RAHN-2, a chromosomal extended-spectrum class A β-lactamase from Rahnella aquatilis

Abstract

Objectives

Rahnella aquatilis is an environmental enterobacterial species with a chromosomal bla_RAHN-1 gene encoding extended-spectrum class A β-lactamase RAHN-1. We describe the diversity of bla_RAHN genes from two groups of strains, G1 and G2, isolated from raw fruits and vegetables, and the new class A β-lactamase RAHN-2.

Methods

MICs were determined by Etest. bla_RAHN genes were amplified by PCR, sequenced, and cloned to produce RAHN-1 and RAHN-2 proteins whose kinetic parameters were determined.

Results

All strains had similar β-lactam resistance patterns. However, isolates of G1 were at least 2-fold more susceptible to piperacillin, amoxicillin, piperacillin/clavulanic acid, piperacillin/tazobactam and cefotaxime. Sequences of bla_RAHN from G1 had <82.9% identity with that of bla_RAHN-1, whereas those of G2 were >92% identical. The RAHN-2 β-lactamase was 89.8% identical to RAHN-1, 5-fold more efficient than RAHN-1 in hydrolysing ticarcillin and 2.5-fold more efficient in cefotaxime and cefuroxime hydrolysis. However, the specific activity of RAHN-1 was 2-fold higher than that of RAHN-2 suggesting that the bla_RAHN genes are regulated differently.

Conclusions

The new class A β-lactamase RAHN-2 is phenotypically difficult to detect and requires MIC determination.

phylogenetic diversity, Rahnella spp., antimicrobial resistance, environment, enterobacteria, class A ESBLs

Introduction

The study of chromosomal extended-spectrum β-lactamases (ESBLs) of class A from environmental enterobacterial species is important since certain of them are considered as the source of the corresponding genes that are found in enterobacteria causing human infections. The most striking example is the bla_CTX-M genes deriving from bla_KLUA genes found in Kluyvera spp., enterobacteria rarely isolated from humans, and widespread in enterobacterial human pathogens.¹

Another example of a class A ESBL gene is bla_RAHN-1 from Rahnella aquatilis,² which has apparently not yet been transferred to other enterobacteria despite the fact that R. aquatilis is widely distributed in nature and may be present in foods.^3,4 To date, the Rahnella genus comprises R. aquatilis, and Rahnella genomospecies 2 and 3. Recently, we have found that in a large sample of 399 raw fruits and vegetables, 13% of products carried Rahnella spp. (51 strains), which grouped into two phylotypes based on analysis of 16S rRNA and rpoB genes.⁴ The phylogenetic separation of these strains suggests there should also be heterogeneity in their chromosomal bla_RAHN genes.

We report the phylogenetic analysis of bla_RAHN genes, and describe the genetic and biochemical characterization of RAHN-2, a new class A ESBL from Rahnella sp.

Materials and methods

Strains and antibiotic susceptibility testing

Fifty-one Rahnella strains isolated from raw fruits and vegetables, and four R. aquatilis reference strains from the Collection Institut Pasteur (CIP 7865T, CIP 103904, CIP 105588 and CIP 105589) were studied.⁴ Susceptibility to antibiotics was determined by disc diffusion as recommended (www.sfm.asso.fr/publi/general.php?pa=1). MICs of antimicrobial agents were determined by Etest (AB Biodisk, Combourg, France).

DNA sequencing and cloning

Internal 721 bp fragments of bla_RAHN genes of the 55 isolates were amplified and sequenced using primers Rahn-up (5′-CTGGAAAAAGAAAGCGGCG-3′) and Rahn-down (5′-TCAATAACCCTGCGTCACA-3′). Genomic DNA from strains 9 and 42 were partially digested with Sau3A, ligated into pACYC184 DNA digested with BamHI, and electrotransformed into Escherichia coli DH10B (Bio-Rad, Marnes-la-Coquette, France). Clones were selected on Mueller–Hinton agar supplemented with 125 mg/L carbenicillin or 50 mg/L amoxicillin and 50 mg/L chloramphenicol. Plasmids pRAHN-1 [pACYC184(+)Ωbla_RAHN-1] and pRAHN-2 [pACYC184(+)Ωbla_RAHN-2] contained a fragment with bla_RAHN from strains 9 and 42, respectively.

Purification and kinetic characterization of RAHN-1 and RAHN-2

bla_RAHN was amplified using primer RahnF (5′-GGTGGTCTCCCATGAAAAATACCCTG-3′) for both strains 9 and 42, and Rahn-1R (5′-CTCCTCGAGATAACCCTGCGTCACAAT-3′) and Rahn-2R (5′-CTCCTCGAGATAACCCGGCGTCACAAT-3′) for strains 9 and 42, respectively. The PCR products were cloned in the pCR-Blunt vector and subcloned in pET28a(+) producing pAT912 [pET28a(+)Ωbla_RAHN-1] and pAT913 [pET28a(+)Ωbla_RAHN-2], respectively. The C-terminal His-tagged RAHN-1 and RAHN-2 proteins were purified as described previously.⁵ The N terminus was determined by microsequencing and the molecular mass by SELDI-TOF mass spectrometry.

The specific activity of RAHN-1 and RAHN-2 in crude extract, expressed as µmol of nitrocefin hydrolysed/min/mg of protein, was determined as described previously.⁶ The specific activity of purified RAHN-1 and RAHN-2 was determined with 100 µM cefalotin as previously used.² Kinetic parameters were determined as described previously.² The enzyme concentrations in the reaction mixture were in the range 0.2–20 µM. The steady-state kinetic parameters were determined using the Hanes–Woolf plot.⁷ The 50% inhibitory concentration (IC₅₀) of β-lactamase inhibitors was determined as described previously.⁸

Nucleotide sequence accession numbers

The sequences of bla_RAHN genes from 55 isolates (GU584880–GU584932) and of the inserts containing bla_RAHN from isolates 9 (GU645205) and 42 (HM114350) have been deposited in GenBank.

Results and discussion

Susceptibility testing

All Rahnella strains were resistant or of intermediate susceptibility to all β-lactams tested except for ceftazidime and imipenem (Table 1). Susceptibility was greater for strains of group 1 (G1) than for those of group 2 (G2). Addition of clavulanic acid or tazobactam reduced the MICs of β-lactams, indicating that RAHN-2 was a clavulanic acid-inhibited ESBL.

Table 1

MICs (mg/L) of β-lactams for the two groups of Rahnella strains in Figure 1, R. aquatilis CIP 7865T, Rahnella strains 9 and 42, and E. coli DH10B with and without plasmids pRAHN-1 and pRAHN-2

	MIC, average (range)
β-Lactam	group 1	group 2	R. aquatilis CIP 7865T	Rahnella strain 9	Rahnella strain 42	E. coli DH10B	E. coli DH10B (pRAHN-1)	E. coli DH10B (pRAHN-2)
Amoxicillin	>256	>256	>256	>256	>256	2	>256	>256
Amoxicillin/clavulanic acid (2/1)	1 (0.5–2)	2 (0.5–4)	1.5	2	0.5	2	3	3
Ticarcillin	>256	>256	>256	>256	>256	1	>256	>256
Ticarcillin/clavulanic acid (2 mg/L)	1 (1–4)	3 (1–4)	1	3	1	1	6	3
Piperacillin	8 (6–16)	12 (8–12)	3	16	8	1	32	16
Piperacillin/tazobactam (4 mg/L)	1 (0.75–2)	1.5 (1.5–2)	1.5	2	1	1	1.5	1.5
Cefotaxime	1 (1–4)	3 (1–4)	0.38	4	1	0.032	3	1
Cefotaxime/clavulanic acid (4 mg/L)	0.032	0.032	0.032	0.032	0.032	0.032	0.032	0.032
Ceftazidime	0.16 (0.094–0.5)	0.75 (0.25–0.75)	0.125	0.25	0.125	0.06	0.25	0.25
Aztreonam	0.16 (0.064–0.1)	0.75 (0.25–1)	0.064	0.75	0.125	0.06	0.38	0.38
Cefepime	0.22 (0.064–0.1)	0.38 (0.094–0.38)	0.047	0.38	0.25	0.032	0.38	0.38
Imipenem	0.25	0.25 (0.19–0.38)	0.38	0.25	0.25	0.06	0.25	0.25

bla_RAHN gene diversity

Phylogenetic analysis revealed that the bla_RAHN genes are separated into two phylogenetic groups (Figure 1): (i) G1 comprised 37 isolates (including CIP 108589) with identity levels with bla_RAHN-1 <82.9%; and (ii) G2 comprised 17 isolates (including CIP 103904 and CIP 105588) with levels of identity with bla_RAHN-1 >92%. This dichotomy was supported by high bootstrap values and was in agreement with the results of phylogenetic analyses obtained from the 16S rRNA/rpoB concatenated sequences of the Rahnella source strains, suggesting co-evolution of 16S rRNA/rpoB and bla_RAHN genes. One strain of each group, strains 42 and 9, was selected for further characterization of their β-lactamase.

Figure 1

Comparison of the unrooted neighbour-joining tree of bla_RAHN genes of 55 Rahnella strains [51 isolated from fruits and vegetables, and 4 reference strains (Rahnella genomospecies 1 CIP 105589, Rahnella aquatilis CIP 7865T, Rahnella aquatilis CIP 103904 and Rahnella genomospecies 2 CIP 105588)] (right) with that of their concatenated 16S rRNA and rpoB sequences (left). Values above the lines are bootstrap values expressed as percentages (only values >90% are shown). Scale bar, accumulated changes per nucleotide.

Cloning and sequencing of bla_RAHN genes

Recombinant plasmids pRAHN-1, and pRAHN-2 contained an insert of 2.3 kb from isolate 9 and of 2.6 kb from isolate 42, respectively. Both inserts contained an 888 bp open reading frame (ORF) with 99.5% and 83.1% identity with bla_RAHN-1 (accession number AF338038), respectively, for isolate 9 and 42. The 295 amino acid protein deduced from bla_RAHN-1 was identical to RAHN-1,² whereas RAHN-2 deduced from bla_RAHN-2 had 89.9% identity with RAHN-1. Apart from RAHN-1, the highest percentage identity was found with SFO-1 (75.2%, accession number BAA76882) from Serratia fonticola.

No other ORF could be identified upstream and downstream of those encoding RAHN-1 and RAHN-2. The 965 bp upstream of bla_RAHN-1 were 99.5% identical to those upstream of bla_RAHN-1 (accession number AF338038) while only 82% identity was found with the 376 bp upstream of bla_RAHN-2. The 479 bp downstream of bla_RAHN-1 had 74% identity with an aldo/keto reductase from Azotobacter vinelandii (accession number CP001157) and the 1332 bp downstream of bla_RAHN-2 had 75% identity with the gene for a putative transcriptional regulator of the GntR family from Serratia proteomaculans (accession number CP000826). No putative LysR-type transcriptional regulator gene, usually present upstream of chromosomal class A β-lactamase genes, has been identified upstream or downstream of either bla_RAHN gene.

Purification and biochemical characterization of RAHN-2

Enzyme purity was estimated to be >99% for RAHN-1 and 95% for RAHN-2, according to SDS–PAGE analysis (data not shown). The specific activity of the purified enzymes was 360 and 538 U/mg of protein for RAHN-1 and RAHN-2, respectively. The N-terminal sequence of both enzymes was QTATKM, indicating that a 28 amino acid leader sequence had been removed to generate the mature β-lactamases. The molecular masses found for RAHN-1 (29 976.8 Da) and RAHN-2 (30 059.7 Da) were consistent with those predicted for the mature proteins.

As expected, RAHN-1 had a substrate specificity profile similar to that described previously² (Table 2). However, catalytic constants (k_cat) were, on average, 20-fold higher than those reported.² This is likely to be due to the low specific activity obtained previously which was 24-fold lower than that of RAHN-1 in our study. In common with RAHN-1, RAHN-2 had strong activity against benzylpenicillin, piperacillin, cefalotin, cefuroxime and ceftriaxone. Unlike RAHN-1, the best substrate for RAHN-2 was cefuroxime (relative k_cat/K_m value of 140). RAHN-2 was 5-fold more efficient than RAHN-1 in hydrolysing ticarcillin, and 2.5-fold more efficient in hydrolysis of both cefuroxime and cefotaxime. RAHN-2 also had activity against cefpirome and cefepime. These results are in contrast to the MICs which showed that strain 9 was more resistant than strain 42. However, the specific activity of RAHN-1 in crude extracts was found to be 35.3 ± 1.45 U/mg whereas that of RAHN-2 was 15.9 ± 0.63 U/mg. These data indicate that RAHN-1 was expressed 2-fold more than RAHN-2, accounting for the higher levels of resistance of the host. Inhibition studies showed that RAHN-2 was strongly inhibited by clavulanic acid (IC₅₀ = 0.025 µM), tazobactam (IC₅₀ = 0.03 µM) and sulbactam (IC₅₀ = 0.02 µM).

Table 2

Kinetic parameters of purified RAHN-1 and RAHN-2^a

	RAHN-1				RAHN-2				kcat/Km (RAHN-1)/kcat/Km (RAHN-2)
Substrate	kcat (/s)	Km (µM)	kcat/Km (/µM/s)	Relative kcat/Km	kcat (/s)	Km (µM)	kcat/Km (/µM/s)	Relative kcat/Km
Benzylpenicillin	50	10	5	100	76	17	4.5	100	1.1
Ticarcillin	14	20	0.7	14	7.7	2.4	3.2	71	0.2
Piperacillin	34	10	3.4	68	44	22	2.0	44	1.7
Cefalotin	144	90	1.6	32	295	69	4.3	96	0.4
Cefaloridine	204	115	1.6	32	92	61	1.5	33	1.2
Cefuroxime	13.5	6	2.3	46	25	4	6.3	140	0.4
Cefoxitin	<0.01	ND	ND	ND	<0.01	ND	ND	ND	ND
Cefotaxime	5	7	0.7	14	11	6	1.8	40	0.4
Ceftazidime	<0.05	ND	ND	ND	<0.05	ND	ND	ND	ND
Ceftriaxone	4	6	0.65	13	5.5	6.5	0.85	19	0.8
Cefpirome	74	598	0.12	2.4	36	335	0.1	2.2	1.2
Cefepime	22	370	0.06	1.2	26	318	0.08	1.8	0.7
Aztreonam	1	510	0.002	0.04	7	356	0.02	0.4	0.1
Imipenem	<0.01	ND	ND	ND	<0.01	ND	ND	ND	ND

ND, not determined.

^aStandard deviations of the kinetic parameters were within 10%.

Conclusions

RAHN-2 has been identified in a phylogenetic group of Rahnella strains separated from strains harbouring RAHN-1. This dichotomy, supported by sequence analysis of 16S rRNA and rpoB genes, suggests that bla_RAHN was acquired by R. aquatilis before the divergence in genomospecies. Although the strains specifying RAHN-2 had the same β-lactam resistance patterns as those harbouring RAHN-1, they were at least 2-fold more susceptible to the β-lactams they hydrolysed. This makes phenotypic detection of RAHN-2 difficult and requires MIC determination. However, even if Rahnella spp. seem to be phenotypically susceptible to cefotaxime, its use to treat Rahnella infections should be avoided.⁹

Funding

This work was supported in part by the French Ministry of Fishery and Agriculture (contract AQS R02/03-921 E 162 RT20), by the CNR ‘Resistance to antibiotics in commensal flora’ (RMES 27), Hôpital Bichat-Claude Bernard, APHP, Paris, France, and by the Institut National de la Veille Sanitaire (INVS).

Transparency declarations

None to declare.

Acknowledgements

We thank M. Dupechez for enzyme production, and P. E. Reynolds for reading the manuscript prior to submission.

Author notes

†

These authors contributed equally to this work.