Representative antimicrobial agent broth microdilution (BMD) results for the C. freundii isolates from the urine, blood, and kidney cultures at both laboratories are shown in Table 1. Results obtained by DD, mCIM/eCIM, and BMD are summarized in Table 1 and Additional file 2: Tables S1 and S2. All isolates were positive for production of serine carbapenemase by mCIM/eCIM.

All isolates were susceptible to meropenem and intermediate to imipenem by the BMD method (Table 1). MICs of ertapenem and meropenem for the blood culture isolates of C. freundii from hospital A were identical to the ertapenem and meropenem MICs of the original urine isolate (susceptible to both drugs) (Additional file 2: Table S1). BC-1 and BC-2, however, had an extended MIC profile suggestive of organisms with a non-induced ampC, i.e., they were susceptible to ceftazidime and cefepime, had variable levels of resistance (ranging from susceptible to resistant) to ceftriaxone, and were susceptible to piperacillin/tazobactam. Isolate BC-3, collected a day later, had a resistance profile closer to that of the urine isolate, i.e., it was resistant to ceftazidime, ceftriaxone, cefepime [one laboratory reported susceptible dose-dependent (SDD)] and resistant to piperacillin/tazobactam. The patient had also developed a kidney abscess and multiple drainage cultures again revealed C. freundii with different morphologies and resistance profiles. The isolates from kidney abscess, K-1 and K-2, presented a resistance profile similar to that of the original urine isolate and BC-3. The AST profiles were intermediate to imipenem. Both isolates were susceptible to ertapenem, although in laboratory B, K-1 was intermediate to ertapenem. All the isolates were resistant to 1st and 2nd generation cephalosporins but showed varying levels of susceptibility to 3rd generation cephalosporins. Isolates BC-1 and BC-2 were susceptible to the 4th generation cephalosporin, cefepime, while the remaining isolates were resistant with one reported as cefepime SDD (Table 1). Variable resistance to aztreonam was also observed among the isolates, with BC-1 and BC-2 showing susceptibility and BC-3, K-1, and showing K-2 resistance. All the isolates were resistant to amoxicillin/ clavulanic acid and ampicillin/sulbactam; however, susceptibilities to ceftolozane-tazobactam and piperacillin-tazobactam were variable. All five isolates were susceptible to ceftazidime/avibactam, meropenem/vaborbactam, and amikacin, although BC-3, K-1 and K-2 displayed varying degrees of resistance to the other aminoglycosides (Table 1). Variable susceptibility patterns were also observed with fluoroquinolones with resistance to ciprofloxacin for all isolates, but with BC-1 and BC-2 showing susceptibility to moxifloxacin and levofloxacin, while BC-3, K-1 and K-2 were resistant to both agents.

The variable resistance profiles to the carbapenems observed with BMD were more evident among the DD results. BC-1 and BC-2 were susceptible to ertapenem and meropenem and intermediate to imipenem, while BC-3, K-1 and K-2 were either intermediate or resistant to the carbapenems (Additional file 2: Table S2). The same resistance patterns were found among cephalosporins and aztreonam.

As expected, the isolates were highly related, with an alignment percentage between 98.99% and 99.40%. Figure 1 groups the five isolates by alignment percentage, relative to type of sample and genotypic and phenotypic results (Fig. 1). All five C. freundii isolates were sequence type (ST)-8 by the Citrobacter spp. PubMLST 7-loci scheme present in CLC Genomic Workbench (CLC Type with MLST scheme 1.3) [10] and each harbored the blaKPC-3 carbapenemase gene, the chromosomal blaCMY-116ampC beta-lactamase gene, and the blaCTX-M-39 extended-spectrum beta-lactamase (ESBL) gene. All five isolates also shared aminoglycoside [aac(6’)-If], tetracycline (tetB), aminocyclitol resistance genes [ant(3″)-Ia], and folate pathway antagonist genes (dfrA1, sul1), which were likely chromosomal (Fig. 1). However, isolates BC-3, K-1, and K-2 harbored additional resistance genes, including the class A carbenicillin-hydrolyzing beta-lactamase, blaCARB-2, the ant(2'') and aadA2 aminoglycoside resistance genes, dfrA19, which produces a trimethoprim-resistant dihydrofolate reductase, the catB3 and cmlA1chloramphenicol resistance determinants, the mph(E) and msr(E) macrolide resistance genes, and qnrA1, which produces a quinolone resistance pentapeptide repeat protein. Isolates BC-1 and BC-2, in turn, carried the qacE gene, which confers resistance to quaternary ammonium compounds. No alterations of the porin genes ompC and ompF were found. PlasmidFinder identified the same two plasmids in all five isolates, namely, IncC (Sequence Type 3 by pMLST 2.0 Server) and pKPC-CAV1321. A BLAST (Basic Local Alignment Search tool, National Library of Medicine) search of the genomic region encompassing the blaCARB-2 gene of isolates BC-3, K-1 and K-2 returned 100% identity to a portion of the antimicrobial resistance island of plasmid pMG252, described in an Escherichia coli isolate with elevated quinolone resistance [11]. The contigs containing resistance genes blaCTX-M-39, aadA2, ant(2'')-Ia, dfrA19, cmlA1, catB3, mph(E), msr(E), qacE, sul1, and qnrA1 also shared 99–100% identity with regions of plasmid pMG252, although only the genomic environment surrounding blaCTX-M-39 was found on the plasmid, but not the actual blaCTX-M-39. All regions showing similarity with plasmid pMG252 are shown in Fig. 2. A BLAST search of the blaKPC-3-containing contigs returned only partial matches to several published Citrobacter spp. plasmids. The ~ 10,000 bp region surrounding the blaKPC-3 gene, however, returned a 99–100% match and 100% coverage to published sequences of a blaKPC-2 or KPC-3-carrying transposon Tn4401 integrated within a Tn2-like element. This transposon is also found on plasmid pKPC_CAV1321 [12], originally identified by CGE PlasmidFinder. Additional file 1: Fig. S1 illustrates the alignment of the five blaKPC-3 genomic environments with the Tn4401b-1 transposon of pKPC_CAV1321-45. The only mismatch is due to the fact that pKPC_CAV1312-45 harbors a blaKPC-2 instead of blaKPC-3.

Selected antimicrobial susceptibility results (broth microdilution) of the five isolates by antimicrobial class with the genes identified by sequencing analysis (ResFinder database). A neighbor-joining (NJ) phylogenetic tree compares the isolates based on whole genome alignment percent. Abbreviations: gentamicin (GM), tobramycin (TM), cefotaxime (CTX), ceftazidime (CAZ), ertapenem (ETP), imipenem (IPM), meropenem (MEM), aztreonam (ATM), piperacillin-tazobactam (TZP), levofloxacin (LVX), moxifloxacin (MXF), tetracycline (TE), minocycline (MI), trimethoprim-sulfamethoxazole (SXT), wildtype (WT)

SnapGene alignment of the contigs of the five isolates harboring the resistance genes blaCARB-2, blaCTX-M-39, aadA2, ant(2'')-Ia, dfrA19, cmlA1,catB3, mph(E), msr(E), qacE, sul1, and qnrA1 with plasmid pMG252 described in E. coli strain J53 (Accession MK638972). Contigs are color-coded by isolate: BC-1 green, BC-2 orange, BC-3 yellow, K-1 light blue, K-2 dark blue. The contigs carrying blaCTX-M-39 aligned with plasmid pMG252, except for the gene itself which was not present on pMG252. Genes are color-coded according to the following key: dark blue, transposase and integrase genes; red, antimicrobial resistance genes; dark red, mercury resistance genes; grey, tra gene complex; dark green, DNA metabolism; brown, plasmid maintenance and replication; white, all other genes