Detection of GES-5 Carbapenemase in Klebsiella pneumoniae, a Newcomer in France

LETTER

Carbapenemase-producing Klebsiella pneumoniae (CPKp) are increasingly reported worldwide and represent a major health threat (1). Carbapenemases frequently encountered in CPKp belong to Ambler's class A (KPC enzymes), class B (NDM, VIM, and IMP enzymes), and class D (OXA-48-like enzymes) (1). GES-1, is an extended-spectrum beta lactamase (ESBL) initially described in 2000 in a K. pneumoniae isolate from French Guiana (2). Hitherto, 31 variants have been identified (http://bldb.eu/BLDB.php?prot=A#GES), including several with carbapenem-hydrolyzing activity (GES-2, -4, -5, -6, -14, -14, -15, -16 -18, -20, and -24), which is due to a replacement of glycine at position 170 by an asparagine or a serine (3–5). GES carbapenemases have rarely been reported in Enterobacteriaceae (GES-5 and GES-6) (3, 6).

Here, we report the first description of a GES-5-producing K. pneumoniae isolate in France. This isolate was recovered from a man previously hospitalized in Bangkok, Thailand, for an acute ischemic cerebrovascular stroke. The patient was transferred to the hospital of Le Havre, France, where a rectal swab upon admission revealed K. pneumoniae isolate MON with reduced susceptibility to carbapenems (MICs of imipenem, meropenem, and ertapenem were 0.25, 0.38, and 0.5 μg/ml, respectively). This isolate was also resistant to penicillins, all cephalosporins, cephamycins, aztreonam, fluoroquinolones, aminoglycosides (gentamicin, netilmicin, and tobramycin), sulfamethoxazole-trimethoprim, chloramphenicol, and tetracycline. This isolate remained susceptible to amikacin (MIC of 1 μg/μl) and colistin (MIC of 0.5 μg/μl). The biochemical test Carba NP revealed a carbapenemase activity in K. pneumoniae MON (7). This carbapenem-hydrolyzing activity was confirmed by several other confirmatory assays, such as by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (8), by the commercially available β Carba test (Bio-Rad, Marnes-la-Coquette, France) (9), and by the recently described carbapenem inactivation method (CIM) (10). However, PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae (bla_NDM, bla_IMP, bla_VIM, bla_KPC, bla_OXA-48), as previously described (11).

Whole-genome sequencing using Illumina's technology was performed (paired-end reads of 150 bp) as previously described (12). A total of 2,352,841 reads were mapped, allowing us to build 206 contigs with an average length of 26,670 bp (from 500 to 509,094 bp), representing 5,493,972 bp (mean coverage, 64-fold). The ResFinder Web server (13) revealed three β-lactamases (SHV-11, SHV-12, and GES-5) and eight aminoglycoside-modifying enzymes (AMEs), as well as genes involved in resistance to phenicols, trimethoprim, sulfonamides, and erythromycin (Table 1). High-level resistance to fluoroquinolones resulted from mutations in chromosomally encoded gyrase (S83F and D87Y in the GyrA subunit) and topoisomerase IV (S79I in the ParC subunit). In addition, a frameshift in the ompK35 gene leading to a stop codon at position 32 (K32Stop) and two insertions in OmpK36 (TS at position 183 and FSGNGE at position 264) likely result in inactive porins, thus resulting in decreased susceptibility to carbapenems, as previously shown (14). Multilocus sequence typing (MLST; https://cge.cbs.dtu.dk/services/MLST/) analysis using the Pasteur Institute's MLST scheme (15) revealed that this isolate belonged to sequence type 42 (ST42; 2-6-1-3-8-1-15), an ST type described in South Asia (16). This ST does not belong to an epidemic clone but seems to have played a role in the genesis of ST258 (17, 18).

TABLE 1.

Resistome of K. pneumoniae MON

Antibiotic family	Resistance gene	% identity	Accession no.	Enzyme		In silico-deduced resistancec	Observed resistance
				Name	Function
β-Lactams	blaGES-5	100.0	AF462395	GES-5	Carbapenemase	All β-lactams except AZT	All beta-lactams
	blaSHV-12	100.0	DQ236171	SHV-12	ESBL	All β-lactams except carbapenems and cephamycins
	blaSHV-11	100.0	X98101	SHV-1	Penicillinase	AMX, TIC
Aminoglycosides	aadA1	100.0	JN815078	ANT(3″)-Ia	Adenyltransferase	STR, SPE	All tested aminoglycosides except amikacin
	strA	100.0	M96392		Phosphotransferase	STR
	strB	100.0	M96392		Phosphotransferase	STR
	aadA2	100.0	JQ364967	ANT(3″)-Ia	Adenyltransferase	STR, SPE
	aph(3′)-Ia	100.0	V00359	APH(3′)-Ia	Phosphotransferase	KAN, NEO
	aacA34	100.0	AY553333	AAC(6′)-I	Acetyltransferase	KAN, TOB, NET, AMI
	aac(3)-IId	99.9	EU022314	AAC(3′)-IIb	Acetyltransferase	GEN, TOB, NET
	aac(6′)-II	99.6	Z54241		Acetyltransferase	GEN, TOB, NET
Quinolones	gyrA	98a	NZ_CDMU01000001	GyrA	Gyrase	CIP, LVX, NA	Fluoroquinolones
	parC	99a	NZ_CDMU01000001	ParC	Topoisomerase	CIP, LVX, NA
Macrolides, lincosamides, and streptogramins	mph(A)	100.0	D16251	MphA	Phosphotransferase	ERY	Macrolidesb
Phenicols	catA1-like	99.9	V00622	CatA1-like	Acetyltransferase	CHL	Chloramphenicol
Sulfonamides	sul1	100.0	CP002151	Sul1	Efflux	SUL	Sulfonamide
Cyclins	tet(A)	99.9	AJ517790	TetA	Efflux	TET	Tetracycline
Trimethoprim	dfrA12	100	AB571791	DfrA12	Dihydrofolate reductase	TRI	Trimethoprim

^aPercent identity to a quinolone-susceptible strain, i.e., Klebsiella pneumoniae ATCC 43816.

^bIntrinsic resistance.

^cAMX, amoxicillin; AZT, aztreonam; CHL, chloramphenicol; CIP, ciprofloxacin; ERY, erythromycin; GEN, gentamicin; KAN, kanamycin; LVX, levofloxacin; NA, nalidixic acid; NEO, neomycin; NET, netilmicin; STR, streptomycin; SPE, spectinomycin; SUL, sulfonamide; TET, tetracycline; TIC, ticarcillin; TOB, tobramycin; TRI, trimethoprim.

Mating-out assays performed as previously described (6) yielded transconjugants possessing both bla_SHV-12 and bla_GES-5 genes or possessing only bla_SHV-12 genes, but none possessed only the bla_GES-5 gene, suggesting that plasmid pMON-GES-5 may be mobilized but not self-transferable. The conjugation frequency for the pMON-SHV-12 plasmid was 1.9 × 10⁻⁴ transconjugants per donor cell, and the presence of the bla_GES-5 gene was found in 42% (21/50) of the transconjugants. Plasmid extraction, performed as previously described (19), revealed the presence of two plasmids of ca. 120 kb (carrying the bla_SHV-12 gene) and 70 kb (carrying the bla_GES-5 gene) in size, determined as described previously (20). Close genetic context analysis revealed that bla_GES-5 was carried by a class 1 integron in the first position. Promoter analysis revealed two promoters, PcH1 (TGGACA-N₁₇ -TAAACT) and P2 (TTGTTA-N₁₄ -TACAGT) upstream of the bla_GES-5 gene. The latter is likely inactive due to a shorter spacer (n = 14).

This report constitutes the first identification of a GES-5-producing Enterobacteriaceae isolate in France (21). This report highlights the usefulness of biochemical tests for the identification of minor carbapenemases (Carba NP test, MALDI-TOF techniques, CIM, or BYG test), as the commercially available tests fail to detect them (22).