Multiplex PCR analysis of virulence genes and their influence on antibiotic resistance in Enterococcus spp. isolated from broiler chicken

Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/µL, 320.0 pg/µL, and 1.6 ng/µL DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.

Antimicrobial Susceptibility and Characterization of Propionibacterium acnes by Multilocus Sequence Typing and Repetitive-Sequence-Based PCR

Propionibacterium acnes, a gram-positive, anaerobic, and aerotolerant bacterium that is found frequently in the skin as part of the human microbiome causes inflammatory acne, shoulder infection, and the contamination of medical devices. The study goals were the antibiotic resistant and molecular epidemiological characterization of the P. acnes isolates in Korea. A total of 22 P. acnes isolates originated from diverse patients were obtained from three National Culture Collections for Pathogens in South Korea. The hemolytic properties and minimum inhibitory concentrations (MIC) of five antibiotics (tetracycline, doxycycline, clindamycin, erythromycin, and minocycline) were determined. Only one isolate showed high MIC values and resistance to all five antibiotics. Genotypic characterization was achieved by multilocus sequence typing (MLST) for eight loci (aroE, guaA, tly, camp2, atpD, gmk, lepA, and sodA) and repetitive-sequence-based PCR (rep-PCR) analysis using the DiversiLab kit. MLST revealed four phylogroups that were type IA₁ (27.3%), type IA₂ (18.2%), type IB (13.6%), and type II (40.9%). Rep-PCR results demonstrated three clusters that were cluster I (39.1%), cluster II (45.5%), and cluster III (13.6%). The isolates of cluster I were part of phylogroup type IA (both IA₁ and IA₂), and the isolates of cluster II belonged to phylogroup type II. All isolates of phylogroup type IB were hemolytic and belonged to cluster III. The results of rep-PCR clustering analysis showed a good correlation with those of MLST phylogroups, suggesting that rep-PCR could be an alternative method to track P. acnes subtype lineages.

Detection of Bacterial Species in Chronic Periodontitis Tissues at Different Stages of Disease Severity

The goal of this research was to determine the relationship between the stage of chronic periodontitis and the presence of six bacterial pathogens (Aggregatibacter actinomycetemcomitans: AA, Fusobacterium nucleatum: FN, Porphyromonas gingivalis: PG, Prevotella intermedia: PI, Enterococcus faecalis: EF, and Parvimonas micra: PM). Forty-six chronic periodontitis patients visiting a dental hospital were included in this investigation. They were classified into four chronic periodontitis stages based on the sulcus bleeding index value and the probing depth. The tissue samples from the periodontal surgery were used for a direct PCR detection assay. A total of 49 samples from 46 patients were collected and classified into four chronic periodontitis groups (N: 6, P1: 13, P2: 18, P3: 12). The PCR assay showed that FN, PI, and PM were involved from the beginning of chronic periodontitis (P1), while AA and PG existed regardless of the disease stages. EF was strongly linked to the P3 stage of the disease. In order to assess the effect of dental treatments on patients with chronic periodontitis, EF should be a critical marker for P3 patients, while FN, PI, and PM would be good indicators for chronic periodontitis.