Characterization and Molecular Epidemiology of Extended-Spectrum β-Lactamases in Clinical Isolates of Enterobacteriaceae in a Tunisian University Hospital

Abstract

One hundred extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae were recovered from the intensive care unit and the urology ward of the University Hospital of Sahloul in Tunisia between May 2005 and May 2006. The majority of strains showed a high level of resistance to cefotaxime and ceftazidime. Double-disk synergy test and E-test strips were used to confirm production of ESBLs. The molecular analysis revealed that the majority of strains (91%) carried genes encoding CTX-M-15. SHV-12 and SHV-2a were produced, respectively, by 9% and 3% of the strains. Pulsed-field gel electrophoresis of ESBL-producing Klebsiella pneumoniae isolates revealed four different clonal groups and three for Escherichia coli, showing the absence of spread of any epidemic clone. The CTX-M-15 ESBL-producing E. coli of the major clonal group belong to the B2 phylogenetic group, to the sequence type 131, and has a high virulence potential. In conclusion, CTX-M-15 ESBLs accounted for the overwhelming majority of ESBL types among Enterobacteriaceae from our hospital. This study confirms the high rate of ESBLs in Tunisia and further demonstrates the worldwide spread of genes coding for CTX-M-15 enzymes in clinical isolates.

Introduction

The production of extended-spectrum β-lactamases (ESBLs) is one of the major sources of resistance to a third-generation cephalosporins in Enterobacteriaceae.³¹ Classically, plasmid-mediated ESBL enzymes have been of the TEM and SHV types, but in recent years the CTX-M-type ESBLs have been increasingly found throughout the world.⁴ This family of enzymes now comprises 12 members, CTX-M-1 to CTX-M-10, Toho-1, and Toho-2.^36,44 These enzymes share 80%–99% amino acid identity. Escherichia coli and Salmonella enterica serotype Typhimurium are the enterobacterial species most frequently reported to produce the CTX-M β-lactamases.^44,48 The CTX-M-type-producing strains are endemic in Latin America, in Japan, and in some areas in eastern Europe.^5,44 Rare reports signal the presence of CTX-M enzymes in enterobacterial isolates from other countries such as Greece (eastern Europe origin), Spain, Taiwan, and France.^16,36,41,44 CTX-M enzymes predominantly hydrolyze cefotaxime but they are weakly active against ceftazidime. However, some CTX-M enzymes display increased hydrolytic activities against ceftazidime, as in the case for CTX-M-15³⁷ and CTX-M-32.⁸ Recently, ESBL production in Enterobacteriaceae has become a real common problem all over the world.⁷ This problem is primarily due to the spread of CTX-M-type ESBLs.^{4,18,24,28,30,32,38,40} In our hospital, a recent study has demonstrated that 20% of Enterobacteriaceae are ESBL producers according to phenotypic screening. No data are available regarding ESBL genes produced by those strains. To grasp further the content and the mode of dissemination of the ESBLs, we collected clinical isolates that showed positive ESBL phenotypes from the intensive care unit and the urology ward. We aim to identify the relative prevalence of ESBL types among ESBL-producing Enterobacteriaceae and to investigate clonality among ESBL-producing E. coli and Klebsiella pneumoniae, elucidating genetic relatedness and genotypes of these isolates.

Materials and Methods

Bacterial strains

During the study period from May 2005 to May 2006, a total of 856 nonduplicate enterobacterial isolates had been identified at Sahloul Hospital, Sousse, Tunisia, a 550-bed university hospital. One hundred and seventy isolates (20%) were ESBL-producing isolates. One hundred nonrepetitive isolates among them had been kept frozen to be used for this study. They were 71 isolates from 2005 and 29 isolates from 2006. Those isolates had been recovered from urine (n = 58) and blood cultures (n = 42). Isolates belonged to the following species: K. pneumoniae (n = 45), E. coli (n = 31), Enterobacter cloacae (n = 16), Klebsiella oxytoca (n = 2), Providencia stuartii (n = 2), Morganella morganii (n = 3), and Citrobacter freundii (n = 1). All strains were collected from the intensive care unit and the urology ward of Sahloul Hospital. They were identified by using the API 20E system (bioMérieux SA, Marcy l'Etoile, France).

Electrocompetent E. coli DH10B was used as recipient strain in electrotransformation experiments. Rifampicin-resistant E. coli J53-2 was used as host in conjugation assays. The quality control strain used for the study of antimicrobial susceptibility was E. coli ATCC 25922.

Antibiotic susceptibility testing and screening for production of ESBLs

The antibiotic susceptibility of strains was determined by disk diffusion method on Mueller-Hinton (MH) agar plates (Bio-Rad, Marnes-la-Coquette, France) with β-lactam and non-β-lactam antibiotic-containing disks (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) according to the Guidelines of Clinical Laboratory Standards Institute.¹² ESBL production was detected by using double-disk synergy test and the E-test strips (AB-Biodisk, Solna, Sweden) for all studied strains. The double-disk synergy test was performed on MH agar plates without or with cloxacillin (250 mg/ml), in the latter case for detecting clavulanic acid-inhibited ESBLs in those strains that also produced AmpC-type enzymes using cefotaxime (30 μg), ceftazidime (30 μg), aztreonam (3 μg), and cefepime (30 μg) disks that are 30 or 20 mm spaced from amoxicillin–clavulanic acid (20/10 μg).²¹ The minimum inhibitory concentrations (MICs) of cefotaxime and ceftazidime were determined using E-test method (AB-Biodisk) in MH agar plates with an inoculum of 10⁴ CFU. E. coli ATCC 25922 was used as control strain.

Characterization of β-lactamases and associated resistance genes

Identification of ESBL-encoding genes was performed using primers specific for bla_TEM, bla_SHV, and bla_CTX-M genes (Table 1), followed by DNA sequencing. Sequences were analyzed with an ABI prism 310 DNA sequencer (Applied Biosystem, Foster City, CA).

Table 1.

Sequences of Primers Used for Polymerase Chain Reaction Assays Carried Out in This Study for Detection of β-Lactamases Genes, Virulence Factors, Resistance Genes, and Phylogenetic Studies

Target	Sequence (5′–3′)	Primer name	Reference
bla _TEM	ATGAGTATTCAACATTTCCG	OT3	12
	CCAATGCTTAATCAGTGAGG	OT4
bla _SHV	TTATCTCCCTGTTAGCCACC	OS5	12
	GATTTGCTGATTTCGCTCGG	OS6
bla_CTX-M (CTX-M-1 type)	GGTTAAAAAATCACTGCGTC	M13 up	12
	TTGGTGACGATTTTAGCCGC	M13 low
tetA	GTTTCGGGTTCGGGATGGTC	tetA up	24
	GCAGGCAGAGCAAGTAGAGG	tetA low
bla _OXA-1	TATCAACTTCGCTATTTTTTTA	OXA-1 up	24
	TTTAGTGTGTTTAGAATGGTGA	OXA-1 low
aac(6′)-Ib	ATGACTGAGCATGACCTT	AAC6′-Ib up	22
	GAAGGGTTAGGCATCACT	AAC6′-Ib low
aac(3)-II	CAATAACGGAGGCAATTCG	AAC3-II up	22
	GATTATCATTGTCGACGG	AAC3-II low
sul1	CGGCGTGGGCTACCTGAACG	Sul1-F	25
	GCCGATCGCGTGAAGTTCCG	Sul1-B
sul2	GCGCTCAAGGCAGATGGCATT	Sul2-F	25
	GCGTTTGATACCGGCACCCGT	Sul2-B
chuA	GACGAACCAACGGTCAGGAT	chuA.1	8
	TGCCGCCAGTACCAAAGACA	chuA.2
yjaA	TGAAGTGTCAGGAGACGCTG	yjaA.1	8
	ATGGAGAATGCGTTCCTCAAC	yjaA.2
TspE4.C2	GAGTAATGTCGGGGCATTCA	TspE4C2.1	8
	CGCGCCAACAAAGTATTACG	TspE4C2.2
papG allele I	TCGTGCTCAGGTCCGGAATTT	AlleleI-f	15
	TGGCATCCCCCAACATTATCG	AlleleI-r
papG allele II	GGGATGAGCGGGCCTTTGAT	AlleleII-f	15
	CGGGCCCCCAAGTAACTCG	AlleleII-r
papG allele III	GGCCTGCAATGGATTTACCTGG	AlleleIII-f	15
	CCACCAAATGACCATGCCAGAC	AlleleIII-r
afa/draBC	GGCAGAGGGCCGGCAACAGGC	Afa f	15
	CCCGTAACGCGCCAGCATCTC	Afa r
fimH	TGCAGAACGGATAAGCCGTGG	FimH f	15
	GCAGTCACCTGCCCTCCGGTA	FimH r
hlyA	AACAAGGATAAGCACTGTTCTGGCT	hly f	15
	ACCATATAAGCGGTCATTCCCGTCA	hly r
cnf1	AAGATGGAGTTTCCTATGCAGGAG	cnf1	15
	CATTCAGAGTCCTGCCCTCATTATT	cnf2
fyuA	TGATTAACCCCGCGACGGGAA	FyuA f	15
	CGCAGTAGGCACGATGTTGTA	FyuA r
iutA	GGCTGGACATCATGGGAACTGG	AerJ f	15
	CGTCGGGAACGGGTAGAATCG	AerJ r
kpsMT II	GCGCATTTGCTGATACTGTTG	kpsII f	15
	CATCCAGACGATAAGCATGAGCA	kpsII r
traT	GGTGTGGTGCGATGAGCACAG	TraT f	15
	CACGGTTCAGCCATCCCTGAG	TraT r
sat	ACTGGCGGACTCATGCTGT	Sat 1	31
	AACCCTGTAAGAAGACTGAGC	Sat 2
iha	CTGGCGGAGGCTCTGAGATCA	IHA f	16
	TCCTTAAGCTCCCGCGGCTGA	IHA r
iroN	AAGTCAAAGCAGGGGTTGCCCG	IRONEC-F	16
	GACGCCGACATTAAGACGCAG	IRONEC-R
sfa/foc	CTCCGGAGAACTGGGTGCATCTTAC	sfa1	15
	CGGAGGAGTAATTACAAACCTGGCA	sfa2
afa1operon	GCTGGGCAGCAAACTGATAACTCTC	Afaclb_1	21
	CATCAAGCTGTTTGTTCGTCCGCCG	Afaclb_2

The nucleotide and the deduced protein sequences were analyzed with software available over the internet (www.ncbi.nlm.nih.gov).

Other antibiotic resistance genes, often found associated with bla_CTX-M-15, are as follows^17,32: bla_OXA-1, aac(3)-II, aac(6′)-Ib, and tetA as well as sul1 and sul2 genes were screened by polymerase chain reaction (PCR) using the primers listed in Table 1.

β-lactam resistance transfer assays

Tests were carried out in trypticase soy broth agar with E. coli J53-2 as recipient strain. Mating broths were incubated at 37°C for 16 hr. Transconjugants were selected on MH agar plates containing rifampicin (250 μg/ml) and cefotaxime (4 μg/ml) or ceftazidime (5 μg/ml).³²

Plasmid extracts of enterobacterial isolates were used for electrotransformation experiments in E. coli DH10B. Transformants were selected on cefotaxime-containing plates (2.5 μg/ml).

Fingerprinting analysis

Repetitive extragenic palindromic sequence PCR (Rep PCR) was performed with primers rep-1R and rep-2T for all the E. coli isolates as previously described.¹⁸ Enterobacterial repetitive intergenic consensus sequence PCR (ERIC PCR) was performed with primer ERIC-2 for all the K. pneumoniae studied isolates as previously described.¹⁸ If isolates had similar patterns, they were subjected to pulsed-field gel electrophoresis (PFGE). Briefly, genomic DNA was digested by XbaI (Ozyme, Saint Quentin en Yvelines, France) at 37°C overnight, and DNA fragments were separated in 1% agarose gel in 0.5× Tris-borate-EDTA buffer by using a gene path system (Bio-Rad). The conditions used were as follows: temperature 14°C, voltage 6 V/cm for 20 hr, with pulse times of 5.3–49.9 sec.³² The same conditions were applied for E. coli and K. pneumoniae strains. The ethidium bromide-stained gel was photographed under ultraviolet illumination. Clonal relationships based on PFGE patterns were interpreted according to previously established criteria by Tenover et al.⁴³

Phylotyping and virulence genotyping of E. coli

To compare the characteristics of epidemic E. coli strains disseminating in our hospital with the ESBL-producing E. coli strains in other countries, we elucidate the phylogenetic group and virulence factors of the examined E. coli isolates. The phylogenetic group was determined by PCR method developed by Clermont et al.,¹¹ using the combination of three markers (chuA, yjaA, and TspE4.C2). All isolates were screened for 16 genes encoding putative virulence factors often found in extraintestinal pathogenic E. coli, namely fimH, sfa/focDE, papG I allele, II and III allele, afa, hlyA, cnf1, fyuA, iutA, kpsM II, traT, sat, iroN, iha, afaclb1/2, using single or multiplex PCR assays^22,23,29,40 and the primers listed in Table 1.

Sequence type determination

Multilocus sequence typing (MLST) was carried out for K. pneumoniae (clone K1 and clone K2) as previously described.¹⁵ The results were analyzed with the K. pneumoniae MLST database available over the internet (www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). E. coli strains belonging to phylogenetic group B2 have been typed by MLST as described by Clermont et al.⁹ (www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html).

Results

Antibiotic susceptibility

One antibiotic resistance phenotype (phenotype I) was the most prevalent (91/100 strains). It was characterized by a resistance to cefotaxime and ceftazidime. A second antibiotic resistance profile (phenotype II) showed a reduced susceptibility to cefotaxime and a resistance to ceftazidime (9/100 strains). Isolates were often resistant to tobramycin (94%), nalidixic acid (90%), tetracycline (87%), ciprofloxacin (86%), nethilmicin (86%), gentamicin (86%), and trimethoprim–sulfamethoxazole (76%). Resistance to chloramphenicol and amikacin was less frequent. It represents 59% and 49% of the isolates, respectively. All of the isolates were susceptible to cefoxitin and imipenem.

Characterization of β-lactamases and associated resistance genes

Ninety-one percent of the isolates gave positive PCR-based amplifications with specific primers of the bla_CTX-M gene. The deduced amino acid sequences corresponded to CTX-M-15 in these isolates. All K. pneumoniae (n = 45), E. coli (n = 35), and M. morganii (n = 3) isolates produce CTX-M-15 enzymes, whereas only seven E. cloacae isolates produce CTX-M-15 enzymes. Those enzymes were absent in K. oxytoca, Providencia rettgeri, and Citrobacter freundii isolates. Most of the strains (82%) were positive for bla_TEM gene. Sequencing identified the narrow-spectrum β-lactamase TEM-1 (Table 2). SHV-type enzymes (n = 13) were SHV-12 from E. cloacae, K. oxytoca, and P. rettgeri, and SHV-2a (n = 4) from E. cloacae and C. freundii. Three E. cloacae had two ESBLs (CTX-M-15 and SHV-2a). The results of PCR experiments using specific primers for the associated resistance genes for all studied isolates are presented in Table 2. The bla_OXA-1 and the aminoglycoside genes aac(3)-II and aac(6′)-Ib were the most prevalent ESBL-associated genes.

Table 2.

Distribution of Extended-Spectrum β-Lactamase Types and Associated Resistance Genes in 100 Extended-Spectrum β-Lactamase-Producing Isolates at Sahloul Hospital, Tunisia, Between May 2005 and May 2006

		Number of isolates with the respective resistance genes
Clinical isolates	Number of isolates	bla_CTX-M-15	bla_TEM-1	bla_SHV-2a	bla_SHV-12	bla_OXA-1	tetA	aac(3)-II	aac(6′)1-b	sul1	sul2
Klebsiella pneumoniae	45	45	38	0	0	45	27	13	45	18	28
Escherichia coli	31	31	24	0	0	31	13	27	31	27	21
Enterobacter cloacae	16	7	14	3	9	11	13	16	16	16	16
Morganella morganii	3	3	2	0	0	3	0	3	3	3	0
Klebsiella oxytoca	2	0	2	0	2	0	0	2	2	2	2
Providencia rettgeri	2	0	1	0	2	1	2	2	2	2	1
Citrobacter freundii	1	0	1	1	0	1	1	1	1	1	0
Total	100	86	82	4	13	92	56	64	100	69	68

Epidemiological results

Forty-five K. pneumoniae isolates yielded four enterobacterial repetitive intergenic consensus sequence PCR patterns, and the 31 E. coli isolates yielded three repetitive extragenic palindromic sequence PCR patterns. Isolates with similar patterns were subjected to PFGE.² Two major clones producing CTX-M-15 and TEM-1 were observed among K. pneumoniae isolates and the designed clones K1 (21 isolates, 48%) and K2 (17 isolates, 37%). Two minor clones were observed: K3 (four isolates) and K4 (three isolates), which produced only CTX-M-15 (Table 3). The K. pneumoniae producing CTX-M-15 and TEM-1 predominated in 2005. K. pneumoniae producing CTX-M-15 only were isolated from the intensive care unit. Three clones of E. coli were observed and designed E1 (23 isolates, 74%), E2 (6 isolates, 19%), and E3 (2 isolates, 6%). The clone E1 predominated in 2005 (20 isolates). bla_OXA-1 and sul1 were associated with the E. coli clonal strain E1 (Table 3). The PFGE patterns showed that the outbreak of CTX-M-15 enzyme was not caused by a single clone of E. coli or K. pneumoniae.

Table 3.

Pulsed-Field Gel Electrophoresis Patterns of Klebsiella pneumoniae and Escherichia coli Producing Extended-Spectrum β-Lactamases at Sahloul Hospital in Sousse

Isolates	Pattern	Number of isolates with respective PFGE
Klebsiella pneumoniae	K1	21
	K2	17
	K3	4
	K4	3
Escherichia coli	E1	23
	E2	6
	E3	2

Table 4.

Phylogenetic Groups and Virulence Factors of Escherichia coli Producing Extended-Spectrum β-Lactamases at Sahloul Hospital in Sousse^a

			Detection of virulence factors
Clone	Number of isolates with respective PFGE pattern	Phylogenetic group	fimH	fyuA	iutA	TraT	sat	IronN	KpsMT II	hlyA	Cnf1
E1	23	B2	+	+	+	+	+	−	+	+	+
E2	6	A	+	−	+	+	−	−	−	−	−
E3	2	D	+	+	+	+	−	+	−	−	−

All strains were negative for papG alleles I, II, and III and sfa/foc, afa, afa clb/1, and iha genes.

PFGE, pulsed-field gel electrophoresis.

Transferability of ESBLs

Twenty K. pneumoniae and 15 E. coli isolates were selected according to their bla_CTX-M gene, their fingerprint, and their antimicrobial resistance pattern: 9, 4, and 2 E. coli isolates with the E1, E2, and E3 fingerprints, respectively, were selected. For K. pneumoniae isolates, 9 with K1, 6 with K2, 3 with K3, and 2 with K4 PFGE types were tested for conjugal transfer of cefotaxime resistance, and 24 (9 E. coli and 15 K. pneumoniae isolates) were positive. Electroporation of plasmid DNA from the other 11 strains into E. coli DH10B successfully transferred cefotaxime and ceftazidime resistance. The E. coli transformant strains exhibited a β-lactam resistance pattern consisting in the expression of an ESBL (CTX-M-15). The ESBL-positive plasmids conferred additional resistances to other antibiotics (tetracycline, gentamicin, amikacin, trimethoprim–sulfamethoxazole).

Phylogenetic analysis and virulence genotyping of E. coli

The results of phylogenetic studies and virulence factor determination are reported in Table 4. All of the 31 isolates examined harbored three virulence genes in common. Two genes encoded siderophores (iutA and fyuA), and one encoded a serum survival factor (traT). None of the papG alleles I, II, and III and sfa/foc, afa, afa clb/1, and iha genes were detected in these 31 isolates. The most prevalent PFGE type E1 belongs to the phylogenetic group B2 according to the criteria described earlier.¹¹ All E. coli strains belonging to this PFGE type were isolated from blood samples (77%). Those strains expressed more virulence factors than the other clones. These virulence factors were often encountered in ExPEC strains. PFGE type E2 was assigned to the phylogenetic group A and expressed only three virulence factors that are in common with the other groups. Finally, the PFGE type E3 strains were related to the phylogenetic group D.

Multilocus sequence typing

When compared by MLST analysis, the selected K. pneumoniae strains with K1 and K2 PFGE types showed two different sequence types (STs). The ST of K. pneumoniae with K1 PFGE type was ST292, and the ST for K. pneumoniae with K2 PFGE type was ST383. For E. coli strains with E1 PFGE type and belonging to the B2 phylogenetic group, the ST was ST131.

Discussion

This study was conducted at Sahloul Hospital in Tunisia between May 2005 and May 2006. The prevalence of ESBL-producing isolates in our hospital was 20% of the Enterobacteriaceae isolates recovered from the intensive care unit and the urology ward of the hospital in this period. This value is approximately the same as in Europe (21%)^39,46 and is higher than in another Tunisian hospital (7, 4%).² All the cefotaxime- and ceftazidime-resistant isolates have the CTX-M-15 enzyme: it was found in 91% of the isolates. Those strains belong to the antibiotic resistance phenotype I. CTX-M-15 is derived from CTX-M-3 by one amino acid substitution at position 240 (Asp-240 → Gly), which confers an increased catalytic activity to ceftazidime.^6,37 Clinical isolates expressing CTX-M β-lactamases often display much higher resistance to cefotaxime than to ceftazidime. In this collection of isolates, all of the cefotaxime-resistant strains and their transformants were also resistant to ceftazidime. Compared with other reports on distribution of ESBL in Enterobacteriaceae, performed in Tunisia, we have shown the same result^26,32; those studies reported that the CTX-M-15 ESBL was the most prevalent ESBL in Tunisia, which is the case in our hospital. K. pneumoniae and E. coli were the most predominant ESBL-producing species among the seven bacteria studied in this report. This result is in accordance with previous studies.^18,24,28,32

The CTX-M-15 ESBL was first described in New Delhi in 1999 and was found to be carried on large plasmids.¹ Since then, clonal outbreaks of CTX-M-15-producing Enterobacteriaceae have been reported in France, Italy, Spain, Portugal, Austria, Norway, the United Kingdom, Tunisia, South Korea, Canada, Lebanon, and Egypt.^{6,17,20,30,31,47} Recent reports indicate that worldwide dissemination of CTX-M-15 is mediated by clonally related E. coli strains, particularly a specific clone—E. coli ST131 of phylogenetic group B2 and with high virulence potential.^{10,13,27,35,42} In accordance with these results, the E. coli ST131 was the most prevalent clone (74%) among our strains and expresses several virulence factors. E. coli ST131 was described previously in Tunisia,²⁸ but our strain expresses more virulence factors than the described one.^28,32

All of the studied strains were multiresistant, producing other β-lactamases (OXA and TEM) and aminoglycoside-modifying enzymes. They were resistant to ciprofloxacin. Multiresistance has often been described for ESBL and especially CTX-M-15-producing clinical isolates^{4,7,18,23,27,31} that carried an important pool of mobile resistances genes including tetA, sul1, sul2, aac(6′)Ib, and aac(3)IIb genes.

The PFGE analysis showed that the spread of the bla_CTX-M-15-containing E. coli and K. pneumoniae isolates was not caused by a single clone. For K. pneumoniae strains, two major clones have been found: K1 and K2 with ST292 and ST383, respectively. Those STs were different from those described in Tunisia by Elhani et al.¹⁹ Twenty-three of 31 CTX-M-15-producing E. coli isolates belong to the major clone E1. The clonality of those strains is evident from their homogeneity with respect to phylogenetic group, ST, and the PFGE profile. This suggested that the clonal dissemination of ESBL-producing strains did not play a predominant role in the overall spread. In this study, ESBL CTX-M-15 demonstrates the ability to spread among different groups of E. coli and K. pneumoniae. Plasmids may have been able to transfer within bacterial populations by means of patient transfer from different wards, which is a common practice in our hospital, or through acquisition from the community. The high prevalence of the ESBL-positive ceftazidime-resistant enterobacterial isolates has induced the spread of ESBL-producing gram-negative bacilli isolates such as Pseudomonas aeruginosa SHV-2a producer strains, which were found disseminating in the different wards of our hospital in the same period of this study.³³

Some dominant SHV types continue to be important, especially SHV-12.¹⁴ SHV-12 and SHV-2a were found in the isolates investigated. These enzymes seem to be common not only in Tunisia but also in many countries in north and central Africa.^3,45 Nine strains produced only SHV-12. Those strains showed a reduced susceptibility to cefotaxime and were resistant to ceftazidime. They belong to the antibiotic resistance phenotype II.

Three E. cloacae carried two ESBLs: CTX-M-15 and SHV-2a. Although this cocarriage of ESBL genes is not a new finding but is uncommon,³⁴ the ESBL-positive plasmids of enterobacterial isolates were transferable. Thus, it is possible that this plasmid has a broad host range and will contribute to the spread of the ESBL genes in enterobacterial and nonenterobacterial gram-negative bacilli.

Conclusion

The current worldwide emergence of multiresistant enterobacterial isolates is mostly associated with ESBL producers. The spread of the CTX-M-15 as the predominant ESBL type is not restricted to Tunisia. In this study, we have also reported the worldwide emergent clone E. coli ST131. This clone represents a real problem for public health.

Footnotes

Acknowledgments

The authors thank Mr. Salem Ben Amor for his endless support. This work was partially funded by grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology and by grants from the University of Medicine Pierre et Marie Curie (site Saint-Antoine), Paris VI.

Disclosure Statement

All authors disclose no commercial associations that might create a conflict of interest in connection with this study.

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