First Detection of Conjugative Plasmid-Borne Fosfomycin Resistance Gene fosA3 in Salmonella Isolates of Food Origin

LETTER

Fosfomycin is a naturally occurring antibacterial agent with a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria (1).

Recently, a fosfomycin resistance gene, fosA3, was detected in Escherichia coli and Klebsiella pneumoniae isolates (2–6). This gene is normally plasmid mediated, surrounded by IS26 transposase genes, and often detectable in CTX-M-producing and multidrug-resistant E. coli isolates (2–6). It has been suggested that the increasing prevalence of fosA3 is due to dissemination of the IncI and IncN plasmids rather than clonal expansion of specific strains (4). This study aimed to determine whether fosA3-borne plasmids can disseminate among other closely related Enterobacteriaceae species such as Salmonella and to identify the underlying mechanisms regulating its dissemination potential.

Two Salmonella isolates, S76 and S79, obtained from meat samples purchased from supermarkets and wet markets in Hong Kong in 2013, were shown to be resistant to quinolones and third-generation cephalosporins (Table 1). Surprisingly, these two isolates were also resistant to fosfomycin. Salmonella strain S76 was determined to be Salmonella enterica serovar Derby; this strain was isolated from chicken product purchased in supermarket on February 22, 2013, whereas strain S79, which was found to be S. enterica serovar Enteritidis, was isolated on the same day from a different chicken product purchased from a different supermarket in Hong Kong. Multilocus sequence typing (MLST) analysis of these two isolates showed that S76 belonged to ST11, whereas S79 belonged to ST460. Previous studies had shown that ST11 was associated with invasive S. Enteritidis and ceftriaxone-resistant S. Enteritidis strains carrying CTX-M-14 and CTX-M-15 on IncI1 and IncFII plasmids, respectively (7–9). In contrast, ST460 had not been reported to be associated with CTX-M-producing S. Derby previously.

TABLE 1.

Characteristics of strains used in this study

Strain	Isolation date (day/mo/yr)	Source	Serotype	MLST	Replicon type	MIC (mg/liter)a
						FOS	CIP	ENR	NAL	OLA	AMP	CTX	CRO	CAZ	CTO	FOX
E. coli J53						<4	<0.125	<0.125	2	1	1	<0.125	<0.125	<0.125	<0.125	<0.125
Salmonella strains
S76	22/02/2013	Chicken	Derby	ST11	FII, FIV	>512	0.25	≤0.25	>64	16	>64	>16	>16	>64	>128	2
TC-S76					FII	>512	0.015	≤0.25	16	8	>64	>16	>16	≤0.5	32	2
S79	22/02/2013	Chicken	Enteritidis	ST460	FII, FIV	>512	1	1	>64	>256	>64	>16	>16	32	>128	2
TC-S79					FII	>512	0.06	≤0.25	>64	128	>64	>16	>16	4	32	2

^aFOS, fosfomycin; CIP, ciprofloxacin; ENR, enrofloxiacin; NAL, nalidixic acid; OLA, olaquindox; AMP, ampicillin; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; CTO, ceftiofur; FOX, cefoxitin.

The gene fosA3 was detectable in both strains by PCR screening of fosfomycin resistance genes and transferrable to E. coli J53 (4, 10). Conjugative plasmids in E coli J53 that harbored the fosA3 gene were shown to be IncFII. S1 pulsed-field gel electrophoresis (PFGE) confirmed that plasmids of two different sizes (∼80 kb and ∼45 kb) were detectable in both isolates, with the conjugative plasmid being the larger one (Fig. 1A). Southern hybridization showed that the fosA3 gene was present in the ∼80-kb conjugative plasmid (Fig. 1A). Genetic-environment analysis showed that the fosA3 gene was surrounded by two IS26 elements with a genetic structure of IS26-fosA3-orf1-orf2-orf3-IS26. An identical structure was first reported for plasmids from clinical E. coli isolates in Japan, South Korea, and Hong Kong (3, 4, 6, 11).

FIG 1.

Analysis of fosA3-borne plasmids and the fosA3-bla_CTX-M-55 genetic environment. (A) Salmonella isolates S76 and S79 and the corresponding transconjugants, TC-S76 and TC-S79, were subjected to S1 PFGE and Southern hybridization analysis using the fosA3 probe. (A) Salmonella isolates S76 and S79 and transconjugants TC-S76 and TC-S79 were subjected to S1 PFGE and Southern hybridization analysis using the bla_CTX-M-55 probe. (C) Representative genetic structures of fosA3 and bla_CTX-Ms in different conjugative plasmids. The genetic structures and the corresponding references are as follows: E. coli 08-642/06-607 (6), pS76 (this study), pHK23a (11), pC121T (GenBank no. JX442753) (3), and ECO96TF/ECO141TF (4).

Importantly, a PCR assay revealed the presence of a β-lactamase gene, bla_CTX-M-55, on the conjugative plasmids recovered from strains S76 and S79; the result was confirmed by Southern hybridization (Fig. 1B). Primers targeting IS26 and bla_CTX-M-55 identified a gene cassette, IS26-bla_TEM-1-orf20-bla_CTX-M-55-IS26, in the conjugative plasmids pS76 and pS79 (Fig. 1C). The genetic structures of fosA3 and bla_CTX-M-55 were very similar to that of a previously identified plasmid, pHK23a, except that the bla_CTX-M-55 and bla_CTX-M-4 elements were found in pHK23a and pS76/pS79, respectively (11). However, the length or nature of linkage between fosA3 and bla_CTX-M-55 genetic structures in pS76 and pS79 was different from that of pHK23a, since an attempt to amplify the linkage region between the fosA3 and bla_CTX-M-55 structures in pS76 and pS79 was not successful. Restriction fragment length polymorphism (RFLP) analysis confirmed that pS76 and pS79 were structurally different from pHK23a (data not shown).

So far, four different genetic structures representing a typical fosA3 cassette, IS26-fosA3-orf1-orf2-orf3-IS26, and cefotaxime resistance determinants, respectively, have been identified. In pHK23a, the IS26-bla_TEM-1-orf20-bla_CTX-M-55-IS26 cassette is located downstream of the fosA3 cassette, whereas in the other two genetic structures, the bla_CTX-Ms cassette was upstream of the fosA3 cassette. In one of the genetic structures, the bla_CTX-M cassette is truncated. These data suggest that a typical fosA3 cassette could be randomly inserted into the adjacent area of the bla_CTX-Ms cassette, forming different genetic structures. In pS76 and pS79, the two cassettes were separated by a distance (Fig. 1C), suggesting that an IS26-flanked antimicrobial resistance cassette can be inserted into the hot spots in plasmids forming multiple resistance gene clusters in the plasmid. In addition to the typical fosA3 cassette IS26-fosA3-orf1-orf2-orf3-IS26, several C-terminally truncated forms, such as IS26-fosA3-orf1-orf2-IS26, IS26-fosA3-orf1-Δorf2-IS26, and IS26-fosA3-Δorf1-IS26, have been detected in plasmids recovered from E. coli isolates, suggesting that IS26 transposition activities mediated the formation of the fosA3 cassette (2, 10).

In conclusion, this study has for the first time identified and characterized a conjugative FII plasmid carrying both IS26-mediated bla_CTX-M-55 and fosA3 gene cassettes in two Salmonella isolates. The fact that this genetic arrangement is similar to that observable in E. coli suggests the possibility that this plasmid can circulate among members of Enterobacteriaceae. The association of the fosA3 gene with bla_CTX-Ms could eliminate the possibility of using fosfomycin as an alternative treatment approach, which may be effective for treatment of multidrug-resistant Salmonella infection. Further monitoring of transmission of the fosA3 gene is necessary.