An investigation of conventional microbial culture for the Naja atra bite wound, and the comparison between culture-based 16S Sanger sequencing and 16S metagenomics of the snake oropharyngeal bacterial microbiota

Abstract

Naja atra is a major venomous snake found in Taiwan. The bite of this snake causes extensive wound necrosis or necrotizing soft tissue infection. Conventional microbial culture-based techniques may fail to identify potential human pathogens and render antibiotics ineffective in the management of wound infection. Therefore, we evaluated 16S Sanger sequencing and next-generation sequencing (NGS) to identify bacterial species in the oropharynx of N. atra. Using conventional microbial culture methods and the VITEK 2 system, we isolated nine species from snakebite wounds. On the basis of the 16S Sanger sequencing of bacterial clones from agar plates, we identified 18 bacterial species in the oropharynx of N. atra, including Morganella morganii, Proteus vulgaris, and Proteus mirabilis, which were also present in the infected bite wound. Using NGS of 16S metagenomics, we uncovered more than 286 bacterial species in the oropharynx of N. atra. In addition, the bacterial species identified using 16S Sanger sequencing accounted for only 2% of those identified through NGS of 16S metagenomics. The bacterial microbiota of the oropharynx of N. atra were modeled better using NGS of 16S metagenomics compared to microbial culture-based techniques. Stenotrophomonas maltophilia, Acinetobacter baumannii, and Proteus penneri were also identified in the NGS of 16S metagenomics. Understanding the bacterial microbiota that are native to the oropharynx of N. atra, in addition to the bite wound, may have additional therapeutic implications regarding empiric antibiotic selection for managing N. atra bites.

Oral Bacteria and Their Antibiotic Susceptibilities in Taiwanese Venomous Snakes

“…In Taiwan, N. atra has the highest infection rate at approximately 80.9% [ 7 ]. Mao et al [ 32 ] identified three important organisms based on comparisons between oral bacteria from N. atra and wound bacterial cultures from envenomed patients, namely M. morganii , P. vulgaris , and Proteus mirabilis. In the current study, these three bacteria were isolated from the oral cavities of venomous snakes, mainly from N. atra and P. mucrosquamatus .…”

The development of surgical risk score and evaluation of necrotizing soft tissue infection in 161 Naja atra envenomed patients

“…These pathogens were also identified in the oropharynx of N . atra [ 13 , 65 , 66 ], which suggested that they may come from the snake’s mouth during envenoming. In addition, a few pathogens were identified only in the later surgeries, suggesting secondary colonization or nosocomial infection.…”

“…In addition, all patients received antibiotics before obtaining the surgical samples, which might have changed the bacterial loads and complexity. Therefore, we suggest a next-generation sequencing technique to better discriminate wound bacteriology [ 13 ]. Nevertheless, we reported only deep tissue cultures, which generally are accepted as the gold standard to diagnose wound infection and as guidance for further antibiotics administration [ 71 ].…”

“…We diagnosed a definite case by examining the culprit snake or laboratory testing of the venom [10][11][12] and a suspected case by having the patient identify the snake in a standard picture exhibited in the emergency department (ED). Some study participants overlapped with previous studies with a different reference period [3,4,13].…”

“…However, a median time of 8.6 h between NSTI diagnosis and surgery has been reported [59], contradicting our study's findings. Our patients' surgeries were delayed, likely because of the aggressiveness of the pathogens and the relatively healthy status of the patients with N. atra bites [13,60]. NSTI has been described as three microbiological classes based on the intraoperative bacterial culture findings [61]: Type I (polymicrobial), Type II (monomicrobial), and Type III (clostridial myonecrosis) [61,62].…”