Macrolide,Lincosamide,and Streptogramin B Resistance in Lipophilic Corynebacteria Inhabiting Healthy Human Skin

Introduction

Macrolides are antibiotics that are commonly used in treatment because of their activity against various bacteria causing pulmonary as well as soft tissue infections, atypical intracellular pathogens, and some eucaryotic parasites such as Entamoeba histolytica or Toxoplasma gondii. Macrolides also have anti-inflammatory characteristics and act as immunomodulators. ⁸ In view of their activity, lincosamides, similar to macrolides, are antibiotics frequently used in anaerobic infections as well as bone and joint infections caused by staphylococci.^6,32 However, the constantly increasing use of these groups of antibiotics has led to the selection of resistant strains. A third antibiotic with a similar mode of action to macrolides and lincosamides is streptogramin B. Although these three groups of antibiotics interact with the 50S subunit of the ribosome and block peptide bond elongation, ¹⁹ it has been possible to confirm several types of bacterial resistance to them, involving many genes: mef(E) and msr(A), which encode efflux pumps, mph(C) and lin(A), which encode the inactivation of the antibiotic by specific enzymes, and erm(A), erm(B), erm(C), and erm(X), which encode modification of the antibiotic target site by methylation of rRNA. The latter is the most common and active mechanism in many bacteria, known as the macrolide-lincosamide-streptogramin B (MLS_B) resistance mechanism.^25,26 A great number of determinants are located on plasmids, transposons, or composite transposons that reveal a wide host range and, hence, may be easily transferred.^19,27,31

The skin is an ecosystem containing microbiota beneficial to the host. Multiresistant skin bacteria are often isolated from clinical samples, and are hence considered opportunistic pathogens. However, the knowledge of the same organisms isolated from healthy human skin is obscure. The Microbiome Project provides a new insight into these bacteria and enhances new studies concerning the characteristics of the skin residents on both the genetic and the phenotypic levels. ¹⁰ Lipophilic corynebacteria have been found to constitute a considerable part of the skin microbiota and are numerously represented on wet surfaces of the skin.^9,14

In our investigation, corynebacteria isolated from the skin of healthy and untreated men, whose residential microbiota was not placed under antibiotic selection pressure, were analyzed. As with coagulase-negative staphylococci, the transfer of these opportunistic bacteria from the skin to areas such as surgical sites is highly probable. Our research highlights the problem of the accumulation of resistance genes in the cells of the bacterial strains colonizing healthy human skin.

Materials and Methods

Results

The screening test for sensitivity to erythromycin and clindamycin was performed using the disc diffusion method as none of the tested lipophilic strains, except from the reference strain, was able to grow on the cation-adjusted M-H broth with lysed horse blood recommended by CLSI. The number of the resistant strains within each species and the type of resistance mechanism are presented in Table 1.

Resistance to examined antibiotics was observed in approximately 80% of the strains, five of which appeared to be resistant only to one antibiotic: two C. jeikeium and one C. tuberculostearicum were resistant to clindamycin, while two C. jeikeium to erythromycin. Seventy-three strains showed resistance to both antibiotics.

The shape of the growth inhibition zones indicated the presence of a constitutive MLS_B resistance type. The bacterial DNA of all 99 strains was amplified with primers for resistance genes to macrolide, lincosamide, and streptogramin antibiotics. In all strains with the MLS_B resistance phenotype, so that in 73 out of 99, the erm(X) gene was present. No amplification products of other tested genes were found (Table 1). MIC determinations for erythromycin and clindamycin with E-test strips confirmed the results of the disc diffusion method; MIC values for the resistant strains were 256 μg/ml or higher.

Identification of the selected group harboring erm(X) gene-resistant strains was confirmed with API Coryne, MALDI-TOF MS, and 16S rDNA sequencing methods. The results are shown in Table 2. Seven strains were identified successfully in all the used methods. In case of one strain, identification was compliant with all methods, but the percentage of homology in 16S rDNA was only 93%. It was not possible to confirm the identification of ZMF 2K37 and ZMF 2K45 previously identified as C. tuberculostearicum and C. jeikeium. The API Coryne and MALDI-TOF methods often provided ambiguous results or did not give a solution. Although in the genetic method the species were pointed, the DNA homology percentage with the typical strain was too small. The method that was based on numerous biological and biochemical tests, which were used for all the 99 investigated strains, was the one that identified the most strains in compliance with at least two other methods. Positive results of three out of four used methods were a basis for claim that most of isolated strains from healthy human skin presenting the MLS_B resistance mechanism belonged to C. tuberculostearicum and C. jeikeium.

Discussion

Our study concerned bacteria isolated from healthy human skin, while previous studies of MLS_B-type resistance in lipophilic corynebacteria were performed with clinical strains, mainly C. jeikeium. C. jeikeium and C. tubeculostearicum, which until 2004 were classified as the Corynebacterium CDC group G, are the two species of lipophilic corynebacteria most frequently isolated from skin.^3,7,11,24 Likewise, these species predominated in the current study, where 73 of 99 isolated strains (74%) demonstrated the MLS_B resistance mechanism, commonly occurring in clinical strains of Corynebacterium spp. Ortiz-Pérez et al. reported the presence of erm(X) in 10 out of 19 (59%) studied clinical strains of C. jeikeium, ²¹ while Rosato et al. in 17 out of 20 (85%). ²⁷ In the present work, 60% (18/30) of C. jeikeium strains demonstrated the MLS_B resistance mechanism. The clinical strains of C. jeikeium investigated by Rosato et al. were found to possess a constitutive MLS_B resistance mechanism. ²⁷

Ortiz-Pérez et al. pointed that erm(X) is the most important gene implicated in the macrolide, lincosamide, and streptogramin B resistance of Corynebacterium species, including C. jeikeium. They detected the erm(B) gene only in two C. urealyticum strains, ²¹ whereas Ojo et al. identified the msr(A) gene in strains of Corynebacterium spp. isolated from human skin. ²⁰ Our results confirmed those of Ortiz-Pérez et al., ²¹ which suggested that the erm(X) gene is important for MLS_B type of resistance, as no amplification products were found for the erm(A), erm(B), erm(C), lin(A), msr(A), or mph(C) genes. The resistance genes found in Corynebacterium species showed high DNA homology with genes in other bacteria, for example, erm(X) of Propionibacterium acnes, Propionibacterium granulosum, Propionibacterium avidum, ²⁸ or msr(A) of Staphylococcus spp.^20,26

The examination of corynebacteria is prone to a wide range of methodological difficulties, two of which were encountered during the present study. The first was related to antibiotic resistance assays. CLSI and EUCAST recommendations do not provide growth requirements for lipophilic bacteria. As the broth proposed by CLSI for the microdilution method was not suitable for skin isolates, the disc diffusion method was used to estimate erythromycin and clindamycin sensitivity. The number of articles describing the use of this method for corynebacteria is limited and no information about the discrepancies of this method for macrolide and lincosamide antibiotics with MIC estimation exists. Only Campanile et al. indicated problems with interpreting the results as intermediate or low resistant. ⁴ The results of the present study showed full compliance between genetic and phenotypic determination of antibiotic resistance, as well as between the results of the E-test and disc diffusion methods. As a consequence, it can be stated that the disc diffusion method, which is considered to be fast, simple and inexpensive, may be an appropriate alternative for the determination of macrolide and lincosamide sensitivity for this lipophilic corynebacteria that are not able to grow on the medium recommended by CLSI.

The second difficulty was the identification of lipophilic corynebacteria. Our research concerned the most frequently identified group of lipophilic corynebacteria. However, the problem is much more complex when all known corynebacteria species are taken into consideration. In many cases, our results obtained by methods commonly used in the laboratories were not comparable with other ones. We found that a multiplot scheme based on various biological methods and biochemical substrates was the most suitable identification method for the vast majority of strains. It appeared to be effective especially for those strains that were slowly reactive and required lipid supplementation. Although this method is rarely used, it is recommended for reference centers. ³ The API Coryne method is claimed to be satisfactory for corynebacteria strains that are reactive and fast growing. ³ In our research, it was found to be helpful, although some results were ambiguous or contradicted those of the other methods. Doubtful reliability of this method was noted also by other researchers.^3,16,22 The MALDI-TOF MS approach for corynebacteria was mostly performed using the Bruker System.^2,15 When the MALDI-TOF/MS Vitek system applied in our experiment with direct colony testing, although considerably fast, presented not fully satisfactory results as identification of 6 strains was not successful and gave misleading results in two cases. Sequencing 16S rDNA is considered to be a gold standard in the identification of various bacterial species, including corynebacteria.^7,11,22 Khamis et al. suggested that such identification provides more reliable results when a 95% threshold of rpoB gene sequence similarity is used.^1,15 To date, a universal sequence has not been discovered for this group of bacteria, which indicates the a wide genetic diversity of corynebacteria. Genetic identification of corynebacteria still remains a challenge as it was also proven by our results. Hence, it can be suggested that strains unable to identify represent yet-to-be described species.^1,30

In summary, the performed unique research shows that percentage of antibiotic-resistant bacteria inhabiting healthy human skin is surprisingly high. Horizontal transfer of genes responsible for MLS_B resistance may lead to the loss of usefulness of this group of antibiotics in therapy. Broadening the knowledge of human skin microbiota, including lipophilic corynebacteria, and improving the methods for their identification are important aims for medical science.