Comparative Analysis of IncHI2 Plasmids Carrying bla_CTX-M-2 or bla_CTX-M-9 from Escherichia coli and Salmonella enterica Strains Isolated from Poultry and Humans

Abstract

Salmonella enterica bla_CTX-M-2 and bla_CTX-M-9 plasmid backbones from isolates from Belgium and France were analyzed. The bla_CTX-M-2-plasmids from both human and poultry isolates were related to the IncHI2 pAPEC-O1-R plasmid, previously identified in the United States in avian Escherichia coli strains; the bla_CTX-M-9 plasmids were closely related to the IncHI2 R478 plasmid.

The plasmid-encoded CTX-M family have recently become dominant extended-spectrum β-lactamases (ESBLs) in human isolates in Europe (13). Significantly, the CTX-M enzymes have emerged in isolates of Escherichia coli and Salmonella spp. from food, companion, and wild animals, raising a potential public health concern (1, 2, 4, 6, 12, 14-16).

In Belgium and France, Salmonella enterica serovar Virchow producing the CTX-M-2 ESBL was firstly identified in poultry flocks, then in poultry meat, and subsequently in humans over the period 2000 to 2003 (3). The chronology of isolation of these strains suggested that these bacteria were transmitted to humans via the food chain, specifically by poultry meat. In French Guiana in 2004, a Salmonella enterica serovar Typhimurium strain producing CTX-M-2 was isolated from a patient with gastroenteritis. In France in 2002, one CTX-M-9-producing S. enterica rough strain was isolated from a patient, and in 2003, S. enterica serovar Virchow strains were isolated from poultry (17).

The objective of this study was to characterize plasmids carrying bla_CTX-M-2 and bla_CTX-M-9 from poultry sources and to compare them with those circulating in humans. CTX-M-positive transconjugants were obtained in E. coli K-12 from CTX-M-2 or CTX-M-9 producers. The following transconjugates were selected for this study: six from the S. enterica serovar Virchow strain of animal origin, one from the rough isolate, and one from the S. enterica serovar Typhimurium strain (Table 1). The parental strains of these transconjugants were representative of CTX-M-2 or -9 producers for country of isolation, source, pulsed-field gel electrophoresis patterns, and resistance profiles, as previously determined (3, 17). The bla_CTX-M-9 integrons in these strains were previously characterized, and gene cassette arrays are reported in Table 1 (17). To determine the structure of the bla_CTX-M-2-carrying integrons, the 8,690-bp and 9,017-bp fragments were amplified and fully sequenced from the plasmids of the S. enterica serovar Virchow p1639-SA-00-1 and S. enterica serovar Typhimurium p04-9275-1 strains, respectively. Sequencing results indicate that the integron in S. enterica serovar Virchow was 99% identical to the CTX-M-2 integron found in the E. coli strain of human origin identified in France in 2006 (7) carrying the dfrA1 and aadA1 gene cassettes (deposited in GenBank under accession no. EF592570). The integron from the S. enterica serovar Typhimurium strain showed a different variable region, including the dfrA12, orfF, and aadA2 gene cassettes (Table 1 [deposited in GenBank under accession no. EF592571]).

TABLE 1.

Plasmid backbone analysis of the IncHI2 plasmids in transconjugants obtained from Salmonella and Escherichia coli CTX-M-2 and CTX-M-9 producers

Plasmid (ESBL)	Parental strain (source)	Country (yr)	HI2 replicon type	PCR result for:												Reference
				smr10-11	smr17-18	smr207-208	smr239-240	O1R_160	terF	arsB	smr201	smr92-93	smr136	tnsD	Integron-borne gene cassettes
p1639-SA-00-1 (CTX-M-2)	S. enterica serovar Virchow (poultry)	Belgium (2000)	HI2-pAPEC-O1-R	+	+	+	+	+	+	−	−	−	−	−	dfrA1-aadA1	This study
p142-SA-01-1 (CTX-M-2)	S. enterica serovar Virchow (poultry)	Belgium (2001)	HI2-pAPEC-O1-R	+	+	+	+	+	+	−	−	−	−	−	dfrA1-aadA1	This study
p03-1902-1 (CTX-M-2)	S. enterica serovar Virchow (poultry product)	Belgium (2003)	HI2-pAPEC-O1-R	+	+	+	+	+	+	−	−	−	−	−	dfrA1-aadA1	This study
p04-9275-1 (CTX-M-2)	S. enterica serovar Typhimurium (human feces)	French Guiana (2004)	HI2-pAPEC-O1-R	+	+	+	+	+	+	−	−	−	−	−	drfA12-orfF-aadA2	This study
pROU-1 (CTX-M-9)	S. enterica rough (human feces)	France (2002)	HI2-R478	+	+	+	+	+	+	+	+	+	−	−	dfrA16-aadA2	This study, 17
p2437-1 (CTX-M-9)	S. enterica serovar Virchow (chicken feces)	France (2003)	HI2-R478	+	+	+	+	+	+	+	+	+	−	−	dfrA16-aadA2	This study, 17
p2731-1 (CTX-M-9)	S. enterica serovar Virchow (retail chicken meat)	France (2003)	HI2-R478	+	+	+	+	+	+	+	+	+	−	−	dfrA16-aadA2	This study, 17
p3464b-1 (CTX-M-9)	S. enterica serovar Virchow (chicken environment)	France (2003)	HI2-R478	+	+	+	+	+	+	+	+	+	−	−	dfrA16-aadA2	This study, 17
112-D-T (CTX-M-9)	S. enterica serovar Virchow (human feces)	Spain (2000)	HI2-R478	+	+	+	+	+	+	+	+	+	−	−	dfrA16-aadA2	8, 9
1185-D-T (CTX-M-9)	E. coli (human feces)	Spain (1998)	HI2-R478	+	+	+	+	+	+	+	+	+	−	−	dfrA16-aadA2	8, 9
1406-D-T (CTX-M-9)	E. coli (human feces)	Spain (1999)	HI2-R478	+	+	+	+	+	+	+	+	+	−	−	dfrA16-aadA2	8, 9
R478a			HI2-R478	+	+	+	+	−	+	+	+	+	+	+	−	10
pAPEC-01-Rb			HI2-pAPEC-O1-R	+	+	+	+	+	+	−	−	−	−	−	aadA1	11

^aExpected PCR result based on the R478 sequence (EMBL accession no. BX664015).

^bExpected PCR result based on the pAPEC-O1-R sequence (EMBL accession no. DQ517526).

Plasmids carrying bla_CTX-M-2 or bla_CTX-M-9 were analyzed by PCR-based replicon typing (PBRT) (5): PBRT indicates that all the strains were positive for the HI2 replicon, thus suggesting the presence of common IncHI2 plasmids in these strains. The HI2 replicon was previously detected on CTX-M-9-positive Salmonella and E. coli strains of human origin from Spain (8, 9). Three transconjugants obtained from representative strains of the Spanish collection (one from an S. enterica serovar Virchow strain and two from E. coli) were also included in this study for comparison (Table 1).

The HI2 amplicon was sequenced to confirm the PBRT result, and two types of DNA sequences were observed: the R478 type, showing 100% identity with the HI2 replicon of the reference plasmid R478 isolated from Serratia marcescens in the United States in 1969 (10); and the HI2-APEC-O1-R type, showing 100% homology with the HI2 replicon of the pAPEC-O1-R plasmid, recently described in avian pathogenic E. coli strains in the United States (11). HI2-R478 and HI2-pAPEC-O1-R differ by 29 nucleotide substitutions located within the cis-repeats (iterons) flanking the replicase (repA) gene (accession no. BX664015 and DQ517526, respectively).

Interestingly, the HI2-R478 replicon type was identified in plasmids carrying the bla_CTX-M-9 gene, while the HI2-pAPEC-O1-R replicon type was identified in plasmids carrying the bla_CTX-M-2 gene (Table 1).

R478 and pAPEC-O1-R plasmids show many common regions, including the previously mentioned HI2 replicon, but also the Tra1 and Tra2 transfer regions and the genes coding for copper, silver, and tellurite resistance (10, 11). The two plasmids diverge in terms of insertion sequences, the presence or absence of a class 1 integron, the Tn10 transposon, and arsenic and mercury resistance loci (11).

To further characterize the CTX-M-2 and CTX-M-9 IncHI2 plasmid backbones, we used a PCR-based scheme, previously applied to the CTX-M-9 producers isolated in Spain (Table 2) (9). This scheme recognizes five DNA targets unique for IncHI2 plasmids and common between R478 and pAPEC (smr10-11, smr17-18, smr207-208, smr239-240 and terF) and five DNA targets that are specific for R478 but negative in pAPEC (smr201, smr92-93, smr136, tnsD, and arsB). An additional PCR is also included here to recognize the O1R_160 gene of pAPEC that is interrupted by a Tn10 insertion in R478 (Table 2). Testing the transconjugants listed in Table 1, we observed that the O1R_160 gene was present in all of the strains, while the smr136 and tnsD genes were absent: these results indicate that both the Tn10 and Tn7 transposons are missing in the CTX-M-2 and -9 plasmids analyzed in this study.

TABLE 2.

Primers used in the plasmid backbone analysis^a

Primer pair	Product of target gene in R478b	Primer sequence	PCR gene name (position of amplicon in R478b sequence)
10Fw	HtdV, putative membrane protein	5′-AATCGCCTGAATCAGCTGG-3′	smr10-11 (6655-7713)
11Rv	HtdT, putative plasmid transfer protein	5′-TTCTTTACTACACCAGAGCC-3′
17Fw	SMR0017, putative lipoprotein	5′-AACTCTTGAAAATCGTGG-3′	smr17-18 (18211-19101)
18Rv		5′-CTTCAGGCTATCGTTTCG-3′
TerFw	Tellurium resistance protein	5′-ATGCAGGCTCAAGGAATCGC-3′	terF (80270-81163)
TerRv		5′-TTCATCGATCCACGGTCTG-3′
92Fw	SMR0092, hypothetical protein	5′-CTATGTAAGCAATGATCCTC-3′	smr92-93 (88861-89862)
93Rv	SMR0093, putative inner membrane protein	5′-TATAGAGAGCACCGAAGG-3′
TnsDAFw	Tn7-like transposition protein	5′-AATCCTTGTTCAGCCGG-3′	tnsD (119360-120825)
TnsDARv		5′-CAAAAGCCAGCCATGCCC-3′
136Afw	SMR0136, hypothetical protein	5′-TACGAAAATGAATTGTGGC-3′	smr136 (120906-121768)
136Arv		5′-AATTTACAATCTGCAGCCC-3′
ArsBFw	Arsenical pump membrane protein	5′-AGTGAAAGACAGACGAAGCG-3′	arsB (159735-160870)
ArsBRv		5′-GGCAGATAGTGTGGAATGCG-3′
201Fw	SMR0201 hypothetical protein	5′-TGTCAGGCTAAGTCACTGG-3′	smr201 (180398-181466)
201Rv		5′-ATTATACGGTAGATCC-3′
207Fw	SMR0207, conserved hypothetical protein	5′-TTTCCCAAATAGGCGACGC-3′	smr207-208 (190238-191131)
208Rv	SMR0208, hypothetical protein	5′-ATGTGAAATTACTATACCGG-3′
239Fw	SMR0239, hypothetical protein	5′-TGGAACGCGTGGTATGTGG-3′	smr239-240 (219372-220364)
240Rv	SMR0240, hypothetical protein	5′-ATACCTGCCGTTTACCC-3′
O1R_160Fw	O1R_160, Tn10 insertion site	5′-TTATGATGCTGGGCGTACC-3′	O1R_160 (202719-212075)c
O1R_160Rv		5′-CACCATTACAATCACCTCC-3′

^aThe DNA from the reference plasmid R478 of the incompatibility group IncHI2 was used as control for PCR assays. Amplification conditions were initial denaturation at 94°C for 5 min and 30 cycles at 94°C for 1 min, annealing at 60°C for 30 s, and elongation at 72°C for 1 min.

^bEMBL accession no. BX664015.

^cPositions 183121 to 182957 in EMBL accession no. DQ517526.

All of the plasmids were also positive for the five targets recognizing both R478 and pAPEC plasmids, while the bla_CTX-M-2-carrying plasmids were negative for the arsB, smr92, and smr201 targets, which were positively detected in the bla_CTX-M-9-carrying plasmids (Table 1). These results provided evidence that the bla_CTX-M-2 and bla_CTX-M-9 plasmids show different plasmid scaffolds despite belonging to the same IncHI2 family: the CTX-M-2 producers from both human and poultry sources are associated with pAPEC-O1-R derivative plasmids, while the CTX-M-9 producers are associated with R478 derivative plasmids. The association between bla_CTX-M-9 and R478 was previously demonstrated for E. coli and S. enterica CTX-M-2 and -9 producers isolated in Spain (9). However, this is the first description of CTX-M-9-R478 derivative plasmids in animal sources.

This is also the first description of pAPEC-O1-R derivative plasmids carrying the bla_CTX-M-2 gene. It is of concern that the pAPEC-O1-R plasmid, largely prevalent in avian pathogenic and commensal E. coli strains in the United States (11), is detected with the bla_CTX-M-2 gene in Salmonella strains of human and animal origins in Europe.

Contamination with ESBL-producing strains of S. enterica has been extensively suggested to occur through the food chain, but very few Salmonella ESBL producers of animal origin have been reported, while these enzymes are frequently detected in human isolates. In this study, plasmid-mediated horizontal transfer of bla_CTX-M-2 and bla_CTX-M-9 genes has been demonstrated between poultry and human S. enterica and E. coli strains isolated in very different geographical regions. This result is of major concern because food animals may represent a large reservoir for a further dissemination of such genetic determinants to human pathogens.