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Microflora of reptiles and amphibians pose a serious threat to the environment and humans, as these bacteria are more resistant to antimicrobials or have more virulence factors. In this study, we focused on the characterization of Gram-negative bacterial isolates obtained from Mauremys rivulata (Western Caspian turtle), Natrix natrix (Grass snake), Natrix tessellata (Dice snake) and Pelophylax bedriagae (Levantine frog) using cultural methods and determine some virulence factors (antibiotic resistance, VanA and TetM resistant genes, biofilm, siderophore and some enzymatic activities etc.). Multiple antibiotic resistance (MAR) and virulence index (VI) of each strain were also calculated. A total of 85 Gram-negative isolates exhibited varying levels of antibiotic resistance and the ability to produce one or more extracellular enzymes such as amylase, lecithinase, protease, lipase, hemolysis, siderophore, amino acid decarboxylase and DNase. Although the TetM has been identified in all 7 isolates, none of the isolates have shown the presence of the VanA gene. According to MAR and VI results, most of the isolates were found to be in the high threat group and these indexes were correlated with antibiotic resistance. The study data revealed that chemicals used in industry and agriculture both trigger environmental pollution and pose risk factors for wildlife.

The evolution of during the modern antibiotic era has been delineated by distinct strain emergence events, many of which include acquisition of antibiotic resistance. The relative high burden of methicillin-resistant (MRSA) in healthcare and community settings is a major concern worldwide. Vancomycin, a glycopeptide antibiotic that inhibits cell wall biosynthesis, remains a drug of choice for treatment of severe MRSA infections. strains exhibiting increased resistance to vancomycin, known as vancomycin intermediate-resistant (VISA) (MIC = 4-8 µg/mL), were discovered in the 1990s. The molecular basis of resistance in VISA is polygenic and involves stepwise mutations in genes encoding molecules predominantly involved in cell envelope biosynthesis. isolates with complete resistance to vancomycin (MIC ≥ 16 µg/mL) are termed vancomycin-resistant (VRSA)-they were first reported in the U.S. in 2002. Resistance in VRSA is conferred by the gene and operon, which is present on a plasmid. Although treatment of VRSA infections is challenging, the total number of human VRSA infections to date is limited (14 in the U.S.). By comparison, the burden of VISA is relatively high and the molecular mechanisms of resistance are less well-defined. VISA are associated with persistent infections, vancomycin treatment failure, and poor clinical outcomes. Here, we review in brief progress made toward understanding the acquisition of antibiotic resistance in , with an emphasis on the molecular mechanisms underlying vancomycin resistance.

Background ESKAPEE pathogens Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , Enterobacter spp. and Escherichia coli are multi-drug resistant (MDR) bacteria that present increasing treatment challenges for healthcare institutions and public health worldwide. Methods 431 MDR ESKAPEE pathogens were collected from Queen Sirikit Naval Hospital, Chonburi, Thailand between 2017 and 2018. Species identification and antimicrobial resistance (AMR) phenotype were determined following CLSI and EUCAST guidelines on the BD Phoenix System. Molecular identification of antibiotic resistant genes was performed by polymerase chain reaction (PCR), real-time PCR assays, and whole genome sequencing (WGS). Results Of the 431 MDR isolates collected, 1.2% were E. faecium , 5.8% were S. aureus , 23.7% were K. pneumoniae , 22.5% were A. baumannii , 4.6% were P. aeruginosa , 0.9% were Enterobacter spp . , and 41.3% were E. coli . Of the 401 Gram-negative MDR isolates, 51% were carbapenem resistant, 45% were ESBL producers only, 2% were colistin resistance and ESBLs producers (2%), and 2% were non-ESBLs producers. The most prevalent carbapenemase genes were bla OXA-23 (23%), which was only identified in A. baumannii , followed by bla NDM (17%), and bla OXA-48-like (13%). Beta-lactamase genes detected included bla TEM, bla SHV , bla OXA , bla CTX-M , bla DHA , bla CMY , bla PER and bla VEB . Seven E. coli and K. pneumoniae isolates showed resistance to colistin and carried mcr-1 or mcr-3 , with 2 E. coli strains carrying both genes. Among 30 Gram-positive MDR ESKAPEE, all VRE isolates carried the vanA gene (100%) and 84% S. aureus isolates carried the mecA gene. Conclusions This report highlights the prevalence of AMR among clinical ESKAPEE pathogens in eastern Thailand. E. coli was the most common MDR pathogen collected, followed by K. pneumoniae , and A. baumannii . Carbapenem-resistant Enterobacteriaceae (CRE) and extended spectrum beta-lactamases (ESBLs) producers were the most common resistance profiles. The co-occurrence of mcr-1 and mcr-3 in 2 E. coli strains, which did not affect the level of colistin resistance, is also reported. The participation of global stakeholders and surveillance of MDR remain essential for the control and management of MDR ESKAPEE pathogens.

High-level, plasmid-based, transferable vancomycin resistance is documented in a community-associated MRSA organism. The vancomycin-resistant strain caused a serious bloodstream infection in a patient in Brazil. Since the description in 2002 of the vanA gene cluster in MRSA as the mechanism of high-level vancomycin resistance, 13 isolates have been reported in the United States, 1 , 2 with others reported in India 3 and Iran. 4 Enterococcus faecalis and E. faecium have been implicated as the donors of the vancomycin resistance genes. 5 , 6 The VRSA isolates were recovered from patients with soft-tissue or skin infections, and multilocus sequence typing has shown that 12 U.S. VRSA isolates belong to clonal complex 5 (including ST5, ST85, ST231, and ST371) 1 and that the 13th isolate belongs to clonal complex 30. Clonal complex 5 . . .

Objective The purpose of this study is to reduce the spread of the v anA gene by curing the vanA -harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system. Methods Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance. Results In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA -harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA -containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19- vanA . Conclusion In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA . This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

The emergence of pandrug-resistant (PDR) and extensive drug-resistant (XDR) methicillin-resistant and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) isolates from bovine milk samples along with biofilm formation ability and harboring various virulence genes complicates the treatment of bovine mastitis and highlights the serious threat to public health. This study investigated for the first time the frequency, antimicrobial resistance profiles, biofilm-forming ability, virulence factors, spa and staphylococcal cassette chromosome mec (SCC mec ) types of MRSA and VRSA isolated from clinical and subclinical bovine mastitis in Egypt. A total of 808 milk samples were collected from each quarter of 202 dairy animals, including 31 buffaloes and 171 cattle. The frequency of mastitis in the collected milk samples was 48.4% (60/124) in buffaloes and 29.2% (200/684) in cattle. A total of 65 Staphylococcus species isolates were recovered, including 27 coagulase-positive S. aureus (CoPS) isolates and 38 coagulase-negative staphylococci (CoNS). The CoNS included 27 mammaliicocci (20 Mammaliicoccus lentus and 7 M. sciuri ) and 11 Non-aureus staphylococci ( S. lugdunensis ) isolates. All the CoPS isolates were mec A positive and resistant to 20–33 tested antimicrobials with multiple antibiotic resistance index ranging from 0.61 to 1. Three isolates were PDR, four were XDR, and 20 were multidrug resistant isolates. VRSA was detected in 85.2% of CoPS isolates with minimal inhibitory concentration (MIC) ranging from 64 to 1024 µg/mL. The van A gene was found in 60.8%, van B in 73.9%, and both genes in 43.5% of VRSA isolates. All the CoPS isolates exhibited biofilm formation ability, with 55.6% being strong, and 44.4% moderate biofilm producers, and harbored ica A (74.1%) and ica D (74.1%) biofilm-forming genes. All S. aureus isolates harbored both beta-haemolysin ( hlb ) and leucotoxin ( luk MF) genes, while 44.4% were positive for toxic shock syndrome toxin ( tsst ) gene. Enterotoxin genes sea , seb , sec , sed , and see were found in 59.3%, 40.7%, 18.5%, 33.3%, and 14.8% of isolates, respectively. Additionally, 70.4% of the isolates had  spa X-region gene, and exhibited eight different MRSA spa types (t127, t267, t037, t011, t843, t1081, t2663, and t1575), with spa t127 being the most common. Three SCC mec types (I, II and III) were identified, with SCC mec I being predominant, and were further classified into subtypes 1.1.1, 1.1.2, 1.n.1, and 4.1.1. The ability of MRSA and VRSA isolates to produce biofilms and resist antimicrobials highlights the serious threat these pathogens pose to bovine milk safety, animal welfare, and public health. Therefore, strict hygiene practices and antimicrobial surveillance are crucial to reduce the risk of MRSA and VRSA colonization and dissemination.

Vancomycin-resistant Enterococci (VRE) are major pathogens causing nosocomial infections globally. This study investigated the genetic characteristics of vancomycin-resistant Enterococcus faecium (VREfm) in Thailand between June and November 2022. Fifty-two clinical VREfm isolates from Bangkok hospitals were analyzed for antimicrobial susceptibility, resistance genes, virulence factors, and genotypes using multilocus sequence typing (MLST). Phylogenetic analysis and goeBURST assessed genetic relationships and population structure. The VRE detection rate was 14.5%, with 97.1% E. faecium and 2.9% E. faecalis , likely reflecting the impact of an active case-finding program. All isolates exhibited resistance to penicillin, ampicillin, vancomycin, levofloxacin, ciprofloxacin, and rifampin. Resistance to erythromycin, high-level streptomycin, teicoplanin, and tetracycline occurred in 98.1%, 53.8%, 51.9%, and 17.3% of isolates, respectively. Chloramphenicol, linezolid, and high-level gentamicin remained effective against all isolates. The van A gene was the sole resistance determinant detected. Virulence genes esp and hyl were present in 100% and 88.5% of isolates, respectively. MLST identified five sequence types (STs), with ST17 (86.5%) as the dominant lineage, followed by ST262 (7.7%), ST202, ST787, and ST80 (1.9% each). All isolates belonged to Clonal Complex 17. Genome analysis revealed various resistance genes (VanHAX, aac (6')-Ii, aad (6), ant(6)-Ia , msr C, and tet M) and virulence factors ( acm , bop D, cps A/ upp S, cps B/ cds A, ebp A, ebp B, ebp C, efa A, esp , sgr A, and srt C). The van A gene primarily drives vancomycin resistance in Thailand’s VREfm. Genome analysis reveals antibiotic resistance genes, virulence factors, and mobile genetic elements that may drive antimicrobial resistance, increase diversity, and support adaptation in hospital settings. Ongoing infection control and active surveillance are essential.

Enterococci are opportunistic zoonotic pathogens. Dairy cattle and farm environments are considered important sources of Enterococcus spp. Here, we detected biofilm-forming Enterococcus faecalis circulating in dairy cattle and farm environments, followed by the detection of their virulence genes, antibiogram phenotype analysis, and genotype characterization. Isolates were cultured and identified by PCR. Ability to biofilm formation was assessed using the Congo red agar test., followed by a disk diffusion test for antibiogram and PCR for virulence and resistance genes detection. Among 150 samples collected from 12 farms, 145 were culture-positive for Enterococci. Among these, 74 were PCR screened, of which 54.05% (40/74, CI 95%: 42.78–64.93) were E. faecalis . About 50% of E. faecalis isolates were strong biofilm formers, 37.5% were intermediate, and 12.5% were weak biofilm formers. In the antibiogram study, 87.5% of isolates were resistant to rifampicin, 75% to erythromycin, 67.5% to vancomycin, and 62.5% to ampicillin. Of the positive isolations of E. faecalis , 80% were positive for the vanA gene, and 50% were positive for the blaTEM resistance gene. Surprisingly, about 70% (28/40) of isolates showed a multidrug resistance phenotype. The Highest levels of multidrug-resistant E. faecalis were present in manure (87.5%) and isolates from Ullapara, Sirajganj. In PCR, 83.33%, 87.50%, 92.67%, 75%, 87.50%, and 58.33% isolates were positive for virulence genes agg, ace, pil, fsrA, fsrB , and gelE . This study marks the first investigation in Bangladesh focused on the molecular identification of biofilm-forming, multidrug-resistant strains of E. faecalis from dairy cattle and farm environments. We recommend implementing a One Health approach with the adoption of effective biosecurity and good farm management to monitor this multi-drug-resistant (MDR) E. faecalis in dairy cattle and farm environments, aiming to effectively tackle the critical challenge of antimicrobial resistance.

Vancomycin-resistant enterococci (VRE) are a major public health concern, yet little is known about their circulation in fish. This study investigated the occurrence, glycopeptide resistance genotypes, virulence characteristics, and sequence types (STs) of VRE isolated from diseased fishes and humans. Isolates were identified using multiplex polymerase chain reaction (PCR) assay and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and tested for antimicrobial susceptibility. VRE isolates were screened for the presence of glycopeptide resistance genes and eight virulence genes. Multilocus sequence typing (MLST) was determined to assess the clonality of VRE isolates from fishes and humans. Among 60 human samples, 20 Enterococcus species isolates (33.33%) including Enterococcus faecalis and Enterococcus faecium (10 of each), were identified. The overall prevalence of E. faecalis was 42.86% (30/70) in Oreochromis niloticus and 48.0% (24/50) in Clarias gariepinus . E. faecium was found in 15.71% (11/70) of Oreochromis niloticus and 14.0% (7/50) of Clarias gariepinus . Over 50% of human isolates were multidrug resistant (MDR) and 30% exhibited an extensive drug resistant (XDR) phenotype. Fish isolates also displayed high MDR (70.83%) and XDR (29.17%) rates. Forty-nine (53.26%; 34 from fish and 15 from human) isolates were VRE including 30 isolates of E. faecalis (VREfs) and 19 isolates of E. faecium (VREfm). The vanA gene was the most frequent among VREfs (83.33%) and VREfm (100%) isolates. The vanB gene was found in 26.67% of VREfs and 15.79% of VREfm. Three out of 10 VREfm (30%) and 2/24 (8.33%) VREfs isolates of fish origin carried both vanA and vanB genes. vanC gene was found in 13.33% (4/30) of VREfs of human and fish origin. One VREfs isolate from human urine carried both vanA and vanC genes. High frequency of the virulence genes  gelE , sprE , asa1 , esp, and cylA were observed;  efa  and ace gene was more associated with VREfs, while hyl  gene was more frequently detected in VREfm. Different combinations of virulence genes suggesting synergistic pathogenic potential. MLST revealed both overlapping and host-specific STs among the examined Enterococcus isolates from humans and fish. Experimental infection of O. niloticus with VREfs and VREfm caused a 100% and 60% mortality rate within 6 days postinfection, respectively with characteristic disease symptoms. The emergence of VRE and the high prevalence of virulence traits could be regarded as an alarming situation. The call for increased infection control and antibiotic stewardship measures is timely and relevant to combat the spread of VRE in fish and humans.

Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn 1546 , which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn 1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012–2015). Genomic information regarding clonality and Tn 1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn 1546 spread accounted for vanA -type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn 1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn 1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn 1546 variant. In 2% of cases, we observed the same Tn 1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA- type resistance.