High Diversity of Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Italian Broiler Flocks

Abstract

We characterized 67 Escherichia coli isolates with reduced susceptibility to cefotaxime or ceftiofur obtained from healthy broilers housed in five Italian farms. The bla_CTX-M-1, bla_CTX-M-32 and bla_SHV-12 β-lactamase genes were identified on IncI1, IncN, or IncFIB plasmids. Considerable genetic diversity was detected among the extended-spectrum β-lactamase (ESBL)-producing isolates, and we identified indistinguishable strains in unrelated farms and indistinguishable plasmids in genetically unrelated strains. The detection of highly mobile plasmids suggests a potential animal reservoir for β-lactamase genes.

Escherichia coli resistant to extended-spectrum cephalosporins through the production of extended-spectrum β-lactamases (ESBLs) is isolated at an increasing rate among different hosts and countries, thus representing a potential public health risk (2, 3). Typically, ESBLs are borne on mobile genetic elements and can therefore spread both clonally and horizontally among different lineages of bacteria (5).

To determine the role of animals as potential reservoirs for ESBL-producing bacteria, there is a need for improved knowledge of the factors that promote dissemination of resistance determinants, including the role of the intestinal flora (4). In this study, we characterized a collection of 67 E. coli isolates showing reduced susceptibility to cefotaxime (MIC ≥ 2 μg/ml) or ceftiofur (MIC ≥ 8 μg/ml). The isolates were obtained during July 2007 by processing fecal samples collected by the “sock method” (1, 22) in a broiler-parent farm and four broiler farms located in northeastern Italy. The farms were ordinary with respect to farm management and biosecurity, thus implying that no contact among the animals reared in different farms could have taken place. One flock (i.e., a group of chickens sharing the same airspace) per farm was sampled (Table 1). All broiler flocks (here identified as flocks 1, 2, 4, and 5) hatched from eggs incubated in the same hatchery, and the parent flock (here identified as flock 6) was among the flocks that provided eggs to that hatchery. Indication of ESBL presence resulted from the ESBL phenotypic detection test performed with the disk method (7, 10). Amplification of the bla_TEM, bla_SHV, and bla_CTX-M genes was done by PCR using primers and conditions previously described (15, 16) on whole-genome bacterial DNA (GenElute bacterial genomic DNA kit; Sigma-Aldrich, Brøndby, Denmark). PCR products were purified (exonuclease I, E. coli, and FastAP thermosensitive alkaline phosphatase; Fermentas, Helsingborg, Sweden) and sequenced (Macrogen Inc., Seoul, South Korea). Comparison of the nucleotide sequences and the derived amino acid sequences with previously described sequences (GenBank database http://www.ncbi.nlm.nih.gov/ and www.lahey.org/studies/webt.html, respectively) showed that the bla_TEM-1, bla_SHV-12, bla_CTX-M-1, and bla_CTX-M-32 genes were present in this collection (Fig. 1). The genetic relatedness of the E. coli isolates was determined by amplified fragment length polymorphism (AFLP) analysis as previously described (1, 6). Using a similarity cutoff value of ≥94% (1), 15 different clonal types were identified (A to O in Fig. 1). Eighteen strains were selected for plasmid analysis by selecting one strain per clonal type from each flock. Within one AFLP type, however, two strains from the same flock were examined to determine the intraclonal diversity. Plasmid DNA (GeneJET plasmid miniprep kit; Fermentas, Helsingborg, Sweden) was used to transform electrocompetent Genehog E. coli (Invitrogen, Taastrup, Denmark). Transformants were selected on brain heart infusion (BHI) agar (Merck, Glostrup, Denmark) plates containing 2 μg/ml of cefotaxime and tested by colony PCR (15, 16) to confirm the presence of ESBL-encoding genes. Plasmid DNA (GeneJET plasmid miniprep kit; Fermentas, Helsingborg, Sweden) from the transformants was analyzed by restriction fragment length polymorphism (RFLP) analysis and PCR-based replicon typing (PBRT) (3). IncN (n = 10), IncI1 (n = 5), or IncFIB (n = 3) plasmids were identified (Fig. 1 and Table 2). Twelve different plasmid profiles were visualized on a 0.8% agarose gel after enzymatic digestion with ClaI and PstI (FastDigest; Fermentas, Helsingborg, Sweden). Notably, indistinguishable band patterns were obtained in (i) two bla_CTX-M-1 IncN plasmids found in two closely related E. coli strains (with 91.6% similarity according to the analysis of the AFLP patterns) from flock 6, (ii) five bla_CTX-M-32 IncN plasmids found in five unrelated E. coli strains from flock 4, and (iii) two bla_SHV-12 IncFIB plasmids found in two clonally related E. coli strains from flocks 1 and 2 (Fig. 1 and Table 2). The disk diffusion test (9, 10) showed that all donors were resistant to at least two additional antimicrobial classes (Table 2). Seventeen (94%) isolates were resistant to tetracycline, and 15 (83%) isolates were resistant to nalidixic acid, 8 (44%) of which were ciprofloxacin resistant. Sulfonamide resistance alone or in combination with trimethoprim resistance was detected in 4 (22%) and 10 (55%) donor isolates, respectively. No resistance against amikacin, colistin, and imipenem was detected. Sulfonamide resistance occurred in seven (39%) transformants and was associated with trimethoprim resistance in one (5%) case (Table 2). The cotransfer of sul2 (n = 1), sul3 (n = 5), and sul1 and sul3 (n = 1) genes was determined in such transformants by the use of PCR as previously described (Table 2) (14). As measured by agar dilution (8), the MIC of ciprofloxacin for all the transformants was <0.03 μg/ml, indicating that possible qnr-mediated resistance to this fluoroquinolone was not cotransferred by the ESBL-producing plasmids.

TABLE 1.

Anamnestic information about the flocks sampled

Flock	Type of animals	Age (days)	Antimicrobial treatment history	Antimicrobial treatment history of the preceding flock	No. of isolates included in the study
1	Broilers	35	None	Oxytetracycline, sulfadiazine-trimethoprim	19
2	Broilers	12	Amoxicillin	Flumequine, amoxicillin, sulfadiazine-trimethoprim, colistin	19
4	Broilers	70	Enrofloxacin, tylosin	None	14
5	Broilers	35	Amoxicillin	Amoxicillin	5
6	Parents	240	Tylosin, tetracycline, colistin	Tylosin, tetracycline, colistin	10

FIG. 1.

Dendrogram showing the genotypic relatedness of 67 Escherichia coli isolates based on AFLP fingerprints. A Dice coefficient and the unweighted-pair group method using average linkages (UPGMA) algorithm were used with a position tolerance of 1%. The scale bar represents the percentage of similarity, and the vertical, dotted line indicates the cutoff value for identifying clonal types. PBRT, plasmid-based replicon typing as determined with a subset of isolates indicated by gray-shaded ovals.

TABLE 2.

Genetic and phenotypic traits of ESBL-producing Escherichia coli from broiler flocks

Flock	Isolate	AFLP clonal type (no. of strains within clonal type)	β-Lactamase gene(s)a	ClaI plasmid RFLP profile	PstI plasmid RFLP profile	Replicon typing	Additional resistancesb	sul genesc
1	3	A (20)	TEM-1, SHV-12	9	l	FIB	TET, SUL, TMP, FFC, CHL, NAL, CIP, STR	sul1, sul3
1	9	H (18)	TEM-1, SHV-12	3	c	I1	TET, SUL, TMP, FFC, CHL, NAL, STR	sul2, sul3
1	10	H (18)	TEM-1, SHV-12	11	n	I1	TET, SUL, TMP, NAL, STR	sul1, sul2
2	15	A (20)	TEM-1, SHV-12	9	l	FIB	TET, SUL, TMP, FFC, CHL, NAL, CIP, STR	sul1, sul3
2	47	B (1)	CTX-M-1	8	i	I1	TET, SUL, TMP, NAL	sul1, sul3
4	18	D (1)	CTX-M-32	5	f	N	TET, NAL
4	30	C (1)	CTX-M-32	5	f	N	TET, NAL
4	31	L (1)	CTX-M-1	2	b	N	SUL, NAL, CIP, STR	sul1
4	49	K (2)	CTX-M-32	6	g	N	TET, SUL, NAL, CIP, SPT	sul2
4	51	M (3)	CTX-M-32	4	d	N	TET, SUL, TMP, CHL, NAL, CIP, STR	sul2
4	52	N (1)	CTX-M-32	5	f	N	TET, SUL, TMP, CHL, NAL, CIP, STR	sul1, sul2
4	53	O (4)	TEM-1, CTX-M-32	5	f	N	TET, SUL, TMP, CHL, NAL, CIP, STR, GEN	sul1, sul2, sul3
4	64	E (1)	TEM-1, CTX-M-32	5	f	N	TET, SUL, CHL, NAL, CIP	sul3
5	21	I (5)	TEM-1, SHV-12	1	a	I1	TET, SUL, TMP, FFC, CHL, STR	sul1, sul2, sul3
6	22	H (18)	TEM-1, SHV-12	7	h	I1	TET, SUL, TMP, FFC, CHL, NAL, STR	sul1, sul2, sul3
6	35	F (7)	CTX-M-1	5	e	N	TET
6	56	G (1)	CTX-M-1	5	e	N	TET
6	57	J (1)	TEM-1, CTX-M-1	10	m	FIB	TET, SUL, CHL, NAL	sul1, sul3

^aβ-Lactamase genes were determined by PCR and by sequencing in the donors. Genes transferred by transformation, as determined by PCR but not by sequencing, are underlined.

^bCHL, chloramphenicol; CIP, ciprofloxacin; FFC, florfenicol; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; SUL, sulfonamides; TET, tetracycline; TMP, trimethoprim. Patterns transferred by transformation are underlined.

^cGenes transferred by transformation are underlined.

We isolated ESBL-producing E. coli in flocks that did not receive treatments with cephalosporins (Table 1). Treatments with other antimicrobials (Table 1) could have coselected for the bacteria described. Although a limited number of flocks and bacterial isolates was included in the study, the genetic environment for the ESBL-encoding genes was highly variable. The bla_SHV-12 gene was associated with three major clones (A, H, and I) detected in four flocks. These strains carried IncI1 (H and I) or IncFIB (A) plasmids. The bla_CTX-M-1 gene and the closely related bla_CTX-M-32 gene were mainly associated with IncN plasmids found in 10 different clonal types in three flocks. As clearly shown in flock 4 (Table 2), IncN plasmids were broadly distributed among genetically unrelated strains. The horizontal mobility of IncN plasmids has already been reported by Moodley and Guardabassi, who described the spread of a bla_CTX-M-1 IncN plasmid among multiple E. coli lineages of porcine and human origin (18). The finding of IncN plasmids that could disseminate by horizontal transmission is worrisome. Our findings concerning the association of ESBL-encoding genes and plasmid replicons are similar to previous findings in humans, chickens, pigs, and dogs (5, 11, 12, 13, 17, 18, 19, 21, 23). The similarity between the ESBL variants we detected and those present in humans in Italy (20) indicates a possible role of poultry in the dissemination of these resistance determinants.