CARB-17 Family of β-Lactamases Mediates Intrinsic Resistance to Penicillins in Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus is commonly resistant to ampicillin, yet the mechanisms underlying this phenomenon are not clear. In this study, a novel class A carbenicillin-hydrolyzing β-lactamase (CARB) family of β-lactamases, bla_CARB-17, was identified and found to be responsible for the intrinsic penicillin resistance in V. parahaemolyticus. Importantly, bla_CARB-17-like genes were present in all 293 V. parahaemolyticus genome sequences available in GenBank and detectable in all 91 V. parahaemolyticus food isolates, further confirming the intrinsic nature of this gene.

TEXT

Vibrio parahaemolyticus is a major causative agent of gastroenteritis in areas with high seafood consumption rates and has recently become pandemic due to the emergence of the serotype O3:K6 (1). In Hong Kong, V. parahaemolyticus is the leading cause of food-borne illnesses due to the high rate of seafood consumption among the population (2). Although most cases of infections are self-limiting, fatality can occur among immunocompromised patients or those with debilitating medical conditions such as liver disease or diabetes (3). Antibiotics such as ciprofloxacin can be used for the treatment of infections caused by V. parahaemolyticus strains, but the choice of antibiotics should be based on the antimicrobial susceptibilities of the organism. V. parahaemolyticus is commonly considered highly susceptible to virtually all antimicrobials except for penicillins. However, mechanisms mediating the development of penicillin resistance in V. parahaemolyticus are not clear. In this study, we identified a novel carbenicillin-hydrolyzing β-lactamase (CARB) from chromosome 2 of V. parahaemolyticus and showed that the product of this gene is responsible for the intrinsic resistance to penicillins in V. parahaemolyticus.

A novel potential β-lactamase gene, bla_V110, with a length of 852 bp was identified through bioinformatics analysis of the whole-genome sequence of V. parahaemolyticus V110, which was shown to be resistant to ampicillin (4). The full-length novel β-lactamase gene was amplified by PCR using primer set F-GCTGAGAGCTCATGAAAAAGTTA, R-CGTAGGATCCTTAACTTTCTTTGTAGTGC and then cloned into Escherichia coli BL21 and tested for the MICs of various β-lactams according to CLSI standards (5). E. coli BL21 isolates expressing the novel β-lactamase gene exhibited MICs of 256, 512, 256, and 1,024 μg/ml toward penicillin G, ampicillin, carbenicillin, and piperacillin, respectively. The Bla_V110 enzyme appears to be susceptible to β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam (Table 1). To verify whether the penicillin resistance phenotype was attributed to the expression of bla_V110, we further purified a truncated form (60 to 852 bp) of this β-lactamase in which the signal peptide was removed and designated mBla_V110. The mBla_V110 protein was purified through several steps, including a Ni-nitrilotriacetic acid (NTA) column, thrombin treatment to remove the His tag, and a size exclusion column as previously described (6) (see Fig. S1 in the supplemental material). The purity of this protein was higher than 99%, and the yield of the purified mBla_V110 was about 2.4 mg/liter. Kinetic constants were determined for mBla_V110 as previously described (7), and very high catalytic activities on ampicillin, penicillin G, carbenicillin, and piperacillin were noted; however, this enzyme exhibited extremely low activities to other β-lactams tested (Tables 2). In general, mBla_V110 exhibited similar K_m values with all penicillins, cefepime, and cefpirome tested but variable k_cat values to these penicillins, some cephalosporins, aztreonam, and imipenem (Tables 2). The kinetic data were highly consistent with the MICs of E. coli isolates carrying bla_V110 genes. Collectively, our data suggested that Bla_V110 is an active β-lactamase that mediates the resistance to penicillins in the V. parahaemolyticus V110 strain.

TABLE 1.

MICs of different β-lactams on V. parahaemolyticus V110 parental strain and E. coli carrying pET28-bla_V110

Antibiotica	MIC (μg/ml) against bacterial strain
	V. parahaemolyticus V110	E. coli pET28-blaV110	E. coli pET-28
Penicillin G	512	256	<1
Ampicillin	128	512	1
AMP/CLA (2:1)	2	<1	<1
AMP/SUL (1:1)	<1	4	<1
Carbenicillin	256	256	2
Piperacillin	256	1,024	<1
PIP/TAZ (10:1)	0.06	1	0.5
Cephalothin	8	1	0.03
Cefuroxime	8	0.25	0.03
Cefotaxime	0.06	0.004	0.004
Cefepime	1	0.06	0.06
Cefpirome	0.25	0.015	0.015
Aztreonam	4	0.008	0.004
Imipenem	0.03	0.25	0.008

^aAMP, ampicillin; CLA, clavulanic acid; SUL, sulbactam; PIP, piperacillin; TAZ, tazobactam.

TABLE 2.

Kinetic constants of mBla_V110 toward different β-lactams

Antibiotic	mBlaV110
	Km (μM)	kcat (s−1)	kcat/Km (s−1μM−1)
Penicillin G	110.4 ± 19.31	2,320 ± 189.2	21.01
Ampicillin	235.8 ± 40.44	2,068 ± 170.9	8.77
Carbenicillin	113.9 ± 28.61	1,233 ± 177.4	10.83
Piperacillin	32.6 ± 8.87	450.7 ± 50.73	13.82
Cefuroxime	NHa	<0.01
Cefotaxime	NH	<0.01
Cefepime	104.3 ± 25.67	0.74 ± 0.08	7.10 × 10−3
Cefpirome	22.6 ± 4.77	0.98 ± 0.07	4.34 × 10−2
Aztreonam	NH	<0.01
Imipenem	NH	<0.01

^aNH, no detectable hydrolysis was observed with 1 μM purified mBla_V110 and up to 500 μM substrate.

Protein BLAST of Bla_V110 showed 99% homology with PSE-4 from V. parahaemolyticus (8, 9) and high homology (54%) with PSE-4 from Pseudomonas spp. PSE-4 is an alternative name for CARB-1, which belongs to the CARB-type family originally identified from Pseudomonas aeruginosa, Acinetobacter, and Vibrio cholerae (10–12). CARBs, also known as carbenicillin-hydrolyzing β-lactamases, have been found to disperse widely among distantly related bacteria, mostly by mobile genetic elements (10, 13, 14). Similarly, Bla_V110 also mediated resistance to ampicillin, penicillin G, carbenicillin, and piperacillin. Therefore, Bla_V110 was designated a novel member of the CARB family, bla_CARB-17 (GenBank accession number KJ934265).

Analysis of its genetic environment showed that the bla_CARB-17 gene was located on chromosome 2 of V. parahaemolyticus V110. Several putative genes including those encoding transporters and enzymes are located upstream and downstream of bla_CARB-17 (see Fig. S2 in the supplemental material). The bla_CARB-17-like genes were also identified in chromosome 2 of 5 V. parahaemolyticus isolates with completed whole-genome sequences in GenBank (see Fig. S2). The genetic environments of the bla_CARB-17-like genes in these isolates were very similar but not identical. There were no mobile genetic elements, such as integrase and transposase, within their genetic environments, and the bla_CARB-17-like genes were not located in any genomic islands, suggesting that bla_CARB-17 genes may be intrinsic to V. parahaemolyticus. Further analyses of the other 292 whole-genome annotation reports of V. parahaemolyticus available in GenBank identified bla_CARB-17-like β-lactamases in 280 out of the 292 V. parahaemolyticus strains. bla_CARB-17-like β-lactamase genes were also identified in the other 12 V. parahaemolyticus strains, but they either showed one nucleotide deletion within the full-length bla_CARB-17-like genes (10 strains) or showed longer nucleotide fragment deletion at the N termini (2 strains). The latter two strains have very low numbers of proteins in their annotation reports, implying that the lack of full-length bla_CARB-17-like genes may be due to the sequencing coverage issue. Taken together, complete genome analysis suggested that all V. parahaemolyticus strains intrinsically harbor bla_CARB-17 and its variants. To further prove the intrinsic nature of bla_CARB-17, 39 and 52 V. parahaemolyticus strains isolated from seafood in Shenzhen and Hong Kong, respectively, were screened for the presence of bla_CARB-17-like genes using primers targeting the full length of bla_CARB-17. These isolates were confirmed to be V. parahaemolyticus through screening for the presence of tlh and atpA genes as well as API20E assays (bioMérieux). All isolates were resistant to ampicillin with the exception of 10 isolates with ampicillin MICs of 16 μg/ml. All strains showed positive amplifications for bla_CARB-17, and these genes were further confirmed to be bla_CARB-17-like genes by sequencing. These data further confirmed the intrinsic nature of bla_CARB-17-like genes in V. parahaemolyticus.

The current nomenclature for the CARB family of β-lactamases is confused with the PSE family in the literature. CARBs are divided into two subgroups, namely the CARB and RTG subgroups (http://www.lahey.org/Studies/). Based on the functional characterization of novel CARB-17 and its variants, the CARB family of β-lactamases can be separated into three distinct squares through further phylogenetic analyses of all CARB β-lactamases. The first square contains CARB-17 and its closely related variants, the second square contains mainly the previously identified narrow-spectrum CARB family from Pseudomonas spp. and V. cholerae, and the third square contains the broad-spectrum RTG subgroup (Fig. 1).

FIG 1.

Phylogenetic tree of CARB family and several related β-lactamases. CARB subgroups, including CARB-1 (PSE-4), CARB-2 (PSE-1), CARB-3, CARB-4, CARB-6, CARB-7, CARB-9, and CARB-11 (PSE-5) to CARB-15, are narrow-spectrum β-lactamases that hydrolyze penicillins (15, 16). RTG subgroups, including CARB-5 (RTG-2), CARB-8 (RTG-3), and CARB-10 (RTG-4), consist of an RTG triad as reported for the GN79 (RTG-1) from Proteus mirabilis (11, 14, 17, 18). Most of the CARB families were not subjected to functional characterization except CARB-10, which has been shown to hydrolyze cefepime and cefpirome and become an extended-spectrum CARB enzyme (14). CARB-17 exhibited 99% homology to PSE-4 from V. parahaemolyticus and approximately 80% to PSE-4 from V. cholerae Henc, as well as VHW-1 and VHH-1 from Vibrio harveyi through BLAST analysis (19). The latter two β-lactamases have been shown to be active on penicillins (19).

In conclusion, this study identified a novel class A β-lactamase, bla_CARB-17, which is responsible for the intrinsic resistance to penicillins in V. parahaemolyticus. The data facilitate clear grouping for the whole family of the CARB class of β-lactamases.