In-vitro efficacy of fluoroquinolones and carbapenems against biofilm-forming and non-forming non-fermenting gram-negative bacteria isolated from clinical specimens

Non-fermenting gram-negative bacteria (NFGNB) pose a public health threat due to their tendency to cause multidrug-resistant and/or biofilm-associated infections. This cross-sectional study analyzed antibiograms, biofilm formation, and minimum inhibitory concentrations (MICs) of quinolones and carbapenems in NF-GNB from clinical specimens in a tertiary care hospital. Clinical specimens were processed for bacterial isolation and identification. Quinolone- and carbapenem-resistant Acinetobacter calcoaceticus-baumannii (ACB) complex and Pseudomonas aeruginosa, confirmed by disc diffusion, were tested for MICs of quinolones and carbapenems using broth microdilution. Biofilm formation was assessed by the microtiter plate method. Statistical analyses were performed in SPSS 17.0. A total of 92 NFGNB were isolated from patients (median age: 24 years, 56.52% female), primarily with urinary tract infections (40.23%). Biofilm formation was detected in 23.94% (17/71) of the ACB complex and 57.14% (12/21) of P. aeruginosa. For P. aeruginosa, the MIC50 against norfloxacin was 8 µg/ml in biofilm non-formers and ≥64 µg/ml in formers, while ≥0.5 µg/ml and ≥1 µg/ml against ofloxacin, respectively. The MIC50 against ciprofloxacin was ≥32 µg/ml for ACB complex (both groups) and ≥4 µg/ml (non-formers) vs. ≥16 µg/ml (formers) for P. aeruginosa. The MIC50 against levofloxacin was ≥16 µg/ml for ACB complex (both groups) and ≥0.5 µg/ml (non-formers) vs. ≥1 µg/ml (formers) for P. aeruginosa. For meropenem, MIC50 was ≥0.5 µg/ml (non-formers) vs. ≥8 µg/ml (formers) in ACB complex and ≥8 µg/ml vs. ≥16 µg/ml in P. aeruginosa. The MIC50 against imipenem for ACB complex was ≥4 µg/ml (non-formers) vs. ≥8 µg/ml (formers). Biofilm non-forming and forming P. aeruginosa exhibited a similar MIC50 value of ≥0.5 µg/ml. Over three-fourths of the NFGNB infections were caused by the ACB complex, with nearly one-fourth involving biofilm-forming strains, necessitating the need for higher MICs of quinolones and carbapenems.

Methods
Patients' demographics and laboratory results, including microbiological analyses, were collected through patient information sheets. The sheet detailed for demographic information, such as age group and gender of the infected patients; clinical information, including clinical samples; microbiological findings comprising bacterial genera, antimicrobial resistance patterns, biofilm formers or non-formers, inhibitory concentrations of fluoroquinolones (norfloxacin, ciprofloxacin, ofloxacin, and levofloxacin) and carbapenems (imipenem and meropenem). Considering that the Patient identification number and Specimen number were human subject data and must be anonymized, these identifiers were removed from the dataset. Exact or direct age is removed and is categorized as an age group in order to anonymize the data. Any missing or unclear records were clarified by communicating with healthcare providers, patients, or families (using the phone numbers from the patient information sheet). The study variables were then recorded in Microsoft Excel, version 13.0.

Data was then entered and analyzed using Statistical Package for Social Sciences (SPSS), version 17.0. Descriptive data were analyzed in terms of frequency and percentage. Quantitative data were reported as mean, median, and interquartile range (IQR). Except for values of inhibitory concentrations, the data were qualitative and were calculated as frequency (percentage) in SPSS version 17.0. Quantitative variables were calculated as median (interquartile range), also referred to as MIC50/MIC90. Qualitative variables were analyzed using the Chi-square test, while quantitative variables were analyzed using the independent student t-test, with statistical significance determined at a p-value of <0.05 within a 95% confidence interval (CI).

非发酵革兰氏阴性菌（Non-fermenting gram-negative bacteria, NFGNB）易引发多重耐药及/或生物膜相关感染，对公共卫生构成威胁。本横断面研究针对某三级医院临床标本分离的非发酵革兰氏阴性菌，分析其药敏谱、生物膜形成能力以及喹诺酮类与碳青霉烯类的最低抑菌浓度（minimum inhibitory concentrations, MICs）。临床标本均完成细菌分离与鉴定。经纸片扩散法确认的喹诺酮类与碳青霉烯耐药鲍曼不动杆菌-醋酸钙不动杆菌复合群（Acinetobacter calcoaceticus-baumannii complex, ACB复合体）及铜绿假单胞菌（Pseudomonas aeruginosa），采用微量肉汤稀释法检测其喹诺酮类与碳青霉烯类的MIC值。生物膜形成能力采用微孔板法进行评估。统计学分析采用SPSS 17.0软件完成。

本研究共从患者中分离得到92株非发酵革兰氏阴性菌，患者中位年龄为24岁，女性占比56.52%，感染类型以尿路感染最为常见（40.23%）。ACB复合体的生物膜形成阳性率为23.94%（17/71），铜绿假单胞菌的生物膜形成阳性率为57.14%（12/21）。

针对铜绿假单胞菌，生物膜阴性株对诺氟沙星的MIC50为8 μg/ml，生物膜阳性株则≥64 μg/ml；对氧氟沙星的MIC50分别为≥0.5 μg/ml与≥1 μg/ml。ACB复合体对环丙沙星的MIC50均≥32 μg/ml（两组），而铜绿假单胞菌的生物膜阴性株与阳性株MIC50分别为≥4 μg/ml与≥16 μg/ml。ACB复合体对左氧氟沙星的MIC50均≥16 μg/ml（两组），铜绿假单胞菌的生物膜阴性株与阳性株MIC50则分别为≥0.5 μg/ml与≥1 μg/ml。针对美罗培南，ACB复合体的生物膜阴性株MIC50为≥0.5 μg/ml，阳性株为≥8 μg/ml；铜绿假单胞菌的两组MIC50分别为≥8 μg/ml与≥16 μg/ml。ACB复合体对亚胺培南的MIC50，生物膜阴性株为≥4 μg/ml，阳性株为≥8 μg/ml。生物膜阴性与阳性的铜绿假单胞菌的MIC50均≥0.5 μg/ml，二者水平相近。

超过四分之三的非发酵革兰氏阴性菌感染由ACB复合体引发，近四分之一的感染由生物膜形成阳性菌株导致，提示临床需使用更高剂量的喹诺酮类与碳青霉烯类药物。

## 研究方法
本研究通过患者信息登记表收集患者人口学资料与实验室检测结果，包括微生物学分析数据。登记表涵盖以下内容：人口学信息，如感染患者的年龄分组与性别；临床信息，如临床标本类型；微生物学检测结果，包括细菌菌属、抗菌药物耐药谱、生物膜形成状态（阳性/阴性）、氟喹诺酮类（诺氟沙星、环丙沙星、氧氟沙星、左氧氟沙星）与碳青霉烯类（亚胺培南、美罗培南）的抑菌浓度。考虑到患者识别号与标本编号属于人体受试者数据，需进行匿名化处理，故本数据集已移除上述标识符。为进一步实现数据匿名化，精确年龄信息被移除，取而代之的是年龄分组。对于存在缺失或表述不清的记录，研究人员通过联系医疗照护人员、患者或家属（使用患者信息登记表留存的电话号码）进行核实确认。随后，研究变量被录入至Microsoft Excel 13.0版本软件。

数据录入与分析均采用社会科学统计软件包（Statistical Package for the Social Sciences, SPSS）17.0版本完成。描述性数据采用频数与百分比进行统计分析。定量数据以均值、中位数及四分位数间距（IQR）进行报告。除抑菌浓度数据外，其余数据均为定性数据，在SPSS 17.0中以频数（百分比）进行计算。定量变量以中位数（四分位数间距）进行统计，即MIC50/MIC90。定性变量采用卡方检验进行分析，定量变量采用独立样本t检验进行比较，以p值<0.05且95%置信区间（CI）作为统计学显著性判定标准。