Chromosome-Based bla_OXA-48-Like Variants in Shewanella Species Isolates from Food-Producing Animals, Fish, and the Aquatic Environment

ABSTRACT

Carbapenems are considered last-resort antibiotics in health care. Increasing reports of carbapenemase-producing bacteria in food-producing animals and in the environment indicate the importance of this phenomenon in public health. Surveillance for carbapenemase genes and carbapenemase-producing bacteria in Dutch food-producing animals, environmental freshwater, and imported ornamental fish revealed several chromosome-based bla_OXA-48-like variants in Shewanella spp., including two new alleles, bla_OXA-514 and bla_OXA-515. Carbapenemase genes were not associated with mobile genetic elements or Enterobacteriaceae.

TEXT

Carbapenemases are extended-spectrum β-lactamases (ESBLs) that hydrolyze carbapenems, last-line therapeutics to treat multidrug-resistant Gram-negative infections (1). Carbapenemase-producing microorganisms are increasingly reported in food-producing animals (2, 3), the food supply (4), and the environment (5, 6). These findings are fueling a debate on the hazards for public health (7, 8), and authorities have rising concerns regarding the appearance of carbapenem resistance in food animal ecosystems (9). The aim of this study is to present the results of the 2013 to 2015 carbapenemase surveillance activities in food-producing animals and environmental freshwater in The Netherlands. Since resistant organisms are not geographically restrained, we also report the results of a pilot study on imported ornamental freshwater fish from other non-European countries, increasingly reported in recent years as sources of multidrug-tolerant bacteria and associated antimicrobial resistance genes (10, 11).

A total of 4,440 fecal samples of broilers, slaughter pigs, veal calves, and dairy cows were collected in 2013 to 2015, as previously described (12). Fifty batches of imported live ornamental fish (2 fish and 1 water sample per batch) from various countries outside the European Union were sampled (from November 2014 to February 2015), together with 24 surface freshwater samples collected from eight Dutch provinces (March 2015). After enrichment, carbapenemase families bla_KPC, bla_NDM, bla_IMP, bla_OXA-48, and bla_VIM were detected by Check-MDR Carba (Check-Points, Wageningen, The Netherlands) as previously described (12, 13). All samples were negative for bla_KPC, bla_NDM, bla_IMP, and bla_VIM. Variants of bla_OXA-48 were identified in 92 samples (Table 1) and confirmed by conventional PCR and sequencing (14): 7 fecal samples (0.16%), 9 surface freshwater samples (37,5%) from five Dutch provinces, and 37 ornamental fish batches (74%), of which water samples were positive (78%) more frequently than fish samples (36%).

TABLE 1.

Characteristics of bla_OXA-48-like genes detected in Dutch freshwater and livestock, imported ornamental fish, and transport water

Gene	No. of samples	Reference	Gene homology (%)	Amino acid differences	Accession no.b	Source (origin/no. of samples)
blaOXA-514	1			A201G (from OXA-416)	KU866382	Ornamental fish (Indonesia)
blaOXA-515	1			G201S (from OXA-252)	KU866383	Freshwater (Netherlands)
blaOXA-48	1	KT265183	99		KU820821	Slaughter pig
blaOXA-48b	7	JX644945	99–100		KU820801 (S), KU820802 (S), KU820804 (S)	Freshwater (Netherlands)
	69		99–100		KU820811, KU820813, KU820814, KU820815, KU820816	Ornamental fish (32) fish transport water (37)
	3		100		KU820805 (S), KU820806 (S), KU820807 (S)	Broiler (2), veal calf (1)
blaOXA-181	3	HM992946	100		KU820809, KU820810, KU820803 (S)	Ornamental fish (Colombia, 2, Singapore, 1)
blaOXA-199	1	JN704570	99		KU820808	Ornamental fish (Congo)
	1		100		KU820819	Freshwater (Netherlands)
blaOXA-252	1	WP_037428895	100a		KU820800 (S)	Slaughter pig
blaOXA-48 family	2	JX644945	99	W222G, V232G	KU820818	Slaughter pig
			99	N179I	KU820820	Broiler
Class D β-lactamase	2	JX644945	94	V21E, N28T, A33T, T104A	KU820812, KU820817	Fish transport water (Israel)

^aAmino acid homology with OXA-252 derived from BLASTX alignment in GenBank, since no gene sequence for bla_OXA-252 is available in GenBank, as of this writing.

^bOnly representative sequences were deposited in GenBank; S, sequenced from Shewanella isolate (see also Table 2).

The most common gene variant was bla_OXA-48b, recovered from ornamental fish, fish transport water, surface freshwater, and livestock (Table 1). Different alleles showed 99% to 100% nucleotide similarity (only silent mutations) to reference genes from Shewanella xiamenensis (15). Alleles that showed up to four amino acid substitutions have been annotated as OXA-48 family class D β-lactamases or class D β-lactamases (<94% nucleotide similarity). Despite the high prevalence of bla_OXA-48b, other bla_OXA-48 variants were also observed, as indicated by a BLASTX search in GenBank. bla_OXA-181 was detected in fish samples from Colombia, bla_OXA-199 was identified in freshwater from Gelderland province (The Netherlands) and ornamental fish from the Democratic Republic of Congo, and bla_OXA-252 was found in one fecal sample from a slaughter pig. Both bla_OXA-181 and bla_OXA-199 progenitors were identified in environmental isolates of S. xiamenensis (15, 16). Finally, bla_OXA-48 found in a fecal sample from one slaughter pig showed close identity to bla_OXA-48 from K. pneumoniae (GenBank accession no. KT265183).

Bacterial isolation was performed on ChromID Carba and ChromID OXA-48 (bioMérieux) and on His agar with 0.125 mg/liter ertapenem (9) on 12 randomly selected batches of ornamental fish, all bla_OXA-48-like-positive Dutch freshwater samples (n = 9), and livestock samples (n = 7), for a total of 28 samples. Shewanella spp. were isolated from 21 samples, whereas no OXA-producing Enterobacteriaceae was isolated (Table 2). Bacterial species identification by 16S rRNA and gyrB gene sequencing (15, 17) showed ≥98% to 100% nucleotide identity to S. xiamenensis or Shewanella oneidensis (GenBank accession no. KC765141.1 and KR732277). Isolates with <98% nucleotide identity were reported as Shewanella spp. PCR amplification and sequencing identified bla_OXA-48b in all but four Shewanella isolates (Table 2). Nucleotide queries using BLASTX identified two genes encoding potential protein products of original sequence: (i) bla_OXA-514, found in Indonesian ornamental fish, whose putative protein displayed one amino acid difference with the protein coded by bla_OXA-416, a gene recently reported in a pediatric case of intestinal carriage of S. xiamenensis acquired from environmental sources (17); and (ii) bla_OXA-515, found in Dutch freshwater, whose putative protein is similar to OXA-252, except for one amino acid difference. Note that molecular detection from water samples was not sufficient to achieve a comprehensive investigation of the presence of bla_OXA-48-like genes, since bla_OXA-181 and bla_OXA-514 were detected only after bacterial isolation with selective media.

TABLE 2.

MICs of Shewanella spp. isolated in this study

Source and isolatea	Species	Origin	Gene	MIC (mg/liter) forb:
				CTX	CTX + CLA	CAZ	CAZ + CLA	FEP	FOX	ETP	IPM	MEM	TRM
Environmental freshwater
W9	S. xiamenensis	Netherlands	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	2	2	0.5	1
W13	S. oneidensis	Netherlands	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	>2	2	1	≤0.5
W14	S. oneidensis	Netherlands	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	2	2	1	≤0.5
W15	S. xiamenensis	Netherlands	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	2	2	1	1
W16	S. oneidensis	Netherlands	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	>2	2	0.5	≤0.5
W17	Shewanella spp.	Netherlands	blaOXA-515c	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	≤0.5	2	2	0.5	≤0.5
W21	S. xiamenensis	Netherlands	blaOXA-48b	≤0.25	0.25/4	≤0.25	0.25/4	0.12	2	>2	4	4	4
Ornamental freshwater fish
10A	S. xiamenensis	Indonesia	blaOXA-514d	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	2	1	1	0.5	≤0.5
13C	S. xiamenensis	Israel	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	≤0.5	2	2	1	≤0.5
25B	Shewanella spp.	Singapore	blaOXA-181e	≤0.25	0.12/4	≤0.25	≤0.12/4	0.12	1	2	2	1	≤0.5
32B	S. xiamenensis	Indonesia	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	2	1	0.5	≤0.5
36B	S. xiamenensis	Singapore	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	2	2	1	0.5	≤0.5
39B	Shewanella spp.	Singapore	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	0.5	2	2	1	0.5	1
45B	Shewanella spp.	Thailand	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	2	1	0.5	1
F2.1-22	S. xiamenensis	Indonesia	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	2	>2	4	2	2
F2.1-24	Shewanella spp.	Indonesia	blaOXA-48b	≤0.25	0.25/4	≤0.25	0.5/4	0.12	2	>2	>16	8	4
F3.2	Shewanella spp.	Indonesia	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	>2	4	2	≤0.5
Slaughter pig
1553/2014	S. xiamenensis	Netherlands	blaOXA-252f	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	2	1	0.5	≤0.5
Broiler
780/2015	S. xiamenensis	Netherlands	blaOXA-48b	≤0.25	≤0.06/4	0.5	≤0.12/4	≤0.06	4	1	2	1	4
1206/2015	S. xiamenensis	Netherlands	blaOXA-48b	≤0.25	≤0.06/4	≤0.25	≤0.12/4	≤0.06	1	2	1	0.5	≤0.5
Veal calf
77/2015	S. oneidensis	Netherlands	blaOXA-48b	≤0.25	0.12/4	≤0.25	≤0.12/4	≤0.06	1	2	2	1	1

^aIsolates were classified as non-wild type susceptible based on EUCAST ECOFFS (www.eucast.org).

^bCTX, cefotaxime; CLA, clavulanic acid; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; ETP, ertapenem; IPM, imipenem; MEM, meropenem; TRM, temocillin.

^cAccession no. KU866383.

^dAccession no. KU866382.

^eAccession no. KU820803.

^fAccession no. KU820800.

Plasmid transformation and conjugation were not successful in transferring the bla_OXA48-like genes from Shewanella spp. to Escherichia coli K-12 recipients, suggesting a chromosomal localization (18, 19). The genetic context of bla_OXA-48-like genes was investigated by PCR using primer pairs oxa48b-Fw (5′-AGCTTGATCGCCCTCGATTT-23′) and lysR-Rev (5′-CGGATAGCCATTCCGGTCTC-3′) and oxa48b-Rev (5′-TGATTTGCTCAGTGGCCGAA-3′) and c15-Fw (5′-AAGCGTACTGGGATCATGGC-3′) designed on the genome sequence of S. xiamenensis S4 (GenBank accession no. JX644945). A conserved genetic arrangement as observed in environmental Shewanella spp. was detected in all isolates (15), with bla_OXA48-like gene downstream of an open reading frame (ORF) coding for a pyroglutamyl peptidase I-like protein and upstream of a putative lysR transcriptional regulator gene. None of the bla_OXA-48-like genes was associated with the epidemic IncL plasmid responsible for the current OXA-48 spread (20), and association with IS1999, ISShes2, and Tn2013 was ruled out (data not shown) (15, 21). Since all bla_OXA-48-like genes were located on the chromosome of waterborne Shewanella spp., thought to be the progenitor of this carbapenemase family (22), and were not associated with mobile genetic elements or Enterobacteriaceae, they were deemed to be of environmental origin and to pose a limited public health risk.

Shewanella isolates were tested for antimicrobial susceptibility using broth microdilution (Sensititre EUVSEC 2) according to ISO 20776-1:2006 (13), and MIC values were slightly increased to ertapenem compared to those of non-OXA-48-like Shewanella isolates (data not shown), with four isolates displaying reduced susceptibility to imipenem and meropenem (Table 2). Interestingly, two of these isolates (F2.1-22 and F2.1-24) were from the same fish sample, with F2.1-24 showing the highest MIC values, likely modulated by additional resistance mechanisms. Similar MIC values were already described in S. xiamenensis producing OXA-416 and OXA-204 (17, 23) and are commonly detected in the aquatic environment, where intrinsically resistant bacteria like Shewanella thrive (6). Given the absence of specific phenotypic tests, high-level temocillin resistance (MIC >64 ml/liter) has been suggested as a first step in identifying OXA-48 producers (9). According to our observations, however, this methodology is likely not applicable to environmental OXA-48 enzymes. Although recognized as β-lactamases with feeble hydrolyzing activity (16, 21), OXA-48 carbapenemases should not be undervalued due to their potential to synergize with ESBLs. Shewanella isolates were susceptible to other β-lactams but were often resistant to ciprofloxacin, nalidixic acid, ampicillin, tetracycline, trimethoprim, sulfonamides, and/or chloramphenicol (data not shown).

In conclusion, the prevalence of carbapenem-resistant bacteria in Dutch food-producing animals was still low, whereas they were more prevalent in environmental freshwater and imported ornamental fish. The fact that carbapenem-resistant genes were only related to naturally resistant Shewanella spp. and no acquired carbapenem resistance mechanism was observed places these microorganisms in a questionably relevant position in regard to human health, contrary to previous suggestions (4). Ongoing carbapenemase monitoring indicates that bla_OXA-48 variants are also present in imported consumption fish and prawns (K.T. Veldman, personal communication). Gene associations to ubiquitous aquatic bacteria that are normally undetected in antibiotic resistance surveillance programs may represent a chance to spread and potentially contribute to the resistome of other clinically relevant bacteria. The location of bla_OXA-48-like genes (on the chromosome or on mobile genetic elements) and the type of bacterial host (environmental bacteria or recognized pathogens) may largely determine the impact of this antimicrobial resistance gene on human and animal health.

Accession number(s).

bla_OXA-48 (KU820821),bla_OXA-48b (KU820801, KU820802, KU820804, KU820811, KU820813, KU820814, KU820815, KU820816, KU820805, KU820806, KU820807), bla_OXA-181 (KU820809, KU820810, KU820803), bla_OXA-199 (KU820808, KU820819), bla_OXA-252 (KU820800), bla_OXA-514 (KU866382), bla_OXA-515 (KU866383), OXA-48 family class D β-lactamases (KU820818, KU820820), class D β-lactamases (KU820812, KU820817); bla_OXA48b, bla_OXA-514, and bla_OXA-515 genomic surroundings (KX060561, KX060562, KX060563, KX060564), and gyrB (KX022127, KX022126).