Molecular Characteristics of Salmonella Genomic Island 1 in Proteus mirabilis Isolates from Poultry Farms in China

Abstract

Six out of the 64 studied Proteus mirabilis isolates from 11 poultry farms in China contained Salmonella genomic island 1 (SGI1). PCR mapping showed that the complete nucleotide sequences of SGI1s ranged from 33.2 to 42.5 kb. Three novel variants, SGI1-W, SGI1-X, and SGI1-Y, have been characterized. Resistance genes lnuF, dfrA25, and qnrB2 were identified in SGI1 for the first time.

TEXT

Genomic islands are discrete DNA segments and have the capacity for integration into the chromosome of bacteria, influencing traits such as antibiotic resistance, symbiosis, fitness, and adaptation (1). Salmonella genomic island 1 (SGI1) is a 42.4-kb mobilizable element initially identified in the multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium phage-type DT104 clone (2, 3). SGI1 consists of a backbone containing 28 open reading frames (ORFs) (S001 to S027 and S044) and an MDR region. The MDR region is a complex class 1 integron named In104 (4), containing five antibiotic resistance genes conferring resistance to ampicillin (Ap), chloramphenicol (Cm) and florfenicol (Ff), streptomycin (Sm) and spectinomycin (Sp), sulfonamides (Su), and tetracycline (Tc) (i.e., ApCmFfSmSpSuTc resistance phenotype) (3). SGI1 variants predominantly result from insertion sequence, homologous recombination, transposition, and loss or exchange of gene cassettes within the MDR region (5).

Proteus mirabilis, a Gram-negative member of the family Enterobacteriaceae, is recognized as an opportunistic pathogen associated with nosocomial infection (6). In 2007, SGI1-L was detected in a P. mirabilis clinical isolate, first confirming SGI1 was present in a genus other than Salmonella (7). Some novel SGI1 variants have been recently described in P. mirabilis (8–11). Three studies have reported that SGI1-containing P. mirabilis could disseminate through meat food consumption, which poses a threat to public health (8, 9, 12), suggesting that SGI1-containing P. mirabilis clinical isolates might partly come from food-producing animals. However, data from animal farms are limited.

In the present study, 64 P. mirabilis strains isolated from the intestinal tracts of chickens among 11 poultry farms in China between March and December 2012 were screened for the presence of SGI1 by PCR with primers chosen in the left and right junction regions between the chromosome and SGI1 (Table S1 in the supplemental material) (2, 8, 9). The left and right junctions of SGI1 were detected in six isolates. They displayed various resistance profiles, as determined by the disk diffusion method according to the CLSI guideline (13). All six SGI1-containing strains harbored different gene cassettes and were not clonally related, as determined by pulsed-field gel electrophoresis. The origins and antibiotic resistance profiles of these strains are listed in Table 1.

TABLE 1.

SGI1-containing P. mirabilis isolates characterized in this study

Strain	Province of isolation	Date of isolation	Antibiotic resistance profilea	Resistance genes in MDR region	SGI1 variants
					Type	Size (kb)
Pm13	Shandong	5 March 2012	AMP, CHL, FFC, NAL, NOR, STR, SPT, DOX, TMP, SUL, SXT	aadA2, floRc, tet(G), blaPSE-1, sul1	SGI1	42.4
Pm107	Anhui	19 March 2012	AMP, CHL, FFC, NAL, STR, SPT, DOX, TMP, SUL, SXT	aadA2, floRc, tet(G), dfrA1, sul1	SGI1-I	42.5
PmC105	Tianjin	21 March 2012	AMP, AMC, NAL, NOR, CIP, STR, SPT, DOX, TMP, SUL, SXT	aadA2, lnuF, sul1	SGI1-W	33.9
PmC162	Hebei	17 October 2012	NAL, NOR, CIP, STR, SPT, DOX, TMP, SUL, SXT	dfrA25, qnrB2, sul1	SGI1-X	38.4
PmX59	Liaoning	1 November 2012	AMP, CHL, NAL, STR, SPT, DOX, TMP, SUL, SXT	dfrA1, sul1	SGI1-O	33.2
PmX60	Sichuan	28 November 2012	AMP, AMC, CTX, CHL, FFC, NAL, STR, SPT, GEN, DOX, TMP, SUL, SXT	aacCA5, aadA7, sul1	SGI1-Y	33.6

^aAMP, ampicillin; AMC, amoxicillin-clavulanic acid; CTX, cefotaxime; CHL, chloramphenicol; FFC, florfenicol; NAL, nalidixic acid; NOR, norfloxacin; CIP, ciprofloxacin; STR, streptomycin; SPT, spectinomycin; GEN, gentamicin; DOX, doxycycline; TMP, trimethoprim; SUL, sulfisoxazole; SXT, trimethoprim-sulfamethoxazole. Resistance to antibiotics conferred by SGI1 is indicated in bold type.

In the six strains, the SGI1 integrated into the specific attachment site attB, corresponding to the last 18 bp at the 3′-end of the chromosomal trmE gene (formerly named thdF) by site-specific recombination between the attB and the SGI1 attP sites (3). The complete SGI1 structures (backbone and MDR region) were established by PCR linkage and sequencing using primers listed in Table S1 in the supplemental material (11). Through PCR mapping, the complete nucleotide sequences of the six SGI1s ranged from 33.2 to 42.5 kb. The SGI1 backbones differed by only several single-base changes from that of S. Typhimurium DT104 (2). The MDR regions were integrated by the class 1 integron belonging to the In4 family and inserted between the backbone genes res (S027) and S044. Three SGI1s (SGI1, SGI1-I, and SGI1-O) have been previously reported (2, 4, 8). Three new variants, SGI1-W, SGI1-X, and SGI1-Y, have been characterized in this study for the first time (Fig. 1).

FIG 1.

Schematic view of three novel SGI1 variants, SGI1-W, SGI1-X, and SGI1-Y. Genes and 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 ends of class 1 integron, respectively; orf, open reading frame; CR1, common region 1.

InSGI1-W (6.54 kb) contained only the aadA2/lnuF cassette, conferring resistance to spectinomycin, streptomycin, and lincosamides. The aadA2/lnuF cassette observed in SGI1-W was found in just two species of Enterobacteriaceae, Escherichia coli (GenBank accession number JF806502) and Salmonella enterica (14). This new cassette in SGI1 might be caused by the homologous recombination of integrons among different species in the intestinal microecological environment. The wide use of lincosamides in animal farms might facilitate its occurrence.

InSGI1-X (11.03 kb) contained the dfrA25 cassette, common region 1 (CR1), and fluoroquinolone resistance gene qnrB2. The major part of this structure harboring qnrB2 differed only by 3 single-base changes from the 5.24 kb of the partial plasmid p137.25 and 7 single-base changes from the 7.45 kb of the partial plasmid pSE936/05 in S. enterica (15, 16), suggesting that the newly found MDR region in SGI1-X might derive from plasmids through homologous recombination of integrons. It is worth noting that SGI1-V recently found in P. mirabilis also contained two new antibiotic resistance genes (bla_VEB-6 and qnrA1), conferring resistance to the third-generation cephalosporins and fluoroquinolones (10), revealing that SGI1 could improve bacterial adaptability to environmental stress through ongoing acquisition of antibiotic resistance genes.

The SGI1 in the PmX60 strain was located between the chromosomal trmE gene and the gene encoding a membrane protein PMI3124 due to the loss of the hipBA module that appeared to occur by homologous recombination between 49-bp direct repeats surrounding the module (17). It contained the aacCA5-aadA7 cassette that was identical to SGI1-PmABB and SGI1-PmMAT (11), but the backbone did not include a deletion from S005 to S009 [aacCA5 has also been called aac(3)-Id]. Therefore, this SGI1 was considered a new variant named SGI1-Y. It might derive from the SGI1-B prototype via the cassette replacement.

In conclusion, this study highlights that poultry farms are an important reservoir of SGI1-containing P. mirabilis. Three novel SGI1 variants were identified in this study for the first time. In addition, it has been demonstrated that some virulence-enhancing properties in Salmonella were associated with the presence of SGI1 (18, 19). Notably, four of the six isolates in this study caused severe peritonitis. The prevalence of SGI1 in animal-origin P. mirabilis poses a threat to public health, as SGI1-producing P. mirabilis from poultry farms could spread to humans through food consumption, and SGI1 in P. mirabilis could spread to Salmonella (12, 20).

Nucleotide sequence accession numbers.

The complete nucleotide sequences of six SGI1s observed in this study were submitted to GenBank and assigned accession numbers KJ186153 (SGI1 in Pm13), KJ186152 (SGI1-I in Pm107), KJ186151 (SGI1-W in PmC105), KJ186154 (SGI1-X in PmC162), KJ186150 (SGI1-O in PmX59), and KJ186149 (SGI1-Y in PmX60).