Genomics of Ochrobactrum pseudogrignonense (newly named Brucella pseudogrignonensis) reveals a new bla OXA subgroup

Ochrobactrum pseudogrignonense (newly named Brucella pseudogrignonensis ) is an emerging pathogen in immunodeficient and immunocompetent patients. Most documented cases associated with Ochrobactrum are frequently catheter-related and exhibit wide-spectrum β-lactam resistance. Misidentification of this pathogen using commercial bacterial identification kits is common. We identified a case of O. pseudogrignonense infection associated with cholelithiasis. The O. pseudogrignonense genome was sequenced and reconstructed using a Nanopore and Illumina hybrid strategy. A novel bla OXA-919 divergent from existing OXA members was identified and subsequent analysis revealed its existence in all available O. pseudogrignonense genomes, which forms a new phylogenetic subgroup distinct from other OXA clusters. Further analysis demonstrated the presence of the novel bla OXA-919 in the chromosome of several other Ochrobactrum species. Our study indicated that Ochrobactrum chromosomes may be a reservoir of bla OXA-919 β-lactamases.


INTRODUCTION
Ochrobactrum has been recognized as an emerging pathogen in immunodeficient and immunocompetent patients. Recently, the genus Ochrobactrum was renamed within Brucella. However, the clinical characteristics are different between the two groups. To date, Ochrobactrum anthropi (newly named B. anthropi), O. intermedium (B. intermedium), O. tritici (B. tritici), O. haematophilum (B. haematophilum) and O. pseudogrignonense (B. pseudogrignonensis) have been reported to cause human infections [1]. Most Ochrobactrum infections are catheter-related, such as central venous vein catheters, drainage tubes and intraperitoneal catheters, because of the ability of the pathogen to adhere to silicone [1]. Ochrobactrum species show wide-spectrum β-lactam resistance. Previous studies reported that β-lactam resistance is associated with production of an inducible chromosomally encoded Amp-C β-lactamase [2]. Misidentification of Ochrobactrum species using commercial bacterial identification kits is common and leads to diagnostic difficulties in clinical settings [1].
O. pseudogrignonense is a gram-negative, non-motile, nonspore-forming and oxidase-positive rod-shaped bacterium [3]. It is a naturally occurring environmental organism found in water and soil [4]. It has been shown to be a pathogen of a fungus [5]. Whole genome sequencing of O. pseudogrignonense was reported in 2016 from Malaysian tropical soil. Clinical cases were reported in Sweden in 1992 and in Norway in 2000, respectively, from blood of a 28-year-old patient and the ear of a newborn [3]. Recently, a human case report in Korea OPEN ACCESS described bacteraemia in a 44-year-old man with extracorporeal membrane oxygenation [6].
In this study, we report the genomic analysis of a clinical O. pseudogrignonense isolate in Taiwan and conduct comparative genomics with publicly available genomes of O. pseudogrignonense (K8, MYb58, CCUG 30717, CCUG43892, MYb37, MYb70 and CIP 109451).

Sample description
An 86-year-old male patient presented with fever and abdominal pain. He had a background of hypertension, history of liver abscess, benign prostatic hyperplasia and pulmonary tuberculosis (for which he received complete treatment at age of 20 years). He received percutaneous transhepatic gallbladder drainage under the impression of gallstone-related acute cholecystitis. After drainage, fever improved. Liver sonography showed bilateral intrahepatic duct dilatation. T-tube cholangiography revealed an obstructive cystic duct. He was referred to a general surgeon and was admitted to the ward for laparoscopic cholecystectomy.
On the first day of hospitalization, he was afebrile, with blood pressure 133/71 mmHg and heart rate 72 b.p.m. Laboratory findings indicated a white blood cell count of 6700 μl -1 with a differential of 60 % neutrophils, a haemoglobin level of 11.5 g dl −1 , a platelet count of 183×10 3 μl -1 , total bilirubin of 0.3 mg dl −1 , direct bilirubin of 0.1 mg dl −1 , AST (aspartate aminotransferase) of 18 U l -1 and ALT (alanine aminotransferase) of 8 U l -1 . He received laparoscopic cholecystectomy on admission day 2. Intravenous antibiotics with cefazolin (1000 mg every 8 h) was started on the day of operation. Operative findings showed gallbladder about 6×13 cm in size with multiple pigmented stones inside and a cystic duct diameter of about 0.84 cm. Bile leakage was observed during the operation, so he received wound care with subsequent wet dressing. The wound was closed on postoperative day 2. Brown colour drainage was observed on postoperative day 3. Because bile leakage was suspected, he received

Impact Statement
Ochrobactrum pseudogrignonense (newly named Brucella pseudogrignonensis) is an emerging pathogen exhibiting wide-spectrum β-lactam resistance, but the underlying resistance determinants are as yet unclear. By sequencing, reconstructing and comparing the O. pseudogrignonense genomes, we found a novel subgroup of OXA β-lactamases, OXA-919, which is found not only in O. pseudogrignonense genomes but also in other Ochrobactrum species. Our results revealed a new OXA cluster. endoscopic retrograde biliary drainage on post-operative day 5. Surgical pathology showed cholelithiasis and chronic active cholecystitis. On post-operative day 5, bile culture yielded morphologically homogeneous colonies which were preliminarily identified as O. anthropi by MALDI-TOF. He received intravenous tigecycline (100 mg loading dose, maintenance dose 50 mg every 12 h) for 7 days and was discharged on day 14. He received biliary drainage removal 2 months after discharge without specific complications.
Whole genome sequencing of the isolate and computation of whole-genome average nucleotide identity (ANI) against other species in the same genus was performed. The results indicated 98.99 % ANI with O. pseudogrignonense CCUG 30717. Antimicrobial susceptibility testing showed that the isolate was sensitive to imipenem, colistin, tigecycline, amikacin and trimethoprim-sulfamethoxazole, but resistant to gentamicin, ampicillin-sulbactam, ceftazidime, cefepime, ceftriaxone, ciprofloxacin and piperacillin-tazobactam.

Genome sequencing. assembly and annotation
The O. pseudogrignonense SHIN genome was deeply sequenced using Nanopore long-read sequencing at 123× and Illumina short-read sequencing at 56×. Adaptor sequences left in long reads were trimmed using Porechop. The remaining reads were hybrid-assembled via Unicycler (v0.4.7) (N50=1 880 620 bp), and classified into two chromosomes and three plasmids. Protein-coding genes, and coding and non-coding RNAs in the chromosomes and plasmids were annotated via the NCBI PGAP pipeline. Antibiotic-resistant genes (ARGs) were predicted by aligning protein-coding genes against the Comprehensive Antibiotic Resistance Database (CARD) using Diamond. Only ARGs with alignment coverage greater than 90 % were retained. Efflux pumps were excluded from ARG analysis.

Motif and phylogenetic analysis of OXA family members
Multiple sequence alignment of OXA-919 and other OXA members was carried out by mega x to identity the conserved motifs. The alignment was used for generating a phylogeny tree by mega x, which was visualized by ITOL in order to construct a circular phylogeny tree.

Data deposition
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under accession PKQI00000000. The version described in this paper is version PKQI00000000.1.

Hybrid genome sequencing revealed beta lactamases of multiples classes
The O. pseudogrignonense SHIN genome was sequenced by Nanopore (123×) and Illumina (56×) (Table S1, available in the online version of this article). Two chromosomes and three plasmids were obtained (Fig. 1, Table S2). The genome encompasses 5199 protein-coding genes, 15 5S/16S/23S rRNAs, 62 tRNAs and four non-coding RNAs. O. pseudogrignonense SHIN was resistant to a wide spectrum of β-lactams, including penicillin, cephalosporins and carbapenems (Table S3). Analysis of the SHIN resistome indicates the presence of 148 ARGs, including three β-lactamases in distinct classes (Table S4). In particular, one novel class D β-lactamase (called bla OXA-919 ) is found on chromosome 1. There are no associated mobile genetic elements or integrative and conjugative elements found around bla OXA-919 . A class C β-lactamase (ampC) is present on chromosome 2. A class B metallo-beta-lactamase, bla Imp-8 , is carried by plasmid 2 (Fig. S1). These chromosomal-encoding β-lactamases along with plasmid-encoding carbapenemase are probably the major resistance factors to a wide spectrum of β-lactams.

The novel class D bla OXA-919 beta lactamase is harboured by all O. pseudogrignonense
The class C β-lactamase, ampC, is well known in Ochrobactrum genomes and also encoded in chromosome 2 of the SHIN genome. To the best of our knowledge, this is the first report of the presence of class D β-lactamases (OXA) in Ochrobactrum (designated as NG_070746.1/ WP_007879679.1 by NCBI). By comparing bla OXA-919 with known OXA members, we found that it is quite dissimilar from others (e.g. AAI=40.16% to OXA-45 and AAI=27.52 % to OXA-54). Nevertheless, bla OXA-919 contains classic OXA signatures of three highly conserved motifs, S-T-F-K, Y-G-N and K-T-G, although the sequence outside the motifs diverges (Fig. 2), implying it is possibly a novel member in the current OXA family. We then used a blast search for the presence of bla OXA-919 in other public O. pseudogrignonense genomes in NCBI (Table S5) and found all of them carry bla OXA-919 (Table S5). By searching the NCBI protein database, bla OXA-919 (WP_007879679.1) is 100 % identical to one annotated protein (AKVI01000111.1/ EMG52215.1) in the Ochrobactrum sp. CDB genome. This indicated that, in addition to ampC, bla OXA-919 is another common β-lactamase encoded under the genus Ochrobactrum. Consequently, Ochrobactrum is probably the reservoir of class C and class D β-lactamases. Unfortunately, the resistance profiles of these public Ochrobactrum genomes are not available. Therefore, we investigated the resistance phenotypes of 13 additional Ochrobactrum samples

DISCUSSION
Ochrobactrum species are emerging pathogens in immunodeficient and immunocompetent patients [7]. We report the first case of O. pseudogrignonense infection and whole genome sequencing in Taiwan.
Ochrobactrum infections are associated with catheters or direct contamination of wounds [8][9][10], intravenous fluid [4], grafts [11] and medical devices [12,13] by the pathogen. Case reports of infections related to Ochrobactrum species have included endocarditis [14][15][16], meningitis [11], brain abscess [8], peritonitis [17,18], endophthalmitis [12,19], osteomyelitis [10], prostatitis [13], septic arthritis [9], urosepsis [20], soft tissue infection [21], pancreatic abscess [22] and pneumonia [23].  (Table 1). Most cases had underlying chronic illness or malignancy and the patient received a procedure during admission (biliary drainage, operation, post-operative drainage tube and endoscopic retrograde cholangiopancreatography) before infection episodes. A pathogen from the environment and adhering to the catheter may be the possible source of infection, similar to most previous case reports. Three cases [18] reported biliary sepsis and spontaneous bacterial peritonitis (SBP) without a drainage tube, demonstrating the possibility of Ochrobactrum species originating from the human gastrointestinal tract or biliary tract. Isolated culture from stool before bacteraemia [24] and antral biopsy [25]  Misidentification of the pathogen using commercial bacterial identification kits also occurred in our case and the literature ( Table 2). Vaidya et al. [26] reported that a case of pelvic abscess due to O. intermedium was incorrectly identified as O. anthropi by API 20NE and 16S RNA gene sequencing analysis. Misidentification of Brucella species as O. anthropi using MALDI-TOF MS and the VITEK 2 system was also reported previously [27,28]. Teyssier et al. [7], who tested the ability of commercial identification systems such as API 20NE and VITEK 2 to identify Ochrobactrum species, showed that commercial kits were not always reliable for genus and species identification. Previous studies reporting the predominant role of O. anthropi in human disease by using non-discriminatory methods and/or before the discovery of other Ochrobactrum species suggested that some infections associated with O. anthropi in the literature should be revised. Further phenotypic features and genotyping methods (ex. recA-based analysis [29]) should be considered for more reliable identification of Ochrobactrum to the species level.
Most Ochrobactrum spcies are resistant to most β-lactams and sensitive to carbapenem, ciprofloxacin, trimethoprim/ sulfamethoxazole or aminoglycoside (Table 3) [30]. Hence, empirical treatment with carbapenem, quinolone, cotrimoxazole and aminoglycoside is feasible. Ochrobactrum exhibited resistance to β-lactam antibacterial agents due to production of an inducible and chromosomally encoded Amp-C β-lactamase [2]. The β-lactamase characterized from O. anthropi was named OCH-1 (gene, bla OCH-1 ) [31]. Alonso et al. found the bla OCH gene in non-O. anthropi species with gene heterogeneity from food animals [32]. The isolated species identified by 16S rDNA sequencing and MALDI-TOF MS included O. intermedium and O. tritici. However, there is limited data regarding antibiotic resistance genes in non-antropi Ochrobactrum species from humans.
A new bla OXA , OXA-919, was found in our isolate and confers resistance to broad-spectrum β-lactam antibiotics. The emergence of OXA enzymes in recent years has caused huge difficulty in treating gram-negative infections. Some intrinsic OXAs have been identified in Acinetobacer species [33]. OXA-51-like β-lactamases are intrinsic to Acinetobacter baumannii [34] and may cause carbapenem resistance. OXA-134a is universal in Acinetobacter lwoffii [35] and may have the potential to cause β-lactam resistance when transferred to other species. We analysed seven O. pseudogrignonense genomes and found all O. pseudogrignonense isolates harboured bla OXA-919 genes. Further investigation identified bla OXA-919 genes in several other Ochrobactrum species. Ochrobactrum may be a reservoir of OXA-919.
O. pseudogrignonense is an emerging pathogen in immunodeficient and immunocompetent patients. Clinical isolates exhibit wide-spectrum β-lactam resistance. Whole genome sequencing shows that Class B, C and D β-lactamases are present in the SHIN genome. A new subgroup of OXA, OXA-919, is identified. O. pseudogrignonense may be a reservoir of multiple β-lactamases, and the impact on antibiotic resistance transfer needs further study. performed the validation; C.L., Y.M., Y.H. and P.L., were involved in the funding acquisition and conceptualization of the study; S.L., drafted the manuscript; S.L., Y.H. and P.L., revised the manuscript.