The genus Corynebacterium
include Gram positive aerobic bacteria, which are widely distributed in the microbiota of humans and animals. Medically relevant Corynebacterium
species consist of Corynebacterium diphtheriae
, the pathogenic bacterium that causes diphtheria, and the non-diphtherial corynebacterial (as Corynebacterium urealyticum
, Corynebacterium striatum
, and Corynebacterium jeikeium
, among others), which are part of the skin and mucous membranes flora [1
]. They are usually not pathogenic but can occasionally opportunistically capitalize on atypical access to tissues or weakened host defenses. A key role of Corynebacterium
species as attenuator of Staphylococcus aureus
virulence in the nose microbiota has been recently suggested [2
]. Corynebacterium urealyticum
is a slow growing, asaccharolytic, and lipophilic microorganism, whose name refers its ability to split urea [3
]. It possesses a strong urease activity that leads to the formation of struvite stones following ammonium magnesium phosphate precipitation due to the increase of urine pH. C. urealyticum
behaves as an opportunistic human pathogen, causing acute and chronic urinary tract infections (UTIs), eventually leading to bacteraemia [3
]. It has also been isolated from the skin of healthy elderly individuals, mainly females [4
]. Some of the risk factors pre-disposing to an infection by C. urealyticum
are prolonged use of urinary catheters, hospitalization for long periods, previous treatment with broad-spectrum antibiotics or immunosuppressants, and history of previous UTIs [5
The majority of C. urealyticum
currently isolated from clinical samples are multidrug resistant, thus potentially limiting effective empirical treatment [1
]. Development of resistance has been observed during treatment with different antimicrobial classes: β-lactams, gentamicin, fluoroquinolones, macrolides, rifampicin, and tetracycline [7
]. However, the resistance mechanisms for most of these compounds have not been described.
] and recent studies [10
] about antimicrobial activity against C. urealyticum
and other Corynebacterium
species have proven that vancomycin and linezolid were uniformly active against these bacteria, whereas most of them displayed high level resistance against quinolones, β-lactams, and macrolides.
The aim of this study was to evaluate the prevalence of multidrug resistant strains as well as determine the resistance mechanisms of C. urealyticum isolates from a Spanish hospital (Santander) during the period 2005–2017. The genomes of five of these isolates have been sequenced and the main antimicrobial resistance determinants identified.
2.1. Susceptibility of C. urealyticum to Antimicrobial Agents
distributions, as well as the percentage of resistance to the different antibiotics for the 40 C. urealyticum
included in this study, are shown in Table 1
Ampicillin (100%), erythromycin (95%), and levofloxacin (95%) showed the highest number of resistant isolates, with a monomodal distribution of their MICs. Interestingly, for each antimicrobial agent, the two susceptible strains were different (VH4696 and VH4851 susceptible to erythromycin, VH6223 and VH4248 susceptible to levofloxacin). All the levofloxacin and erythromycin-resistant isolates had high level resistance (MIC > 32 mg/L and MIC > 256 mg/L, respectively). Concerning ampicillin-resistant isolates, 39 isolates showed a MIC > 256 mg/L whereas one isolate showed a MIC = 24 mg/L (the urine isolate VH2234).
Gentamicin and tetracycline also showed high number of resistant C. urealyticum strains, with 82.5% and 50% of isolates, respectively. In both cases, a bimodal MICs distribution was observed. The MICs values for gentamicin were in the range 1.5–256 mg/L, and between 3 and 256 mg/L for tetracycline, with relatively low MIC90 values, 9 and 4 mg/L, respectively.
Rifampicin and linezolid were the compounds with the lowest percentage of resistance, with only two and one resistant strains, respectively. Rifampicin-resistant isolates showed a high level resistance (MICs > 32 mg/L), whereas the MIC of the linezolid resistant isolate was 3 mg/L. Vancomycin was the only compound uniformly active against all tested isolates.
On the whole, multidrug resistance, defined as nonsusceptibility to at least one agent in three or more antimicrobial categories [11
], was observed in 39 out of 40 isolates.
All the strains were resistant to at least two antimicrobial compounds. They presented nine different resistance profiles, the resistance combination for levofloxacin (LVX), ampicillin (AMP), gentamicin (GEN) and erythromycin (ERY) being the most prevalent (LVX-AMP-GEN-ERY = 14 isolates), as well as in combination with tetracycline (TET) resistance (LVX-AMP-GEN-ERY-TET = 13 isolates). The relationship between antibiotic resistance profiles and sample origin could not be established.
Strain VH4549, isolated from a urine sample, was resistant against six of the eight tested compounds (it showed LVX-AMP-GEN-ERY-TET-RIF profile), being the isolate with less therapeutic options. On the other hand, the strain VH6223, from placenta, was the most susceptible isolate, showing resistance only against ampicillin and erythromycin.
The susceptibility of the five C. urealyticum
whose genomes were sequenced against the eight compounds tested is shown in Table 2
2.2. Detection of Resistance Genes by PCR and Genome Sequencing
The 38 C. urealyticum
resistant to erythromycin carried the ermX
gene. The PCR amplification product of strain VH2234 was sequenced and compared with the ermX
gene of C. urealyticum
DSM 7109 [4
], showing a 95% identity. Whole genome sequencing of five erythromycin-resistant C. urealyticum
confirmed the presence of the ermX
Thirty-eight out of 40 C. urealyticum were resistant to levofloxacin. The 38 resistant strains showed a MIC of levofloxacin >32 mg/L. The sequences of the QRDR region of the gyrA gene of 28 isolates categorized as resistant and one isolate categorized as susceptible were compared to the sequence of this region in the gyrA gene of C. urealyticum DSM 7109 (quinolone-susceptible). Twenty-two levofloxacin-resistant isolates showed the double mutation Ser-90→Val and Asp-94→Tyr, whereas in three resistant strains Asp-94 was replaced by Ala. Three levofloxacin-resistant strains were single Ser-90→Val mutants. The levofloxacin susceptible strain VH4248 did not show mutations at residues 90 and 94, as the reference strain DSM 7109. One strain resistant to levofloxacin showed no mutation at the QRDR region of gyrA, suggesting a different resistance mechanism.
Two of our isolates (VH3073 and VH4549), as well as C. urealyticum
DSM 7109, were resistant to rifampicin (MIC > 32 mg/L). Rifampicin resistance is nearly always due to a genetic change in the β subunit of RNA polymerase (RpoB). Alignment of the RpoB sequences of these three rifampicin-resistant strains with the corresponding proteins of three rifampicin-susceptible C. urealyticum
revealed non-conservative changes in Ser-444 (VH4549 and DSM 7109) or Gln-511 (VH3073), which can be related with the rifampicin-resistant phenotype (Figure 1
The 40 C. urealyticum
strains were ampicillin-resistant. The high ampicillin resistance phenotype (MIC90
> 256 mg/L) was associated with the presence of the blaA
gene. Ampicillin-resistant C. urealyticum
rendered the expected 0.8 Kb PCR product when amplified with blaA
specific primers. Conversely, C. urealyticum
18408721, a clinical isolate susceptible to ampicillin, was negative by the blaA
-based PCR. The C. urealyticum blaA
gene encodes a serine hydrolase belonging to the class A β-lactamase protein family. In order to know the genomic context of the resistance genes and inquiry about their transfer mechanisms, we sequenced the genomes of five C. urealyticum
. Genome analysis revealed that the region containing the blaA
gene is highly conserved among the five C. urealyticum
and the previously sequenced strains C. urealyticum
DSM 7109 [4
] and DSM 7111 [12
] (Figure 2
A). This region spans 20 Kbp of the assembled genomes, including a tnp
gene (transposase), lysR
(transcriptional regulator), and a Penicillin-Binding-Protein (PBP) type 1 gene. A similar genomic organization can be found in C. striatum
KC-Na-01 (Figure 2
B). We compared the amino acid sequence encoded by the C. urealyticum blaA
gene with its counterparts in other species and we found that it is highly conserved in C. striatum
strain KC-Na-01 (NCBI’s protein accession #WP_049063072), in C. jeikeium
K411 (#WP_034987125), in C. amycolatum
SK46 (#WP_076773763.1), as well as in C. resistens
DSM 45100 (#WP_042378726.1) [13
]. There is a particular region in the BlaA sequence (between amino acid positions 157–164) that concentrates most of the variability when comparing all species.
Thirty-three of the C. urealyticum
showed low level gentamicin resistance. The presence of the gene aac(3)-XI
encoding for an aminoglycoside 3-N acetyltransferase was evaluated by PCR using primers based on the C. striatum aac(3)-XI
], giving negative results in all strains. However, analysis of the five C. urealyticum
sequenced genomes revealed the presence of a 447-bp open reading frame showing 79% identity with the C. striatum aac(3)-XI
gene in four strains but not in strain VH4248 (Figure 3
). Homology search in databases revealed that the C. urealyticum aac(3)-XI
orthologous encodes an aminoglycoside 3-N acetyltransferase also present in C. coyleae
(#WP_092102070.1) (80% identity), C. fournierii
(#WP_085957501) (75% identity), and several Corynebacterium
spp. The aac(3)-XI
gene is flanked by arfB
, encoding the peptidyl-tRNA hydrolase ArfB, at the upstream region, and the luxR
-family two-component transcriptional response regulator, at the downstream region (Figure 3
). On the other hand, a search for additional aminoglycoside resistance genes revealed the presence of the gene aph(3′)-Ic
, encoding resistance to kanamycin and other aminoglycosides rarely used in clinical practice, and the pair of genes aph(3″)-Ib
, conferring streptomycin resistance, in four strains, but not in strain VH4248, which does not present any of these genes.
Twenty of our C. urealyticum
were resistant to tetracycline. Tetracycline-resistant strains showed a bimodal MICs distribution: strains VH638 and VH2234 showed high resistance level (MICs ≥ 256 and 32 mg/L, respectively), whereas 18 strains showed low resistance level (MICs range = 3–6 mg/L). The C. urealyticum
reference strain DSM 7109 is tetracycline-resistant (MIC = 32 mg/L) and this resistance is associated to the tetAB
]. The tetAB
genes were neither detected by genome analysis of the strains VH4549, VH5757, and VH5913, nor by PCR analysis of the remaining 17 tetracycline-resistant strains, which suggests the existence of alternative resistance mechanisms. When the tetracycline MICs of seven of our tetracycline-resistant strains were measured in the presence of the efflux-pump inhibitor Phe-Arg-β-naphthylamide (PAβN), a dramatic increase of tetracycline susceptibility was observed (Table 3
). However, the tetracycline MIC of strain DSM 7109 remained unchanged.
2.3. Molecular Epidemiology of the C. urealyticum Isolates
The PFGE method displayed a high typeability and discriminatory power. XbaI
digestion of the 40 C. urealyticum
isolates revealed 39 distinct PFGE patterns which were labelled from 1 to 39 (Figure 4
). The reference strain DSM 7109 PFGE pattern was also included in the dendrogram. Only two strains were assigned into the same PFGE pattern (VH4696 and VH4851), designated as pattern 18. These two strains were isolated from urine samples taken from the same patient at an interval of eight days, and showed the same antibiotic resistance profile (LVX-AMP-GEN-TET). The relationship between PFGE patterns and antibiotic resistance profiles or sample origin could not be established.
The high number of pulsotypes obtained in our C. urealyticum isolates highlighted the elevated genetic diversity in this specie. Among 40 strains, 39 were unrelated, producing sporadic infections.
Management of C. urealyticum
infections usually relies on glycopeptides (vancomycin or teicoplanin), to which this microorganism is uniformly susceptible [15
]. All our C. urealyticum
were susceptible to vancomycin. In two previous studies, we have reported full activity of vancomycin [16
] and teicoplanin [17
] against C. urealyticum
. In a more recent report including 52 C. urealyticum
clinical strains, vancomycin was also active against all the isolates [10
]. In vitro studies indicated that linezolid can also be effective [5
], and subsequent studies confirmed that linezolid was fully active against C. urealyticum
]. The data presented in this work basically agree with linezolid susceptibility data from the mentioned studies, highlighting its option as a therapeutic alternative for vancomycin. However, one of our isolates showed low level resistance to this compound (MIC = 3 mg/L), which raises the need of maintaining surveillance strategies among this multidrug resistant pathogen as well as defining its resistance profile before treatment.
Isolates resistant against erythromycin, levofloxacin, and ampicillin showed a monomodal distribution of their MICs, which suggests the existence of a unique or major mechanism of resistance for each antimicrobial. Erythromycin was inactive against the majority of our C. urealyticum
, in accordance with previously reported data [19
]. All the erythromycin-resistant C. urealyticum
carried the ermX
gene. It is well established that the ermX
gene encodes an N-6-methyltransferase that modifies an adenine of the 23S rRNA, conferring resistance against erythromycin [4
]. Whole genome sequencing of five C. urealyticum
confirmed the presence of the ermX
Fluoroquinolones have been extensively used in the empirical treatment of urinary tract infections. Upon antibiotic administration, these drugs tend to accumulate in the organs of the body leading to the selection of spontaneous mutants in large bacterial populations, including those that colonize the skin and mucous membranes such as corynebacteria. In fluoroquinolone-resistant Corynebacterium
spp. mutations are circumscribed to the gyrA
gene (QRDR region), since these bacteria lack the parC
gene. Thirty-eight of our 40 C. urealyticum
(95%) showed high level resistance to levofloxacin. López-Medrano et al. reported that 79% of their isolates were resistant to ciprofloxacin [18
]. Sequencing of the QRDR region of the gyrA
gene of 29 levofloxacin-resistant C. urealyticum
revealed that resistance is associated with single or double amino acid substitutions in residues Ser-90 and Asp-94 (C. urealyticum
numbering). Ramos et al. have recently reported three C. urealyticum
strains showing high level quinolone resistance associated to double amino-acid substitutions in Ser-90 and Asp-94 [20
]. However, in three of our strains, one amino acid replacement (Asp-94 by Tyr) was enough to display high resistance level to levofloxacin. In one C. urealyticum
, no link between mutations in this region and levofloxacin-resistant phenotype could be established, suggesting the existence of additional resistance mechanisms.
Rifampicin has been used as complementary agent for the management of C. striatum
]. Rifampicin showed good activity against our C. urealyticum
, since only two out of 40 isolates were resistant. Of note, the C. urealyticum
reference strain (DSM 7109) is also rifampicin-resistant. A recent study including 52 C. urealyticum
in Canada showed the same MIC50
values for rifampicin (≤0.05 mg/L) [10
]. Resistance to this compound typically results from the substitution of some highly conserved residues in the RNA polymerase β subunit [23
]. In Mycobacterium tuberculosis
, more than 96% of rifampicin-resistant strains have mutations within the 81-bp rifampicin resistance-determining region (RRDR) of the rpoB
gene (codons 507–533) [24
]. We compared RpoB sequences of the three rifampicin-resistant C. urealyticum
with that of susceptible strains. Considering the presumptive location of the RpoB active site and discarding the influence of conservative replacements in rifampicin susceptibility, we propose that the high rifampicin MICs can be explained by amino acid replacements in RpoB of the two rifampicin-resistant strains (Ser-444→Phe in VH4549 and Gln-511→Lys in VH3073) (Figure 1
). We have also identified the substitution Ser-444→Asn in DSM 7109.
All our C. urealyticum
displayed high level resistance to ampicillin (MIC90
> 256 mg/L). Hydrolysis of β-lactam antibiotics by β-lactamases is the most common mechanism of β-lactam resistance in clinically relevant bacteria. The ampicillin-resistant C. urealyticum
were positive for the blaA
-based PCR whereas a susceptible strain was negative. Whole genome analysis of five C. urealyticum
isolates confirmed the presence of the blaA
gene, flanked by transposase encoding genes (Figure 2
A). The blaA
gene encodes a serine hydrolase belonging to the class A β-lactamase protein family, which is highly conserved in several Corynebacterium
species. In seven C. urealyticum
strains and in C. striatum
KC-Na-01 the blaA
gene is in close vicinity to a transposase encoding genes (Figure 2
B), suggesting that it has been horizontally propagated.
Aminoglycosides are complementary antibiotics for the treatment of infections caused by Corynebacterium
spp. However, it has been reported that C. urealyticum
is mostly resistant to aminoglycosides [25
]. The MICs of C. urealyticum
DSM 7109 for kanamycin and streptomycin are >256 and >128 mg/L, respectively [4
]. Thirty-three out of our 40 C. urealyticum
were resistant to gentamicin, with MIC50
values of 4 and 9 mg/L, respectively, indicating a high prevalence but a low level of resistance (range tested 0.016–256 mg/L). The gene aac(3)-XI
, encoding an aminoglycoside 3-N acetyltransferase, which confers resistance to gentamicin and other aminoglycosides, has been recently identified in C. striatum
], whose presence was correlated to low level of resistance to gentamicin [26
]. Search by PCR with primers based on C. striatum aac(3)-XI
sequence gave negative results in our 40 C. urealyticum
, since these primers did not match with the C. urealyticum aac(3)-XI
gene. However, by means of whole genome sequencing, we detected this gene as part of a highly conserved region in strains VH3073, VH4549, VH5757, and VH5913, but not in strain VH4248. We hypothesize that low level gentamicin resistance in these four strains is related with the presence of the aac(3)-XI
gene, whereas in strain VH4248 is due to another mechanism. This analysis also revealed the presence in these four strains of a region including the gene aph(3′)-Ic
(related to kanamycin resistance) and the pair of genes aph(3″)-Ib
(related to streptomycin resistance), which is also present in C. urealyticum
DSM 7109 [4
] as well as in other Corynebacterium
Fifty percent of our C. urealyticum
were resistant to tetracycline. Tetracycline-resistant strains showed a bimodal MICs distribution, which suggests the existence of different resistance mechanisms. In C. striatum
, tetracycline resistance is mediated by an ATP gradient efflux mechanism encoded by the pair of genes tetA-tetB
], which is also found in C. urealyticum
DSM 7109 [4
]. However, in our C. urealyticum
, the tetA-tetB
genes were not detected. The remarkable decrease of tetracycline MICs of seven of our C. urealyticum
observed in presence of PAβN, a broad-spectrum efflux pump inhibitor, indicates that tetracycline resistance is mediated by an efflux mechanism.
PFGE is considered as the “gold standard” technique to assess epidemiological relationships for most clinically-relevant bacteria [28
]. Our results showed almost the same number of isolates as PFGE patterns, with only two strains sharing the same pulsotype. This high diversity among C. urealyticum
isolates revealed that they are not related but causing sporadic infections. While whole genome sequencing provides more detailed and accurate information, its use is not still viable in the daily clinical routine. Thus, PFGE remains as an important tool at epidemiological level. However, increasing the number of sequenced strains will provide more information, such as antimicrobial resistance and virulence, in comparison to PFGE, which will improve, in turn, our knowledge about the resistance and virulence mechanisms of this pathogen.
4. Materials and Methods
4.1. Bacterial Strains and Growth Conditions
Forty C. urealyticum isolated from clinical samples at Clinical Microbiology Laboratory, Hospital Universitario Marqués de Valdecilla (HUMV), Santander (Spain), during the period 2005–2017, were used in this study. C. urealyticum DSM 7109 was also included as the reference strain. The origin of the samples was diverse: urine (25), abdominal drainage (3), surgical wound (3), blood (3), skin ulcer (2), urinary stone (1), non-surgical wound (1), diabetic foot ulcer (1), and placenta (1). They were initially identified by the API Coryne system (bioMérieux, Marcy l’Etoile, France) and confirmed by MALDI-TOF mass spectrometry using the Vitek MS (bioMérieux) platform, according to manufacturer’s instructions. C. urealyticum 18408721, isolated at Hospital Universitario Central de Asturias (HUCA), Oviedo (Spain), was used as an ampicillin-susceptible control strain. All strains were grown in blood agar (BA) plates at 37 °C for 72 h and kept frozen at −80 °C in Brain Heart Infusion (BHI) broth with 20% glycerol until use.
4.2. Antimicrobial Susceptibility Assays
To study the activity of eight antimicrobial compounds (ampicillin, erythromycin, gentamicin, levofloxacin, linezolid, rifampicin, tetracycline, and vancomycin) against the 40 C. urealyticum, minimal inhibitory concentrations (MICs) were determined using Etest® strips (bioMérieux) on Mueller-Hinton (MH) agar plates supplemented with horse blood and β-NAD (Oxoid, Madrid, Spain). Briefly, agar plates were inoculated with a 100 μL aliquot of a bacterial suspension at OD600 = 0.1, and incubated for 48 h. Tetracycline MICs were also determined in presence of PAβN (50 mg/L).
Clinical categories were established according to the breakpoints for the microdilution susceptibility assay defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org
) guidelines [29
]. EUCAST defined Corynebacterium
spp. specific breakpoints for vancomycin, linezolid, tetracycline, and rifampicin; for ampicillin, gentamicin, and levofloxacin we considered EUCAST PK-PD breakpoints. For erythromycin, Staphylococcus
spp. cut-offs defined by EUCAST were used.
4.3. Search of Resistance Genes by PCR
The 40 C. urealyticum
were screened for the presence of resistance genes commonly found in Corynebacterium
spp. and other Gram positive multidrug resistant bacteria. Specific primers are listed in Table 4
. The gyrA
genes were amplified and sequenced for mapping mutations associated with levofloxacin and rifampicin resistance, respectively. C. urealyticum
DNA for PCR reactions was prepared using InstaGeneTM
Matrix (Bio-Rad, Madrid, Spain) following manufacturer’s instructions.
4.4. PCR Products Sequencing
PCR products were purified with silica gel columns using NucleoSpin® Gel and PCR clean-up kit (Macherey-Nagel, Düren, Germany). Purified DNA was sequenced by Macrogen (Madrid, Spain) with the primers outlined in Table 4
. Mutations in gyrA
genes and amino acid changes in their corresponding proteins were identified by pairwise and multiple alignment of sequences between resistant and susceptible isolates using MEGA7 [33
] and Clustal W [34
4.5. Genome Sequencing and Analysis
For whole genome sequencing, genomic DNAs of strains VH3073, VH4248, VH4549, VH5757, and VH5913 (selected on the basis of their resistance profile), were extracted using the NucleoSpin® Microbial DNA kit (Macherey-Nagel). Library preparation followed the NEBNext Fast DNA Fragmentation and Library Preparation Kit (New England Biolabs, Beverly, MA, USA) protocol and sequencing was performed in an Illumina HiSeq 2500 machine, at above 1000× coverage for all strains. De novo genome assembly was done with the SPAdes assembler and built-in on the PATRIC assembly server [35
]. Structural and functional annotations were performed in RAST server (https://rast.nmpdr.org
]. Prediction of antimicrobial resistance profiles and comparative genomic analyses were performed using the bioinformatic platforms PATRIC (https://www.patricbrc.org/
] and EDGAR [37
]. Multiple alignments were performed using T-coffee server in its variant M-coffee [38
4.6. Pulsed-Field Gel Electrophoresis (PFGE)
PFGE was performed with a CHEF-DRIII system (Bio-Rad). Bacteria were grown in BHI broth with shaking at 37 °C for 48−72 h. Cultures were adjusted to OD600 = 2.0, cells from 250 μL were pelleted and resuspended in 300 μL of TE buffer (10 mM Tris, 1 mM EDTA) containing 2 mg/mL lysozyme. This suspension was incubated at 37 °C for 1 h, inverting the tubes every 10 min. An equal volume of 2% LM agarose (Pronadisa, Madrid, Spain) in TE buffer containing 1% SDS and 0.2 mg/mL proteinase K was added, and plugs were cast with a standard casting tray. After the plugs solidified, they were incubated overnight at 55 °C with shaking in 4 mL of TE buffer containing 1% sarcosyl and 0.15 mg/mL proteinase K. The plugs were washed six times with pre-warmed TE buffer and then digested with 30 U of XbaI at 37 °C overnight. Electrophoresis was performed in a 1.2% agarose gel at 6 V/cm and at 14 °C with 0.5× TBE buffer (0.5 mM Tris, 45 mM boric acid, 0.5 mM EDTA). Pulse times ramped from 0.1 to 5 s for 24 h. Low range PFGE marker (New England Biolabs) was used as the molecular size marker.
Cluster analysis was performed with Fingerprinting II v4.5 software (Bio-Rad) by using the Dice similarity coefficient and the Unweighted Pair Group Method with Arithmetic means (UPGMA), with 1.3% of optimization and tolerance. Isolates were classified as indistinguishable if they showed 100% similarity, as closely related subtypes if they showed 95–99% similarity, and as different strains if they showed <95% similarity.
4.7. Data Availability
The whole genomes of the five C. urealyticum have been deposited in the Integrated Microbial Genomes Database under the following accession numbers: VH3073 (2833973259); VH4248 (2833948465); VH4549 (2833950470); VH5757 (2833952634); VH5913 (2830819286). Four of them have also been deposited in GenBank under the following accession numbers: VH3073: GCA_008244525.1; VH4248: GCA_008180085.1; VH4549: GCA_008180045.1; VH5913: GCA_008180065.1.