A 67-year-old woman with a history of rectal cancer and recently diagnosed bladder cancer experiencing anuria presented to the clinic for ureteral stent removal approximately 1 month post bladder surgery, cystectomy and ileal conduit ( Figure 1 ). She was referred to the ER for evaluation and was admitted due to hematuria, chills, nausea, abdominal pain and severe sepsis on the same day. A primary bacterial culture (Isolate 10) was recovered from the aerobic blood culture bottle and identified as Klebsiella pneumoniae by MALDI-TOF. Antimicrobial susceptibility testing was performed using BMD but wells for ceftriaxone and ceftolozane-tazobactam displayed an indeterminate growth pattern in which the MIC could not be interpreted accurately. Upon repeat testing by DD, a single colony (Isolate 1B) was noted within the zone of inhibition for ceftriaxone and was sub-cultured for further workup.
Further phenotypic testing revealed two sub-populations in the primary culture, one susceptible to ceftriaxone and all other 3rd generation cephalosporins (Isolate 1A) and the other resistant to most cephalosporins and ertapenem (Isolate 1B) ( Table 1 ). Isolate 1A showed pan susceptibility to all antibiotics tested except fluoroquinolones. Isolate 1B was resistant to most beta-lactams tested, except ceftazidime-avibactam, cefepime, and meropenem. The contrasting differences in beta-lactam susceptibility results between the two sub-populations in the primary culture prompted us to perform carbapenemase assays and WGS for further investigation.
Carbapenemase was tested by MHT, mCIM, and CarbaR PCR assays. No carbapenemase activity was detected in any isolate by MHT. However, Isolate 1B was mCIM positive, while Isolate 1A was negative. CarbaR PCR test, which detects KPC, NDM, VIM, OXA-48-group and IMP genes, was negative in all isolates. Further phenotypic testing was performed to determine possible AmpC activity with cefotaxime adjacent to imipenem or cefoxitin disks on Isolate 1A and 1B ( Figure 2 ). Cefoxitin and imipenem were both able to induce resistance against cefotaxime in Isolate 1B but not in Isolate 1A as noted by truncation of the zone, suggesting a possible inducible AmpC resistance mechanism.
Whole genome sequencing was performed on the two sub-populations and the primary mixed culture for comparison to determine genetic relatedness and to identify drug resistance genetic elements. All isolates were identified as Sequence Type (ST) 11 based on MLST analysis and all closely related to the same strain Klebsiella pneumoniae strain KP38731 by KmerFinder analysis. Variant analysis was performed using KP38731 as the reference genome and showed no single nucleotide polymorphisms (SNPs) among the four isolates, indicating they are of the same bacterial lineage. Drug resistance genetic analysis using ResFinder identified numerous genes conferring resistance to beta-lactams, aminoglycosides, fluroquinolones, rifamycins, tetracycline, phenicol, macrolides, and fosfomycin in Isolate 10, 1B ( Table 2 ). In contrast, 1A had much fewer resistance genes identified ( Table 2 ), with only blaSHV-182, oqxA, oqB, and fosA, which are known to be chromosomally encoded in K. pneumoniae. In addition, in silico plasmid identification analysis by PlasmidFinder detected the presence of two plasmid types, IncFIB(K) and IncR in primary Isolate 10 and Isolate 1B. The InR-type plasmid was not detected in Isolate1A, which was the isolate that demonstrated susceptibility to most antibiotics, further supporting that the primary culture was mixed with two-subpopulations.
Further BLAST analysis identified the InR-plasmid in Isolate 10 and 1B to be closely related to a previously published InR pKPS30 plasmid (NC_023314.1) with 98.3% pairwise identity and 100% coverage. This 61,288-bp plasmid was initially described in a K. pneumoniae ST11 strain isolated in France. It carries several mobile genetic elements (integron and transposons) with multiple resistance genes, including blaDHA-1, blaOXA-30, aac(6'))-Ib-cr, aphA1, arr-3, catB3, mph(A), qnrB4, aac(6'))-Ib-cr, sul1, and tet(A), all of which were also identified in Isolate 10 and 1B. Notably, both blaDHA-1 and ampR, along with sul1, are within a class-1 integron that was originally discovered in a DHA-1-Producing Klebsiella spp. in France over 15 years ago.