A 63-year-old female housewife from Narayanganj district of Dhaka division in Bangladesh attended the outpatient department of a local private hospital on January 2022. Her main complaints were dysuria, fever with chills, lethargy and mild lower abdominal pain for 6 days. She had a history of hypertension and T2D for 4 years. She was on medication for hypertension [Bisoprolol Hemifumarate (5 mg)] and diabetes [Linagliptin plus Metformin Hydrochloride (2.5 mg plus 500 mg)]. On consultation, the patient was found febrile with a 101 F body temperature, and her pulse, respiratory rate, blood pressure and oxygen saturation were detected to be 109 pulses/min, 20 breaths/min, 128/84 mmHg, and 97% on room air, respectively. She did not have a recent history of a hospital visit, travel, contact with the pet, or ingesting raw food. The patient provided a blood sample for a laboratory investigation. An initial hematological assessment of the blood specimen demonstrated that the patient's hemoglobin was 10.3 g/dl and the erythrocyte sedimentation rate was 50 mm/h. In particular, the number of leukocytes was 13,000 cells/mm3, with 81% neutrophils, 26% lymphocytes, 3% eosinophils, and 2% monocytes. The platelet counts, as well as other blood cell variables, were normal. Blood biochemistry analysis showed random blood glucose raised at 10.3 mmol/L and C-reactive protein elevated at 85 mg/ml with normal hepatorenal function parameters. A COVID-19 screening test was done per the hospital requirements and a negative result was obtained. Preliminarily, it was suspected to be a case of UTI. Therefore, the patient was empirically treated with intravenous ceftriaxone (500 mg/8 h) for 7 days. However, she did not respond to the treatment and symptoms like increased frequency of painful and burning micturition, fever, and abdominal pain continued while she was admitted to the hospital (Table 1). Therefore, a clean catch midstream urine sample was obtained and sent to the laboratory for routine microscopic examination and microbiological investigation. The urine sample was turbid and revealed the presence of 20 to 30 pus cells per high-power field. The urine sample was inoculated onto 5% sheep blood agar, MacConkey's agar, and CHROMagar Orientation agar plates and incubated the plates at 37 C in an aerobic environment. After overnight incubation, a significant (>105 CFU/ml) growth of single-type colonies was observed on all culture plates. The bacteria produced 2-3mm, circular, raised, mucoid, pale pink color colonies on MacConkey agar, gray-white colonies on a blood agar plate with gamma hemolysis and metallic blue pigmented colonies on a CHROMagar Orientation plate. Based on the colony morphology on MacConkey and CHROMagar Orientation agar plates, we suspected it would be either Klebsiella or Enterobacter species. Gram staining revealed gram-negative bacilli. Further, biochemical characterization was carried out as described previously. The bacterium was finally identified as K. aerogenes based on its gram-negative morphology, motility, utilization of citrate, decarboxylation of Ornithine, reduction of nitrate and fermentation of glucose and lactose with the production of acid and gas (Table 2). The strain was further tested with the API 20E test (BioMerieux, France), and it confirmed the strain as Enterobacter aerogenes with 96% certainty. Antibiotic susceptibility testing followed the Kirby-Bauer disc diffusion method using 21 antibiotic disks representing 11 classes. The K. aerogenes (RD99) strain was resistant to most antibiotic classes tested, including aminoglycosides, beta-Lactams (penicillin), beta lactams (cephalosporins), amphenicol, fluoroquinolones, folate drugs, tetracyclines, phosphonic acid, and glycycline. However, the strain was susceptible to carbapenems and polymyxins (Table 3).
The K. aerogenes (RD99) strain was further subjected to bacterial whole-genome sequencing (WGS) using an Illumina NextSeq 500 platform at icddr,b using an established protocol. The raw genome sequence data is publicly available at GenBank with accession number: JAQQRH000000000. The quality control, assembly of sequenced data into draft genomes and annotation were performed using established bioinformatic pipelines. The FastANI tool was used for taxonomy identification, and K. aerogenes was confirmed with a 98.81 Average Nucleotide Identity (ANI) value.
The in-silico analysis predicted sequence types (ST) and clonal complex using the PubMLST database (https://pubmlst.org/kaerogenes/), capsule polysaccharides using the Kaptive database, and plasmid replicons using Plasmid Finder 2.1. The virulence genes were identified using Virulence Factor Database (VFDB). The resistance genes were identified by ABRicate tool v1.0.1 (https://github.com/tseemann/abricate) using Resfinder, and NCBI AMR Finder database. Mobile genetic elements using Mobile Element Finder v1.0.3, point mutations using PointFinder software, prophage using PHASTER, and integrons using IntegronFinder 2.0. Default parameters were applied for all software unless otherwise mentioned. We aligned six K. aerogenes isolates (including RD99), 15 publicly available Klebsiella species, and 5 Enterobacter species genomes with K. pneumoniae ATCC 25955 as reference genome using Snippy v4.4.0 and Gubbins v3.2 5. A phylogenetic tree was constructed using RaxML v8.2.12, employing the Generalized Time Reversible substitution model and the GAMMA distribution for rate heterogeneity. The tree was visualized using iTOL.
The K. aerogenes strain (RD99) genome sequence is 5,490,860 bp with a GC content of 54.8%. It comprises 73 contigs, having 5,348 genes, of which 5,224 coding DNA sequences (CDSs) and 124 RNAs. In silico profiling revealed that RD99 is a K. aerogenes strain and belonged to ST4 and the CC1 clonal complex group. More than 60% (108/175) of the reference virulence genes were detected in the RD99 strain. It harbored the fimA-K operon, involved in adherence to human mucosal or epithelial surfaces, iutA, basG, ent, fep, and iro, which encodes aerobactin, acinetobactin, enterobactin and salmochelin siderophores, rcsAB, regulators of mucoid phenotype A. We also found SenB, an intrinsic gene encoding toxin (Table 4).
A total of 10 prophage elements were detected, including 3 intact, 5 incomplete and 2 questionable phages and a Class 1 integron was identified. The strain RD99 harbored 17 antibiotic resistance genes that included aminoglycosides resistance genes aph(3')-Ib, aph(6)-Id, aac(3)-Ile, aac(6')-Ib-cr, beta-Lactams resistance genes, blaTEM - 1B, blaCTX - M-15, blaOXA - 1, blaampC, folate pathway antagonists resistance genes, dfrA14, sul2, plasmid-mediated fluoroquinolones resistance genes, oqxA, oqxB, amphenicol resistance gene catB3, fosfomycin resistance gene fosA, and tetracyclines resistance gene tet(D). The efflux pumps AcrAB-associated genes acrA and acrB were also detected in this strain that would mediate tigecycline resistance. The genes blaampC and fosA were located on the chromosome. Other resistance genes were located on plasmids, including blaTEM - 1B, blaCTX - M-15, and blaOXA - 1. We also identified the TN2 transposon element linked with the blaTEM - 1B gene. In addition, we identified amino acid substitutions at codon positions S83I in gyrA and S80I in parC. We also found mutations in ompK36 gene. No mutations were detected in ompK35 and ompK37 genes. In agreement with the phenotypic results, we did not detect any genes conferring resistance to polymyxins and carbapenem antibiotics. We identified five plasmid replicons in RD99 genome, which were Col440I, IncFIB(K), IncFII(K), IncN, IncR (Table 2). The strain RD99 demonstrated OL102 and KL186 antigens.
We conveyed the results of the above analysis, particularly its phenotypic and genotypic features, to the physician. Based on this, the therapy course was changed to meropenem (500 mg/8 h) for 14 days. After 8 days of effective antibiotic treatment and appropriate management of diabetes, the patient became afebrile with no complaints of dysuria and abdominal pain. She was discharged from the hospital and advised to complete the course of antibiotics to avoid the recurrence of the infection. After 14th day the patient recovered completely and demonstrated a negative urine and blood culture. A monthly urine culture was advised for 3 months (one urine CS/month) to assess the efficacy of treatment and the recurrence of infection. All monthly follow-ups (for 3 months) of urine culture and sensitivity tests were negative, and the patient did not experience infection recurrence.