Investigation of in vitro susceptibility and resistance mechanisms to amikacin among diverse carbapenemase-producing Enterobacteriaceae.

OBJECTIVE: This study aims to assess the in vitro drug susceptibility of various Carbapenemase-Producing Enterobacteriaceae (CPE) genotypes and elucidate the underlying mechanisms of amikacin resistance. METHODS: A total of 72 unique CPE strains were collected from the Second Hospital of Jiaxing between 2019 and 2022, including 51 strains of Klebsiella pneumoniae, 11 strains of Escherichia coli, 6 strains of Enterobacter cloacae, 2 strains of Klebsiella aerogenes, 1 strain of Citrobacter freundii, and 1strain of Citrobacter werkmanii. Among these strains, 24 carried blaKPC gene, 20 carried blaNDM gene, 23 carried blaOXA-48-like gene, and 5 carried both blaKPC and blaNDM. We measured the in vitro activity of amikacin and other common antibiotics. Strains carrying blaOXA-48-like gene were selected for whole genome sequencing (WGS) via next-generation sequencing to identify genes related to antimicrobial resistance (AMR) and virulence factor (VF). RESULTS: Out of the 72 CPE strains tested, 41.7% exhibited resistance to amikacin. The drug resistance rates for K. pneumoniae, E. coli, and Enterobacter spp. were 51.0%, 27.3%, and 10.0%, respectively. The majority of the CPE strains (> 90%) displayed resistance to cephalosporins and carbapenems, while most of them were sensitive to polymyxin B and tigecycline (97.2% and 94.4%). The amikacin resistance rate was 100% for strains carrying blaOXA-48, 20.8% for those with blaKPC, 5.0% for those with blaNDM, and 20.0% for those with both blaKPC and blaNDM. These differences were statistically significant (P < 0.05). Through sequencing, we detected aminoglycoside resistance genes rmtF and aac(6')-Ib, VF genes iucABCD and rmpA2 in OXA-48-producing multidrug resistance and highly virulent strains. These genes were located on a IncFIB- and IncHI1B-type plasmid, respectively. Both plasmids were highly homologous to the plasmid from OXA-232 strains in Zhejiang province and Shanghai province. Integration of these resistance genes into the IncFIB plasmid, facilitated by the IS6 and/or Tn3 transposons, resulted in OXA232-producing K. pneumoniae with amikacin resistance. CONCLUSION: This study identified significant amikacin resistance in CPE strains, particularly in those carrying the blaOXA-48 gene. Resistance genes rmtF and aac(6')-Ib were identified on plasmids. These results highlight the need for careful monitoring of amikacin resistance.

Metallo-ß-lactamase-producing Pseudomonas aeruginosa Isolates from Two Tertiary Care Centres in a District of Nepal: A Descriptive Cross-sectional Study.

INTRODUCTION: Pseudomonas aeruginosa isolates producing metallo-ß-lactamase have caused nosocomial outbreaks, severe infections, and ineffective carbapenem therapy worldwide since 1991. Due to their prevalence, hospital infection control techniques are difficult. This study aimed to find out the prevalence of metallo-ß-lactamase among P. aeruginosa isolates from two tertiary care hospitals in Kathmandu. METHODS: A descriptive cross-sectional study was conducted at the Department of Microbiology and Department of Pathology of two tertiary care centres in Kathmandu from 7 December 2021 to 6 April 2023, after receiving ethical approval from the Ethical Review Board. Isolated strains were identified and tested for antibiotic susceptibility by modified Kirby-Bauer Methods. Metallo-ß-lactamase presence was confirmed using an imipenem-imipenem/ ethylenediaminetetraacetic acid disc. A convenience sampling method was used. The point estimate was calculated at 95% Confidence Interval. RESULTS: Among 255, Pseudomanas aeruginosa isolates, the distribution of metallo-ß-lactamase-producing Pseudomanas aeruginosa was 103 (40.39%) (34.32-46.69 at 95% Confidence Interval). Multidrug resistance categories included multidrug resistance 74 (71.80%), extensively drug resistance 32 (31.10%), P. aeruginosa difficult-to-treat 16 (15.53%) and carbapenem-resistant P. aeruginosa was determined to be 82 (79.60%). CONCLUSIONS: The study found a high prevalence of metallo-ß-lactamase-producing Pseudomanas aeruginosa isolates, requiring early identification, infection control measures, and an all-inclusive antimicrobial therapy protocol to reduce their spread in medical settings.

Investigation on the mechanisms of carbapenem resistance among the non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae.

Background: In Malaysia, an increase in non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) has been observed over the years. Previously, four NC-CRKP with increased susceptibility to ciprofloxacin in the presence of phenylalanine-arginine ß-naphthylamide (PAßN) were identified. However, no contribution of the PAßN-inhibited efflux pump to carbapenem resistance was observed. All four NC-CRKP harboured non-carbapenemase ß-lactamase, with two also exhibiting porin loss. In this study, we further investigated the genomic features and resistance mechanisms of these four isolates. Methods: All four NC-CRKP were subjected to whole-genome sequencing, followed by comparative genomic and phylogenetic analyses. Results: Multi-locus sequence typing (MLST) analysis divided the four NC-CRKP into different sequence types: ST392, ST45, ST14, and ST5947. Neither major nor rare carbapenemase genes were detected. Given the presence of non-carbapenemase ß-lactamase in all isolates, we further investigated the potential mechanisms of resistance by identifying related chromosomal mutations. Deletion mutation was detected in the cation efflux system protein CusF. Insertion mutation was identified in the nickel/cobalt efflux protein RcnA. Missense mutation of ompK36 porin was detected in two isolates, while the loss of ompK36 porin was observed in another two isolates. Conclusions: This study revealed that NC-CRKP may confer carbapenem resistance through a combination of non-carbapenemase ß-lactamase and potential chromosomal mutations including missense mutation or loss of ompK36 porin and/or a frameshift missense mutation in efflux pump systems, such as cation efflux system protein CusF and nickel/cobalt efflux protein RcnA. Our findings highlighted the significance of implementing whole-genome sequencing into clinical practice to promote the surveillance of carbapenem resistance mechanisms among NC-CRKP.

The enhanced antibacterial and antibiofilm properties of titanium dioxide nanoparticles biosynthesized by multidrug-resistant Pseudomonas aeruginosa.

The emergence of Multidrug-resistant (MDR) bacteria are becoming a major worldwide health concern, encouraging the development effective alternatives to conventional antibiotics. The study identified P. aeruginosa and assessed its antimicrobial sensitivity using the Vitek-2 system. Carbapenem-resistant genes were detected through Polymerase chain reaction (PCR). MDR- P. aeruginosa isolates were used to biosynthesize titanium dioxide nanoparticles (TiO2NPs) and characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM). A study involving 78 P. aeruginosa isolates revealed that 85.8% were MDR, with meropenem and amikacin showing effectiveness against 70% of the isolates. The most prevalent carbapenemase gene was blaOXA-48, present in 83% of the isolates. Majority of the isolates formed biofilms, and biosynthesized TiO2NPs were able to reduce biofilm formation by 94%. TiO2NPs exhibited potent antibacterial action against MDR-Gram-negative bacilli pathogens and showed synergistic activity with antibiotics, particularly piperacillin, with a significant fold increase in areas (283%). A new local strain of P. aeruginosa, identified as ON678251 in the World GenBank, was found capable of producing TiO2NPs. Our findings demonstrate the potential of biosynthesized TiO2NPs to manage antibiotic resistance and regulate the formation of biofilms. This presents a promising direction for the creation of novel antimicrobial agents or substitutes for use in clinical settings, particularly in the management of isolates capable of resisting multiple drugs.

Antimicrobial resistance profile and associated factors of hospital-acquired gram-negative bacterial pathogens among hospitalized patients in northeast Ethiopia.

BACKGROUND: Antimicrobial resistance is a major global public health issue. Infections caused by resistant species are associated with higher mortality rates, longer hospital stays, medication failure, and rising medical costs. The World Health Organisation has declared multidrug resistance-associated infections as an epidemic of public health concern. OBJECTIVE: This study aimed to evaluate the antimicrobial resistance profile and associated factors of hospital-acquired Gram-negative bacterial pathogens among hospitalized patients in Northeast Ethiopia. MATERIALS AND METHODS: A health facility-based cross-sectional study was conducted among hospitalized patients from March 2021 to February 2022. About 810 clinical specimens were collected, transported, and processed from admitted patients following the standard bacteriological procedures. The clinical samples were inoculated onto blood agar, MacConkey agar, and chocolate agar. Furthermore, the species identification was done using gram reactions, colony morphology, and color and biochemical tests. Antimicrobial susceptibility tests, extended-spectrum beta-lactamase, and carbapenemase production were performed as per the clinical laboratory standard institute guidelines. For analysis, the information was entered into Epi-data and exported to SPSS. A P value of < 0.05 with a 95% confidence interval was considered as a statistically significant association. RESULTS: Out of 810 clinical specimens, 285/810 (35.2%) developed bacterial infections. From the isolated bacteria, E. coli was the predominant bacteria accounting for 78/285 (27.4%) followed by K. pneumoniae, 69/285(24.42%), whereas P. vulgaris accounted for the least, 7/285 (2.5%). Overall, 132/285 (46.3%) and 99/285 (34.7%) of culture-positive patients were infected by extended-spectrum beta-lactamase and carbapenemase-producing bacteria. The overall multidrug resistance rate of the isolated bacteria was 89.4%. The highest antibiotic resistance rates were detected for doxycycline (92.9%), amoxicillin-clavulanic acid (83.9%), and ampicillin (93%). The least antibiotic resistance rate was observed for meropenem at 41.1% and amikacin at 1.7%, respectively. CONCLUSIONS AND RECOMMENDATIONS: In the study area, significant health concerns include a range of hospital-acquired bacterial infections associated with elevated rates of multidrug resistance, Extended-spectrum beta-lactamase (ESBL), and carbapenemase-producing bacterial pathogens. Consequently, it is recommended to conduct drug-susceptibility testing of isolates and molecular detection at a national level to optimize antibiotic usage for treating prevalent bacterial infections in this area.

The role of early use of Carbapenems perioperatively for urolithiasis with ESBL-producing Escherichia coli.

BACKGROUND: Urolithiasis combined with ESBL-producing E. coli is often difficult to control and leads to higher postoperative infection-related complications. This study was aim to explore the efficacy and necessity for early use of carbapenem antibiotics perioperatively in urolithiasis patients with urinary tract infections caused by ESBL-producing E. coli. METHODS: The study included a total of 626 patients who were separated into two groups: Group I (the ESBL-producing E. coli group) and Group II (the non-ESBL-producing E. coli group). Antibiotic susceptibility testing was performed and the two groups induced postoperative infection-related events were recorded. the efficacy of perioperative antibiotics was evaluated. RESULTS: All strains of E. coli in our research were sensitive to Carbapenems antibiotics. In addition to Carbapenems, the resistance rates of ESBL-producing E. coli to 6 other commonly used antibiotics were higher than those of non-ESBL-producing strains. Based on the preoperative antibiotic susceptibility test for the ESBL-producing E. coli group and the qSOFA score, the Carbapenems were more effective than the ß-lactamase inhibitors (p = 0.08), while for the non-ESBL-producing E. coli group, there was no difference in the treatment effects between Carbapenems, ß-lactamase inhibitors, Ceftazidime and Quinolones (p = 0.975). CONCLUSIONS: Carbapenem antibiotics significantly reduced the incidence of postoperative infection-related events compared with other types of antibiotics for ESBL-producing E. coli infections in patient with urolithiasis.

Effectiveness of selective antibiotics use in ESBL-related UTIs.

BACKGROUND: Urinary tract infections (UTIs) are the second most common infection, affecting 150 million people each year worldwide. Enterobacteriaceae species expressing extended-spectrum ß-lactamases (ESBLs) are on the rise across the globe and are becoming a severe problem in the therapeutic management of clinical cases of urinary tract infection. Knowledge of the prevalence and antibiogram profile of such isolates is essential to develop an appropriate treatment methodology. This study aimed to investigate the prevalence of Enterobacteriaceae isolates exhibiting ESBL and their selective oral antibiogram profile at the district general hospital, Polonnaruwa. RESULTS: A total of 4386 urine specimens received to the Microbiology Laboratory during the study period. Among them, 1081 (24.6%) showed positive results for urine culture while 200/1081 specimens showed ESBL isolates. Out of the selected 200 specimen's majority (67.5%) of samples received from the In-Patient Department. There were 200 patients and reported that 115 (57.5%) were females and 85 (42.5%) were males. The majority (51%) of the patients belong to the age group of 55-74 years. Among the ESBLs positive specimens, the majority 74.5% (n = 149) identified organisms were E. coli followed by Klebsiella spp.17.5% (n = 35), Enterobacteriaceae 7% (n = 14) and only1% (n = 2) isolate of Proteus spp. Mecillinam (87.92%) and Nitrofurantoin (83.2%) showed higher effectiveness against E. coli. Nitrofurantoin showed the highest effectiveness against Klebsiella spp. (40%), other Enterobacteriaceae spp. (100%). Proteus spp. showed 100% effectiveness and resistance respectively against Ciprofloxacin, Cotrimoxazole and Nitrofurantoin. CONCLUSION: The most predominant ESBLs producing uro-pathogen was the E. coli in the study setting and E. coli had higher sensitivity rate against Mecillinam. Among currently used oral antibiotics Nitrofurantoin was the best choice for UTIs caused by ESBL producers.

First report of a bla NDM-producing extensively drug resistant Klebsiella pneumoniae ST437 in Italy.

Carbapenemase-producing Klebsiella pneumoniae strains (CP-Kps) have recently been observed to spread rapidly worldwide. New Delhi metallo-ß-lactamase (NDM) producing clones of Klebsiella pneumoniae (K. pneumoniae) cause a significant healthcare burden, particularly in Indian sub-continent, where this clone is circulating widely. However, in Italy, data on the incidence of these new clones is limited, and an ST437 NDM-producing K. pneumoniae strain has not been reported to date. A sacral ulcer infection caused by a K. pneumoniae strain was identified in an 85-year-old Italian male patient with several comorbidities. Antimicrobial susceptibility testing revealed an extensive resistance to a wide range of antimicrobials, including novel agents such as cefiderocol and ceftazidime/avibactam. Genomic analysis identified the pathogen as an ST437 K. pneumoniae strain harboring bla NDM-5, bla OXA-232 and bla CTX-M-15 genes. Following the identification of this first case, several infection control measures were implemented in healthcare settings, including direct precautions and reinforcement of standard cross-transmission control measures. The emergence of pathogenic microbial clones carrying new genetic determinants, particularly in a little city, requires prompt diagnosis and therapeutic protocols. An effective infection control system for the early detection and/or control of the transmission of NDM-producing Enterobacteriaceae is also needed. Further investigations are required to better understand the potential transmission routes and evolution of these clones.

Understanding epistatic networks in the B1 ß-lactamases through coevolutionary statistical modeling and deep mutational scanning.

Throughout evolution, protein families undergo substantial sequence divergence while preserving structure and function. Although most mutations are deleterious, evolution can explore sequence space via epistatic networks of intramolecular interactions that alleviate the harmful mutations. However, comprehensive analysis of such epistatic networks across protein families remains limited. Thus, we conduct a family wide analysis of the B1 metallo-ß-lactamases, combining experiments (deep mutational scanning, DMS) on two distant homologs (NDM-1 and VIM-2) and computational analyses (in silico DMS based on Direct Coupling Analysis, DCA) of 100 homologs. The methods jointly reveal and quantify prevalent epistasis, as ~1/3rd of equivalent mutations are epistatic in DMS. From DCA, half of the positions have a >6.5 fold difference in effective number of tolerated mutations across the entire family. Notably, both methods locate residues with the strongest epistasis in regions of intermediate residue burial, suggesting a balance of residue packing and mutational freedom in forming epistatic networks. We identify entrenched WT residues between NDM-1 and VIM-2 in DMS, which display statistically distinct behaviors in DCA from non-entrenched residues. Entrenched residues are not easily compensated by changes in single nearby interactions, reinforcing existing findings where a complex epistatic network compounds smaller effects from many interacting residues.

Evaluation of virulence determinants and cell surface properties associated with biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) Escherichia coli from livestock and poultry origin.

Antibiotic resistance poses a persistent threat to modern medicine due to the emergence of novel antibiotic-resistant strains. Therefore, a timely understanding of antibiotic resistance and the virulence biology of pathogenic bacteria, particularly those of public health significance, is crucial for implementing effective mitigation strategies. This study aimed to investigate the virulence profiles of ten S. aureus isolates (NDa to NDj) and ten E. coli isolates (ND1 to ND10) originating from livestock and poultry, and to assess how various cell surface properties and biofilm formation abilities influence antibiotic resistance phenotypes. Antibiotic resistance profiling through phenotypic (AST) and genotypic methods (PCR) confirmed that NDa to NDe were methicillin-resistant S. aureus (MRSA) and ND1 to ND5 were extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Virulence properties such as hemolytic activity, coagulase activity, and nuclease activity were found to be independent of the antibiotic resistance phenotype in S. aureus. In contrast, biofilm formation phenotype was observed to influence antibiotic resistance phenotypes, with MRSA and ESBL E. coli isolates demonstrating higher biofilm formation potency. Chemical and enzymatic analysis of S. aureus and E. coli biofilms revealed proteins and polysaccharides as major components, followed by nucleic acids. Furthermore, cell surface properties such as auto-aggregation and hydrophobicity were notably higher in isolates with strong to medium biofilm-forming capabilities (ESBL and MRSA isolates), corroborated by genomic confirmation of various genes associated with biofilm, adhesion, and colonization. In conclusion, this study highlights that surface hydrophobicity and biofilm formation ability of MRSA (NDa to NDe) and ESBL E. coli (ND1 to ND5) isolates may influence antibiotic resistance phenotypes.

Carbapenemase and extended-spectrum beta-lactamase-producing bacteria in waters originating from a single landfill in Slovenia.

Groundwater, rainwater, and leachate associated with a single landfill were analysed to detect extended-spectrum beta-lactamase (ESBL)-producing and carbapenemase (CP)-producing bacteria. After cultivation on three commercial selective-differential media, 240 bacterial isolates were obtained and identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Isolates from clinically relevant species were further genotyped by enterobacterial repetitive intergenic consensus polymerase chain reaction, and tested for antibiotic susceptibility and presence of CPs and ESBL enzymes. Two ESBL-producing isolates and two isolates producing CPs were detected in rainwater, groundwater, and leachate: Klebsiella oxytoca complex with the gene for the ESBL enzyme CTX-M-1 and the gene for the CP OXA-48, Serratia fonticola with the gene for the ESBL enzyme FONA-2, and Pseudomonas aeruginosa with the gene coding Verona integron-encoded Metallo-beta-lactamases (VIM) metallo-beta-lactamase. Our study indicates that bacteria with ESBL and CP genes can be present in landfill-associated waters.

Private benefit of ß-lactamase dictates selection dynamics of combination antibiotic treatment.

ß-lactam antibiotics have been prescribed for most bacterial infections since their discovery. However, resistance to ß-lactams, mediated by ß-lactamase (Bla) enzymes such as extended spectrum ß-lactamases (ESBLs), has become widespread. Bla inhibitors can restore the efficacy of ß-lactams against resistant bacteria, an approach which preserves existing antibiotics despite declining industry investment. However, the effects of combination treatment on selection for ß-lactam resistance are not well understood. Bla production confers both private benefits for resistant cells and public benefits which faster-growing sensitive cells can also exploit. These benefits may be differentially impacted by Bla inhibitors, leading to non-intuitive selection dynamics. In this study, we demonstrate strain-to-strain variation in effective combination doses, with complex growth dynamics in mixed populations. Using modeling, we derive a criterion for the selection outcome of combination treatment, dependent on the burden and effective private benefit of Bla production. We then use engineered strains and natural isolates to show that strong private benefits of Bla are associated with increased selection for resistance. Finally, we demonstrate that this parameter can be coarsely estimated using high-throughput phenotyping of clonal populations. Our analysis shows that quantifying the phenotypic responses of bacteria to combination treatment can facilitate resistance-minimizing optimization of treatment.

Methodological Evaluation of Carbapenemase Detection by Different Methods.

The global proliferation of carbapenemase-producing bacteria (CPB) has garnered significant attention worldwide. Early diagnosis of CPB and accurate identification of carbapenemases are crucial for preventing the spread of CPB and ensuring targeted antibiotic therapy. Therefore, efficient and accurate identification of carbapenemases is paramount in clinically treating diseases associated with CPB. In this study, 58 CPB strains were collected and detected using the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method, a rapid detection platform based on CRISPR-Cas12a gene editing and isothermal amplification. Additionally, four conventional methods (the APB/EDTA method, PCR, NG-test Carba 5, and GeneXpert Carba-R) were employed and compared against whole genome sequencing (WGS) results, considered the gold standard, to evaluate their efficacy in detecting carbapenemases. Detection by the APB/EDTA method revealed that 29 strains were positive for Class A serine endopeptidases, while 29 strains were positive for Class B metalloenzymes. The classification of these zymotypes was consistent with the sequencing result. All target carbapenemases for KPC were identified with 100% sensitivity using NG-test Carba 5, PCR, DETECTR, and GeneXpert Carba-R. In the case of NDM, both Xpert Carba-R and DETECTR showed a sensitivity of 100%. In contrast, NG-test Carba 5 and PCR had a slightly lower sensitivity of 96.7%, each missing one target carbapenemase. n this study, the APB/EDTA method is capable of identifying the zymotype classification but not the specific resistant genes, while Xpert Carba-R and DETECTR are able to detect all target carbapenemases.

High qnrS retention of ESBL-producing and mcr-harbouring colistin-resistant Escherichia coli in Vietnamese food products.

Plasmid-mediated antibiotic-resistant bacteria's transmission is fatal and a major threat to public health. This study aimed to clarify the presence of plasmid-mediated quinolone resistance（PMQR）genes in extended-spectrum ß-lactamase（ESBL）-producing or/and mcr-harbouring colistin（COL）-resistant Escherichia coli（ESBL-COL-EC）isolates from Vietnamese and Japanese chicken meat. Resistance towards ciprofloxacin（CIP）was examined in 308 ESBL-COL-EC isolates; CIP-resistant ESBL-COL-EC isolates were examined for the PMQR gene. Approximately, 71.1% and 38.1% of ESBL-COL-EC and ESBLproducing E. coli isolates from Vietnamese and Japanese chicken meat were CIP-resistant, respectively. Multiplex PCR led PMQR detection showed that 35.2% of CIP-resistant ESBL-COL-EC isolates from Vietnamese food contained PMQR gene, whereas CIP-resistant ESBL-COL-EC isolates from Japanese chicken meat did not. Conjugation assays showed that the transmission of qnrS gene carried by E. coli to Salmonella. In conclusion, ESBL-COL-EC isolates from Vietnamese food are associated with a high frequency of fluoroquinolone resistance and a high distribution of the qnrS gene.

FosA3 emerging in clinical carbapenemase-producing C. freundii.

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales, but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii. The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3, blaKPC-2, and blaVIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3-harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales, affecting even low-virulence pathogens, as C. freundii.

Prevalence, risk factors, and characterisation of extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) in horses entering an equine hospital and description of longitudinal excretion.

BACKGROUND: Extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) are important zoonotic pathogens that can cause serious clinical infections, also in horses. Preventing the spread of ESBL-E, especially in the equine hospital environment, is key to reducing the number of difficult-to-treat infections. Estimating the local prevalence of ESBL-E in horses is crucial to establish targeted infection control programs at equine hospitals. We conducted a prevalence and risk factor study in equine patients on admission to an equine teaching hospital in Finland through a rectal ESBL-E screening specimen of the horse and a questionnaire. RESULTS: The prevalence of ESBL-E in admitted horses was 3% (5/161, 95% CI 1-7%); none of the tested factors remained statistically significant in multivariate analysis, although antimicrobial treatment within three months was borderline significant (p = 0.052). Extended-spectrum ß-lactamase -producing Klebsiella pneumoniae ST6179:CTX-M-15 was detected in three horses using whole-genome sequencing, which in combination with patient records suggested nosocomial transmission. Escherichia coli isolates were ST1250:CTX-M-1 (n = 1), ST1079:CTX-M-1 (n = 1), and ST1245:CTX-M-14 (n = 1). Multiple virulence genes were detected in the ESBL-E isolates. In the ESBL-E positive horses enrolled in a one-year follow-up study, ESBL-E were unlikely to be isolated in rectal screening specimens after the initial positive specimen. CONCLUSIONS: The prevalence of ESBL-E in horses visiting a veterinary teaching hospital in Finland is low, indicating an overall low prevalence estimate in the country's equine population. No statistically significant risk factors were identified, likely due to the low number of cases. The duration of ESBL-E carriage is likely to be very short in horses.

Identifying Artifacts from Large Library Docking.

While large library docking has discovered potent ligands for multiple targets, as the libraries have grown the hit lists can become dominated by rare artifacts that cheat our scoring functions. Here, we investigate rescoring top-ranked docked molecules with orthogonal methods to identify these artifacts, exploring implicit solvent models and absolute binding free energy perturbation as cross-filters. In retrospective studies, this approach deprioritized high-ranking nonbinders for nine targets while leaving true ligands relatively unaffected. We tested the method prospectively against hits from docking against AmpC ß-lactamase. We prioritized 128 high-ranking molecules for synthesis and testing, a mixture of 39 molecules flagged as likely cheaters and 89 that were plausible inhibitors. None of the predicted cheating compounds inhibited AmpC detectably, while 57% of the 89 plausible compounds did so. As our libraries continue to grow, deprioritizing docking artifacts by rescoring with orthogonal methods may find wide use.

Emergence and transmission of the high-risk ST78 clone of OXA-48-producing Enterobacter hormaechei in a single hospital in Taiwan.

Carbapenem-resistant Enterobacter cloacae complex is a significant global healthcare threat, particularly carbapenemase-producing Enterobacter hormaechei (CPEH). From January 2017 to January 2021, twenty-two CPEH isolates from a regional teaching hospital in central Taiwan were identified with the carriage of carbapenemase genes blaKPC-2, blaIMP-8, and predominantly blaOXA-48. Over 80% of these CPEH strains clustered into the high-risk ST78 lineage, carrying a blaOXA-48 IncL plasmid (pOXA48-CREH), nearly identical to the endemic plasmid pOXA48-KP in ST11 Klebsiella pneumoniae. This OXA-48-producing ST78 lineage disseminated clonally from 2018 to 2021 and transferred pOXA48-CREH to ST66 and ST90 E. hormaechei. An IMP-8-producing ST78 strain harbouring a blaIMP-8-carrying pIncHI2 plasmid appeared in 2018, and by late 2020, a KPC-2-producing ST78 strain was identified after acquiring a novel blaKPC-2-carrying IncFII plasmid. These findings suggest that the high-risk ST78 lineage of E. hormaechei has emerged as the primary driver behind the transmission of CPEH. ST78 has not only acquired various carbapenemase-gene-carrying plasmids but has also facilitated the transfer of pOXA48-CREH to other lineages. Continuous genomic surveillance and targeted interventions are urgently needed to control the spread of emerging CPEH clones in hospital settings.

Gut microbiota-derived butyrate selectively interferes with growth of carbapenem-resistant Escherichia coli based on their resistance mechanism.

We investigated consequences of resistance acquisition in Escherichia coli clinical isolates during anaerobic (continuous culture) growth and examined their sensitivity to butyrate, a hallmark metabolite of healthy gut microbiota. Strains were stratified based on carrying either a carbapenemase (CARB) or displaying porin malfunctioning (POR). POR displayed markedly altered growth efficiencies, lower membrane stability and increased sensitivity to butyrate compared with CARB. Major differences in global gene expression between the two groups during anaerobic growth were revealed involving increased expression of alternative substrate influx routes, the stringent response and iron acquisition together with lower expression of various stress response systems in POR. Longitudinal analyses during butyrate wash-in showed common responses for all strains as well as specific features of POR that displayed strong initial "overshoot" reactions affecting various stress responses that balanced out over time. Results were partly reproduced in a mutant strain verifying porin deficiencies as the major underlying mechanism for results observed in clinical isolates. Furthermore, direct competition experiments confirmed butyrate as key for amplifying fitness disadvantages based on porin malfunctioning. Results provide new (molecular) insights into ecological consequences of resistance acquisition and can assist in developing measures to prevent colonization and infection based on the underlying resistance mechanism.

Extended-spectrum beta-lactamase (ESBL)- and non-ESBL producing Escherichia coli surveillance in surface water sources in Edo State, Nigeria: a public health concern.

This research explores the antimicrobial resistance (AMR) profiles and prevalence of extended-spectrum beta-lactamase (ESBL) and non-ESBL-producing Escherichia coli in Ojerame Dam and Ovokoto Spring, Edo State, Nigeria. Over 12 months, water was systematically sampled to accommodate seasonal variations and analyzed by employing an ESBL-selective medium for bacterial species. Additionally, bacterial isolates underwent identification and characterization using polymerase chain reaction (PCR) and disk diffusion methods to evaluate their susceptibility to antimicrobials. Results indicated significant prevalence of ESBL-producing E. coli, which exhibited complete resistance to common antimicrobials like ceftriaxone, ceftazidime, cefotaxime, and ampicillin while demonstrating 100% sensitivity to ertapenem, imipenem, meropenem, and nitrofurantoin. Non-ESBL-producing E. coli were resistant to ampicillin but sensitive to other antimicrobials mentioned earlier. Furthermore, both ESBL and non-ESBL-producing E. coli displayed multidrug resistance to varying degrees. Specific ESBL genes, including blaTEM, blaCTX-M-1, and blaCTX-M-15, were identified, alongside resistance genes like tetA, tetM, sul1, sul2, sul3, qnrA, qnrB, and qnrS in E. coli. This study pioneers the documentation of ESBL-producing E. coli in surface water in the region. This signals impending health risks associated with water being a reservoir of resistant genes while emphasizing the urgency for further research and public awareness concerning the quality of surface water.