The continued rise in carbapenem-resistant Enterobacter cloacae (CREC) detections indicates that current control measures are insufficient, potentially due to underappreciated biological contributors. Rapid growth phenotypes are thought to facilitate the spread of drug-resistant bacteria. Yet, the growth fitness, phenotypic stability, and mechanisms underlying the rapid growth phenotype of CREC (RG-CREC) remain unclear. Forty RG-CREC isolates collected from eight hospitals in Guangdong Province, China (2022–2024) were defined as the study group. Enterobacter cloacae ATCC 23,355 was included as the standard strain. Compared with the standard strain, the study group exhibited significantly higher maximum and average growth rates (µₘ, r) and shorter lag and generation time (λ, G) across 30 serial passages (P < 0.05), while maintaining a stable rapid growth phenotype and carbapenem resistance. Exploratory proteomic and metabolomic profiling of the standard strain and study group identified 3,479 differentially expressed proteins and 147 differentially accumulated metabolites, with the mannose-specific phosphotransferase system (PTSₘₐₙ) showing exploratory enrichment in both omics datasets at raw P-values < 0.05. Upregulation of key phosphotransferase system components, including ptsI and manX were validated using qRT-PCR. In eugenol growth inhibition assays, the study group showed higher µₘ and r, lower λ and G, and increased ptsI expression after treatment (P < 0.05). Genetic analysis confirmed a functional role for manX, showing the manX-deletion mutant (RG-CREC-ΔmanX) lost the growth advantage in LB and M9 + 0.4% mannose, which was restored in the complemented strain; no differences were observed in M9 + 0.4% others alternative monosaccharides (P > 0.05). A GFP reporter demonstrated higher activity of an RG-CREC manX promoter than the standard strain promoter in LB and M9 + 0.4% mannose (P < 0.05). These exploratory findings suggest that 40 RG-CREC exhibits enhanced growth fitness and genetic stability under laboratory conditions. Among the potential contributing factors, the PTSₘₐₙ (via elevated ptsI and manX) appears to play a critical role, with manX upregulation likely driven by increased promoter activity.

Rapid growth phenotype of carbapenem-resistant Enterobacter cloacae: growth fitness, stability, and mechanistic insights

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2026年2月7日

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Junlin Feng,
Chunqing Bai,
Yingnan Deng,
Rong Fu,
Xinliang Yan,
Mufa Cai,
Jun Liu

摘要

The continued rise in carbapenem-resistant Enterobacter cloacae (CREC) detections indicates that current control measures are insufficient, potentially due to underappreciated biological contributors. Rapid growth phenotypes are thought to facilitate the spread of drug-resistant bacteria. Yet, the growth fitness, phenotypic stability, and mechanisms underlying the rapid growth phenotype of CREC (RG-CREC) remain unclear. Forty RG-CREC isolates collected from eight hospitals in Guangdong Province, China (2022–2024) were defined as the study group. Enterobacter cloacae ATCC 23,355 was included as the standard strain. Compared with the standard strain, the study group exhibited significantly higher maximum and average growth rates (µₘ, r) and shorter lag and generation time (λ, G) across 30 serial passages (P < 0.05), while maintaining a stable rapid growth phenotype and carbapenem resistance. Exploratory proteomic and metabolomic profiling of the standard strain and study group identified 3,479 differentially expressed proteins and 147 differentially accumulated metabolites, with the mannose-specific phosphotransferase system (PTS^ₘₐₙ) showing exploratory enrichment in both omics datasets at raw P-values < 0.05. Upregulation of key phosphotransferase system components, including ptsI and manX were validated using qRT-PCR. In eugenol growth inhibition assays, the study group showed higher µₘ and r, lower λ and G, and increased ptsI expression after treatment (P < 0.05). Genetic analysis confirmed a functional role for manX, showing the manX-deletion mutant (RG-CREC-ΔmanX) lost the growth advantage in LB and M9 + 0.4% mannose, which was restored in the complemented strain; no differences were observed in M9 + 0.4% others alternative monosaccharides (P > 0.05). A GFP reporter demonstrated higher activity of an RG-CREC manX promoter than the standard strain promoter in LB and M9 + 0.4% mannose (P < 0.05). These exploratory findings suggest that 40 RG-CREC exhibits enhanced growth fitness and genetic stability under laboratory conditions. Among the potential contributing factors, the PTS^ₘₐₙ (via elevated ptsI and manX) appears to play a critical role, with manX upregulation likely driven by increased promoter activity.