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Tingting Li,* Zhaoyinqian Li,* Li Huang, Jingyang Tang, Zixuan Ding, Zhangrui Zeng, Yao Liu, Jinbo Liu Department of Laboratory Medicine, the Affiliated Hospital of Southwest Medical University, Lu Zhou, Sichuan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jinbo Liu, Department of Laboratory Medicine, the Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, 646000, People’s Republic of China, Tel +86 08303165730, Email liujb7203@swmu.edu.cnObjective: Diabetic foot ulcers (DFUs) and ESKAPE pathogens have attracted attention globally, but the role of ESKAPE pathogens in diabetic foot infection is not well described. The purpose of this study was to evaluate the clinical features, antimicrobial resistance, and risk factors for ESKAPE infection in patients with DFUs.Methods: A retrospective study was conducted on 180 patients with diabetic foot infection admitted to The Affiliated Hospital of Southwest Medical University (Luzhou, China), from January 2017 to April 2021. Antimicrobial susceptibilities of all isolates were determined. Multivariate logistic regression analysis was performed to analyze the independent risk factors for ESKAPE infection, multidrug-resistant (MDR)-ESKAPE infection, MDR-pathogen infection, and severe group in patients with DFUs.Results: A total of 206 isolates were collected, of which 42.2% were ESKAPE pathogens. The independent risk factors for ESKAPE infection were cigarette smoking (OR = 1.958; 95% CI, 1.015– 3.777) and peripheral vascular disease (OR = 2.096; 95% CI, 1.100– 3.992), while alcohol consumption (OR = 2.172; 95% CI, 1.104– 4.272) was the independent risk factor for MDR-pathogen infection. Additionally, the independent risk factors for severe DFU group were invasive treatment (OR = 326.642; 95% CI, 76.644– 1392.08), the duration of systemic antibiotic treatment (OR = 0.918; 95% CI, 0.849– 0.992), and length of hospital stay (OR = 1.145; 95% CI, 1.043– 1.256). No independent risk factors for MDR-ESKAPE infection were found.Conclusion: Our data established the microbiological features of ESKAPE pathogens and clinical manifestations of diabetic foot infection, and provide support for monitoring and management of ESKAPE infection in patients with DFUs in southwest China.Keywords: diabetic foot ulcers, ESKAPE pathogens, infection, antimicrobial resistance, risk factors

Zhaoyinqian Li,* Jingling Xie,* Jiaxin Yang, Siyi Liu, Zixuan Ding, Jingchen Hao, Yinhuan Ding, Zhangrui Zeng, Jinbo Liu Department of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University, Luzhou, 646000, Sichuan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jinbo LiuDepartment of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University, Taiping Street, Luzhou, 646000, Sichuan, People’s Republic of ChinaTel +86 0830 3165730Email Liulab2019@163.comObjective: This study aimed to determine the clinical manifestations, antimicrobial resistance, molecular characteristics, and risk factors for ESKAPE pathogens infection in burn patients.Methods: A retrospective study of 187 burn patients infected with ESKAPE pathogens was conducted at the Department of Plastic and Burn Surgery of the Affiliated Hospital of Southwest Medical University (Luzhou, China) from October 2018 to June 2021. All strains were identified using a MicroScan WalkAway 96 Plus System, and antimicrobial susceptibilities were determined using the VITEK system or the disk diffusion method. The antimicrobial resistance genes of multi-drug resistant ESKAPE (MDR-ESKAPE) were detected by polymerase chain reaction (PCR). The multivariable logistic regression analysis was used to estimate the risk factors for ESKAPE infection and MDR-ESKAPE infection.Results: A total of 255 strains were isolated in various types of clinical specimens from 187 burn patients, of which 47.5% were ESKAPE pathogens (121/255). Among these, MDR-ESKAPE pathogens accounted for 55% (67/121). Additionally, aph3ʹIII, mecA, blaSHV, blaTEM, blaPDC, and blaSHV were the most prevalent genes detected in Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., respectively. The independent risk factors for ESKAPE infection were total body surface area (TBSA) > 30– 50% (odds ratio [OR] = 10.428; 95% confidence interval [CI], 2.047 to 53.108), TBSA > 50% (OR = 15.534; 95% CI, 1.489 to 162.021), and parenteral nutrition (OR = 3.597; 95% CI, 1.098 to 11.787). No independent risk factors were found for MDR-ESKAPE infection.Conclusion: Clinical staff should be alert to the risk of nosocomial infection with ESKAPE pathogens in burn patients receiving parenteral nutrition and under TBSA > 30%. Full attention should also be paid to the ESKAPE resistance, strict adherence to infection control protocols for the rational use of antimicrobial agents, and enhanced clinical standardization of antimicrobial agents management.Keywords: burn, ESKAPE pathogens infection, antimicrobial resistance, risk factors

Zhaoyinqian Li,* Zixuan Ding,* Jia Yang,* Yao Liu, Xinrui Jin, Jingling Xie, Tingting Li, Yinhuan Ding, Zhangrui Zeng, Jinbo Liu Department of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University, Luzhou, 646000, Sichuan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jinbo LiuDepartment of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University, Luzhou, 646000, Sichuan, People’s Republic of ChinaTel/Fax +86 830 3165730Email Liulab2019@163.comBackground: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection has attracted worldwide concern and became a serious challenge for clinical treatment. The aims of this study were to evaluate the molecular characteristics and risk factors for CRKP infection.Methods: All the CRKP strains were screened for antimicrobial resistance genes, virulence genes, and integron by polymerase chain reaction (PCR). Plasmid typing was performed by plasmid conjugation assay and PCR-based replicon typing (PBRT). The genetic environments of blaKPC-2 and blaNDM-1 were analyzed by using overlapping PCR and molecular typing was performed by multi-locus sequence typing (MLST). Risk factors for CRKP infection were analyzed by logistic regression model.Results: All the 66 CRKP isolates were multidrug-resistant, but all of them were susceptible to tigecycline and polymyxin B. Among the CRKP isolates, 42 blaKPC-2-positive strains were identified carrying IncFII plasmids. Meanwhile, 24 blaNDM-positive strains were found on lncX3 plasmids, including 20 blaNDM-1 isolates and 4 blaNDM-5 isolates. Most of CRKP isolates contained several virulence genes and the class I integron (intl1). The genetic environments of blaKPC-2 and blaNDM-1 revealed that the conserved regions (tnpA-tnpR-ISkpn8-blaKPC-2) and (blaNDM-1-bleMBL-trpF-tat) were associated with the dissemination of KPC-2 and NDM-1. ST11 was the most common type in this work. Hematological disease, tracheal cannula, and use of β-lactams and β-lactamase inhibitor combination were identified as independent risk factors for CRKP infection.Conclusion: This study established the resistance pattern, molecular characteristics, clonal relatedness, and risk factors of CRKP infection. The findings of the novel strain that co-harboring blaNDM-5 and blaIMP-4, and the novel ST4495 indicated that the brand-new types have spread in Southwest China, emphasizing the prevent and control the further dissemination of CRKP isolates are highly needed.Keywords: carbapenem-resistant Klebsiella pneumoniae, molecular characteristics, genetic environments, plasmid, risk factors

Zhaoyinqian Li,* Zixuan Ding,* Yao Liu, Xinrui Jin, Jingling Xie, Tingting Li, Zhangrui Zeng, Zhibin Wang, Jinbo Liu Department of Laboratory Medicine, the Affiliated Hospital of Southwest Medical University, Luzhou, 646000, Sichuan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jinbo LiuDepartment of Laboratory Medical, Affiliated Hospital of Southwest Medical University, 25 Taiping Street, Luzhou, 646000, Sichuan, People’s Republic of ChinaTel/Fax +86 0830 3165730Email Liulab2019@163.comBackground: Acinetobacter baumannii is an important pathogen in clinical infections, and biofilm formation is an effective way for A. baumannii to survive under external pressures. In this study, the aims were to examine the antimicrobial resistance, biofilm formation, and biofilm-specific resistance in clinical isolates of A. baumannii.Materials and Methods: A total of 104 clinical A. baumannii isolates were collected from a large teaching hospital in Southwest China. The antibiotics susceptibilities were tested, and biofilm-forming ability was evaluated by crystal violet staining by confocal laser scanning microscopy (CLSM). Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) of ciprofloxacin, meropenem, and ceftazidime were tested on selected strains by broth microdilution method. Biofilm-associated genes were detected by polymerase chain reaction (PCR), and expression of genes at planktonic stage and biofilm stage were analyzed by real-time reverse transcription PCR (RT-PCR).Results: Multidrug-resistant (MDR) isolates accounted for 65.4%, but no strain was resistant to tigecycline and polymyxin B. Moreover, non-MDR strains tended to form stronger biofilms than MDR strains, and a negative correlation between biofilm-forming ability and resistance profiles to each of tested antimicrobials were observed. The MBECs and MBICs of ciprofloxacin, ceftazidime, and meropenem were evidently increased compared with MICs and MBCs among all tested strains. Additionally, the biofilm formation ability of the csuD-positive strains was stronger than that of the csuD-negative strains. The strains in MDR group had higher carrying rate of csuA and csuD genes than non-MDR group, while non-MDR strains possessed more ompA gene than MDR group. Finally, abaI gene was significantly up-regulated after biofilm formation.Conclusion: These results revealed valuable data for the negative correlation between antimicrobial resistance and biofilm formation, as well as phenotypic and genotypic characteristics of biofilm formation in A. baumannii.Keywords: Acinetobacter baumannii, biofilm formation, antimicrobial resistance, gene expression

Background: The emergence of multidrug resistance (MDR) and extensively drug-resistance (XDR) of Klebsiella pneumoniae strain has brought great threaten to traditional antibiotics. Previous studies showed that plant-derived flavonoids have inhibitory functions on pathogens. However, In K. pneumoniae, the antibacterial activities of different flavonoids on the growth and biofilm formation remains a mystery. The aim of the present study is to evaluate the antioxidant abilities of different flavonoids and to identify their inhibitory effects on K. pneumoniae growth and biofilm formation. Results: Totally 10 flavonoids representing 4 major categories were screened and used in this study. The antioxidant capacity of each flavonoid was evaluated through a DPPH assay. Rutin showed highest free radical scavenging capacity, followed by kaempferol, luteolin, quercetin, apigenin, hesperidin, sinensetin, naringenin, naringin and 3,5,6,7,8,3',4'-heptamethoxyflavone. The inhibitory effect of rutin and naringin on bacteria growth were compared. The antibacterial activity of flavonoid is highly correlated with the antioxidant capacity. Among these flavonoids, rutin showed the best inhibitory capacity against both the growth curve and biofilm production. The lowest value of MIC was found in rutin against K. pneumoniae ATCC700603 (1024μg/mL) and E.coli ATCC25922 (512μg/mL). The MBIC were not found. The expression profiles of 15 genes were analyzed in biofilm cells both with and without rutin treatment. The correlation analysis showed the mrkA gene expression was positively correlated with the biofilm biomass accumulation. Conclusions: Our study indicated that flavonols showed stronger antibacterial activities. The biofilm production is correlated with several gene expression rather than one. Rutin is a potential agent to inhibit the K. pneumoniae biofilm formation.

Background: Klebsiella pneumoniae is one of the most common Enterobacteriaceae. In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) has become one of the most important carbapenem-resistant Enterobacteriaceae. CRKP are usually resistant to antibiotics. Up to this day, the emergence of carbapenemase-producing K. pneumoniae has been a challenge for treatment of clinical infection.Methods: (i) 66 non-repetitive clinical CRKP isolates were identified by matrix-assisted laser analytical ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and drug sensitivity analysis was performed by Vitek2 Compact. EDTA-synergy test and mCIM / eCIM test were used to detect drug-resistant phenotypes. (ii) Carbapenemase genes, extended-spectrum β-lactamase genes (ESBLs), cephalosporinase gene (AmpC), virulence genes, integron and resistance gene cassettes were amplified by PCR. (iii) Plasmid typing was performed by plasmid conjugation assay and PCR-based replicon typing (PBRT) method. (iv) The genetic environments of KPC-2 and NDM-1 were analyzed by using overlapping PCR. (v) MLST was used to analyze the molecular epidemiological characteristics of CRKP. (vi) Risk factors of CRKP infection by logistic regression model.Results: Our study revealed that 42 of the 66 CRKP isolates obtained from patients were identified as blaKPC-2, 24 blaNDM-1-positive strains were identified (20 blaNDM-1 and 4 blaNDM-5), of which 18 were from the neonatal departments. And CRKP strains were ESBL (extended-spectrum β-lactamases) and AmpC enzymes producer, Notably, we found two CR-hvKp (carbapenem-resistant hypervirulent klebsiella pneumoniae) strains, which contains blaKPC-2 gene and other resistant genes. Two of the 42 KPC-2-producing CRKP strains were positive for transconjugants, and the plasmid typing was the IncFII type. And two NDM-producing CRKP strains tested positive for transconjugants, which belonged to the lncX3 plasmid. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (tnpA-tnpR-ISkpn8-blaKPC-2) and conserved regions (blaNDM−1-bleMBL-trpF-tat) are associated with the dissemination of KPC-2 and NDM-1. Intl1 carrying drug resistance gene cassettes were widely distributed in CRKP. According to the MLST results, a total of 13 ST types were measured in 66 CRKP strains, ST11 and ST4495 were the main ST types, and the latter was the newly discovered ST type. Hematological disease, tracheal cannula and prior use of β-lactams and β-lactamase inhibitor combination were identified as independent risk factors for CRKP infections.Conclusion: These findings manifested the need for intensive surveillance and precautions to monitor the further spread of KPC and NDM in southwest China.

Hypervirulent Klebsiella pneumoniae (hvKp) has been globally disseminated recently, especially in Asia. The purpose of this study was to identify the molecular characteristics, clinical manifestations, and clinical risk factors of hvKp infections among patients in a large teaching hospital. A retrospective study was conducted in 123 patients infected with K. pneumoniae at the Affiliated Hospital of Southwest Medical University (Luzhou, China) from October 2016 to November 2018. An isolate that positive for both PCR amplification of aerobactin gene and Galleria mellonella infection model was defined as hvKp. Overall, 43.1% (53/123) of K. pneumoniae isolates were hvKp. String tests were performed on all isolates, and MLSTs of all hvKp were conducted. The K1 ST23 isolates were the dominant clone of hvKp (35.8%). Univariate analysis revealed the following risk factors for hvKp: hepatic abscess (OR = 41.818 [95% CI, 5.379–335.086]), bacteremia (OR = 19.94 [95% CI, 5.565–71.446]), metastatic spread (OR = 19.938 [95% CI, 6.344–62.654]), CRP (OR = 1.008 [95% CI, 1.001–1.015]), nitroimidazole treatment (OR = 7.907 [95% CI, 1.652–37.843]), diabetes (OR = 3.067 [95% CI, 1.38–6.817]), and admission to positive culture interval (OR = 3.636 [95% CI, 1.524–8.678]). Moreover, Multivariate analysis implicated hepatic abscess (OR = 74.332 [95% CI, 3.121–1769.588]), bacteremia (OR = 28.388 [95% CI, 3.039–264.200]), and metastatic spread (OR = 19.391 [95% CI, 3.633–103.498]) as independent risk factors for hvKp infections. Thirteen of twenty-one tested antibiotics were founded resistance to non-hvKp, which is significantly greater than hvKp. Importantly, the ESBL-hvKp and MDR-hvKp were responsible for 7.5% and 15.1% in the hvKp group, respectively.