Antimicrobial susceptibility and multilocus sequence typing of Clostridium perfringens isolated from yaks in Qinghai-Tibet plateau, China

Abstract

Clostridium perfringens (C. perfringens) is an opportunistic pathogen that cause necrotic enteritis, food poisoning and even death in animals. In this study, we explored the prevalence, antibiotic resistance and genetic diversity of Clostridium perfringens isolated from yak in the Qinghai-Tibet plateau, China. A total of 744 yak fecal samples were collected and assessed for toxin genes, antimicrobial susceptibility and multilocus sequence typing (MLST). Results indicated that 144 out of 744 (19.35%) yak fecal samples were tested to be positive for C. perfringens, 75% (n = 108, 108/144) were C. perfringens type A, 17.36% (n = 25, 25/144) were C. perfringens type C, 2.78% (n = 4, 4/144) were C. perfringens type D, and 4.86% (n = 7, 7/144) were C. perfringens type F. In addition, 2.78% (n = 4, 4/144) of the isolates were positive for cpb2 toxin gene. Antimicrobial susceptibility testing revealed that 98.61% (142/144) of the isolates showed multiple-antibiotic resistance. According to MLST and phylogenetic tree, 144 yak-derived C. perfringens isolates had an average of 12.95 alleles and could be divided into 89 sequence types (STs) and clustered in 11 clonal complexes (CCs). The most of isolates belong to type A with a considerable genetic diversity, having Simpson index up to 0.9754. MLST and phylogenetic analysis showed that the isolates under the same clade came from multiple regions. Cross-transmission among isolates and interconnectedness were observed in the genetic evolution. According to the study, the most of the isolates exhibited broad-spectrum antibacterial resistance, diverse alleles, and multiple lethal toxin genes of C. perfringens.

Keywords: antibiotic resistance; clostridium perfringens; multilocus sequence typing; toxin genes; yak.

Figure 1

Geographical locations from where samples were collected (Marked as: ▴).

Figure 2

Heat map of antibiotic resistance of 144 Clostridium perfringens isolates from the Qinghai-Tibet Plateau. The different regions are listed on the left vertical axis and antibiotics (A–Z) are marked on the horizontal axis. Colored scale bars represent the zone of inhibition ranges between 0 and 40 mm. (A) Vancomycin; (B) Minocycline; (C) Doxycycline; (D) Ofloxacin; (E) Cefotaxime; (F) Cefazolin; (G) Ampicillin; (H) Cefuroxime; (I) Chloramphenicol; (J) Ceftazidime; (K) Florfenicol; (L) Penicillin; (M) Cephalexin; (N) Oxacillin; (O) Tetracycline; (P) Ciprofloxacin; (Q) Mideamycin; (R) Clindamycin; (S) Furazolidone; (T) Bacitracin; (U) Gentamicin; (V) Polymyxin B; (W) Erythromycin; (X) Kanamycin; (Y) Sulfamethoxazole; (Z) Streptomycin.

Figure 3

Drug resistance of 144 yak-derived Clostridium perfringens in Qinghai-Tibet Plateau. The left side is the number of resistant strains of the corresponding antibiotics; the right side are the Isolates in corresponding antibiotic resistance profiles showing resistance to this antibiotic, the top is the number of isolates resistant to multiple antibiotics and the bottom is the main yak-derived isolates in antibiotic resistance spectrum.

Figure 4

Resistance of yak-derived Clostridium perfringens to different antibiotics in different regions.

Figure 5

(A) Minimal evolutionary tree generated with different Source characteristics. (B) Minimal evolutionary tree generated with different Toxin types as features. A circle represents a sequence type, the size of the circle represents the number of strains, the number on the circle represents the name of the ST type. The different connecting lines between the two circles represent the difference between the two ST types, the thick solid line indicates that there is one locus difference between the two ST types, the thin solid line indicates that there are two or three different loci, the thin dotted line indicates that there are four different loci, and the remaining lines indicates more than four different loci. Strains in the same shade represent the same Clonal complex (CC).

Figure 6

Phylogenetic tree and allelic profiles of 144 yak-derived Clostridium perfringens sequence types (STs).