Our pipeline achieved a genotype-phenotype balanced accuracy (bACC) of 85% and 81% across 10 clinically relevant antibiotics when tested against the Global and Validation Datasets, respectively, vs. just 56% and 54% with abritAMR, 58% and 54% with AMRFinderPlus, and 60% and 53% with ResFinder. ARDaP's superior performance was predominantly due to the inclusion of chromosomal AMR variants, which are generally not identified with most AMR identification tools. CONCLUSIONS: Our ARDaP software and associated AMR variant database provides an accurate tool for predicting AMR phenotypes in P. aeruginosa, far surpassing the performance of current tools. Implementation of ARDaP for routine AMR prediction from P. aeruginosa genomes and metagenomes will improve AMR identification, addressing a critical facet in combatting this treatment-refractory pathogen. However, knowledge gaps remain in our understanding of the P. aeruginosa resistome, particularly the basis of colistin AMR.