ABSTRACT
Whole-genome sequence data for clinically relevant Gram-negative bacteria from the African continent are scarce. In this report, we present the draft genome sequence data and antibiograms of four species, namely, Kerstersia gyiorum, Providencia vermicola, Providencia stuartii, and Alcaligenes faecalis, that were recovered from human soft tissue biopsy samples.
ANNOUNCEMENT
Whole-genome sequencing technologies have emerged as important methodologies for diagnosis and surveillance of antimicrobial resistance (1). Here, we report on the draft genome sequences and antibiograms of four Gram-negative bacteria species, namely, Kerstersia gyiorum, Alcaligenes faecalis, Providencia stuartii, and Providencia vermicola, that were recovered from human soft tissue biopsy samples. Kerstersia gyiorum represents opportunistic bacteria (2) that are frequently resistant to chloramphenicol and ciprofloxacin (3, 4). Alcaligenes faecalis has been associated with severe clinical conditions in humans (5, 6). Providencia stuartii infections are difficult to manage (3) and are often resistant to multiple antibiotics (7–9). The NDM-1 carbapenemase gene has been detected in Providencia vermicola isolates (10, 11).
The study was approved by the Noguchi Memorial Institute for Medical Research Institutional Review Board (Ethical code number: FW00001824) and University of Ghana Ethical and Protocol Review Committee, College of Health Sciences (Protocol number: CHS-Et/M.I-P.2.13/2017-2018). Soft tissue biopsy samples that had been aseptically obtained from patients (n = 50) with diabetic foot ulcers were placed in 10 mL thioglycolate broth, incubated for 18 to 24 h at 37°C, and plated on 5% sheep blood agar (Oxoid, Basingstoke, UK) and MacConkey agar (Oxoid). Isolates recovered were identified to the species level using matrix-assisted laser desorption ionization–time of flight mass spectrometry (Bruker Daltonics, Germany).
The MICs for 12 antibiotics were determined with an AutoSCAN-4 system (American MicroScan, Mahwah, NJ), with the quality control strain ATCC 25922. The results were interpreted using CLSI guidelines (12).
Genomic DNA extraction and purification were performed using the QIAamp DNA minikit (Qiagen, Germany). The concentration of extracted DNA was determined using a Qubit 4.0 fluorometer. Following the manufacturer’s instructions, DNA libraries were prepared using the DNA preparation (M) tagmentation kit (Illumina Inc., San Diego, CA, USA). This process involved DNA fragmentation, tagmentation, addition of index sequences, and amplification of indexed fragments. The quality and concentration of the libraries were assessed with the 2100 Bioanalyzer system (Agilent) and quantitative PCR (qPCR) (KAPA SYBR Fast qPCR kit), respectively. Libraries were diluted to a concentration of 2 nM, pooled, and sequenced on the MiSeq platform (Illumina) using 2 × 300-bp chemistry. Indexes and reads with quality scores of <20 were trimmed using Trimmomatic v.0.39 (13), and quality control checks were performed with FastQC v.1.0. (14). Trimmed reads were assembled using Unicycler v.0.4.9 (15) and evaluated using QUAST v.5.2.0 (16). All genomes passed the basic quality metrics of Q scores of >30, minimum coverage of 20×, and minimum contig size of 200 bp. Assembled sequence data were analyzed using kmerFinder v.4.1 (https://cge.food.dtu.dk/services/KmerFinder) (17) to determine bacterial species, CARD v.3.0.9 (https://card.mcmaster.ca/analyze/rgi) (18) and ResFinder v.4.1 (https://cge.food.dtu.dk/services/ResFinder) (19) to identify resistance genes, PlasmidFinder v.2.1 (https://cge.food.dtu.dk/services/PlasmidFinder) (20) to detect plasmids, VirulenceFinder v.2.0 (https://cge.food.dtu.dk/services/VirulenceFinder) (21) to detect virulence genes, and multilocus sequence typing (MLST) v.2.0 (https://cge.food.dtu.dk/services/MLST) (22) to determine sequence types. The draft genomes were annotated using Prokka v.1.14.6 (23). Table 1 summarizes the results.
TABLE 1.
Antimicrobial resistance profiles and genomic characteristics of isolates
| Characteristic | Data fora: |
|||
|---|---|---|---|---|
| Providencia stuartii | Kerstersia gyiorum | Providencia vermicola | Alcaligenes faecalis | |
| MIC (mg/L) | ||||
| Amikacin | ≤16 (S) | ≤16 (S) | >32 (R) | ≤16 (S) |
| Aztreonam | 8 (S) | 8 (S) | >16/8 (R) | >8 (R) |
| Cefepime | 8 (S) | 4 (S) | >8 (R) | >8 (R) |
| Ceftazidime | 4 (S) | ≤1 (S) | NT | >16 (R) |
| Ciprofloxacin | >2 (R) | >2 (R) | >2 (R) | >2 (R) |
| Gentamicin | 8 (I) | ≤4 (S) | >8 (R) | >8 (R) |
| Imipenem | 4 (S) | ≤1 (S) | >8 (R) | ≤1 (S) |
| Levofloxacin | >4 (I) | ≤2 (S) | >4 (R) | >4 (R) |
| Meropenem | ≤1 (S) | ≤1 (S) | >8 (R) | ≤1 (S) |
| Piperacillin-tazobactam | >64 (R) | ≤16 (S) | >64 (R) | >64 (R) |
| Tobramycin | >8 (R) | ≤4 (S) | >8 (R) | >8 (R) |
| Trimethoprim-sulfamethoxazole | >2/38 (R) | ≤2/38 (S) | NT | >2/38 (R) |
| Resistance genes | ||||
| Aminoglycosides | aac(2′)-Ia, aadA1, aph(3′)-Ia | ND | aac(6')-Ib-cr, aph(3′)-VI, aadA1, rmtC, ant(3″)-IIa | aac(6')-Ib-cr, aac(6')-Ib3, aadA1, ant(2″)-Ia, aph(3″)-Ib, aph(6)-Id |
| Sulfonamides | sul3 | ND | sul1 | sul1 |
| β-Lactams | ND | ND | ||
| Cephalosporins | bla DHA-1 | |||
| Carbapenems | blaOXA-10, blaNDM-1 | bla CARB-4 | ||
| Tetracyclines | tet(B) | ND | ND | tet(A), tet(G) |
| Diaminopyrimidines | dfrA1 | ND | dfrA14 | dfrA15, dfrA17 |
| Fluoroquinolones | qnrD1 | ND | qnrA1, aac(6′)-Ib-cr | aac(6')-Ib-cr |
| Phenicols | catA3 | catB3 | ND | catB3, floR |
| Rifamycins | ND | ND | arr-2 | ND |
| Disinfecting agents and antiseptics | qacL, rsmA | ND | qacE | qacE |
| Macrolides | crp | ND | ND | ND |
| Nucleosides | sat-2 | ND | ND | ND |
| Genome size (bp) | 4,329,306 | 3,983,113 | 4,455,003 | 4,464,976 |
| Sequence type | Unknown | Unknown | Unknown | Unknown |
| N50 (bp) | 187,893 | 485,120 | 642,172 | 282,357 |
| GC content (%) | 41.2 | 62.5 | 40.8 | 56.8 |
| No. of reads | 808,942 | 621,880 | 782,484 | 989,950 |
| No. of contigs | 106 | 17 | 63 | 66 |
| Size of largest contig (bp) | 650,510 | 1,055,846 | 1,087,565 | 915,131 |
| No. of coding sequences | 3,990 | 3,466 | 3,988 | 4,191 |
| No. of rRNAs | 4 | 3 | 5 | 3 |
| No. of tRNAs | 71 | 52 | 70 | 52 |
| No. of transfer-messenger RNAs | 1 | 1 | 1 | 1 |
| Coverage (×) | 38 | 26 | 35 | 41 |
| Genome accession no. | JANKKP000000000 | JANKLF000000000 | JANKLM000000000 | JANKKY000000000 |
| BioSample accession no. | SAMN29222022 | SAMN29222006 | SAMN29221999 | SAMN29222013 |
| SRA accession no. | SRR21354960 | SRR21354962 | SRR21354959 | SRR21354961 |
NT, not tested; R, resistant; I, intermediate; S, susceptible; ND, not detected. No virulence genes were determined for all organisms.
Data availability.
Assembled data were deposited in the National Center for Biotechnology Information (NCBI) database under the BioProject accession number PRJNA851374. This whole-genome shotgun project was deposited in DDBJ/ENA/GenBank under the accession numbers JANKKP000000000, JANKLF000000000, JANKLM000000000, and JANKKY000000000, as listed in Table 1.
ACKNOWLEDGMENTS
This study was funded by the Department of Health and Social Care, managed by the Fleming Fund, and performed under the auspices of the SEQAFRICA project. The Fleming Fund is a £265 million UK aid program supporting up to 24 low- and middle-income countries to generate, share, and use data on antimicrobial resistance and works in partnership with Mott MacDonald, the management agent for the Country and Regional Grants and Fellowship Program. The views expressed in this publication are those of the authors and not necessarily those of the UK Department of Health and Social Care or its Management Agent, Mott MacDonald.
Contributor Information
Beverly Egyir, Email: beverlyegyir@gmail.com, begyir@noguchi.ug.edu.gh.
Frank J. Stewart, Montana State University
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Assembled data were deposited in the National Center for Biotechnology Information (NCBI) database under the BioProject accession number PRJNA851374. This whole-genome shotgun project was deposited in DDBJ/ENA/GenBank under the accession numbers JANKKP000000000, JANKLF000000000, JANKLM000000000, and JANKKY000000000, as listed in Table 1.