Dominance of bla_CTX-M within an Australian Extended-Spectrum β-Lactamase Gene Pool

Abstract

bla_CTX-M genes, particularly bla_CTX-M-15, are the dominant extended-spectrum β-lactamase (ESBL) genes among clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sydney, Australia, where we also found one example of bla_CTX-M-62, encoding a novel enzyme conferring ceftazidime resistance. ESBL genes were present in diverse community isolates and in a variety of associated conjugative plasmids.

The dominant mechanism of resistance to expanded-spectrum cephalosporins and monobactams among members of the family Enterobacteriaceae is the production of Ambler class A extended-spectrum β-lactamases (ESBLs), with more than 200 variants described (23). SHV-type ESBLs have been sporadically reported in Klebsiella pneumoniae isolates from Australia (24, 27), and a single isolate from Queensland, Australia was reported to carry a bla_CTX-M-3-like gene in a study of ESBLs in invasive K. pneumoniae from 1996 to 1997 (24). SHV- and TEM-type ESBLs were dominant all over the world in members of the family Enterobacteriaceae during the 1990s (3, 5) but now appear less important than the widely distributed CTX-M enzymes (1, 5).

Reduced susceptibility (MIC ≥ 2 μg/ml) (Phoenix NMIC/ID-101 panel; Becton, Dickinson & Co., Franklin Lakes, NJ) to cefotaxime (CTX) and/or ceftazidime (CAZ) was observed in 206 of 9,946 Escherichia coli (2.1%) and 64 of 1,391 K. pneumoniae clinical isolates (4.6%) submitted to our laboratory from four regional hospitals and two associated community clinics in the western Sydney area of New South Wales, Australia, from March 2005 to May 2007. Of these, 81 randomly selected isolates (61 E. coli and 20 K. pneumoniae isolates) from different patients had been stored and were screened for bla_CTX-M, bla_SHV, and bla_TEM genes by PCR (Table 1).

TABLE 1.

PCR primers used in this study

Primer	Sequence (5′-3′)a	Target	GenBank accession no.	Position	Reference
CTX-M-U1	ATGTGCAGYACCAGTAARGTKATGGC	blaCTX-M genes	AY458016	20993-20218	18
CTX-M-U2	TGGGTRAARTARGTSACCAGAAYCAGCGG			20426-20454
CTXM1-F	AAAAATCACTGCGCCAGTTC	blaCTX-M-1 group	AY458016	21202-21221	30
CTXM1-R	AGCTTATTCATCGCCACGTT			20807-20826
CTXM2-F	CGACGCTACCCCTGCTATT	blaCTX-M-2 group	X92507	49-67	30
CTXM2-R	CCAGCGTCAGATTTTTCAGG			581-600
CTXM8-F	TCGCGTTAAGCGGATGATGC	blaCTX-M-8 group	AF189721	285-304	30
CTXM8/25-R	AACCCACGATGTGGGTAGC			954-973
CTXM9-F	CAAAGAGAGTGCAACGGATG	blaCTX-M-9 group	AF174129	6343-6364	30
CTXM9-R	ATTGGAAAGCGTTCATCACC			6528-6547
CTXM25-Fb	GCACGATGACATTCGGG	blaCTX-M-25 group	AF518567	2673-2689	30
42bp-F	GGATTGACCGTATTGGGAGTT	blaCTX-M-9 group	AF252622	1716-1736	This work
5′orf903-R	CGGTTGATGAGGGCTTTATT	IS903	AF252622	2738-2757	This work
5′orf3-R	GGCGGAAACAATGAGAAAAC	ORF3	AF252622	7478-7497	10
orf477-F	GGTGGCATAATTTTTGAAGT	ORF477	AY458016	20151-20170	This work
ISEcp1IR-F	CAATGTGTGAGAAGCAGTCTAAA	Near IRR ISEcp1	AY458016	21332-21354	This work
CR1-F	ACAAATCGGAAGGTCTCG	ISCR1	AF174192	5702-5719	11
SHV-F	CGCCGGGTTATTCTTATTTGTCGC	blaSHV and adjacent regions	X98101	3-27	21
SHV-R	TCTTTCCGATGCCGCCGCCAGTCA			995-1018
FIN	ATTCTTGAAGACGAAAGGGC	blaTEM and adjacent regions	AY458016	23841-23860	4
DEB	ATGAGTAAACTTGGTCTGAC			24913-24912
blaTEM-F	GAGTATTCAACATTTTCGT	blaTEM	AY458016	24052-24070	16
blaTEM-R	ACCAATGCTTAATCAGTGA			24890-24908
VEB-F	CGACTTCCATTTCCCGATGC	blaVEB	AF010416	343-362	19
VEB-R	GGACTCTGCAACAAATACGC			985-966
GES-1A	ATGCGCTTCATTCACGCA	blaGES	AF156486	1332-1349	25
GES-1B	CTATTTGTCCGTGCTCAG			2195-2178
BES-1F	AGCGGCGAGAGTTACAGCTA	blaBES	AF234999	343-362	This work
BES-1R	AGAGGATGGCGATATCGTTG			931-912
SFO-F	GTTCGGTAGCGCACCATTAT	blaSFO	AB003148	1477-1496	This work
SFO-R	TTGCCCAAAGTTAGGGTTTG			2028-2009
PER-UF	CCTGACGATCTGGAACCTTT	blaPER	Z21957	645-666	This work
PER-UR	TCATCGASGTCCAGTTTTGA			1055-1036
CA1	ATGTCGCASAYHGAAAATGC	IncFII copA or oriR	AY458016	88558-88577	22
OR1	CCTTGCAGTTWWHTGTGRRTAA			90150-90171

^aH = A, C, or T; K = G or T; M = A or C; R = A or G; S = C or G; W = A or T; Y = C or T.

^bPaired with CTXM8/25-R.

The majority (50 of 61 E. coli isolates and 10 of 20 K. pneumoniae isolates) yielded amplicons with bla_CTX-M universal primers (18). Subsequent multiplex PCR (30) and analysis of sequences (ABI PRISM 3100 genetic analyzer; Applied Biosystems, Foster City, CA) revealed genes encoding CTX-M-3 (n = 4), CTX-M-15 (n = 33), and CTX-M-62 (n = 1) from the CTX-M-1 group and genes encoding CTX-M-9 (n = 3), CTX-M-14 (n = 17), CTX-M-24 (n = 2), and CTX-M-27 (n = 1) from the CTX-M-9 group (Table 2) (bla_CTX-M-9 and bla_CTX-M-14 coexisted in one isolate).

TABLE 2.

Isolates with ESBL genes

Speciesa	Isolate (JIE)b	Drug resistance profilec	blaTEM variantd	blaSHV variant	Inc groupe
Isolates with blaCTX-M-15 (n = 33)
E. coli B2	101	CTX CAZ GENTOBSXT CIP	1b		FIA+FIB+FII+N
E. coli B2	118,157,224	CTXCAZGENTOBSXT CIP	−, 1b, 1b		FIA+FII
E. coli B2	186/295	CTXCAZGENTOB SXT CIP	1b/1b		FII
E. coli B2	250	CTXCAZGENTOB FOX SXT CIP	1b		FIA+FIB+FII
E. coli D	085,166,204	CTXCAZGENTOB FOX SXT CIP	1b, −, −		FIA+FIB+FII
E. coli B2	106, 110	CTX CAZ GEN TOB SXT	1a, 1b		−
E. coli D	236/242	CTXCAZGEN TOB SXT	1b/1b		I1
E. coli D	174	CTX CAZ GEN TOB CIP	1b		I1
E. coli A	189, 291	CTX CAZ GEN TOB CIP	−, 1i		−
E. coli B2	188	CTX CAZ GEN TOB CIP	1b		−
E. coli B2	100	CTXCAZ CIP	1b		FII
E. coli B2	143	CTXCAZ CIP	1b		ND
E. coli A	222	CTX CAZ CIP	1b		−
E. coli B2	097/154/286	CTXTOBSXT CIP	1b/−/−		FIA+FII
E. coli D	134	CTXCAZGENTOB FOX CIP			FIA+FIB+FII
E. coli D	098	CTXCAZGEN TOB FOX CIP	1i		FIA+FIB+FII
E. coli B1	113	CTXCAZ SXT CIP	1b		I1
E. coli D	139	CTXCAZ SXT CIP	1b		I1
E. coli B2	289	CTX GEN TOB CIP			−
K. pneumoniae	120/127,146	CTXCAZGENTOBSXT CIP	1b (all)	11 (all)	ND
K. pneumoniae	162f	CTX CAZ GEN TOB FOX SXT CIP	1b	11, 12	−
Isolates with blaCTX-M-3 (n = 4)
E. coli D	161	CTX GEN TOB FOX SXT CIP	1b		FII+B
E. coli D	095	CTX GEN TOB FOX SXT	1b		−
E. coli D	077	CTX GEN FOX SXT CIP	1b		N
E. coli B2	251	CTX GEN CIP	1b		FII+Y
Isolate with blaCTX-M-62 (n = 1)
K. pneumoniae	137	CAZSXT	1b	1	ND
Isolates with blaCTX-M-14 (n = 17)
E. coli A	081	CTX GEN TOB SXT	1b		FII
E. coli D	084g, 110b, 196	CTX GEN TOB SXT	1b, 1b, 1i		FII
E. coli D	182	CTX GEN TOB SXT	1b		B
E. coli D	153, 180	CTX SXT CIP	−, 1b		FII
E. coli D	201	CTX GEN TOB FOX SXT CIP	1b		K
E. coli D	168	CTX GEN TOB SXT CIP	1b		ND
E. coli D	121	CTX GEN TOB FOX CIP	1b		−
E. coli A	088	CTX GEN CIP	1b		I1
E. coli B2	052	CTX CAZ GEN TOB SXT	1b		B
K. pneumoniae	014/021/025/056	CTXGEN TOB SXT	1b (all)	11 (all)	ND
K. pneumoniae	223	CTX		11	FII
Isolates with blaCTX-M-9 (n = 3)
E. coli D	059/084g/277	CTX GEN TOBSXT	−/1b/−		FIB
Isolates with blaCTX-M-24 (n = 2)
E. coli D	216	CTX GEN TOB FOX SXT CIP	1b		FII
E. coli B2	298	CTX			FII
Isolate with blaCTX-M-27 (n = 1)
E. coli A	058	CTXCAZGEN TOB FOX SXT CIP	1b		FII
Isolates with blaSHV-12 as the only ESBL gene (n = 6)
E. coli D	163	CAZSXT	1b	12	FIB
E. coli B2	038	CAZ SXT	1b	12	−
E. coli D	119	CTX CAZ GEN TOB FOX SXT CIP	1b	12	−
E. coli B2	124	CAZGENTOBSXT		12	A/C
K. pneumoniae	024	CTX CAZ GEN TOB FOX SXT CIP	1b	1, 12	−
K. pneumoniae	205	CTX CAZ GEN TOB FOX SXT CIP	1b	11, 12	−

^aE. coli phylogenetic groups are shown.

^bJIE isolates (shown in the table without JIE prefix) with identical DNA fingerprints (pulsed-field gel electrophoresis) are separated by slashes (e.g., 097/154/286), and JIE isolates with dissimilar DNA fingerprints are separated by commas. JIE isolates from which the ESBL gene was transferred to E. coli by conjugation are underlined. −, absence of bla_TEM gene.

^cIsolates were not susceptible by NCCLS/CLSI guidelines (20) to drugs unless left blank. None were resistant to imipenem or amikacin. Resistance phenotypes transferred to E. coli by conjugation are underlined. CTX, cefotaxime; CAZ, ceftazidime; GEN, gentamicin; TOB, tobramycin; FOX, cefoxitin; SXT, trimethoprim-sulfamethoxazole; CIP, ciprofloxacin.

^dbla_TEM-1i is identical to bla_TEM in GenBank accession no. EF035590 (E. coli, India), a C228T variant of bla_TEM-1a (e.g., EMBL accession no. X54604); slashes and commas reflect identity relationships as in footnote a above.

^eInc group, incompatibility groups of conjugative plasmids; ND, Inc group not determined; −, no plasmid transferred.

^fNeither bla_CTX-M-15 nor bla_SHV-12 was transferred from isolate JIE162 by conjugation.

^gThe JIE084 isolate carried both bla_CTX-M-9 and bla_CTX-M-14; only bla_CTX-M-14 was found in the IncFII-positive transconjugant.

The CTX-M-3 enzymes identified in this study are encoded by the first reported and now widespread bla_CTX-M-3 gene (e.g., GenBank accession no. Y10278) (here designated bla_CTX-M-3a) (12), which is closely related to bla_CTX-M-15. A novel variant of CTX-M-3 (Pro167Ser) has the CAZ resistance characteristic of this substitution (26) and was designated CTX-M-62. Ceftazidime resistance, but not cefotaxime resistance, was transferred to E. coli with bla_CTX-M-62 on a conjugative plasmid, but the incompatibility group of the plasmid could not be determined. bla_CTX-M-62 is a G509T variant of a bla_CTX-M-3 gene, here designated bla_CTX-M-3b (e.g., GenBank accession no. AB059404), previously reported from Asia, which differs from bla_CTX-M-3a at 8 nucleotide positions (Table 3). Additional novel (silent) bla_CTX-M variants were seen (Table 3): bla_CTX-M-9b is a C109T variant of all previously deposited bla_CTX-M-9 sequences (e.g., GenBank accession no. AF174129) and bla_CTX-M-24 variants, including bla_CTX-M-24a (e.g., GenBank accession no. AY143430) and the novel bla_CTX-M-24e with AGG at codon 275. Using primers located in ISEcp1 (ISEcp1IR-F) and ISCR1 (CR1-F) combined with primers located in bla_CTX-M-1 group genes (CTXM1-R) and bla_CTX-M-9 group genes (CTXM9-R), bla_CTXM-9 was found adjacent to ISCR1, while all other bla_CTX-M genes were associated with ISEcp1, as expected.

TABLE 3.

CTX-M gene variants

blaCTX-M gene varianta	Original GenBank accession no.b	Reported location(s)	Variation(s)c
3a	Y10278	Various
3b	AB059404	Japan, Taiwan	8 nt
15a	AY044436	Various
62	EF219134	Australia
9a	AF174129	Various
9b	EU418915	Australia	C109T
14	AF252622	Various
24a	AY143430	Mainland China, Taiwan	823-825 CGC
24b	AJ972953	France	823-825 CGT
24c	DQ343293	Mainland China	823-825 AGG; G153A
24d	EF374096	Latin America	823-825 AGA
24e	EU418918	Australia	823-825 AGG
27	AY156923	France, Australia

^aLetters designated in order of identification; other variants of some genes exist but are not relevant here.

^bAccession nos. listed in http://www.lahey.org/Studies/ are in bold typeface.

^cnt, nucleotides. Nucleotides 823 to 825 encode Arg at Ambler position 275 in CTX-M-24.

Four E. coli isolates yielded amplicons with bla_SHV primers, all found to be the ESBL gene bla_SHV-12. Sequencing of amplicons obtained from all 20 K. pneumoniae isolates suggested single bla_SHV genes in 17 (3 bla_SHV-1, 11 bla_SHV-11, and 1 each of bla_SHV-27, bla_SHV-28, and bla_SHV-109). SHV-109 is a novel variant most similar to SHV-61 (Thr268Met) and SHV-11 (Thr268Met with Leu10Arg in the signal peptide). The remaining three isolates appeared to have bla_SHV-12 plus another bla_SHV gene, with uncut and cut amplicons evident on electrophoresis after digestion with NheI (New England Biolabs, Ipswich, MA), which cuts at the position of a relevant sequence variation in bla_SHV-12. Separate sequencing of purified uncut and cut bands revealed that one isolate had both bla_SHV-12 and bla_SHV-1 and that two isolates had both bla_SHV-12 and bla_SHV-11. Fifty-nine isolates also had bla_TEM genes, all encoding (non-ESBL) TEM-1.

The 66 isolates (54 E. coli and 12 K. pneumoniae isolates) with bla_CTX-M (n = 60) and/or bla_SHV-12 (n = 7) (one isolate, JIE162, had both bla_SHV-12 and bla_CTX-M-15), were subjected to pulsed-field gel electrophoresis after XbaI (New England Biolabs) restriction of DNA purified from whole-cell extracts (14), and E. coli phylogenetic groups were assigned (7) (Table 2). Forty-eight unique strains were identified in this way among the 54 E. coli isolates, and 8 unique strains were identified from the 12 K. pneumoniae isolates.

Most (47/60) bla_CTX-M genes transferred on conjugative plasmids to rifampin-resistant E. coli DH5α(ΔlacZ) selected with rifampin (80 or 200 μg/ml) (Sigma, St. Louis, MO) plus ampicillin (80 μg/ml), CTX (2 μg/ml), or CAZ (2 μg/ml) by filter (29) and/or broth mating methods (9). Plasmid replicon typing of transconjugants as previously described (6), with an additional PCR for IncFII (22), revealed significant plasmid diversity (Table 2). Consistent with previous reports, multiple plasmid replicons were present in some transconjugants but IncF plasmids were numerically most important (13). All non-IncF amplicons and several IncF amplicons were sequenced, confirming the specificity of PCR typing. Several different HpaI (New England Biolabs) restriction patterns were observed among bla_CTX-M-15 and bla_CTX-M-14 plasmids (IncF and IncI1) extracted from transconjugants by alkaline lysis (28), but none matched the recently described epidemic IncFII plasmids in Europe (8) (not shown).

Three-quarters of the 66 isolates were not susceptible to gentamicin or tobramycin, and most were resistant to both (Table 2). Three-quarters were also resistant to trimethoprim-sulfamethoxazole. Aminoglycoside resistance was cotransferred with bla_CTX-M-15 particularly. Although more than 60% of the original isolates were ciprofloxacin resistant, this phenotype was not transferred to transconjugants (Table 2). Variable associations of bla_CTX-M-15 with genes conferring β-lactam and aminoglycoside resistance have been previously documented (2, 8), and further investigation is ongoing.

Nearly three-quarters of the 66 isolates with ESBL genes were recovered from urine. Two-thirds (35/54) of the E. coli isolates were from community-acquired infections, almost all of unique clonal type. K. pneumoniae isolates were more commonly (8/12) collected in the hospital setting and were less diverse (Table 2).

We detected no ESBL-type bla_SHV, ESBL-type bla_TEM, or bla_CTX-M in 15 isolates. Despite having reduced susceptibility to CTX or CAZ (MIC ≥ 2 μg/ml), there was no zone enhancement to suggest an ESBL in any of these isolates by disk approximation test (15, 17), and none of the several less common ESBL genes were detected by PCR (Table 1). The majority (13/15) were cefoxitin resistant, and most carried either a plasmid-borne ampC gene (bla_DHA or bla_CMY-2-like; n = 7) or a metallo-β-lactamase gene (bla_IMP-4; n = 3).

In summary, bla_CTX-M genes are well-established in the general community here, and bla_CTX-M-15 (and, to a lesser extent, bla_CTX-M-14) is particularly dominant despite the presence of novel local variants. Our data indicate that these genes, including bla_CTX-M-15, are associated with a variety of plasmid replicons and are present in a wide range of bacterial strains.

Nucleotide sequence accession numbers.

The nucleotide sequences of bla_CTX-M and bla_SHV genes from representative isolates have been submitted to GenBank under accession nos. EU418908 to EU418920. The bla_CTX-M-62 sequence is available under GenBank accession no. EF219134.