Extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae, implicated in urinary tract infections, represent a major challenge and limit therapeutic options. Our study sought to characterize the genes of resistance to IMP and NDM in urine samples from patients in the city of Ouagadougou by real-time PCR. This cross-sectional study focused on 130 bacterial isolates collected from February to July 2023 at HOSCO and CERBA. The determination of the enterobacterium species was followed by the performance of an antibiogram. Gene detection consisted of the extraction of bacterial DNA and its amplification by real-time PCR. 60 isolates came from male patients (46.2%) and 70 from female patients (53.8%), for a sex ratio (M/F) of 0.85 and an age between 51 days and 83 years, including 87.7% over 20 years. The most frequent species were respectively Escherichia coli (64.6%), Klebsiella sp. (30.8%), Proteus sp. (3.1%) and Enterobacter cloacae (1.5%). Resistance to imipenem for all Enterobacteriaceae strains was 19.2% and the majority gene was NDM (70%). The IMP gene was rare with only one positive case (0.8%) which was a coexistence of the 2 genes IMP and NDM. The NDM gene could be a cause of resistance to antibiotics, particularly imipenem, and special attention should be paid to Escherichia coli in the fight against the emergence of extended-spectrum β-lactamases. Real-time PCR could serve as a routine analytical technique to facilitate obtaining more reliable results in a short timeframe.

Background: Antibiotic resistance in Enterobacteriaceae is a major global threat, particularly in hospitalized children, where it leads to difficult-to-treat infections and prolonged hospital stays. The gastrointestinal tract serves as a key reservoir for transmission, and limited pediatric data, including in Togo, highlights the need for further research. Aims: To estimate the prevalence of digestive carriage of multidrug-resistant Enterobacteriaceae (MDR-E) upon admission and after antibiotic treatment among children hospitalized in the paediatric department of the Sylvanus Olympio Teaching Hospital, Lomé, Togo, in 2023. Study Design: This was a cross-sectional before-and-after study involving children who received empirical antibiotic therapy. Place and Duration of Study: Intensive care units of the paediatric department, and microbiology laboratory of the of the Sylvanus Olympio Teaching Hospital in Lomé (Togo). The study took place from June 6 to September 11, 2023. Methodology: 203 rectal swabs were collected at admission, and 132 rectal swabs after empirical antibiotic therapy at discharge in hospitalized children. Screening for resistance was performed using media made selective through the addition of antibiotics. MacConkey medium (Oxoid) was made selective by the addition of cefotaxime (CTX) at a ratio of 20 ml of MacConkey medium+ 20 µl/ml of CTX for ESBL screening, and by adding ertapenem (ERT) at a ratio of 20 ml of MacConkey medium +0.5 µl/ml of ERT to screen for carbapenem-resistant strains. Results: Rectal swab at admission revealed a carriage rate of 52.7% for Extended-Spectrum Beta-Lactamase-producing Enterobacteriaceae (ESBL-E) and 15.3% for Carbapenem-Resistant Enterobacteriaceae (CRE). The most frequently isolated strain was Escherichia coli in 61.2% of cases. Rectal swab at discharge isolated 84.1% of ESBL strains and 48.5% of carbapenem-resistant strains. The overall carriage rate of MDR-E (multidrug-resistant Enterobacteriaceae) was 53.2% at admission and 84.8% at discharge. An MDR-E acquisition rate of 74.2% was observed. The acquisition of MDR-E strains was significantly associated with the length of hospital stay (p=0.003) and age (p=0.0001). Conclusion: The faecal carriage rate of multidrug-resistant Enterobacteriaceae strains in hospitalized children in this study is very high. It is therefore urgent to take measures to raise awareness about the spread of multidrug-resistant strains among children in Togo.

Urban rivers are increasingly facing contamination and degradation due to unregulated human activities and are becoming significant reservoirs for pollutants, including toxic heavy metals (HMs) and antibiotics. This co-occurrence of antibiotics and HMs in water bodies may pose greater health and environmental concerns as HMs can co-select for antibiotic-resistant bacteria, leading to a higher prevalence of antibiotic resistance (ABR) genes/bacteria. This study investigates the co-selection of HMs and ABR in Enterobacteriaceae strains isolated from the polluted urban stretch of the Mula-Mutha River flowing through Pune Metropolitan (India). Water samples were collected from five different sites of the river, and physicochemical parameters and metal concentrations were assessed, along with microbiological profiling. Elevated levels of toxic HMs, including arsenic (As,1010µg/L), lead (Pb, 90µg/L), cadmium (Cd, 42µg/L), and chromium (Cr, 163µg/L) were recorded at one of the urban sites (Mhatre Bridge), surpassing the World Health Organization (WHO) thresholds, marking it as a contamination hotspot. Eighty-eight Enterobacteriaceae isolates were obtained, of which 41 (46.5%) were multidrug-resistant (MDR), while one was extensively drug-resistant (XDR). Out of these, 33 isolates were used for further investigation. ABR levels were highest against cephalosporins (93.9%) and β-lactam/β-lactamase inhibitor combinations (67.7%). Thirteen isolates (39.3%) exhibited multiple antibiotic resistance (MAR) indices ≥ 0.2, suggesting exposure to high-risk environments. Heavy metal resistance (HMR) profiling revealed minimum inhibitory concentrations (MICs) exceeding 1500mg/L for Mn, Cr, As, and Pb in multiple isolates, while the multiple HM resistance (MHMR) index reached a value of up to 1.0 at two urban sampling sites. Pearson correlation analysis revealed strong associations between HMR and ABR, including arsenic-lomefloxacin (r = 0.43), copper-cefotaxime (r = 0.46), and lead-piperacillin tazobactam (r = 0.365), suggesting co-selection via shared resistance mechanisms. These findings hold significance and highlight the convergence of chemical and biological pollution in shaping resistant microbial communities and emphasize the urgent need for integrated river basin monitoring and pollution control strategies.

SummaryCurrent microbiome studies lack real-time or spatial information, often requiring large cohorts for longitudinal investigations. Real-time, in vivo molecular imaging would alleviate these shortcomings but is hampered by the limited labeling performance of current strategies. Here, we use the azido-modified sugar 8-Azido-3,8-dideoxy-D-manno-octulosonic acid (Kdo-N3) to metabolically engineer the core oligosaccharide of Gram negative lipopolysaccharide, enabling conjugation with DBCO-fluorophores. We extend this approach by systematically screening multiple Enterobacteriaceae strains, optimizing labeling conditions, validating core-oligosaccharide-specific labeling using LPS knockout mutants, and demonstrating in vivo species-specific MOE-BCC labeling with Kdo-N3 inside the murine gastrointestinal tract and subsequent in vivo imaging. Beyond Salmonella Typhimurium and Escherichia coli Nissle 1917, a broad range of Enterobacterales species were amenable to Kdo-N3 sugar incorporation, potentially unlocking species-specific studies of bacterial interaction in vitro and in vivo.

Latamoxef has been used to treat various bacterial infections. To provide data supporting the rational clinical use of latamoxef, bacterial resistance over 10 years was analyzed at multiple centers throughout China. The results were used to develop tentative epidemiological cut-off values (TECOFFs) for latamoxef. A total of 46,716 strains of common Enterobacteriaceae were collected from patients with bloodstream infections at 72hospitals in 21 provinces of China between 2014 and 2023. The in vitro antimicrobial activities of latamoxef were compared with those of other commonly used cephalosporin and carbapenem. The distribution of minimum inhibitory concentrations was subjected to cumulative log-normal fitting to obtain the TECOFFs of latamoxef for common Enterobacteriaceae. The sensitivities of Escherichia coli (E. coli), Salmonella species, Serratia marcescens (S. marcescens) and Proteus mirabilis (P. mirabilis) to latamoxef ranged from 94.37% to 98.08%. Klebsiella oxytoca (K. oxytoca) and Enterobacter aerogenes (E. aerogenes) had sensitivities of 89.91% and 87.21%, respectively, whereas Klebsiella pneumoniae (K. pneumoniae) and Enterobacter cloacae (E. cloacae) had lower sensitivities. The in vitro activity of latamoxef against these bacteria, especially extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, was similar to the activities of ertapenem and meropenem and significantly higher than the activities of ceftriaxone, ceftazidime, and cefepime. The sensitivities of ESBL (+) E. coli and ESBL (+) P. mirabilis to latamoxef were similar to their sensitivities to ertapenem and meropenem. ESBL (+) K. pneumoniae and ESBL (+) K. oxytoca exhibited comparable but lower rates of sensitization to latamoxef, ertapenem, and meropenem. Latamoxef had TECOFFs of 2µg/mL for E. coli, K. pneumoniae, K. oxytoca, P. mirabilis, E. aerogenes, Salmonella, and S. marcescens, and 4µg/mL for E. cloacae. Latamoxef has good and stable in vitro antimicrobial activity against Enterobacteriaceae, including ESBL-producing Enterobacteriaceae. The calculated TECOFFs of latamoxef provide an important reference for subsequent use of this antibiotic against Enterobacteriaceae.

The JinEr mushroom (“Golden Ear”), a globally rare edible and medicinal macrofungus, comprises a symbiotic complex formed by the symbiotic association of Naematelia aurantialba (Tremellomycetes) and Stereum hirsutum (Agaricomycetes). However, the interactions between these fungi and their associated microbiome remain poorly understood. This study employed high-throughput amplicon sequencing, in situ microbial isolation and culture, and microbial confrontation assays to analyze microbial diversity, community structure, and potential functional roles of the endomycotic bacterial community within JinEr basidiomata and its cultivation substrate. Molecular analysis confirmed the heterogenous composition of the basidiomata, revealing N. aurantialba constitutes less than 20% of the fungal biomass, while S. hirsutum predominates, accounting for approximately 80%. Endomycotic fungi accounted for 0.33% (relative abundance) of the fungal community. Prokaryotic analysis identified Delftia and Sphingomonas as the dominant endomycotic bacterial genera within basidiomata, comprising 85.42% of prokaryotic sequences. Endomycotic bacterial diversity differed significantly (p < 0.05) between basidiomata and substrate, indicating host-specific selection. Cultivation-based approaches yielded 140 culturable bacterial isolates (spanning four families and seven genera) from basidiomata core tissues. In vitro co-culture experiments demonstrated that eight representative bacterial strains exhibited compatible growth with both hosts, while one Enterobacteriaceae strain displayed antagonism towards them. These findings confirm that the heterogeneous JinEr basidiomata harbor a specific prokaryotic assemblage potentially engaged in putative symbiotic or commensal associations with the host fungi. This research advances the understanding of microbial ecology in this unique fungal complex and establishes a culture repository of associated bacteria. This collection facilitates subsequent screening for beneficial bacterial strains to enhance the JinEr cultivation system through the provision of symbiotic microorganisms.

IntroductionAntimicrobial resistance in the Enterobacteriaceae poses a global health concern by jeopardizing the effectiveness of antibiotics. The scarcity of new antibiotics has prompted increased interest in natural bioactive secondary metabolites derived from microbial sources and their co-action with existing antimicrobials.MethodsIn this study, we investigated the bioactivity of crude extracts from Pseudomonas aeruginosa MC9 (accession no. MK530186) and evaluated the in-vitro antimicrobial-augmenting efficacy of its quorum sensing (QS) effectors against multidrug-resistant strains of Salmonella Typhi (S. Typhi-29C), Salmonella Typhimurium (S. Typhimurium-W20), and Escherichia coli (E. coli SS1). Mass spectrometry was used to identify secondary metabolites, and combination assays followed by growth curve analysis were performed to assess interaction effects under sub-inhibitory conditions.ResultsThe MC9 extract exhibited inhibition zones of 26±1.5, 24±1, and 19±1.5 mm, with minimum inhibitory concentrations of 16, 32, and 256 mg/mL against S. Typhimurium-W20, S. Typhi-29C, and E. coli-E92, respectively. Mass spectrometric analysis revealed the presence of 5-methyl-1(5H)-phenazinone (pyocyanin), rhamnolipids, 4-hydroxy-2heptylquinoline (PQS), and 2-heptyl-3-hydroxy-4(1H)-quinolone (HHQ). Notably, pyocyanin and rhamnolipids exhibited significant antimicrobial activities across a concentration range from 0.04 mg/mL to 50 mg/mL, whereas HHQ and PQS showed no anti-Enterobacteriaceae activity up to 5 mg/mL. Combination assays demonstrated that all four QS effectors potentiate the activity of conventional antibiotics. Pyocyanin showed the highest synergistic effect, with a 300% increase in the inhibition zone when combined with sulfamethoxazole/trimethoprim (23.75/1.25 µg/mL) against S. Typhimurium-W20. Rhamnolipids exhibited a 106% increase in synergy with ceftriaxone (30 μg/mL) against E. coli-SS1, whereas HHQ (10 μg/mL) showed a 257% increase with ampicillin (10 μg/mL) against E. coli-SS1. PQS displayed the highest synergistic effect of 109% with amoxicillin clavulanic acid (30 μg/mL) against E. coli-SS1. Moreover, growth curve analysis revealed a dose-dependent reduction in bacterial growth with sub-inhibitory concentrations of antimicrobials, particularly for the combinations exhibiting the highest synergy across the QS effectors.DiscussionThese findings demonstrate the potential of the QS effectors in reducing the required dosage of antibiotics against resistant Enterobacteriaceae strains and highlight the need to develop a comprehensive understanding of the underlying mechanisms for the co-action of antimicrobials and QS mediators.

The epidemiology of ESBL infection varies widely from one region to another and rapidly evolves across hospital and community boundaries. Therefore, obtaining an updated local picture of the epidemiology of ESBL-producing microorganisms and analyzing trends in their dissemination is essential. The main aim of this study was to assess the prevalence and the antibiotic resistance profiles of ESBL-producing Enterobacteriaceae (ESBL-E) at Ibn Tofail Hospital in Marrakesh. To our knowledge, the present study is the first to conduct a descriptive analysis from January 1, 2010, to December 31, 2022, including all ESBL-E strains isolated in the microbiology laboratory. Antimicrobial susceptibility testing was determined using the standardized Kirby-Bauer disk diffusion method on Mueller-Hinton agar. The double-disc synergy test confirmed the presence of ESBL. Among 3672 Enterobacteriaceae strains isolated, 20% were ESBL producers. Klebsiella pneumoniae accounted for 45.9% of ESBL-E, followed by Escherichia coli (25.3%) and Enterobacter cloacae (12.8%). The surgical and intensive care units were most affected. Urine samples were the most common source (42.4%). ESBL-E strains exhibited high resistance to tobramycin (80.3%), gentamicin (72.9%), and ciprofloxacin (73.7%), but maintained sensitivity to imipenem (15.6% resistance) and amikacin (21.9%). Significant differences were detected between non-ESBL and ESBL regarding all tested antibiotics. Male patients were significantly more affected by ESBL-E than females. The increasing incidence of ESBL-E has become a significant concern. Monitoring their epidemiological and resistance profiles is crucial for guiding antibiotic therapy and preventing the development of further resistant strains.

Providencia, as an opportunistic pathogen, can cause serious infections and increasingly poses a life-threatening risk to public health. Carbapenems were initially considered effective agents conquering such infectious; however, the emergence of carbapenem-resistant Providencia strains has been attributed to the rapid spread of carbapenemase genes. In this study, we first reported a uniquely blaNDM-1 genetic structure that can evolve 'circular DNA'. Among 901 carbapenem-resistant Enterobacteriaceae strains examined, only one P. huaxiensis strain was identified using VITEK MS, 16S rRNA sequencing, average nucleotide identity (ANI) and the DNA-DNA hybridization (DDH). Susceptibility testing using the broth microdilution method revealed that the P. huaxiensis strain was highly resistant to carbapenems, cephalosporin, quinolone and aminoglycosides. By whole-genome sequencing (WGS) and large fragment PCRs, we characterized more than 7 resistance genes including blaNDM-1, blaOXA-10, qnrB, aadB, sul1, tetA and aadA1 in the P. huaxiensis strain PH1503, in which 2 copies of each the blaNDM-1 and blaOXA-10 genes were identified to be integrated into 3 plasmids. The blaNDM-1 gene was integrated into a novel structure of ISCR1-trpF-bleMBL-blaNDM-1-CatB3-Arr-3-DfrA27-AadA16-qacEdelta1-Sul1, which was present in both pPH1503-1-NDM and pPH1503-5-NDM. Moreover, the DNA sequences of the novel structure and pPH1503-5-NDM was 100% identical to each other, indicating that the novel structure may cyclize into a 'circular DNA'. To the best of our knowledge, this is the first description of a circular DNA carrying the blaNDM-1 gene, which was identified in clinically isolated P. huaxiensis strains and appears to be stably maintained.

Precise microbiota modulation towards improving immune function and metabolic homeostasis is a major goal in clinical research. It is also critical for reducing pathogen invasion or pathobiont expansion, contributors to epidemic Inflammatory Bowel Diseases (IBD), where recurrent antibiotic treatments often exacerbate microbiota imbalances. Within the thousands of strains of a natural gut microbiota, we previously identified a specific Klebsiella strain, ARO112, capable of promoting resistance to, and clearance of, pathogenic Enterobacteriaceae. Here, we assess its therapeutic potential using a comprehensive genomic and phenotypic analysis and experiments in mouse models of IBD. We demonstrate that ARO112 not only exhibits a safety profile comparable to the widely used probiotic Escherichia coli Nissle 1917, but also has a reduced capacity to acquire antibiotic resistance, via horizontal gene transfer, and to capture iron, thereby bypassing major concerns associated with pathogenic Enterobacteriaceae strains. In antibiotic-treated, genetically predisposed IBD mice, ARO112 accelerates pathobiont clearance, promotes the recovery of microbiota diversity, elevates intestinal butyrate concentration, and prevents mild inflammation. Moreover, even in the absence of pathogen infection, ARO112 prevents severe inflammation-driven pathology in a chemically-induced colitis model. Our findings highlight ARO112 as a potential biotherapeutic agent that disrupts inflammation-treatment-infection cycles characteristic of chronic gut inflammatory diseases.

BackgroundInfected wounds and chronic infections have become a global issue and aetiology is complicated by the increase of antibiotic resistance in bacteria. The Gram-negative bacteria Enterobacter cloacae complex has become an emerging threat and are routinely associated with non-healing wounds and biofilm related infections. Phage therapy has been used for more than a century to treat bacterial infections that are caused by MDR organisms.MethodsThis study investigated the efficacy of a cocktail of two lytic bacteriophages (vB_EluM_RZH and vB_EholP_HHH) isolated from the River Chelt and the antibiotic meropenem in inhibiting and degrading the biofilms produced by certain strains of MDR Enterobacteriaceae expressing carbapenemases. Phages underwent physical and genomic characterization, then validated by the NCBI. Biofilm metabolic activity and biomass was measured in a modified chequerboard assay using MBEC format. Phage–antibiotic synergy was calculated and harmonized using synergy scoring models HSA, Loewe, Bliss and Zip. The influence of phages on human fibroblasts cells was evaluated through cell metabolic activity and the release of lactate dehydrogenase (LDH).ResultsBoth phages showed broad-host range, producing bacteriolytic activities against strains of E. cloacae complex and Escherichia coli. They showed short replication times, large burst sizes with rapid adsorptions. They exhibited significant stability under various environmental conditions. Genomic analysis revealed that both phages are genetically distinct phages, belonging to the Tesseptimavirus and Karamivrus genus, with the lack of toxin, virulence, lysogeny and antibiotic resistance genes. Checkboard assay results indicated phage and antibiotic synergy (PAS) against 24 h and 48 h biofilms when compared to the control or a single agent alone . Synergy scores from all models indicated a top score of 30 (combination of antibiotic at 128–256 μg/ml and phage cocktail 6–7 PFU/ml) demonstrated the best results. Traditional fractional inhibitory concentration (FIC) calculations also indicated a synergy effect (≤0.5). When human fibroblasts were treated with the phages, no differences in LDH levels or metabolic activity were observed when compared to the untreated control (P<0.001). Furthermore, phages and phage–antibiotic combinations could significantly reduce the bacterial load (cfu/mL) when targeted against infected fibroblasts, without causing damage to the dermal cells, however, antibiotic alone did not significantly reduce the bacterial load and indicated a slight cytotoxic effect on the cells.ConclusionsThese findings suggest bacteriophage cocktail combined with meropenem improved the inhibition and destruction of biofilms when compared to testing the agents alone, moreover the combination demonstrated a positive outcome when tested with fibroblasts. These results contribute to the expanding field of biofilm dynamics and the potential of bacteriophages as a novel treatment strategy

Pathogenic Enterobacteriaceae (PE) pose significant public health threats, particularly with the emergence of antibiotic-resistant isolates that frequently cause foodborne infection outbreaks through the consumption of contaminated leafy vegetables. However, there is currently a lack of comprehensive genomic analysis for PE in leafy vegetables, hindering insights into the distribution patterns and resistance capacity of these PE in diverse agricultural settings around the world. This study addresses this gap by performing a genomic analysis of PE related to leafy vegetables, encompassing 458 publicly available genomes and 49 newly sequenced genomes from our study. The newly sequenced genomes correspond to PE isolates from organic fertilizer, fertilizer-applied soils, and various parts of Brassica rapa grown in fertilizer-applied soils (31 from B. rapa plants, 18 from organic fertilizer and soils), for which we also performed antibiotic susceptibility testing as a supplement. Results showed extensive multidrug resistance among PE isolates (>95%), and a significant progressive increase of high-risk antibiotic resistance genes (ARGs) from roots to leaves. We established a genome dataset comprising 489 PE isolates (458 public isolates plus 31 B. rapa isolates from our study) from leafy vegetables worldwide and identified Escherichia coli as harboring substantially higher numbers of both high-risk ARGs (mean 9.71 per genome) and virulence factor genes (mean 72.45 per genome) compared to other PE taxa. This study provides a valuable genomic resource for investigating PE contamination in leafy vegetables and underscores the urgent need for enhanced surveillance targeting antibiotic-resistant PE, particularly E. coli, in leafy vegetables to mitigate emerging public health threats.

Background: The emergence of multidrug-resistant (MDR) bacterial pathogens is a global problem for medicine despite the vast number of available antimicrobials. Novel effective antimicrobials and various approaches are required to overcome this challenge. The non-proteinogenic amino acids and biphenyl-containing compounds are widely used in drug design. The incorporation of the biphenyl group into amino acid side chains appears to be a promising approach for developing effective antimicrobials. Bacterial extracellular proteases are virulent factors that can be detrimental, destroying host tissue and compromising the immune system during infection. The treatment of infectious diseases with protease inhibitors, in combination with antibiotics, has shown promising results. Objective: To reveal the effective antimicrobial compounds, the influence of biphenyl-containing amino acids on the growth of antibiotic-resistant bacteria, and the activity of bacterial proteases involved in the progression of infections were studied. Methods: Susceptibility of the strains was assessed by serial dilutions in the presence of the studied compound in a broth at final concentrations ranging from 0.005 mM to 0.5 mM. The optical density of bacterial cultures was measured. The MIC was determined as the lowest concentration of the compound that prevented visible bacterial growth after incubation. Proteinase activity was measured by the method of free amino groups determined by the OPA reagent. Results: The non-proteinogenic amino acids containing biphenyl groups in the side chain were tested on their ability to inhibit the growth of antibiotic-resistant P. aeruginosa 80 (clinical isolate), K.pneumonia 63 (clinical isolate), E.coli ESBL 64 (clinical isolate), S.maltophilia MDC 9288, S. aureus MDC 5233 strains, and bacterial protease activities. According to the results obtained, (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid and (S)-3-([1,1':4',1''-terphenyl]-4-yl)-2-aminopropanoic acid demonstrate antibacterial activity against Gram-negative Enterobacteriaceae strains and Gram-positive S. aureus MDC 5233 strain with MIC values ranging from ≤ 0.5mg/L to 14.4 mg/l. The non-proteinogenic amino acid (S)-3-([1,1':4',1''-terphenyl]-4-yl)-2-aminopropanoic acid inhibits proteinase K activity. Conclusions: This study demonstrated that (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid and (S)-3-([1,1':4',1''-terphenyl]-4-yl)-2-aminopropanoic acid showed a high level of antibacterial activity against Gram-negative Enterobacteriaceae strains and Gram-positive S. aureus MDC 5233 strain. Thus, these non-proteinogenic amino acids can be considered novel, promising antimicrobials and recommended for the treatment of infections. Novel inhibitor of Proteinase K and collagenase activity - (S)-3-([1,1':4',1''-terphenyl]-4-yl)-2-aminopropanoic acid was revealed. Novelty: Novel non-proteinogenic amino acids – biphenyl derivatives possessing antibacterial activity towards antibiotic-resistant strains have been revealed in this study. According to the results obtained, (S)-3-([1,1':4',1''-terphenyl]-4-yl)-2-aminopropanoic acid is a novel inhibitor of proteinase K and clostridial collagenase. Keywords: multi-drug resistance, biphenyl derivatives, antimicrobials, virulence factors, protease inhibitors.

Emergence and multiplication of antibiotic-resistant bacteria is a public health problem. It appears necessary to regularly update data on the resistance of germs isolated in practitioners&#39; environments. Among pathogenic bacteria, Enterobacteriaceae represent a major clinical threat due to their multidrug resistance, driven in large part by the production of &beta;-lactamases. The aim of this study was to investigate the antibiotic resistance of Enterobacteria strains isolated at the Center for Diagnosis and Research on Acquired Immunodeficiency Syndrome (AIDS) and Other Infectious Diseases (CeDReS) at University Hospital Center of Treichville, from November 2012 to November 2019. A total of 2661 strains of Enterobacteriaceae were isolated. The most frequent species were, in order: Escherichia coli (65.6%), Klebsiella pneumoniae (27.9%) and Enterobacter cloacae (6.5%). Imipenem, latamoxef, amikacin, and colistin exhibited the highest efficacy against E. coli and K. pneumoniae. Significant proportions of extended-spectrum beta-lactamases (ESBLs) were detected at rates of 36.7 and 56%, respectively. The activity of imipenem, amikacin and colistin was very good on E. cloacae strains, with 33.5% of ESBL. These results suggest the need for ongoing monitoring of bacterial resistance to antibiotics. Key words: Enterobacteriaceae, antibiotics, bacterial resistance.

Serratia marcescens is an emerging opportunistic pathogen with high genetic diversity. The emergence and prevalence of carbapenem-resistant S. marcescens poses a major health threat due to its intrinsic resistance to multiple antibiotics, which severely restricts the selection and treatment of antibiotics for S. marcescens infection. This study presents the first documented case in China of a bloodstream infection caused by Staphylococcus epidermidis and S. marcescens strain (designated S96) co-producing blaNDM-1, blaKPC-2, and blaSRT-2. Strain S96 exhibited resistance to nearly all categories of β-lactam antimicrobials, β-lactam/inhibitor combinations, aminoglycosides, quinolones, and other clinical antibacterial agents, with the exception of tigecycline. Our main objective was to characterize the genetic mechanisms underlying its carbapenem resistance and plasmid transfer potential. Whole-genome sequencing revealed blaKPC-2 on a 44,047 bp "IncX6-like" plasmid and blaNDM-1 on a 100,081 bp IncFII(Yp)-type plasmid, alongside chromosomal blaSRT-2 and aac(6')-Ic. "IncX6-like" and IncFII(Yp)-type plasmids are widely distributed among carbapenem-resistant Enterobacteriaceae strains globally. Conjugation experiments demonstrated that the blaNDM-1-carrying plasmid could be successfully transferred to recipient Escherichia coli 600, with no significant fitness cost observed (P > 0.05). The experimental results demonstrate that carbapenem-resistant genes can disseminate among Enterobacteriaceae via plasmid-mediated horizontal transfer between bacterial cells. Comparative genomic analysis revealed plasmid structural homology with global counterparts, demonstrating IS-mediated recombination and horizontal gene transfer. The low adaptive cost of plasmid carriage and multidrug resistance phenotype pose significant challenges for clinical management. This study highlights the need for enhanced clinical surveillance and antibiotic stewardship to curb the spread of such multidrug-resistant pathogens.IMPORTANCECarbapenem resistance in Serratia marcescens is primarily mediated by Klebsiella pneumoniae carbapenemase (KPC), with New Delhi metallo-β-lactamase (NDM) being a relatively uncommon alternative resistance mechanism. KPC-2 and NDM-1 coexisting in S. marcescens is extremely rare clinically. This study reports the first clinical isolate of S. marcescens in China co-harboring blaNDM-1, blaKPC-2, and blaSRT-2. The isolate exhibits multidrug resistance to nearly all β-lactam antibiotics and β-lactam/inhibitor combinations, with low adaptive costs and high dissemination potential. The potential spread of resistance genes through mobile genetic elements poses a serious public health risk. The study underscores the need for enhanced surveillance, rational antibiotic use, and novel strategies to combat resistance. It also provides insights into the evolutionary mechanisms of bacterial resistance, emphasizing the urgent need for interventions to address the growing threat of antimicrobial resistance.

In Enterobacteriaceae, antibiotic susceptibility is frequently influenced by mechanisms such as membrane modifications, target site mutations, and enzymatic resistance barriers. Recently, there has been a notable rise in Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae isolates exhibiting antibiotic resistance in hospital settings. Of particular concern, some resistant isolates employ membrane-associated resistance mechanisms that significantly lower intracellular antibiotic concentrations, reducing them below the threshold required for therapeutic efficacy. Advancements in methods for quantifying drug accumulation within bacterial cells have provided critical insights into these resistance mechanisms. A key step in these studies relies on cell lysis to release intracellular contents including antibacterial molecules for precise quantification. However, current lysis methods are often time-consuming, underscoring the need for a robust, efficient approach to accurately measure intracellular antibiotic concentrations in isolates exhibiting various levels of efflux pump activity. In this study, we developed a rapid and reliable lysis protocol that minimizes the risk of drug alteration while enabling precise and reproducible measurement of intracellular antibiotic concentrations allowing an evidence-based study of efflux in resistant clinical strains of Enterobacteriaceae. This approach holds significant promise for enhancing our understanding of membrane-associated resistance mechanisms and for informing the optimization of treatment strategies.

IntroductionThe transmission of antimicrobial resistance (AMR), particularly the antimicrobial resistance gene in Enterobacteriaceae, presents a critical challenge to global public health. Sichuan province is the largest producer and consumer of rabbit meat in China. However, few studies have focused on AMR surveillance in rabbits.MethodsEnterobacteriaceae strains were isolated and identified by MALDI-TOF. The minimum inhibitory concentration (MIC) was determined according to the Clinical and Laboratory Standards Institute. Whole-genome sequencing was performed using the Illumina and Oxford Nanopore Technologies (ONT) platforms.Results and discussionA total of 73 Enterobacteriaceae strains were isolated, including Klebsiella pneumoniae, Salmonella enterica, Enterobacter hormaechei, and Escherichia coli. Resistance rates to tetracycline, ciprofloxacin, nalidixic acid, sulfamethoxazole-trimethoprim, and ampicillin exceeded 60%. For Escherichia coli isolates showed that ST328, ST22, and ST29 were the primary sequence types, with O178:H7 being the predominant serotype. Remarkably, 48% (35/73) of the isolates carried the mcr-1.1 gene, and among these, 82.9% (29/35) mcr-1.1-positive isolates contained the IncI2 plasmid replicon. The mcr-1.1 gene in Klebsiella pneumoniae, Salmonella enterica and Escherichia coli transferred to a recipient strain. Furthermore, the genetic environment of the mcr-1.1 gene showed that it was flanked by PAP2 and a relaxase. Comparative analysis indicated that the mcr-1.1-positive plasmid exhibited high sequence identity to plasmids from human, porcine, and bovine sources. Notably, a phylogenetic analysis based on core single nucleotide polymorphisms demonstrated that certain rabbit-derived mcr-1-positive Escherichia coli strains clustered within the same evolutionary branch as humanderived strains. These findings indicated that smaller-scale breeding operations, such as rabbit farming, could serve as underrecognized reservoirs of AMR determinants, particularly the mcr-1.1 gene, thus requiring systematic assessment.

Carbapenem-resistant Enterobacteriaceae (CRE) poses a significant challenge in treating infections, leading to increased mortality and healthcare burden. Early and accurate identification of CRE is crucial for appropriate antibiotic selection. This study aimed to evaluate the prevalence of CRE isolated from ICU patients and carbapenemase gene profiles among Klebsiella pneumoniae isolates. A cross-sectional study was conducted on Enterobacteriaceae isolates from 53 ICU patients from March to May 2023. Among 125 initial isolates, 89 non-duplicated Enterobacteriaceae strains were included. Carbapenem resistance was determined from Kirby-Bauer disk diffusion susceptibility testing results. Carbapenemase-producing phenotype and carbapenemase classification were classified from BD Phoenix M50 results using the NMIC-500 CPO panel. Carbapenemase-encoding gene were identified from amplification results of carbapenemase-encoding genes in K. pneumoniae strains using multiplex real-time PCR. Of 89 isolates, 51 (57.3%) were carbapenem-resistant, predominantly K. pneumoniae (34/48, 70.8%) and Serratia marcescens (12/13, 92.3%). All isolates were carbapenem-resistant due to carbapenemase production. Class D carbapenemase was the most prevalent (39/51, 76.5%). Among K. pneumoniae isolates, 12/34 harboured single carbapenemase genes: blaOXA-48-like (17.7%), blaKPC (14.7%), and blaNDM-1 (2.9%); Sixteen isolates carried two genes: blaNDM-1 +blaOXA-48-like and blaKPC+blaOXA-48-like; Three isolates possessed three genes: blaKPC+blaNDM-1+blaOXA-48-like. Multiple gene carriage was significantly associated with increased antibiotic resistance (p&lt;0.01). This study revealed a high prevalence of carbapenemase-producing, CRE. This significantly increased the risk of spreading multidrug-resistant pathogens. Therefore, stronger infection control measures are in demand. Additionally, automated susceptibility testing and carbapenemase class identification are crucial for guiding treatment decisions.

Invasive species represent a significant threat to ecological balance and the maintenance of native populations. Besides, these have been associated with the emergence of pathogens of public health importance, including multidrug-resistant bacteria. This study aimed to screen and describe the antimicrobial resistance profile of clinically important Enterobacteriaceae species isolated from whistling frogs (Eleutherodactylus johnstonei), an invasive anuran species in São Paulo, Brazil. Clinically relevant Enterobacteriaceae strains (n = 35) were isolated from oral and skin swabs of 19 whistling frogs and tested for antimicrobial susceptibility and antimicrobial resistance encoding genes. Resistance to amoxicillin + clavulanate and cefoxitin were the most frequent (16.67%; 4/24), followed by cefotaxime (5.71%; 2/35), ceftriaxone (2.86%; 1/35), and tetracycline (2.86%; 1/35). Among the antimicrobial resistance genes screened, blaCTX-M group 8, blaTEM, and blaCMY were identified. The whole genome of the blaCTX-M group 8-positive E. coli strain was assessed and confirmed blaCTX-M-8 presence and phylogenetic analysis. Given the synanthropic behavior of whistling frogs, these amphibians may act as carriers of antimicrobial-resistant bacteria.

Objective To analyze the clinical status and risk factors of patients with blood infection caused by Klebsiella pneumoniae.Methods 110 patients with blood infection in clinical departments of our hospital from May 2022 to May 2023 were reviewed, and 130 strains of Enterobacteriaceae bacteria were isolated from blood flow samples,110 of which were Klebsiella pneumoniae (cKP).Staphylococcus aureus ATCC29213, Streptococcus pneumoniae ATCC49619, Escherichia coli ATCC25922,Enterococcus faecalis ATCC29212 and Pseudomonas aeruginosa ATCC27853 were used as quality control strains to analyze the distribution, clinical characteristics, risk factors and drug resistance of Klebsiella pneumoniae.Results Among 130 strains of Enterobacteriaceae,110 strains of Klebsiella pneumoniae (cKP)were mainly distributed in hematology, surgery and other departments. Compared with other departments, the number of Klebsiella pneumoniae in department of hematology and surgery was significantly increased, and the difference was statistically significant (P&lt;0.05).There was no significant difference in the distribution of Klebsiella pneumoniae among other departments (P&gt;0.05). A total of 110 cases of Klebsiella pneumoniae infection were included in this study, of which 60.91% were male, 59.09% were over 60 years old, 33.64% had blood infection as the primary focus, and the infection method was mainly hospital-acquired, accounting for 69.09%. The results of single factor analysis showed that the risk factors of Klebsiella pneumoniae infection were primary lesion, treatment, acquisition, underlying disease,urinary tract intubation and blood type. Multivariate Logistic regression analysis showed that nosocomial infection,liver disease,diabetes,urinary tract intubation and blood type were independent risk factors for bloodstream infection of Klebsiella pneumoniae.The resistance rate of Klebsiella pneumoniae to piperacillin was the highest (39.92%). The resistance rate of the first to fourth generation of cephalosporins was 6.21% to 41.25%, of which the lowest resistance rate of cefotetan was 6.21%, and the highest resistance rate of cephalosporin was 41.25%. 42 ESBLs strains were detected among 110 Klebsiella pneumoniae strains, and the positive rate was 38.18%.Conclusion Physicians should pay special attention to the infection of Klebsiella hyperpneumoniae caused by diabetes,blood type,urinary tract intubation, and pay attention to the diagnosis, treatment and infection control.