Two Novel Salmonella Genomic Island 1 Variants in Proteus mirabilis Isolates from Swine Farms in China

Abstract

Four different Salmonella genomic island 1 (SGI1) variants, including two novel variants, were characterized in one Salmonella enterica serovar Rissen sequence type ST1917 isolate and three Proteus mirabilis isolates from swine farms in China. One novel variant was derived from SGI1-B with the backbone gene S021 disrupted by a 12.72-kb IS26 composite transposon containing the dfrA17-aadA5 cassettes and macrolide inactivation gene cluster mphA-mrx-mphR. The other one was an integron-free SGI1 and contained a 183-bp truncated S025 next to IS6100 and S044.

TEXT

Salmonella enterica is a zoonotic pathogen and is one of the primary causes of human infections worldwide. Salmonella genomic island 1 (SGI1) is an integrative 42.4-kb chromosomal element first identified in the multidrug resistance (MDR) S. enterica serovar Typhimurium phage type DT104 clone that has been epidemic among humans and domestic animals since the 1990s (1, 2). The MDR region in SGI1 is a complex In4-type class 1 integron (named In104) clustering five antibiotic resistance genes that confer resistance to ampicillin, chloramphenicol and florfenicol, streptomycin and spectinomycin, sulfonamides, and tetracycline (3). SGI1 was unexpectedly detected in a Proteus mirabilis clinical isolate in 2007 (4). Sequence analysis showed that SGI1 in S. enterica and P. mirabilis had the same chromosomal integration site, corresponding to the last 18 bp of the 3′ end of the trmE (also named thdF) gene (5). It has been confirmed that SGI1 in S. enterica could be transferred by conjugation with the help of the IncA/C plasmid (6, 7).

Many SGI1 variants result from the homologous recombination of gene cassettes within the MDR regions (3, 8–11). A few variations in the SGI1 backbone are also described due to deletion, insertion, and transposition (3, 9, 10, 12–15). Furthermore, several novel resistance genes, including the extended-spectrum β-lactamase (ESBL) gene bla_VEB-6 and the fluoroquinolone resistance genes qnrA1 and qnrB2, have been reported in SGI1 (9, 11), suggesting that SGI1 could act as a mobilizable element to disseminate the critical resistance genes. In the present study, we characterized SGI1 among S. enterica and P. mirabilis isolates from swine farms in China.

A total of 24 S. enterica and 61 P. mirabilis strains were isolated from samples of swine stools and diseased tissues in 35 swine farms from 16 provinces in China between May 2012 and February 2014. Antimicrobial susceptibility testing was performed by the disc diffusion method according to the CLSI guidelines (16). Primers used in this study are listed in Table S1 in the supplemental material. The left and right junctions of SGI1 were detected in one S. enterica serovar Rissen and three P. mirabilis strains. The multiple locus sequence typing for SGI1-containing S. Rissen strain Z4 showed that the types of the seven housekeeping genes, not reported to date, were 92 (aroC), 137 (dnaN), 8 (hemD), 524 (hisD), 206 (purE), 313 (sucA), and 330 (thrA). It was submitted to the website http://mlst.warwick.ac.uk/mlst/dbs/Senterica and assigned as a new sequence type, ST1917. To the best of our knowledge, this is the first report of the SGI1 in S. Rissen. Three SGI1-containing P. mirabilis strains belonged to different clusters by pulsed-field gel electrophoresis after SmaI digestion (see Fig. S1 in the supplemental material). The origin, antibiotic resistance profiles, and cassette genes of the four SGI1-containing strains are listed in Table 1.

TABLE 1.

SGI1-containing strains characterized in this study

Strain	Province of isolation	Date of isolation	Origin	Integron cassette(s)	Antibiotic resistance profilea
S. Rissen Z4	Anhui	28 October 2012	Liver	aadA2, dfrA1-orfC	CHL, FFC, STR, SPT, DOX, TMP, SUL, SXT
P. mirabilis SC17	Sichuan	12 April 2013	Liver	dfrA17-aadA5, blaPSE-1	AMP, CHL, FFC, NAL, STR, SPT, DOX, TMP, SUL, SXT
P. mirabilis SC42	Sichuan	15 November 2013	Stool	dfrA17-aadA5	CHL, FFC, NAL, CIP, STR, STP, GEN, DOX, TMP, SUL, SXT
P. mirabilis XJF	Henan	09 January 2014	Stool	aacA5-aadA7	AMP, STR, STP, GEN, DOX, TMP, SUL, SXT

^aAMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; DOX, doxycycline; FFC, florfenicol; GEN, gentamicin; NAL, nalidixic acid; SPT, spectinomycin; STR, streptomycin; SUL, sulfizoxazole; SXT, trimethoprim-sulfamethoxazole; TMP, trimethoprim.

Four different SGI1 variants were identified through PCR mapping and sequencing (Table 2). Two known SGI1 variants, SGI1-I and SGI1-PmABB, have previously been reported (10, 17). Two novel SGI1 variants, SGI1-B2 (45.93 kb) in P. mirabilis strain SC17 and SGI1-Z (24.30 kb) in P. mirabilis SC42, were characterized for the first time in this study (Fig. 1).

TABLE 2.

Four different SGI1 variants characterized in this study

Strain	Amplification of PCR product (bp) in:a					Resistance genes in MDR region	SGI1 variants
	S005−S010	S020−S024	S024−S025	res-intI1	S024−S044		Type	Size (kb)
S. Rissen Z4	4,793	3,598	3,579	1,417	ND	aadA2, floRc, tet(G), dfrA1, sul1	SGI1-I	42.48
P. mirabilis SC17	4,793	NE	3,579	1,417	ND	dfrA17, aadA5, blaPSE-1, sul1, mphA, mrx, mphR	SGI1-B2	45.93
P. mirabilis SC42	4,793	3,598	NE	NE	3,399	None	SGI1-Z	24.30
P. mirabilis XJF	3,272	3,598	3,579	1,417	ND	aacCA5, aadA7, sul1	SGI1-PmABB	32.02

^aPrimers are listed in Table S1 in the supplemental material. ND, not detected; NE, negative.

FIG 1.

Schematic view of the two novel SGI1 variants characterized in this study. Genes and open reading frames (orfs) are shown as arrows, and their orientations of transcription are indicated by the arrowheads. DR-L and DR-R represent the 18-bp direct repeats at the ends of SGI1. CS, conserved segment; IRi and IRt, inverted repeats defining the left and right hands of the integron.

Two gene cassettes, bla_PSE-1 (1.20 kb) and dfrA17-aadA5 (1.66 kb), were detected in P. mirabilis SC17. However, the MDR region in SGI1-B2 contained only the bla_PSE-1 cassette identical to that in SGI1-B (3). The PCR amplicon was negative by using primers S020-R and S024-outF in the PCR mapping of the SGI1-B2 backbone. Through primer walking and PCR linkage, a 12.72-kb IS26 composite transposon, not reported to date in SGI1, was found inserted in the backbone gene S021 (Fig. 1). The transposition event occurred in the region between 128 bp and 175 bp of the S021 gene, resulting in a 48-bp target site duplication surrounding the IS26-composite transposon. It contained the dfrA17-aadA5 cassettes and the macrolide inactivation gene cluster mphA-mrx-mphR, which differed only by 4 single-base changes from the corresponding regions in the Escherichia coli plasmid pEK499 (18). Therefore, SGI1-B2 was derived from SGI1-B with S021 disrupted by an IS26 composite transposon.

SGI1-Z (24.30 kb) contained backbone genes S001 to S024 and the 183-bp truncated S025 next to IS6100 and S044. Although P. mirabilis SC42 harbored the dfrA17-aadA5 cassettes, they were not located in SGI1. The 183-bp truncated S025 was directly next to the left 14-bp inverted repeat region of IS6100. So SGI1-Z is an integron-free SGI1 and does not contain any resistance genes. We hypothesize that an IS6100-mediated transposition event might have occurred in the backbone gene S025 and the homologous recombination happened subsequently between the two copies of IS6100, resulting in the loss of the integron.

The mobility and stability of the SGI1 variants have previously been confirmed in S. enterica (6, 19, 20). Many SGI1 variants can be excised from the chromosome, and the resulting free circular form may be transferable with the helper plasmid (6, 20). The circular extrachromosomal form was detected in all four SGI1s after two rounds of PCR amplification by using the same circ1/2 primers (see Fig. S2 in the supplemental material) (6), implying the mobility of the SGI1. Four SGI1-containing strains were propagated, lasting for 20 days (40 passages) in the absence of antimicrobial pressure. No SGI1-negative clone was detected from the 827 clones picked (about 200 clones were picked for each strain) in the 41st passage, suggesting that SGI1 was stable in both S. enterica and P. mirabilis. Nevertheless, homologous recombination within SGI1 integrons could occur in a few clones. Four SGI1-I variants changed to SGI1-C, containing only the aadA2 cassette in the MDR region already described (19, 20). The exchange of gene cassettes between the two integrons in SGI1-B2 was detected in nine clones, generating a new SGI1 MDR region containing the dfrA17 and aadA5 cassettes (see Fig. S3 in the supplemental material).

In conclusion, four SGI1 variants, including two novel variants, were characterized in S. enterica and P. mirabilis isolates from swine farms in China. The dfrA17 and aadA5 cassettes, as well as the mphA-mrx-mphR cluster, were reported in SGI1 for the first time. The persistence of SGI1 in S. enterica and P. mirabilis might threaten public health, given that the SGI1-containing strains could spread from animal farms to humans through meat consumption (5, 8, 21).

Nucleotide sequence accession numbers.

The complete nucleotide sequences of four SGI1 variants detected in this study were submitted to GenBank and assigned accession numbers KM234279 (SGI1-I in S. Rissen Z4), KP116299 (SGI1-B2 in P. mirabilis SC17), KP057606 (SGI1-Z in P. mirabilis SC42), and KP313760 (SGI1-PmABB in P. mirabilis XJF).