In recent years, New Delhi metallo-beta-lactamases 1 (bla NDM-1) has been reported with increasing frequency and become prevalent. The present study was undertaken to investigate the epidemiological dissemination of the bla NDM-1 gene in Enterobacter cloacae isolates at a teaching hospital in Yunnan, China. Antimicrobial susceptibility testing was performed using VITEK 2 system and E test gradient strips. The presence of integrons and insertion sequence common region 1 were examined by PCR and sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of plasmids. Ten E. cloacae isolates and their Escherichia coli transconjugants were exhibited similar resistant patterns to carbapenems, cephalosporins and penicillins. 8 (80%) of E. cloacae isolates carried class 1 integron and 1 (12.5%) carried class 2 integron. Integron variable regions harbored the genes which encoded resistance to aminoglycosides (aadA1, aadA2, aadA5, aadB, aac(6′)-Ib-cr), sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Six E. cloacae isolates belonged to ST74 and exhibited highly similar PFGE patterns. Each isolate shared an identical plasmid with ~33.3 kb size that carried the bla NDM-1 gene, except T3 strain, of which the bla NDM-1 gene was located on a ~50 kb plasmid. Our findings suggested that plasmid was able to contribute to the dissemination of bla NDM-1. Hence, more attention should be devoted to monitor the dissemination of the bla NDM-1 gene due to its horizontal transfer via plasmid. In addition, nosocomial surveillance system should actively monitor the potential endemic clone of ST74 to prevent their further spread.

Introduction. The bla _NDM-1 -positive Enterobacter cloacae has led to limited therapeutic options for clinical treatment. Hypothesis/Gap Statement. Analysing the antimicrobial resistance and molecular typing of bla _NDM-1-positive E. cloacae is of great significance. Meanwhile, the effect of the bla _NDM-1 gene on the virulence and pathogenicity of E. cloacae remains unclear and should be assessed. Aim. To understand bla _NDM-1-positive E. cloacae from different perspectives. Methodology. The PCR was used to screen bla _NDM-1-positive E. cloacae , then, antimicrobial susceptibility tests and multilocus sequence typing (MLST) were performed on them; sixty-nine strains of bla _NDM-1-negative E. cloacae were collected as the controls, 28 pairs of virulence-related genes' carriage and biofilm-forming ability were detected for preliminary evaluation of the virulence phenotype of the strains; to gain insight into the effect of the bla _NDM-1 gene on the virulence and pathogenicity of E. cloacae , the bla _NDM-1-positive E. cloacae T2 (NDM-1), the T2 bla _NDM-1 knockout strain (ΔNDM-1) and ATCC13047 (ST) were studied, compared the motility, anti-serum killing ability, and virulence to cells. Then, the mice intraperitoneal infection model was established, the survival curve, histopathological characteristics, bacterial load in spleen and the contents of cytokines were compared. Results. (1) Thirty-five bla _NDM-1-positive E. cloacae exhibited multidrug resistance. MLST distinguished 12 STs, ST74 was the most common clonal type (11/35), followed by ST114 (10/35). (2) The detection rates of virulence genes clpB, icmf, VasD/Lip and acrA in the bla _NDM-1-positive E. cloacae were significantly higher than those in bla _NDM-1-negative E. cloacae (P<0.05), while there was no significant difference in the amount of biofilm formation between two groups. (3) The presence of bla _NDM-1 gene attenuated the motility diameter of E. cloacae , but had no significant effect on their ability to resist serum killing, and the virulence to cells. The survival rate, histopathological changes, bacterial load in spleen and inflammatory cytokines were not significantly affected. Conclusions. (1) The bla _NDM-1-positive E. cloacae exhibited multidrug resistance, and the MLST typing was mainly ST74 and ST114, with a small-scale clonal spread of the ST114 strain in the hospital NICU ward. (2) The bla _NDM-1 gene did not affect the virulence and pathogenicity of E. cloacae .

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in hospitals and the issues arising from their rapid spread have become significant public health challenges. Therefore, a new class of effective and safe antibacterial drugs is urgently required. In this study, we identified an antimicrobial peptide in the cecropin family, cecropin-TY1 (CEC-TY1), from the salivary glands of the horsefly. CEC-TY1 showed significant antibacterial activity against gram-negative bacteria, at concentrations up to 100 μg/mL, with no observed cytotoxicity or hemolytic activity against cells. Moreover, CEC-TY1 protected mice from lethal infections of CRKP in vivo , improving the survival rate of the infected mice. This peptide also alleviated excessive and harmful inflammatory responses by inhibiting the production of interleukin (IL)-6, tumor necrosis factor-α, and IL-1β. IMPORTANCE: Antimicrobial peptides are a promising alternative to antibiotics in the current antibiotic resistance crisis. Specifically, the antimicrobial peptide cecropin-TY1 (CEC-TY1), identified in the salivary glands of horseflies, showed significant antibacterial activity against carbapenem-resistant Klebsiella pneumoniae (CRKP), both in vitro and in vivo . CEC-TY1 also has selective toxicity and low hemolytic activity and is therefore a potential therapeutic agent for acute CRKP infection.

The cyclic AMP receptor protein (CRP) is a highly conserved global transcriptional regulator that integrates carbon metabolism and environmental adaptation in bacteria. However, its systematic role in the regulation of virulence in K. pneumoniae remains poorly understood; In this study, we constructed a crp deletion mutant (Δcrp) and a complemented strain (c-Δcrp) and employed a combination of in vitro virulence assays, in vivo infection models, transcriptomic profiling, and functional metabolic analyses to dissect the CRP-mediated metabolism–virulence regulatory axis; We show that crp deficiency does not significantly alter susceptibility to clinically relevant antibiotics but markedly impairs biofilm formation, motility, and host cell adhesion and invasion. In murine infection models, the Δcrp strain exhibits significantly reduced pulmonary colonization and lethality. Transcriptomic analysis reveals broad downregulation of phosphotransferase system (PTS)-associated genes, including srlA/srlB/srlE, mtlA and malX. Functional assays further demonstrate that crp loss severely compromises growth on multiple host-relevant carbon sources and is accompanied by aberrant accumulation of intracellular ATP and NADH, indicative of disrupted metabolic homeostasis; Collectively, these findings identify crp as an important regulator associated with PTS-mediated carbon metabolic balance, and virulence-related phenotypes in K. pneumoniae. Accordingly, targeting the CRP–PTS axis may offer a theoretical basis for metabolism-oriented anti-virulence interventions against K. pneumoniae by attenuating pathogenicity.