Novel spore-forming species exhibiting intrinsic resistance to third- and fourth-generation cephalosporins and description of
Tigheibacillus jepli
gen. nov., sp. nov.
Authors: Miliotis Georgios , Sengupta Pratyay , Hameed Asif , Chuvochina Maria , McDonagh Francesca , Simpson Anna C. , Parker Ceth W. , Singh Nitin K. , Rekha Punchappady D. , Morris Dearbháile , Raman Karthik , Kyrpides Nikos C. , Hugenholtz Philip , Venkateswaran Kasthuri 
Published: March 2024

A comprehensive microbial surveillance was conducted at NASA’s Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HS
T
, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HS
T
is most closely related to
Virgibacillus halophilus
5B73C
T
,
sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus
Virgibacillus
is polyphyletic, strain 179-BFC-A-HS
T
is proposed as a novel species of a novel genus,
Tigheibacillus jepli
gen. nov., sp. nov (type strain 179-BFC-A-HS
T
= DSM 115946
T
= NRRL B-65666
T
), and its closest neighbor,
V. halophilus
, is proposed to be reassigned to this genus as
Tigheibacillus halophilus
comb. nov. (type strain 5B73C
T
= DSM 21623
T
= JCM 21758
T
= KCTC 13935
T
). It was also necessary to reclassify its second closest neighbor
Virgibacillus soli,
as a member of a novel genus
Paracerasibacillus
, reflecting its phylogenetic position relative to the genus
Cerasibacillus
, for which we propose
Paracerasibacillus soli
comb. nov. (type strain CC-YMP-6
T
= DSM 22952
T
= CCM 7714
T
). Within
Amphibacillaceae
(
n
= 64),
P. soli
exhibited 11 antibiotic resistance genes (ARG), while
T. jepli
encoded for 3, lacking any known β-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy.
P. soli
was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins.

IMPORTANCE

The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species
Tigheibacillus jepli
, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the
Amphibacillaceae
family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.