Plasmid-Mediated Florfenicol Resistance Encoded by the floR Gene in Escherichia coli Isolated from Cattle

Abstract

A florfenicol resistance gene almost identical to floR of Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coli isolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104.

Florfenicol (FFC) is a fluorinated derivative of chloramphenicol (CM) approved for use in cattle in Europe since 1995. Ffc is active against CM-resistant isolates coding for either CM acetyltransferases (6) or CM exporters, such as CmlA (3). The first Ffc resistance gene, pp-flo, was detected in 1996 on a plasmid in the fish pathogen Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) (9). Ffc resistance has gained interest with its description in the multidrug-resistant Salmonella enterica serovar Typhimurium phage-type DT104 worldwide epidemic strains (1, 2, 4, 5). Serovar Typhimurium DT104 strains harbor a chromosomal locus in which the floR gene (1) and the tetracycline resistance operon tetR-tet(G) are bracketed by two integron structures. This antibiotic resistance gene cluster including the floR gene has recently been found in serovar Agona isolates from poultry, suggesting the mobility of this locus (7). In this study, we investigated FFC-resistant Escherichia coli isolates from France and Germany for the presence of the floR gene and its transferability. Analysis of the floR flanking areas was performed to obtain information on whether the floR gene area in E. coli might have originated from Photobacterium or Salmonella.

All 31 French E. coli isolates included in this study were from cattle, while the 13 German isolates included 11 isolates from cattle and single isolates from a sheep and a cat living on the same farm as one of the diseased cattle. All isolates were multidrug resistant, and the FFC MICs varied between 64 and 256 μg/ml. Conjugation experiments with the E. coli strain BM14 (pro met azi) as the recipient confirmed the FFC resistance genes to be located on conjugative plasmids. The plasmids of the original E. coli isolates as well as those of all FFC-resistant transconjugants were subjected to PCR with internal primers of the floR gene (1, 2, 7). The floR-specific internal 496-bp amplicon was seen in all cases. Two PCR products were sequenced and showed only one 1-bp difference as compared to the respective part of the serovar Typhimurium DT104 floR sequence (1). Southern blot hybridization of the plasmids extracted from the transconjugants with a floR probe (1, 2, 7) confirmed the location of the floR gene on plasmids ranging in size from 110 to 125 kb (data not shown). Four transconjugant strains from the French isolates were selected for further study on the basis of their antibiotic resistance profiles and their FFC MICs (Table 1). They showed four different types of FFC resistance plasmids which varied in their sizes and their additional resistances but also in their EcoRI restriction patterns and the corresponding floR hybridization (Table 1; Fig. 1). A fifth type of FFC resistance plasmid was detected in all 13 German E. coli isolates (Table 1; Fig. 1). A recent report (D. G. White, L. Bolton, J. Maurer, T. Foley, and J. Sherwood, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. A-80, p. 17, 1999) described a possible chromosomal location of the floR gene in bovine E. coli. Their FFC-resistant strains did not share common plasmids, and FFC resistance was not transferable by conjugation. Recently, Keyes et al. (8) also reported FFC resistance due to the floR gene in avian E. coli isolates. The FFC MICs were 32 to 64 μg/ml, and the floR gene was located on larger plasmids of 186 to 204 kb.

TABLE 1.

Characteristics of the E. coli strains used in this study

Straina	Originb	Serotype	Antibiotic resistance profilec	MIC (μg/ml) of FFC	floR PCR	floR hybridization	Plasmid size (kb)	EcoRI plasmid restriction profile	floR hybridization EcoRI fragment size (kb)
BN10337	F	K99+	Ap Cm Ffc Sm Su Tc Tp	256	+	+
BN10337-1	F	K99+	Cm Ffc Su	64	+	+	120	A	5.5
BN10507	F	K99−	Ap Cm Ffc Sm Su Tc Tp	128	+	+
BN10507-1	F	K99−	Cm Ffc Su	32	+	+	115	B	9
BN10660	F	K99−	Ap Cm Ffc Sm Su Tc Tp	128	+	+
BN10660-1	F	K99−	Cm Ffc Su Tp	64	+	+	110	C	5.7
BN10870	F	K99−	Ap Cm Ffc Sm Su Tc Tp	256	+	+
BN10870-1	F	K99−	Ap Cm Ffc Sm Su Tp	64	+	+	120	D	9
EC-23-99	G	K99−	Ap Cm Ffc Sm Su Tc Tp	256	+	+
EC-23-99-1	G	K99−	Ap Cm Ffc Sm Su Tc	256	+	+	125	E	12.4

^aFor each strain the transconjugant is indicated by the suffix “1” at the end of the strain number. 

^bF, France; G, Germany. 

^cAntibiotics; Ap, ampicillin; Cm, chloramphencol; Ffc, florfenicol; Sm, streptomycin; Su, sulphonamides; Tc, tetracyclines; Tp, trimethoprim. 

FIG. 1.

(A) EcoRI restriction profiles of plasmids extracted from the transconjugants BN10337-1 (lane 2), BN10507-1 (lane 3), BN10660-1 (lane 4), BN10870-1 (lane 5), and EC-23-99-1 (lane 6). Lanes 1 and 7, DNA ladder. (B) Southern blot hybridization with the floR probe of the EcoRI-digested plasmids extracted from the transconjugants BN10337-1 (lane 2), BN10507-1 (lane 3), BN10660-1 (lane 4), BN10870-1 (lane 5), and EC-23-99-1 (lane 6). Lanes 1 and 7, DNA ladder.

To assess the genetic environment of the floR gene, several PCR assays which amplified segments flanking the floR gene in strains of the serovar Typhimurium DT104 and serovar Agona (2, 7) were used. The absence of amplification products indicated that the genetic environment of the floR gene in the E. coli plasmids was different. Therefore, the floR-carrying EcoRI fragment of the plasmid from transconjugant BN10660-1 was cloned in plasmid pGEM-7Zf and sequenced (Fig. 2). Comparative sequence analysis confirmed that the E. coli floR gene was almost identical to the pp-flo gene of P. damselae subsp. piscicida and the floR gene of serovar Typhimurium DT104 (>98% identity). A database search for homologies confirmed that the flanking regions of the E. coli floR gene matched almost exactly (98% identity) with that of the 3.7-kb fragment of plasmid pSP92088 of P. damselae subsp. piscicida containing the pp-flo gene (10) (Fig. 2). The floR locus also revealed homology to a sulII gene carrying a fragment of plasmid pSP9351 from P. damselae subsp. piscicida (11) (Fig. 2). Part of the sequences which exhibited homology to the two plasmids from P. damselae were found to be repeated in the floR upstream region (Fig. 2). The 1,978-bp segment that showed homology to the serovar Typhimurium DT104 antibiotic resistance gene cluster included only the floR gene, the upstream region of floR with its putative promoter region, and a stretch of 99 bp which is repeated in serovar Typhimurium DT104 downstream of the floR gene and connects floR with the tetR-tet(G) region of the antibiotic resistance gene cluster (1, 5). The floR gene was bracketed by two closely related open reading frames, orfA′ and orfA (Fig. 2). Comparative analysis of the deduced amino acid sequence of orfA revealed homology to putative transposases (GenBank database accession no. AB020531, AF028594, and AF109307). In comparison to orfA, the 5′ end of orfA′ appeared to be deleted. Thus, the floR gene might be part of a transposable element. This would explain its mobility and its location on structurally different plasmids but also might have played a role in the formation of the DT104 antibiotic resistance gene cluster.

FIG. 2.

Structural organization of the 6,522-bp floR locus of the plasmid from E. coli isolate BN10660. Regions which exhibit homology to the pp-flo-carrying plasmid pSP92088 (GenBank accession no. D37826) and the sulII-carrying plasmid pSP9351 (GenBank accession no. D37825), both from P. damselae subsp. piscicida, but also to the serovar Typhimurium DT104 antibiotic resistance gene cluster, to plasmid ColIb-P9 from Shigella sonnei, and to the enterobacterial plasmid pKM101 are indicated. The numbers of the homologous segments refer to their position within the sequence of the floR locus. The extent and the direction of transcription of the floR, orfA, and orfA′ reading frames are marked by arrows. The black box upstream of floR indicates the 99-bp direct repeat. Restriction sites are abbreviated as follows: Ba (BamHI), D (DraI), EV (EcoRV), H (HpaI), K (KpnI), P (PstI), S (SmaI), and X (XhoI). Distances (in kilobases) are given for constructs shown below the map of the floR locus.

In conclusion, the occurrence of the floR gene on conjugative plasmids could explain its distribution in several bacterial species from different animal hosts. Analysis of the floR flanking regions suggested that the occurrence of the floR gene in E. coli plasmids is more likely to derive from P. damselae subsp. piscicida plasmids than from the antibiotic resistance gene cluster detected in serovar Typhimurium DT104 (1, 2) and serovar Agona (7). The observation that the floR gene was located on plasmids which usually carry other resistance genes also implicates that the spread of the floR gene might be the result of coselection in the presence of antimicrobials other than FFC. Moreover, the proposal that presumptive identification of serovar Typhimurium DT104 could be rapidly made based on the presence of the floR gene or its resulting phenotype (4) appears to be a questionable approach in light of the occurrence of floR genes in bacteria other than serovar Typhimurium DT104.

Nucleotide sequence accession number.

The sequence of the EcoRI floR containing fragment from plasmid of transconjugant BN10660-1 has been deposited in GenBank under accession no. AF231986.