Date Published: January 30, 2017
Publisher: Public Library of Science
Author(s): Aneta Nowakiewicz, Grażyna Ziółkowska, Przemysław Zięba, Sebastian Gnat, Aleksandra Trościańczyk, Łukasz Adaszek, Massimiliano Galdiero.
The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson’s index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of isolates based on phenotypic resistance and analysis of resistance and virulence genes (Wallace coefficient 1.0). This feature seems to be the most useful for epidemiological purposes and short-term analysis.
Bacteria from the genus Enterococcus constitute an important part of the intestinal biota both in humans and in animals. Among enterococcal species, mainly E. faecium and E. faecalis have the largest epidemiological importance since they are recognized as major nosocomial pathogens. From a medical perspective, the greatest risk associated with these species is the phenomenon of easily acquired resistance to many groups of antimicrobials, particularly to ampicillin, and high-level aminoglycoside and glycopeptide resistance, which significantly reduces the therapeutic alternatives and limits treatment to antimicrobials of last resort .
In total, 321 isolates belonging to the genus Enterococcus were obtained, with 40% (128 strains) classified as E. faecalis (16S-23S rRNA REA). Using the MIC value, seven distinct phenotypic resistance profiles were separated in the pool of the isolates tested (data not shown).
The prevalence of antimicrobial resistance in commensal gut bacteria including Enterococcus is a good indicator of the selective pressure caused by the use of antimicrobials in farm animals. Enterococcus bacteria constitute a significant proportion of the gastrointestinal part of animal microbiota and fecal contamination of carcasses during slaughter processes [3,28]. Direct and indirect contact with animals may favor the spread of bacteria. Enterococci originating from animals (including pigs) are also considered as an important reservoir of resistance genes, which may be transferred to other human pathogens [28–31]. Special attention should be paid to multidrug resistant strains of Enterococcus, and especially those belonging to the species E. faecalis, which is the third most prevalent nosocomial pathogen worldwide . Therefore, multidrug resistant (MDR) E. faecalis strains isolated from farm animals were chosen for our study. In order to evaluate most reliably the usefulness and discriminatory potential of typing methods, we selected potentially closely related strains (the analysis was limited to the local range), and the phenotypic resistance profile was used as a main criterion.
The MALDI -TOF MS analysis showed higher discrimination power in diversification of multidrug resistant strains of E. faecalis (initially classified into three groups according to the resistance profile) than ADSRRS (0.941 vs. 0.884, respectively). However, the ADSRRS technique allowed reliable reproduction of the clustering pattern of isolates obtained with the technique of phenotypic analysis of resistance profiles and the analysis of resistance and virulence genes (Wallace coefficient 1.0). This feature seems to be the most applicable for epidemiological purposes and short-term analysis. Moreover, the agreement of the results from ADSRRS is at a higher level (50–90%) than in the case of MALDI (40–85%). This is probably related to the fact that both methods are based on completely different methodological bases. Since ADSRRS-fingerprinting is based on analyzing total genomic DNA diversity by detecting polymorphism restriction sites and MALDI -TOF MS spectra correspond rather to housekeeping proteins, it would be more appropriate to compare the results of the MALDI -TOF MS analysis with the results of multilocus sequence typing (MLST). This phenomenon has been confirmed by many authors who observed greater compatibility between MSP spectra and the type of ST [9,72–74] rather than the PFGE profile [21,75].