First Extended-Spectrum-β-Lactamase (CTX-M-15)-Producing Salmonella enterica Serotype Typhimurium Isolate Identified in Lebanon

In Salmonella, most extended-spectrum β-lactamases (ESBLs) are derivatives of TEM and SHV β-lactamase families, although other groups, including PER and CTX-M, have been described recently (1, 2). CTX-M-type ESBLs display levels of resistance to cefotaxime and ceftriaxone significantly higher than those to ceftazidime (2). Since 1995, CTX-M-type ESBLs have disseminated dramatically in several parts of the world (1).

In January 2004, a Salmonella enterica serotype Typhimurium isolate, CAM18, was recovered from a stool specimen collected from a 6-year-old child upon admission to a hospital in Northern Lebanon. The child had no history of travel. The isolate had a high resistance level to cefotaxime and ceftazidime and was also resistant to several aminoglycosides, sulfamethoxazole-trimethoprim, and tetracycline. It was susceptible to cefoxitin, imipenem, quinolones, and chloramphenicol. ESBL production was detected by the double-disk synergy test (6).

The ESBL phenotype was transferred to Escherichia coli K-12 resistant to nalidixic acid with an efficiency of 10⁻² per donors. Certain transconjugants also acquired resistance to tetracycline and kanamycin (TC-pCAM18-1), while others acquired resistance to tetracycline alone (TC-pCAM18-2). The addition of clavulanic acid reduced the MICs of all β-lactams tested (Table 1).

TABLE 1.

Characteristics of S. enterica serotype Typhimurium CAM18, E. coli K-12, and two transconjugants

Strain	MIC (μg/ml)a				β-Lactamase		Plasmid size (kb)	Resistanceb
	CT/CTL	TZ/TZL	PM/PML	TX	pI valuec	bla gene		Tc	K
Salmonella serotype Typhimurium CAM18	>256/0.19	128/0.75	>256/0.064	>256	5.4	blaTEM	∼20	R	R
	7.3	blaOXA-30	∼60
					8.6	blaCTX-M-15
E. coli
TC-pCAM18-1d	>256/0.064	32/0.75	128/<0.064	>256	5.4	blaTEM	∼20	R	R
					7.3	blaOXA-30	∼60
					8.6	blaCTX-M-15
TC-pCAM18-2	>256/0.064	32/0.75	128/<0.064	>256	7.3	blaOXA-30	∼60	R	S
					8.6	blaCTX-M-15
K-12	0.19/0.19	0.19/0.19	0.047/<0.064	0.047				S	S

^aAbbreviations: CT, cefotaxime; CTL, cefotaxime-clavulanate; TZ, ceftazidime; TZL, ceftazidime-clavulanate; PM, cefepime; PML, cefepime-clavulanate; TX, ceftriaxone.

^bAbbreviations: Tc, tetracycline; K, kanamycin; R, resistant; S, susceptible.

^cpI values in bold type indicate the β-lactamases that were positive in the bioassay with cefotaxime as a substrate.

^dTransconjugants indicated by TC prefix in the strain designation.

Results of isoelectric focusing (8) and of amplifications using TEM, SHV, CTX-M, and OXA-1 primers (3, 10, 11) are shown in Table 1. The β-lactamase with a pI of 5.4 was most probably TEM-1. Sequencing of bla_OXA-1 amplicons revealed the presence of OXA-30, a β-lactamase with a pI of 7.3 differing from OXA-1 by one amino acid and recently reported in E. coli (4) and S. enterica serotype Typhimurium (5). Sequencing of bla_CTX-M amplicons revealed that coding regions were 100% identical to the coding region of the bla_CTX-M-15 gene (GenBank accession number AY044436). Amplification and sequencing of the entire coding sequence of bla_CTX-M-15 (14) confirmed that the β-lactamase with a pI of 8.6 was CTX-M-15, an enzyme that has been previously identified by several researchers (for a recent review, see reference 1). Unlike most CTX-M enzymes, CTX-M-15, an Asp-240-Gly variant of CTX-M-3, increased the catalytic efficiency against ceftazidime (12), as observed with MIC results (Table 1).

The presence of ISEcp1, a mobile sequence located upstream of several CTX-M genes, was investigated by a PCR assay as previously described (13). Sequencing of PCR products obtained with isolate CAM18 and transconjugants revealed that the ISEcp1 element was located in the same position as that found in Indian and Turkish isolates (7, 9). Moreover, the 160-bp sequence upstream of bla_CTX-M-15 was 100% identical to the corresponding sequences of Indian (GenBank accession number AY044436) and Canadian (accession number AY458016) isolates.

Kanamycin resistance was associated only with strain TC-pCAM18-1, while tetracycline resistance was exhibited by both transconjugants. This result in addition to plasmid purification revealed that bla_TEM and kanamycin resistance genes reside on a plasmid of 20 kb and bla_CTX-M-15, bla_OXA-30, and tetracycline resistance genes reside on a larger plasmid of 60 kb.

We report for the first time the isolation of a CTX-M-type ESBL in Lebanon. In addition, this is also the first report of an ESBL-producing Salmonella in Lebanon. A study done in 2003 at St. George hospital (Beirut, Lebanon) on 49 Salmonella strains showed 100% susceptibility to ceftazidime and cefotaxime (Z. Daoud, personal communication). The appearance of the CTX-M-15-producing S. enterica serotype Typhimurium in the community in 2004 is a serious threat to public health.