Molecular and Biochemical Characterization of a Novel Class A β-Lactamase (HER-1) from Escherichia hermannii

Abstract

Escherichia hermannii showed a low level of resistance to amoxicillin and ticarcillin, reversed by clavulanate, and a moderate susceptibility to piperacillin but was susceptible to all cephalosporins. A bla gene was cloned and encoded a typical class A β-lactamase (HER-1, pI 7.5), which shares 45, 44, 41, and 40% amino acid identity with other β-lactamases, AER-1 from Aeromonas hydrophila, MAL-1/Cko-1 from Citrobacter koseri, and TEM-1 and LEN-1, respectively. No ampR gene was detected. Only penicillins were efficiently hydrolyzed, and no hydrolysis was observed for cefuroxime and broad-spectrum cephalosporins. Sequencing of the bla gene in 12 other strains showed 98 to 100% identity with bla_HER-1.

Escherichia hermannii was initially considered to be an Escherichia coli-like biogroup, but was subsequently distinguished from E. coli on the basis of DNA-DNA homology, conventional biochemical tests (4), electrophoretic polymorphism of enzymes (11), and ribosomal DNA restriction fragment length polymorphism (22). In the clinical microbiology laboratory, E. hermannii can be distinguished from E. coli by its production of yellow pigment and by various biochemical characteristics (4, 10). Moreover, E. hermannii is distinguished from other Escherichia species by its natural resistance to penicillins, ampicillin, and carbenicillin and its sensitivity to other commonly used β-lactam antibiotics (10); this is also the case of other Enterobacteriaceae, such as Klebsiella pneumoniae and Klebsiella oxytoca (16). In a preliminary study, β-lactamases of E. hermannii were characterized by their susceptibility to inhibition by clavulanate and their pI values (10). We report the biochemical properties of the purified E. hermannii enzyme (HER-1). The nucleotide sequence of the gene, its variability, and the deduced amino acid sequence were determined and compared to those of other class A β-lactamases.

Bacterial strains.

Thirteen strains of E. hermannii were studied (Table 1). All the strains were identified by their biochemical characteristics using the API 20E System (bioMérieux, Marcy l'Etoile, France) and by 16S ribosomal DNA sequencing of PCR products obtained with the universal 16S rRNA primers Ad and rB (24).

TABLE 1.

Characteristics, source, and pI of β-lactamase of E. hermannii strains

Strain	Relevant characteristic, origin	Sourcea	pI (β-lactamase)
CIP 103176 T	Reference strain	P. Grimont, Institut Pasteur	7.5 (HER-1)
CIP 104945	Reference strain	P. Grimont, Institut Pasteur	7.5 (HER-2)
134-87	Clinical isolate	P. Grimont, Institut Pasteur	7.5 (HER-1)
50-82	Clinical isolate	P. Grimont, Institut Pasteur	7.7 (HER-4)
1200-74	Clinical isolate	P. Grimont, Institut Pasteur	7.5 (HER-1)
5402-97	Clinical isolate	P. Grimont, Institut Pasteur	7.5 (HER-1)
9904662	Clinical isolate	P. Grimont, Institut Pasteur	7.7 (HER-5)
1-84	Shrimp (Thailand)	P. Grimont, Institut Pasteur	8.0 (HER-8)
12-65	Clinical isolate, Saint-Etienne (France)	P. Grimont, Institut Pasteur	7.2 (HER-7)
1-87	Clinical isolate, Paris (France)	P. Grimont, Institut Pasteur	7.5 (HER-2)
2-89	Clinical isolate, Creteil (France)	P. Grimont, Institut Pasteur	8.1 (HER-6)
RAV	Clinical isolate, Paris (France)	Saint-Louis Hospital	7.5 (HER-3)
REG	Clinical isolate, Paris (France)	Saint-Louis Hospital	7.5 (HER-2)

^aAll are in Paris, France.

Antibiotic susceptibility testing, conjugation experiments, and plasmid analysis.

MICs were determined on Mueller-Hinton agar (Bio-Rad SA, Marnes-la-Coquette, France) containing serial twofold dilutions of antibiotics. Plates were inoculated with a Multipoint Inoculator A400 (Denleytech, Woking, United Kingdom) and an inoculum of 10⁵ CFU per spot. The plates were incubated at 37°C for 18 h. The MICs of some β-lactams were determined both alone and in combination with clavulanate (2 μg/ml) or tazobactam (4 μg/ml). All the strains showed low-level resistance to amoxicillin (MICs, 8 to 16 μg/ml) and ticarcillin (MICs, 16 to 64 μg/ml) and a moderate susceptibility to piperacillin (MICs, 0.25 to 1 μg/ml) (Table 2). Combination of amoxicillin or ticarcillin with clavulanate was highly synergistic, with MICs falling by 64- to 128-fold as previously described (10); tazobactam also reduced piperacillin MICs. All the strains were susceptible to cephalothin, cefuroxime, cefoxitin (Table 2), and broad-spectrum cephalosporins (MICs, <0.06 μg/ml), aztreonam (MICs, <0.06 μg/ml), and imipenem (MICs, 0.06 to 0.12 μg/ml). The β-lactam susceptibility pattern of E. hermannii was similar to that of K. pneumoniae, K. oxytoca, Citrobacter koseri, and Serratia fonticola (16, 19, 21). Susceptibility to cefuroxime distinguished E. hermannii from Rahnella aquatilis, Citrobacter sedlakii, and Proteus vulgaris (2, 20, 21). These results suggested that E. hermannii produces a class 2a β-lactamase (5).

TABLE 2.

MICs of 6 β-lactams for the differents strains of E. hermannii, E. coli XL-1 recipient strain (R−), and E. coli XL-1 harboring the recombinant plasmid pBK-EH-2 (R+)

β-Lactam(s)a	No. of isolates for which MIC (μg/ml) was:												MIC (μg/ml) for E. coli XL-1
	≤0.06	0.125	0.25	0.5	1	2	4	8	16	32	64	128	R+	R−
Amoxicillin								8	5				1,024	4
Amoxicillin + CA	1	11	1										16	1
Ticarcillin									1	8	4		2,048	0.5
Ticarcillin + CA	1	5	5	2									32	0.125
Piperacillin			3	4	6								16	1
Piperacillin + TZ	11	2											0.25	0.12
Cephalothin			2	9	2								8	0.5
Cefuroxime		9	4										2	1
Cefoxitin		7	4	2									2	1

^aCA, clavulanic acid (2 μg/ml); TZ, tazobactam (4 μg/ml).

The technique of Clowes and Hayes (7) was used for conjugation assays to transfer the bla gene from E. hermannii to the rifampin-resistant E. coli strain J53-2 (F⁻ met pro Rif^r). Repeated mating-out experiments on Drigalski agar (Bio-Rad SA) supplemented with ticarcillin (25 μg/ml) and rifampin (250 μg/ml) failed to transfer the β-lactam resistance to E. coli.

The plasmid DNA content of E. hermannii strains was studied by using the procedures of Birnboim and Doly (3), Kado and Liu (14), and Takahashi and Nagano (26). No plasmid was detected in any E. hermannii strain, suggesting that the β-lactamase gene was located on the chromosome.

Genetic characterization of the E. hermannii β-lactamase.

Genomic DNA from E. hermannii (strain CIP 103176 T) was digested with BamHI and ligated into the BamHI site of the pBK-CMV phagemid Kan^r (Stratagene, Amsterdam, The Netherlands). The recombinant plasmids were then transformed into E. coli XL1 (Stratagene). Transformants were selected on Drigalski agar (Bio-Rad) supplemented with kanamycin (25 μg/ml) and ampicillin (50 μg/ml). Recombinant plasmid DNA was recovered on Qiagen (Courtaboeuf, France) columns.

The recombinant plasmid DNA (pBK-EH-2) conferring a β-lactam resistance pattern compatible with that of the parental strain (Table 2) was selected. The insert of about 5.2 kb was sequenced on both strands by using the ABI prism dye terminator cycle sequencing ready reaction kit and ABI cycle sequencer A373 (Applied Biosystems/Perkin-Elmer, Foster City, Calif.).

The nucleotide and deduced amino acid sequences were analyzed with the BLASTN and BLASTP programs through the BLAST program of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Multiple sequence alignment of the deduced peptide sequence was carried out by using the Clustal W program (http://www.ebi.ac.uk). A phylogenetic tree was also derived from this alignment by using the Clustal W program. DNA sequence analysis revealed five open reading frames (ORF). ORF 1 (<1 to 570) showed 83% identity to a gene encoding a putative inner transmembrane protein of Salmonella choleraesuis. ORF 2 (210 to 1175) encoded a putative protein sharing less than 30% identity with a UV endonuclease of Bacillus cereus and Bacillus subtilis. ORF 4 (2501 to 3562) encoded a putative protein that shared 43% identity with a Salmonella enterica serovar Typhimurium DNA-repair ATPase. ORF 5 (4101 to 4727) had no homology with GenBank DNA sequences. ORF 3 (873 bp, nucleotides 1315 to 2188), encoded a 290-amino-acid protein (Fig. 1). Within this protein, a serine-threonine-phenylalanine-lysine tetrad (STFK) was found at position 70 to 73 (standard numbering system of Ambler), and the motif KTG was found at position 234 to 236. These sequences (STFK and KTG) are characteristic of β-lactamases possessing a serine-active site (1). Two other structural elements were also found: serine-aspartic acid-asparagine (SDN) in position 130 to 132 and glutamate-X-X-leucine-asparagine (EXXLN) in position 166 to 170, a region that is part of the omega loop of class A β-lactamases. E. hermannii strain CIP 103176T produced a typical Ambler class A β-lactamase (1) that we designated HER-1. As observed in TEM-1, TEM-2, LEN-1, OXY-1, and OXY-2 β-lactamases, an Ala residue was found in position 237. On the other hand, some TEM-type and SHV-type extended-spectrum β-lactamases, CTX-M variants, and chromosome-encoded enzymes of Kluyvera ascorbata (KLUA-1), Kluyvera cryocrescens (KLUC-1), Kluyvera georgiana (KLUG-1), P. vulgaris (RO104), S. fonticola (CUV), C. sedlakii (Sed-1), Citrobacter diversus (CdiA), R. aquatilis (RHAN-1), and Erwinia persinica (ERP-1) possess a Ser residue in this position, which is involved in extended activity towards cefuroxime and extended-broad-spectrum cephalosporins (2, 9, 12, 19, 20, 21, 23, 28). Moreover, HER-1 possess two cysteines in positions 77 and 123 as in TEM and SHV β-lactamases and as in few others; a disulfide bridge is also likely to be present in the E. hermannii β-lactamase.

FIG. 1.

Multiple alignment (according to the Ambler's numbering system) of the eight deduced HER amino acid sequences. Identical amino acids are indicated by dashes. The sites STFK, SDN, and KTG are boxed.

No putative LysR-type regulator gene was identified upstream of the gene encoding HER-1, whereas such chromosome-encoded AmpR regulators were divergently transcribed upstream of the gene coding for the class A β-lactamase of C. sedlakii, C. diversus, and P. vulgaris (8, 13, 21).

HER-1 showed less than 45% identity with other class A β-lactamases. The strongest identity was with AER-1 from Aeromonas hydrophila (45%) (25), MAL-1/Cko-1 from C. koseri (44%), TEM-1 (41%), LEN-1 from K. pneumoniae (40%), carbenicillin-hydrolyzing β-lactamases, and VHH-1 or VHW-1 (41 to 37%) (27) (Fig. 2). Identity with other chromosomal β-lactamases in enterobacteriaceae was less than 37%.

FIG. 2.

Schematic dendrogram obtained from 22 representative chromosomally encoded and plasmid-encoded class A β-lactamases. Percentages in brackets are percent identities between the indicated amino acid sequence and that of HER-1.

We then studied the variability of the bla gene of the E. hermannii strains by using two primers designed to amplify and the entire coding region: E.h. upper (5′ TAATGAAGCCGTTTTGCC 3′) and E.h. lower (5′ GTGTGACGCCTGTAGCC 3′). The PCR products were sequenced in both strands using PCR primers. Analysis of the deduced amino acid sequences showed little interstrain variability (≥98% identity); seven HER-1 variants, designated HER-2 to HER-8, were nonetheless identified (Table 1; Fig. 1).

Preparation of β-lactamase crude extracts and analytical isoelectric focusing.

Preparation of crude extracts and analytical isoelectric focusing were carried out as previously described (17, 18). The β-lactamase was revealed in the gel by the nitrocefin procedure or by the iodine procedure in the presence of benzylpenicillin (18). The pI of the enzyme was determined by using SHV-1 (p453; pI, 7.6), SHV-3 (pHuc; pI, 7), SHV-4 (pUD21; pI, 7.8), and SHV-5 (pAFF5; pI, 8.2) as reference β-lactamases. Analytical isoelectric focusing detected in all strains a single band of β-lactamase activity with a pI of ≥7.2 (7.2 to 8.1) (Table 1). The variability of the pIs was less extensive than that observed by Fitoussi et al. (10). Alcaline pIs were also observed for chromosomally encoded class A β-lactamases in enterobacteriaceae, such as K. pneumoniae (7.6), C. sedlakii (8.6), R. aquatilis (7.2), or Erwinia persicina (8.1).

β-Lactamase purification and kinetic measurements.

In order to determine the kinetic constants of HER-1, the cloned β-lactamase of E. hermannii CIP 103176T produced in E. coli XL1(pBK-EH-2) was overproduced. The enzyme was purified as previously described (18). The purification factor was about 100, and the purity of the enzyme was higher than 95%. The kinetic constants K_m and k_cat for substrates were determined in a computerized microacidimetric assay at pH 7.0 and 37°C in 0.1 M NaCl as described by Labia et al. (15).

One β-lactamase activity unit is defined as the amount of enzyme that hydrolyzes 1 μmol of benzylpenicillin per min at pH 7 and 37°C. We used an initial benzylpenicillin concentration of 500 μM. Enzyme kinetics was determined with the highly purified enzyme (Table 3). The enzyme was a narrow-spectrum β-lactamase, as only penicillins were efficiently hydrolyzed. Surprisingly, the corresponding K_m values were low, suggesting high affinity, with values ranging from 5 to 15 μM, i.e., two to four times lower than those reported for TEM-1 (6). Low hydrolytic activity was found for cephalothin, cephaloridine, and cefoperazone, with high K_ms for cephalothin and cephaloridine and a low K_m for cefoperazone. No significant hydrolysis was observed with cefuroxime and broad-spectrum cephalosporins, as suggested by the deduced amino acid sequence of HER-1. Similarly, carbapenems were not hydrolyzed (data not shown). Fifty-percent inhibitory concentrations determined after 10 min of incubation using benzylpenicillin (500 μM) as a reporter substrate showed that HER-1 activity was efficiently inhibited by clavulanic acid (fifty-percent inhibitory concentration = 0.08 μM). The experimental results were comparable to those reported for TEM-1 (6).

TABLE 3.

Kinetic parameters of purified β-lactamase HER-1

Substrate	kcat (s−1)a	Km (μM)	kcat/Km (μM s−1)
Benzylpenicillin	400b	9.0	44
Amoxicillin	250	14.6	17
Ticarcillin	185	5	37
Piperacillin	108	5.7	18.9
Cephalothin	6.8	850	0.008
Cefoperazone	5.2	10.7	0.5
Cefuroxime	—c	>500	NAd
Cefotaxime	—c	>500	NAd
Ceftazidime	—c	>500	NAd

^aFor compounds with a k_cat lower than 10 s⁻¹, K_i values were determined instead of K_m values, using benzylpenicillin as the substrate.

^bStandard deviations for k_cat values were 15%, and standard deviations were about 20% for K_m values. Each determination was made at least in triplicate.

^ck_cat of <0.05 S⁻¹.

^dNA, not applicable.

In conclusion, this work identified a novel chromosome-encoded class A β-lactamase from E. hermannii, with a narrow spectrum limited to penicillins which shared less than 45% amino acid identity with other chromosome-encoded class A β-lactamases from enterobacteriaceae.

Nucleotide sequence accession numbers.

The bla_HER nucleotide sequences have been submitted to the EMBL nucleotide sequence database. Their accession numbers are the following: AF311385 for bla_HER-1 (CIP 103176T), AF398334 for bla_HER-2 (CIP 104945), AF 398335 for bla_HER-3 (clinical isolate RAV), AJ536088 for bla_HER-4 (strain 50-82), AJ536089 for bla_HER-5 (strain 9904662), AJ536090 for bla_HER-6 (strain 2-89), AJ536091 for bla_HER-7 (strain 12-65), and AJ536092 for bla_HER-8 (strain 1-84).