Surface sterilization methods impact measures of internal microbial diversity in ticks

Abstract

Background Ticks are obligate blood feeders transmitting major pathogens worldwide. Over the past few years, considerable research efforts have focused on the diversity, distribution and impact of gut and intracellular bacterial symbionts on tick development and tick-borne pathogen transmission. The study of this internal microbiome requires the use of a sterilization method to remove external (i.e. cuticular) microbes present on the tick’s surface and to avoid any further contamination. Several sterilization methods exist, including ethanol- or bleach-based treatments that are both effective in killing microbes but with different potential effects on DNA denaturation. Methods We examined how these different sterilization methods impact the measure of internal microbial diversity hosted by the Cayenne tick Amblyomma cajennense ( sensu stricto ). Bacterial barcoding investigations based on 16S rRNA gene sequences were conducted on two batches of 50 individuals each: Ticks of the first batch were sterilized with bleach diluted at 1% and the second batch with 70% ethanol. Tick external microbiome was also determined from cuticle smearing and water samples used for tick washing. Results Bacterial barcoding investigations showed major differences between ethanol- and bleach-treated specimens. Both methods led to the detection of major intracellular bacteria associated with A. cajennense ( s.s. ) but ethanol-treated ticks always harbored a higher bacterial diversity than bleach-treated ticks. Further examinations of tick gut and tick external microbiome revealed that ethanol-based surface sterilization method is inefficient to eliminate the DNA of external bacteria. Conclusions We herein provide evidence that studies investigating the internal microbiome of ticks should consider bleach as the gold standard to efficiently remove cuticular bacterial DNA. Indeed, this method does not impact the internal bacterial diversity hosted by ticks and is thus a better method than the ethanol-based one for studying the internal microbiome. Electronic supplementary material The online version of this article (10.1186/s13071-019-3517-5) contains supplementary material, which is available to authorized users.

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“…Probably both. Indeed, Binetruy et al [66], demonstrated that the tick surface sterilization using ethanol (as we did in the present study) did not efficiently remove bacterial DNA from the tick cuticle compared to bleach. They also showed that environmental taxa, only detected in ethanol cleaned entire ticks, belonged to the same family than those directly detected on the tick cuticle by swabbing.…”

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“…Particular attention should be payed to the washing and surface sterilization steps prior to the extraction to avoid as much as possible external contaminants. Even though the effect of surface microbes in the whole microbiome assessment may be minimal as well (Hammer et al, 2015), the method used for surface sterilization can impact the microbiome diversity retrieved (Binetruy et al, 2019).…”

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“…Likewise, Tokarz et al disputed the identification of Bartonella as a component of the I. scapularis microbiome, attributing the mistake to a lack of specificity in primer design (Tokarz et al, 2019). Another factor that can inflate bacterial diversity estimates is the method of tick sterilization employed prior to sequencing, where ethanol-based surface sterilization of Amblyomma cajennense ticks correlated with reports of higher internal bacterial diversity than studies that used bleach-based methods, likely due to cuticular contaminants that were not removed by ethanol (Binetruy et al, 2019). Finally, detection of microbial DNA does not in and of itself prove that a specific species is a component of the microbiome.…”