Emergence of Salmonella Genomic Island 1 Variant SGI1-C in a Multidrug-Resistant Clinical Isolate of Klebsiella pneumoniae ST485 from Egypt

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Salmonella genomic island 1 (SGI1) is a 42.2-kb integrative mobilizable element (IME) that was originally identified in Salmonella enterica serovar Typhimurium DT104 (1). SGI1 consists of a 27.4-kb backbone (28 open reading frames [ORFs]: S001(intSGI1) to S027(resG) and S044) with a multidrug-resistant (MDR) region consisting of a 15-kb complex class 1 integron of the family In104 (1). More than 30 SGI1 variants were previously detected in S. enterica and Proteus mirabilis (2, 3). Additionally, SGI1-W, -L, and -B variants were recently found in Providencia stuartii, Morganella morganii, and Escherichia coli, respectively (4–6). SGI1 integrates specifically at the 3′ end of the chromosomal trmE (tRNA modification GTPase) gene (1–6). Numerous IMEs related to SGI1 have been identified, e.g., SGI0 (lacking class 1 integron) (2), SGI2 (integron located at S023) (7), and SGI-V (8). Klebsiella genomic island (KGI1) is another SGI1-related element (SGI1-RE) lacking integrons and detected during an in silico analysis in Klebsiella pneumoniae isolates from the United Kingdom (9). Interestingly, the A/C conjugative plasmid was involved in the specific mobilization of SGI1 due to the presence of the transcriptional activator AcaCD and its binding site on SGI1 (10–12). Here, we report the emergence of the SGI1-C variant in an MDR clinical isolate of K. pneumoniae from Egypt.

The strain, designated Kpu48, was isolated in March 2014 from a urine sample of an adult female patient in a public hospital in Kafr El-Sheikh, Egypt (13). Kpu48 was found to carry two class 1 integrons with two different gene cassettes, aadA2 and dfrA7, which prompted us to perform PCR screening for SGI1 as previously reported (3, 4). The strain was resistant to ampicillin (MIC, >512 μg/ml), kanamycin (MIC, >256 μg/ml), nalidixic acid (MIC, >64 μg/ml), tetracycline (MIC, 128 μg/ml), tigecycline (MIC, 8 μg/ml), and fosfomycin (MIC, >256 μg/ml) but susceptible to aztreonam (MIC, <0.25 μg/ml), ciprofloxacin (MIC, 1 μg/ml), cefotaxime (MIC, <0.25 μg/ml), chloramphenicol (MIC, 8 μg/ml), colistin (MIC, <0.125 μg/ml), doripenem (MIC, <0.125 μg/ml), ertapenem (MIC, <0.125 μg/ml), gentamicin (MIC, 1 μg/ml), and meropenem (MIC, <0.125 μg/ml), according to Clinical and Laboratory Standards Institute guidelines (14).

Kpu48 was subjected to complete genome sequencing using the Illumina MiniSeq and Oxford Nanopore sequencing platforms. Hybrid assembly performed using Unicycler software (15) identified the SGI1 variant on a 5,154,267-bp chromosome. The identified SGI1 variant in the K. pneumoniae strain was SGI1-C, designated SGI1-C-Kp (32,990 bp in size with an average G+C content of 46.4%). The MDR region was 4,880 bp, with the context of intI1-aadA2b-qacEΔ1-sul1-orf5-orf6-IS6100 conferring resistance to spectinomycin, streptomycin, and sulfonamides (Fig. 1). SGI1-C-Kp was integrated between the 3′ end of the trmE gene and a gene encoding N-acetyltransferase (Fig. 1). Whole-sequence queries of SGI1-C-Kp against the National Center for Biotechnology Information (NCBI) GenBank database showed that it is highly similar to other SGI1 structures, e.g., the SGI1-C variant from P. mirabilis strains CA150801 and CA141651 isolated from urinary tract infections in Beijing, China, in 2014 and 2015 (100% query coverage and 99.99% sequence identity; GenBank accession no. MH990680.1 and MH990676.1) (16) and an SGI1-C variant from S. enterica subsp. enterica strain PNCS014858 isolated in Canada (100% query coverage and 99.79% sequence identity; GenBank accession no. CP039582.1). Interestingly, BLAST analysis identified the presence of an SGI1 variant in the chromosome of K. pneumoniae strain 2018C01-046 isolated in 2018 in Taiwan (GenBank accession no. CP044368.1). The SGI1 variant in that strain contained an MDR region with a class 1 integron sparing any gene cassettes. Compared with the SGI1-C variant (accession no. MH990680.1), SGI1-C-Kp showed four single nucleotide polymorphisms (SNPs) within the ORF S025 encoding toxin SgiT (2,322 bp) and the dihydropteroate synthase gene sul1 (201 bp) resulting in no amino acid changes; the other two SNPs were located at positions 12 (G→C) and 15 (G→A) in the right direct repeat 5′-TTCTGTATCGGCAAATAA-3′.

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FIG 1

Schematic representation of SGI1-C-Kp from K. pneumoniae strain Kpu48. Sequence comparison between SGI1-C-Kp, SGI1-C (GenBank accession no. MH990680), and Klebsiella genomic island 1 (KGI1) (GenBank accession no. MN708012). SGI1-C-Kp is inserted between the chromosomal genes (trmE and N-acetyltransferase encoding gene). KGI1 is another SGI1-related element lacking integrons and detected during an in silico analysis in K. pneumoniae isolates from the United Kingdom. Genes and ORFs are shown as arrows, with their transcriptional orientations indicated by arrowheads. Right direct repeat (DR-R) and left direct repeat (DR-L) are shown as 18 bp flanking SGI1. Figure was drawn using the EasyFig tool (http://mjsull.github.io/Easyfig/).

The circular form was detected after two steps of DNA amplification using primers SGI1circ1 and SGI1circ2 with the expected target size (364 bp) as previously described (3, 4). Furthermore, a mobilization assay of SGI1-C-Kp to the azide-resistant E. coli strain J53 (A/C plasmid-carrying transconjugant) was successful (4). This was illustrated by the presence of four intact ORFs (ORF S005 encoding TraN, ORFs S006 and S007 encoding homologs of FlhDC_SGI1, and ORF S0012 encoding TraH), resulting in the transfer and spread of SGI1-C-Kp.

Some SGI1 variants were detected that carried resistance genes to frontline and clinically significant antibiotics, e.g., bla_NDM-1 (carbapenem resistance) (17), bla_VEB-6 (8), bla_CTX-M-15 (cephalosporin resistance) (18), and rmtC (aminoglycoside resistance) (19), posing a serious public health threat. Besides detection of SGI1-C-Kp, we reported in Egypt (i) an SGI1-W variant in a P. mirabilis strain from the same hospital (3), (ii) clonally related SGI1-W-carrying P. mirabilis strains isolated from chickens and patient (20), and (iii) the first SGI1-W-positive P. stuartii isolate (4). These findings indicated that SGI1 variants might be horizontally circulated in Egypt among Enterobacterales and may be transferred to other Gram-negative bacterial species.

Analyzing the whole-genome sequence of Kpu48 at the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/) using ResFinder 3.2, MLST 2.0 software, and PlasmidFinder identified the following, besides the resistance genes encoded on SGI1-C-Kp: (i) chromosomal genes, e.g., bla_SHV-27, oqxAB, and fosA conferring resistance to β-lactams, quinolones, and fosfomycin, respectively; (ii) the IncFIB(K):FII(K) plasmid (222,698 bp in size) carrying resistance genes against sulfonamides (sul1 and sul2), tetracycline [tet(D)], trimethoprim (class 1 integron carrying dfrA7), and aminoglycosides [aph(3′')-Ib, aph(3′)-Ia, and aph(6)-Id]; and (iii) an IncR plasmid (30,445 bp in size) carrying resistance genes against β-lactams (bla_TEM-1B) and tetracycline [tet(A) and tet(R)]. Additionally, the strain belongs to ST485.

In conclusion, we report the emergence of an SGI1-harboring K. pneumoniae human clinical isolate; this strain carries an SGI1-C variant. Implementation of screening strategies for SGI1 variants and other SGI1-REs among Gram-negative bacteria (not only Salmonella spp. or P. mirabilis) is essential to prevent the spread of this IME to other human pathogens.