Biofilm formation and antibiotic sensitivity in Elizabethkingia anophelis

Abstract

Elizabethkingia anophelis has recently gained global attention and is emerging as a cause of life-threatening nosocomial infections. The present study aimed to investigate the association between antimicrobial resistance and the ability to form biofilm among E. anophelis isolated from hospitalized patients in China. Over 10 years, a total of 197 non-duplicate E. anophelis strains were collected. Antibiotic susceptibility was determined by the standard agar dilution method as a reference assay according to the Clinical and Laboratory Standards Institute. The biofilm formation ability was assessed using a culture microtiter plate method, which was determined using a crystal violet assay. Culture plate results were cross-checked by scanning electron microscopy imaging analysis. Among the 197 isolates, all were multidrug-resistant, and 20 were extensively drug-resistant. Clinical E. anophelis showed high resistance to current antibiotics, and 99% of the isolates were resistant to at least seven antibiotics. The resistance rate for aztreonam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, cefepime, and tetracycline was high as 100%, 99%, 99%, 99%, 99%, 95%, and 90%, respectively. However, the isolates exhibited the highest susceptibility to minocycline (100%), doxycycline (96%), and rifampin (94%). The biofilm formation results revealed that all strains could form biofilm. Among them, the proportions of strong, medium, and weak biofilm-forming strains were 41%, 42%, and 17%, respectively. Furthermore, the strains forming strong or moderate biofilm presented a statistically significant higher resistance than the weak formers (p < 0.05), especially for piperacillin, piperacillin-tazobactam, cefepime, amikacin, and ciprofloxacin. Although E. anophelis was notoriously resistant to large antibiotics, minocycline, doxycycline, and rifampin showed potent activity against this pathogen. The data in the present report revealed a positive association between biofilm formation and antibiotic resistance, which will provide a foundation for improved therapeutic strategies against E. anophelis infections in the future.

Keywords: Elizabethkingia anophelis; biofilm formation; biofilm-specific resistance; multidrug-resistant; nosocomial infections.

Figure 1

Scanning electron microscopy imaging analysis of four E. anophelis biofilm formations. The four strains were as follows: weak biofilm-forming strain SKL 051060 (A, B); moderate biofilm-forming strain SKL014219 (C, D); strong biofilm-forming strain SKL067015 (E, F); and SKL068512 (G, H). Among them, the left part of the figure (A, C, E, G) is the imaging result at 5,000-fold magnification (5.00k SE), bar = 10.0 µm; the right part of the figure (B, D, F, H) is the image result of 10,000-fold magnification (10.00k SE), bar = 5.00µm. The data show that the cell morphology is intact and densely stacked, and layered bacteria can be observed.

Figure 2

A bar graph displays the relationship between biofilm formation intensity and antimicrobial resistance. (A) Strong or moderate biofilm-forming strains presented a significantly higher average number of resistances than the weak producers. (B) The discrepancy in antimicrobial resistance among weak, moderate, and strong biofilm-producing isolates. W, weak biofilm formation; M, moderate biofilm formation; S, strong biofilm formation; S/W, strong or moderate biofilm formation. ** means significant at 0.01 level, *** means significant at 0.001 level.

Figure 3

The frequency of antibacterial resistance in strong/moderate biofilm formation and weak biofilm producer E. anophelis isolates. S/W, strong or moderate biofilm formation; W, weak biofilm formation. PIP, piperacillin; TZP, piperacillin-tazobactam; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem; MEM, meropenem; AZT, aztreonam; GEN, gentamicin; AMK, amikacin; MNO, minocycline; DOX, doxycycline; TCY, tetracycline: TGC, tigecycline; CIP, ciprofloxacin; LVX, levofloxacin; SXT, trimethoprim-sulfamethoxazole; RFP, rifampin; VA, vancomycin; and CHL, chloramphenicol.

Figure 4

The correlation between antibiotic resistance and biofilm-forming capacity of clinical E. anophelis isolates to five antibiotics. (A–E) For piperacillin (PIP), piperacillin-tazobactam (TZP), cefepime (FEP), amikacin (AMK), and ciprofloxacin (CIP). Susceptible isolates tended to form weaker biofilms than non-susceptible isolates. Significant differences were detected between groups. The p-values obtained by Mann–Whitney analysis were as follows: piperacillin (p < 0.0001), piperacillin-tazobactam (p = 0.0088), cefepime (p = 0.0022), amikacin (p = 0.0442), and ciprofloxacin (p = 0.0461). * means significant at 0.05 alpha level, ** means significant at 0.01 level, *** means significant at 0.001 level.