Isolation, Whole-Genome Sequencing, and Annotation of Two Antibiotic-Producing and -Resistant Bacteria, Enterobacter roggenkampii RIT 834 and Acinetobacter pittii RIT 835, from Disposable Masks Collected from the Environment

The COVID-19 pandemic has led to a global increase in the use of disposable masks and other personal protection equipment (1). This practice poses risks not only to the environment by increasing pollution from discarded masks, but also to human health, since masks are potential reservoirs for resistant bacteria that may be pathogenic (2). To evaluate the risk of bacterial resistance spread through disposable masks, we isolated, sequenced, and annotated the genomes of Enterobacter roggenkampii RIT 834 and Acinetobacter pittii RIT 835 from surgical masks collected from the environment near the Rochester Institute of Technology (RIT) on 3 February 2022. Enterobacter species are generally considered human pathogens (3), and E. roggenkampii in particular has been shown to harbor antibiotic resistance genes (4). Small squares of four different masks were incubated in tryptic soy broth (TSB) and Reasoner’s 2A (R2A) broth at 30°C under aerobic conditions, followed by dilution and plating. Four distinct colonies were chosen from each mask for an antibiotic susceptibility screen against seven commonly used antibiotics. Both E. roggenkampii RIT 834 and A. pittii RIT 835 were isolated from the same mask and selected for further studies due to their high antibiotic resistance profiles (Fig. 1A and B). They form white colonies on R2A agar and upon electron microscopy examination (5), show both individual and clumps of rod-shaped cells, about 1 to 2 μm in diameter (Fig. 1C and D). Ethyl acetate spent medium extracts of both bacteria were tested for antimicrobial activity using a disk diffusion inhibitory assay (6) against Escherichia coli ATCC 25922 (Fig. 1E and F). E. roggenkampii RIT 834 showed higher bactericidal activity than A. pittii RIT 835 (Fig. 1G).

Genomic DNA (gDNA) was isolated from a 5-mL single colony cultured in R2A medium using the GenElute bacterial genomic DNA isolation kit (Sigma-Aldrich, USA) according to the manufacturer’s protocol. For sequencing, the gDNA was quantified using a NanoDrop spectrophotometer. The library preparation for Illumina sequencing was performed using the Nextera XT library preparation kit (Illumina Inc., USA) following the manufacturer’s instructions. The fragment size distribution was checked using a high-sensitivity DNA kit on an Agilent 2100 Bioanalyzer. The libraries were quantified using a Qubit 3.0 fluorometer and diluted to 16 pM. Sequencing was performed on an Illumina MiSeq (v3 chemistry, 2 × 300 cycles) instrument at the Genomics Lab at RIT.

The demultiplexed FASTQ files were processed for quality control using the program fastp (7). We performed de novo assembly of the filtered data using Unicycler v0.5.0 (8), which uses SPAdes v3.15.4 (9) to assemble the short reads. The results of the genome assembly are presented in Table 1. The quality of the genome assemblies was assessed using QUAST (10), and all metrics were computed for contigs that exceeded 500 bp. The assignment of genomes to genera and species was performed using a fast bacterial genome identification platform (fIDBAC; http://fbac.dmicrobe.cn/). We used the combination of genome-wide average nucleotide identity (gANI) and alignment fraction (AF) with a cutoff of 96.5% (gANI) and 0.6 (AF) for species assignment. The genomic sequencing reads were annotated using the Prokaryotic Genome Annotation Pipeline (PGAP), which is a component of the Read Assembly and Annotation Pipeline Tool (RAPT; https://www.ncbi.nlm.nih.gov/rapt). We present the results of the annotation in Table 1.

We acknowledge ongoing support from the Gosnell School of Life Science (GSoLS) and the College of Science (COS) at Rochester Institute of Technology (RIT).