Clonal Diversity, Biofilm Formation, and Antimicrobial Resistance among Stenotrophomonas maltophilia Strains from Cystic Fibrosis and Non-Cystic Fibrosis Patients

Abstract

The intrinsic antibiotic resistance of Stenotrophomonas maltophilia, along with its ability to form biofilm both on abiotic surfaces and host tissues, dramatically affects the efficacy of the antibiotic therapy. In this work, 85 S. maltophilia strains isolated in several hospital of central Italy and from several clinical settings were evaluated for their genetic relatedness (by pulsed-field gel electrophoresis, PFGE), biofilm formation (by microtiter plate assay), and planktonic antibiotic resistance (by Kirby-Bauer disk diffusion technique). The S. maltophilia population showed a high genetic heterogeneity: 64 different PFGE types were identified, equally distributed in cystic fibrosis (CF) and non-CF strains, and some consisted of multiple strains. Most of the strains (88.2%) were able to form biofilm, although non-CF strains were significantly more efficient than CF strains. CF strains produced lower biofilm amounts than non-CF strains, both those from respiratory tracts and blood. Non-CF PFGE types 3 and 27 consisted of strong-producers only. Cotrimoxazole and levofloxacin were the most effective antibiotics, being active respectively against 81.2% and 72.9% of strains. CF strains were significantly more resistant to piperacillin/tazobactam compared to non-CF strains (90% versus 53.3%), regardless of sample type. Among respiratory strains, cotrimoxazole was more active against non-CF than CF strains (susceptibility rates: 86.7% versus 75%). The multidrug resistant phenotype was significantly more prevalent in CF than non-CF strains (90% versus 66.7%). Overall, the multidrug-resistance level was negatively associated with efficiency in biofilm formation. Our results showed, for the first time, that in S. maltophilia both classical planktonic drug resistance and the ability of biofilm formation might favor its dissemination in the hospital setting. Biofilm formation might in fact act as a survival mechanism for susceptible bacteria, suggesting that clinical isolates should be routinely assayed for biofilm formation in diagnostic laboratories.

Keywords: Stenotrophomonas maltophilia; antibiotic resistance; biofilm formation.

Figure 1

Distribution of antibiotic resistance rates and profiles. S. maltophilia strains were evaluated for in vitro susceptibility to six antibiotics: SXT, cotrimoxazole; CHL, chloramphenicol; LVX, levofloxacin; CPX, ciprofloxacin; TZP, piperacillin/tazobactam; MER, meropenem. The frequencies of (A) antibiotic resistances, and (B) multiresistance profiles (non-MDR, MDR, XDR, and PDR) were calculated for cystic fibrosis (CF) (n = 40), non-CF (n = 45), and overall (n = 85) strains. (C) Resistance rates were calculated according to the capability to form biofilm: Non-producers (NP; n = 10) or biofilm-producers (n = 75; gathering strong, moderate, and weak-producers). Significance level at Fisher’s exact test: * p < 0.05; *** p < 0.001. Resistance profiles according to Magiorakos et al. [24]: MDR, multidrug-resistant strains; XDR, extensively drug-resistant strains; PDR, pandrug-resistant strains.

Figure 2

Biofilm formation according to patient and sample types. Biofilm biomass, assessed by spectrophotometric assay after crystal violet assay, was stratified according to (A) patients with (CF; n = 40) or without (non-CF; n = 45) cystic fibrosis, and (B) sample type (CF, n = 40; respiratory non-CF, n = 29; blood, n = 11). Results are shown as box and whiskers: the ends of the whiskers represent the minima and the maxima of all the data; the box always extends from the 25th to 75th percentiles, while the line in the middle of the box is plotted at the median. Significance level from Mann–Whitney test: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Biofilm formation and multidrug-resistant phenotypes. Biofilm formation was assessed by spectrophotometric assay after crystal violet assay and categorized according to Stepanovic et al. [23]: Non-producers, weak-producers, moderate-producers, and strong-producers (left-to-right, in each series of histograms). Susceptibility tests were performed using Kirby-Bauer disk diffusion agar. Resistance profiles according to Magiorakos et al. [24]: MDR, multidrug-resistant strains; XDR, extensively drug-resistant strains; PDR, pandrug-resistant strains. Significance level at Fisher’s exact test: *** p < 0.001; **** p < 0.0001.

Figure 4

Biofilm formation according to antibiotic resistance and patient type. Biofilm biomass formation, spectrophotometrically assessed by crystal violet assay, was stratified on each antibiotic tested—according to susceptibility (S) or resistance (R)—and patient type. Results are shown as scatter plots, with horizontal lines indicating the median values. Significance level at Mann–Whitney test: * p < 0.05; *** p < 0.0001.

Figure 5

Biofilm formation according to the antibiotic resistance level. Biofilm formation was measured by crystal violet assay, and antibiotic resistance by disk diffusion technique. Biofilm formation was stratified according to (A,C,E) the resistance phenotype (non-MDR, MDR, XDR, PDR)— showing results as scatter plot, with the horizontal line indicating the median value—and (B,D,F) the number of resistances to each antibiotic tested. Significance level: ** p < 0.01, Mann-–Whitney test; r²: 0.067, p = 0.024, linear regression analysis.