ESBL-producing Enterobacteriaceae, Central African Republic

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Vol. 12, No. 5
May 2006

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Letter

Extended-spectrum β-Lactamase–producing Enterobacteriaceae, Central African Republic

Thierry Frank,* Guillaume Arlet,†‡ Valerie Gautier,† Antoine Talarmin,* and Raymond Bercion*
*Institut Pasteur de Bangui, Bangui, Central African Republic; †Université Pierre et Marie Curie (Paris VI), Paris, France; and ‡Hôpital Tenon AP-HP, Paris, France

Suggested citation for this article

To the Editor: Since the early 1980s, extended-spectrum β-lactamases (ESBLs) have been the largest source of resistance to broad-spectrum oxyimino-cephalosporins among Enterobacteriaceae (1). Molecular analysis techniques suggest that many ESBLs are derived from mutations in TEM-1, TEM-2, and SHV-1 β-lactamases and that these ESBLs can hydrolyze the extended-spectrum cephalosporins (particularly ceftazidime) and aztreonam (1). Members of a new group of ESBLs have been recently identified (1). Among them, CTX-M–type ESBLs are rapidly expanding and are derived from chromosomal class A β-lactamases of Kluyvera spp. (1,2). The CTX-M enzymes are not related to TEM or SHV enzymes, as they share only 40% identity with these ESBLs (2).These ESBLS are usually characterized by a higher level of resistance to cefotaxime than ceftazidime, except for CTX-M-19 (2). Most organisms that harbor ESBLs are also resistant to other classes of antimicrobial drugs, such as aminoglycosides, fluoroquinolones, chloramphenicol, and tetracyclines (1,2).

Reports concerning the existence of ESBL-producing Enterobacteriaceae in sub-Saharan Africa are scarce. We therefore conducted a study in the Central African Republic to determine the frequency of ESBLs in Enterobacteriaceae isolated at the Institut Pasteur de Bangui and to characterize their bla_TEM, bla_SHV, and bla_CTX_-M genes.

From January 2003 to March 2005, all Enterobacteriaceae isolated from human specimens at the Institut Pasteur de Bangui were screened for ESBLs. Antimicrobial drug susceptibility was determined by using the disk diffusion method (Bio-Rad, Marnes la Coquette, France) on Mueller-Hinton agar (MHA) and interpreted according to the recommendations of the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM) (http://www.sfm.asso.fr). ESBL-producing Enterobacteriaceae were selected by the following criteria: susceptibility to cefoxitin; decreased susceptibility to cefotaxime (30 μg), ceftazidime (30 μg), or cefepime (30 μg) (zone diameter <21 mm); and enhanced susceptibility in the presence of clavulanic acid by the double disk synergy test (3). For suspected ESBLs, the MICs of broad-spectrum cephalosporins were determined by using the agar dilution method.

We screened 450 Enterobacteriaceae for ESBLs during the study. We isolated and identified 17 (4%) ESBL-producing strains (Table). These strains were associated with urinary tract infection, pneumonia in an AIDS patient, wound infection, vaginal or intestinal colonization, and ear infection. We found that 11 isolates were more resistant to cefotaxime (MIC >256 μg/mL) than to ceftazidime (MIC <128 μg/mL), which suggests CTX-M–type enzymes. Enterobacteriaceae strains that harbor ESBLs were frequently associated with resistance to aminoglycosides and ciprofloxacin (Table).

The conjugal transfer of the resistance determinants was carried out in trypticase soy (TS) broth with rifampin-resistant Escherichia coli J53-2 as the recipient. Mating broths were incubated at 37°C for 18 h. Transconjugants were selected on MHA plates containing rifampin (250 μg/mL) and cefotaxime (2.5 μg/mL). If conjugal transfer failed, plasmid DNA was extracted from donors with the Qiagen Plasmid Mini Kit (Qiagen, Courtaboeuf, France); 20 μL of E. coli DH10B cells were transformed with plasmid DNA by electroporation according to the manufacturer's instructions (Bio-Rad). Transformants were incubated for 1.5 h at 37°C in TS broth and then plated on MHA plates supplemented with 2.5 μg/mL cefotaxime.

Plasmid-encoded β-lactamase genes were detected on clinical isolates and their tranconjugants or transformants by polymerase chain reaction with oligonucleotide primer sets specific for the bla_TEM, bla_SHV, and bla_CTX-M genes (4). PCR assays were performed on total DNA extracted by using the commercial Qiagen DNA Mini Kit. The 3 β-lactamase genes were detected in different clinical isolates (Table). PCR results showed that the strains were harboring >2 different types of β-lactamases.

Plasmid-encoded β-lactamase genes were characterized by direct DNA sequencing with PCR primers. The nucleotide sequences were analyzed by the BLASTN (nucleotide basic local alignment search tool) program. For ESBLs, the gene types (SHV-2a, SHV-12, CTX-M-15, and CTX-M-3) were identified from different Enterobacteriaceae (Table). Only 1 strain (Enterobacter aerogenes) harbored 2 different ESBLs (CTX-M-3 and SHV-12). We identified TEM-1 and CTX-M15 enzymes, which are the most prevalent β-lactamases detected in our strains.

ESBL-producing Enterobacteriaceae have been previously described in South Africa (5), Kenya (6), Senegal (7), Cameroon (8), Tanzania (9), and Nigeria (10). As described in these countries, we found that CTX-M-15, SHV-2a, and SHV-12 were the most prevalent enzymes. CTX-M-15, the most recently described ESBL type, is particularly common in Bangui and seems to be closely related to E. coli, as was previously observed in Tanzania (9). This finding is also the first report of CTX-M-3 in sub-Saharan Africa.

Multidrug resistance profiles involving non–β-lactam antimicrobial drugs coselected these ESBL-producing isolates. We suggest that the misuse of antimicrobial drugs in the Central African Republic and the migratory flux of regional populations could result in emergence and selection of these ESBL phenotypes in the community. We could not establish a relationship between the different strains isolated in hospitalized and ambulatory patients. Because of the implications for treating such infections, particularly in developing countries, the spread of ESBL-producing Enterobacteriaceae merits close surveillance in the Central African Republic.

This work was financed by grants from Institut Pasteur de Bangui, Faculté de Médecine Pierre et Marie Curie, Université Pierre et Marie Curie (Paris VI), and the European Community, contract LSHM-CT 2003-503335.

References

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Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C, Ould-Hocine Z, et al. Dissemination of CTX-M–type β-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother. 2004;48:1249–54.
Pitout JDD, Thomson KS, Hanson ND, Ehrhardt AF, Moland ES, Sanders CC. β-lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa. Antimicrob Agents Chemother. 1998;42:1350–4.
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Weill FX, Perrier-Gros-Claude JD, Demartin M, Coignard S, Grimont P. Characterization of extended–spectrum β-lactamase (CTX-M-15) producing strains of Salmonella enterica isolated in France and Senegal. FEMS Microbiol Lett. 2004;238:353–8.
Gangoue-Pieboji J, Miriagou V, Vourli S, Tzelepi E, Ngassam P, Tzouvelekis LS. Emergence of CTX-M-15-producing enterobacteria in Cameroon and characterization of a bla_CTX-M-15–carrying element. Antimicrob Agents Chemother. 2005;49:441–3.
Blomberg B, Jureen R, Manji KP, Tamim BS, Mwakagile DSM, Urassa WK, et al. High rate of fatal cases of pediatric septicemia caused by gram-negative bacteria with extended-spectrum beta-lactamases in Dar es Salaam, Tanzania. J Clin Microbiol. 2005;43:745–9.
Soge OO, Queenan AM, Ojo KK, Adeniyi BA, Roberts MC. CTX-M-15 extended-spectrum β-lactamase from Nigerian Klebsiella pneumoniae. J Antimicrob Chemother. 2006;57:24–30. Epub 2005 Nov 30.

Table. Characteristics of extended-spectrum β-lactamase–producing Enterobacteriaceae in Bangui, Central African Republic

Strain†	Patient hospitalized	Results of sequencing			MICs of β-lactams (μg/mL)*							Resistance

		bla_CTX-M	bla_SHV	bla_TEM	AMC	CTX	CAZ	CRO	FEP	CPO	ATM

K. pneumoniae 022	N		SHV-2a	TEM-1	16	16	16	16	8	8	2	KGT
K. pneumoniae 043	Y		SHV-12	TEM-1	16	16	256	32	8	8	256	KGTNC
K. pneumoniae 106	Y	CTX-M-15	SHV-12	TEM-1	8	256	128	256	64	256	64	None
K. pneumoniae 047	Y	CTX-M-15	SHV-2a	TEM-1	64	16	16	16	8	16	32	None
E. coli 272	Y	CTX-M-15		TEM-1	32	256	128	256	128	256	128	KGTNC
E. coli 065	Y	CTX-M-15		TEM-1	20	256	128	256	64	256	128	C
E. coli 047	N	CTX-M-15		TEM-1	16	256	32	256	32	128	64	KGTC
E. coli 010	N	CTX-M-15		TEM-1	32	256	128	256	128	256	256	KGT
E. coli 073	N	CTX-M-15		TEM-1	16	256	128	256	128	256	128	KGTC
E. coli 059	Y	CTX-M-15		TEM-1	19	256	128	256	8	256	256	C
E. coli 064	N	CTX-M-15		TEM-1	128	256	128	256	64	256	64	C
E. coli 070	N	CTX-M-15		TEM-1	128	256	128	256	64	256	128	C
E. coli 054	N	CTX-M-15		TEM-1	128	256	32	256	64	256	32	KGTC
E. coli 026	N	CTX-M-15		TEM-1	32	256	64	256	128	256	256	KGTC
E. cloacae 081	Y		SHV-12	TEM-1	32	16	256	16	0.125	1	256	KGTN
E. cloacae 106	Y		SHV-12	TEM-1	128	16	256	16	32	8	256	KGT
E. aerogenes 014	Y	CTX-M-3	SHV-12	TEM-1	128	256	256	256	32	256	128	KGTN

*AMC, amoxicillin + clavulanic acid (2 μg/mL); CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; CPO, cefpirome; ATM, aztreonam; K, kanamycin; G, gentamicin; T, tobramycin; N, netilmicin; C, ciprofloxacin.
†On Klebsiella pneumoniae strains, polymerase chain reaction and sequencing for bla_SHV genes were studied on Escherichia coli transconjugant or electroporant.

Suggested citation for this article:
Frank T, Arlet G, Gautier V, Talarmin A, Bercion R. Extended-spectrum β-lactamase–producing Enterobacteriaceae, Central African Republic [letter]. Emerg Infect Dis [serial on the Internet]. 2006 May [date cited]. Available from http://www.cdc.gov/ncidod/EID/vol12no05/05-0951.htm

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