Biofilm formation and plasmid-mediated quinolone resistance genes at varying quinolone inhibitory concentrations in quinolone-resistant bacteria superinfecting COVID-19 inpatients

The likelihood of treatment failure in COVID-19 patients with bacterial
superinfection stems from phenotypic, viz., biofilms, and genotypic
mechanisms. This cross-sectional study aimed to determine the inhibitory
concentrations of quinolones—nalidixic acid, norfloxacin, ciprofloxacin,
ofloxacin, and levofloxacin—in biofilm formers [minimum biofilm inhibitory
concentration (MBIC)] and non-formers [minimum inhibitory concentration
(MIC)] as well as correlate the quinolones’ folds with the presence of
plasmid-mediated quinolone-resistance (PMQR) genes in quinolone-resistant
bacteria isolated from COVID-19 inpatients. Quinolone-resistant isolates
(n=193), verified through disc diffusion, were tested for quinolone
inhibitory concentrations and biofilm formation with broth microdilution
and microtiter plate methods, respectively. Polymerase chain reaction was
used to detect PMQR genes. MIC to MBIC median increase in folds for
ciprofloxacin, ofloxacin, and levofloxacin was 128 (2-8,192), 64
(4-1,024), and 32 (4-512) in gram-positive cocci (GPC), respectively,
while it was 32 (4-8,192), 32 (4-2,048), and 16 (2-1,024) in
fermentative-gram-negative bacilli (F-GNB), and 16 (4-4,096), 64 (2-64),
and 16 (8-512) in non-fermentative-gram-negative bacilli (NF-GNB).
Biofilm-forming F-GNB (32/126) and NF-GNB (10/24)
harbored qnrB [11/32 versus (vs.)
3/10], aac(6')-Ib-cr (10/32 vs. 4/10),
and qnrS (9/32 vs. 0/10) genes, respectively. A 32-fold
median increase in ciprofloxacin was significantly associated
with qnrA and qnrS in F-GNB and
NF-GNB, respectively. F-GNB and NF-GNB biofilms were significantly
associated with aac(6')-Ib-cr and qnrS genes,
respectively. Nearly one-third of the superinfecting bacteria in
COVID-19 patients formed biofilms, and had at least one PMQR gene,
increasing the need for quinolone inhibitory concentrations.

合并细菌二重感染的新冠肺炎患者出现治疗失败的风险，源于表型（如生物膜）与基因型两类致病机制。本横断面研究旨在明确喹诺酮类药物——萘啶酸、诺氟沙星、环丙沙星、氧氟沙星及左氧氟沙星——对生物膜形成菌的最低生物膜抑菌浓度（minimum biofilm inhibitory concentration, MBIC）与非形成菌的最低抑菌浓度（minimum inhibitory concentration, MIC），并分析分离自新冠肺炎住院患者的喹诺酮耐药菌中，喹诺酮类药物的抑菌浓度升高倍数与质粒介导喹诺酮耐药（plasmid-mediated quinolone-resistance, PMQR）基因携带情况的相关性。
本研究共纳入经纸片扩散法验证的喹诺酮耐药分离株（n=193），分别采用肉汤微量稀释法与微孔板法检测其喹诺酮类抑菌浓度及生物膜形成能力；通过聚合酶链反应（polymerase chain reaction, PCR）检测PMQR基因携带情况。
革兰阳性球菌（gram-positive cocci, GPC）中环丙沙星、氧氟沙星及左氧氟沙星的MIC较MBIC的中位数升高倍数分别为128（范围2~8192）、64（范围4~1024）及32（范围4~512）；发酵型革兰阴性杆菌（fermentative-gram-negative bacilli, F-GNB）中对应数值分别为32（范围4~8192）、32（范围4~2048）及16（范围2~1024）；非发酵型革兰阴性杆菌（non-fermentative-gram-negative bacilli, NF-GNB）中则分别为16（范围4~4096）、64（范围2~64）及16（范围8~512）。
生物膜形成型F-GNB（32/126）与NF-GNB（10/24）分别携带qnrB[11/32 vs. 3/10]、aac(6')-Ib-cr[10/32 vs. 4/10]及qnrS[9/32 vs. 0/10]基因。环丙沙星中位数升高32倍的表型，分别与F-GNB的qnrA基因及NF-GNB的qnrS基因显著相关；F-GNB与NF-GNB的生物膜形成能力，分别与aac(6')-Ib-cr基因及qnrS基因显著相关。
近三分之一的新冠肺炎患者合并感染细菌可形成生物膜，且至少携带一种PMQR基因，这一情况使得临床对喹诺酮类药物抑菌浓度的需求进一步提升。