Improved culture enrichment broth for isolation of Arcobacter-like species from the marine environment

Arcobacter-like species are found associated with many matrices, including shellfish in marine environments. The culture media and conditions play a major role in the recovery of new Arcobacter-like species. This study was aimed to develop a culture media for isolation and enhanced growth of Arcobacter-like spp. from marine and shellfish matrices. For this purpose, 14 different Arcobacter-like spp. mostly isolated from shellfish, were grown in 24 different formulations of enrichment broths. The enrichment broths consisted of five main groups based on the organic contents (fresh oyster homogenate, lyophilized oyster either alone or in combination with other standard media), combined with artificial seawater (ASW) or 2.5% NaCl. Optical density (OD420nm) measurements after every 24 h were compared with the growth in control media (Arcobacter broth) in parallel. The mean and standard deviation were calculated for each species in each broth and statistical differences (p < 0.05) among broths were calculated by ANOVA. The results indicated that shellfish-associated Arcobacter-like species growth was significantly higher in Arcobacter broth + 50% ASW and the same media supplemented with lyophilized oysters. This is the first study to have used fresh or lyophilized oyster flesh in the enrichment broth for isolation of shellfish-associated Arcobacter-like spp.

Growth of Arcobacter-like spp. in oyster homogenate with conventional broth media group. This group consisted of five different types of broth. The results (Fig. 2) from this media group demonstrated lower growth than from group 1, with an OD 420nm below 0.8. The growth of Arcobacter-like spp. was comparatively higher in the enrichment broth ABOHSW than in ABOHN, but the difference among the growth was not significant (p > 0.05) when compared with the AB control broth media. Candidatus 'A. salitolerans' F166- 33 T and P. aquimarinus W63 T was higher (no significant difference as p > 0.05) in ABOHSW than ABOHN. While M. marinus W132-33, A. butzleri F170G17, and P. aquimarinus F185-17 have comparatively higher growth (no significant difference as p > 0.05) in ABOHN (Arcobacter broth + 2.5% NaCl supplemented with Oyster homogenate) than ABOHSW. There was no significant difference among all 14 Arcobacter-like species when compared with control AB enrichment broth.
Growth of Arcobacter-like species in oyster homogenate enrichment broth group. The enrichment broth group consisted of four enrichment broth media prepared only with oyster homogenate i.e. OH, OHNC, OHSW and OHSW100, without addition of any conventional media components or carbon source. Almost all the strains showed growth (Fig. 3) in these different enrichment broth media but with lower levels than the control broth AB. One exception was for the strain A. butzleri LMG 10828 T , which showed comparatively higher growth than in the control AB broth in comparison to all other strains, but the growth was not significantly higher (p > 0.05 Growth of Arcobacter-like species in conventional broth media supplemented with powder from lyophilized oysters. This media group consisted of five types of enrichment broth media prepared by adding to the conventional broth media powder from lyophilized oysters. In this group of enrichment broths ABLSW was the most favourable enrichment broth followed by ABLNC in which almost all Arcobacter-like species showed significantly higher growth (p < 0.05) compared to the control AB media except for A. butzleri F170G17, A. butzleri LMG10828 T , A. hispanicus F164-18 and P. aquimarinus W63 T (Fig. 4) Growth of Arcobacter-like species in salty water or artificial seawater supplemented with powder from lyophilized oysters. This group of media were prepared with lyophilized oyster powder in distilled water, alone or with a basal solution of 2.5% NaCl, 50% ASW or 100% ASW. This group consisted of four types of broth media. Growth was observed in almost all four types of broth included in this group for all Arcobacter-like species tested ( Fig. 5)

Discussion
Some Arcobacter-like species are considered emerging foodborne pathogens 3 . They were initially reported from terrestrial environments, food and dairy products but many were later on found associated with seafood 9,24,30 . Shellfish have been considered as reservoirs for pathogenic Arcobacter-like species for humans 26,44 . These gramnegative rod-shaped bacteria, previously known as the genus Arcobacter, have been recently re-classified into seven genera 2 . This new classification is partially supported by the different nutritional requirements for the growth of some of the new genera like the growth in the presence/absence of NaCl 30 . It had been demonstrated that the addition of salt to the culture media favoured the recovery of many new species from shellfish. However, to our knowledge so far it had not been evaluated if the addition of shellfish-based tissues and inter-valval liquid to the enrichment broth could be an organic component that would favour the growth, isolation and recovery of Arcobacter-like species from shellfish. For the isolation of bacteria from its natural reservoirs, it is always a strategy by environmental microbiologists to provide, with the best possibility, the same nutrients and environmental conditions found in the sampling site. Microbial interaction among microbial communities in nature and their metabolism is dependent on several parameters like pH, nutrients, osmotic pressure, temperature and www.nature.com/scientificreports/ several other factors 55 . The failure in isolation of bacteria in many culture media is attributed to inappropriate culture conditions 56 . Therefore, in order to improve isolation of Arcobacter-like spp. related to shellfish and/or their environment we aimed to incorporate into the standard Arcobacter broth media, a series of enrichments to better mimic the marine and the shellfish environment. The latter were assayed independently or combined and are the following: seawater (ASW), oyster homogenate (OH), lyophilized oyster (LO) and artificial seawater (ASW). Overall, there were 24 different combinations of enrichment broth, which were divided into five groups of enrichment broth media each with different basal ingredients. The composition of oyster meat is 77-83% moisture content, 9-13% proteins, 1-3% fats, and 1-5% glycogen as a main carbohydrate 57 . According to the work of Zhu et al. 58 , Pacific oyster (Crassostrea gigas) contains 79-80% moisture and the composition by percent dry weight was 50-53% proteins, 3-5% fats, 16-22% glycogen and 9-10% ash after freeze drying. The OH or LO materials were provided in a specific quantity in the enrichment broth in order to get an amount of approximately 0.5% organic content from oyster in combination with conventional enrichment broth (group b and d), or 1% organic contents from oyster (in group c and e enrichment broths). These percentages were calculated based on the organic contents of the conventional broth media i.e. Arcobacter broth, tryptic soy broth, heart infusion and marine broth. From the oyster, organic content 0.5% was used where there was an organic content added from the conventional broth media, while 1% was used in the absence of any organic source other than oyster. The different Arcobacter-like species grew in almost all types of broth media, but with large variation between the five groups of enrichment broth. The conventional broth group also comprised three derivatives of Arcobacter broth by adding salts, i.e., Arcobacter broth supplemented with 2.5% NaCl (ABNC), (used previously for isolation of marine Arcobacter-like spp 30,59 .) and two concentrations of ASW i.e. 50% and 100% (ABSW and ABSW100 respectively). The growth Arcobacter-like spp. was comparatively higher in ABSW than previously used ABNC 30 except P. aquimarinus F185-17 and M. canalis F138-33 T where the growth was higher in ABNC.
The major non-essential amino acids were aspartic acid, glutamic acid, arginine, taurine and alanine, which makes about 50% of the total protein content in oyster meat. Leucine, valine, lysine, isoleucine, phenylalanine, methionine and threonine are also present as essential amino acids in the composition of oyster (C. gigas) meat. Saturated and unsaturated fatty acids like palmitic acid are also found in C. gigas meat. Calcium is the most abundant macro mineral followed in C. gigas by Mg along with Zn, Cu, Fe and Se 58 . The results obtained from these experiments shows that Arcobacter-like species nutritional requirements vary widely. The overview of results from all the species indicates that seawater has a very positive influence on enhancing growth of Arcobacter-like species in the conventional broth media group and in enrichment broth combined with LO (group 3a). The enhanced recovery of Arcobacter-like spp. by the addition of 2.5% NaCl in Arcobacter broth resulted in the recovery of 40% more Arcobacter-like spp. 30 , compared to the previously used Arcobacter broth. Recently, a similar approach used Arcobacter broth + 75% ASW for the isolation of Arcobacter-like species from abalone (Haliotis gigantea) samples and this resulted in the recovery of two previously unknown Arcobacter isolates 27 . The higher growth shown by the species such as Candidatus 'A. salitolerans' F166-33, M. marinus W132-33, M. halophilus F166-43, www.nature.com/scientificreports/ M. molluscorum F189-DG33, H. bivalviorum F159-36 and M. canalis F138-33 T in ABSW (group 1) compared to control broth AB, indicates that the growth of these species is significantly influenced by the presence or absence of seawater. These results clearly indicate that using seawater in the media preparation improve their isolation and enhance cultivation of Arcobacter-like species from marine environments. In addition to Na and Cl, other ions though collectively in extremely low concentrations, can be critically important for marine organisms 60 .
Tyler et al. 61 demonstrated that 35 out of 96 marine bacterial isolates had been isolated from marine samples by adding all 4 major salts i.e. NaCl, MgCl 2 , MgSO 4 , and KCl in 1% (w/v) trypticase broth media. Neither of these isolates could be recovered in the absence of any of these salts. This may be due to the inability of marine bacteria to grow on the substrate or media combination provided as we have very little knowledge of the organic substrate and its concentrations available to those bacteria in the marine environment 62 . Marine bacteria need Na + for their growth, as the ion is necessary for enzyme activity, membrane stability, active transport, respiration-dependent primary Na + -pumps and many other functions in bacterial cells [63][64][65][66][67] . The genome analyses of many human and animal bacterial pathogens show that they encode Na + based enzymes i.e. decarboxylases, oxidoreductases and permeases as well as membrane primary Na + pumps instead of, or in addition to H + pumps, and these might be important virulence factors for several pathogenic bacteria 68 . Further, the presence of ions can affect the mechanical properties of biofilms and serves as a cross linker in the attachment during biofilm formation 69,70 . The complete elemental requirements for cell growth (i.e. which elements and in which amounts/ concentration) for Arcobacter-like species remains an area for future studies. Although, the 14 species used in this study (out of 29 Arcobacter-like species known) were originally isolated from the marine environment, we still lack complete knowledge on the diversity and presence of commensals or pathogenic species among those genera found in marine invertebrates 27 . The approaches followed by Salas-Masso et al. 30 , Mizutani et al. 27 and Kim et al. 71 resulted in enhanced recovery of Arcobacter-like species from shellfish, abalone and food samples. These studies indicate that still there is a great potential for isolation of new species, especially from the marine environment.
The supplementation of shellfish tissue (OH and LO) into the enrichment broth significantly enhanced the growth of Arcobacter-like species (p value < 0.05) especially in the ABLSW enrichment broth (Arcobacter broth + Lyophilized Oysters + 50% ASW) when compared to the control broth AB. The supplementation of only LO did not improve the growth in the absence of 50% ASW, which may suggest a synergy between the ASW and the LO supplement for the enhancement of the recovery of strains from marine environments. While the lowest growth was observed in ABOH enrichment broth for most species, the addition of only OH into Arcobacter broth did not have a promising effect on the growth either. The control AB broth when supplemented with LO flesh (i.e. ABL broth) does not seem to improve growth, and even decreased growth for most of the species. However, when ABL was supplemented with salts such as 2.5% NaCl and 50% ASW, there were significant improvements in growth for most of Arcobacter-like spp. mentioned above.
Through successive studies, a more efficient broth ASB II (Arcobacter selective broth II) has been adopted for the enrichment of Arcobacter-like spp. 72 . However, fastidious microorganisms such as Gemmata obscuriglobus and Gemmata massiliana have been successfully grown using supplements, such as ground fresh sponge tissues, in culture media 56 . Similarly, the results herein indicate that Arcobacter broth + 50% ASW (ABSW) and Arcobacter broth + Lyophilized Oyster + 50% ASW (ABLSW) are a better enrichment broth for the isolation of Arcobacter-like species from shellfish and marine environments, as compared to the Arcobacter broth used in many studies. Therefore, these protocols should be adopted in future studies for the isolation of Arcobacter-like species from shellfish samples. Unidentified specific components of oyster flesh/ASW may contain key trace compounds necessary for fulfilling nutritional requirements and enhancing growth of some Arcobacter-like spp. The biochemical composition of C. gigas flesh is a rich source of proteins, essential and non-essential fatty acids, and macro and micro minerals 58 . Preferably, lyophilized oysters should be used in the media along with ASW, rather than oyster homogenate because this confers to the broth media a dark green colour and increases turbidity thereby obscuring observations.
The results obtained from this study suggest that marine-derived Arcobacter-like species have somewhat specific nutritional requirements. The approach used in this study should result in more efficient isolation and cultivation of marine-derived Arcobacter-like species in the future and could help enhance the description of diversity of species obtained from shellfish. These finding suggests that Arcobacter broth + 50% artificial seawater (ABSW) is the best enrichment broth medium for the growth of marine based Arcobacter-like spp. A more detailed work is required to refine the lyophilized oyster material used for supplementation and study the effect of different biochemical components in the lyophilized oyster on the growth of individual species or on the different genera as target groups.

Material and methods
Bacterial isolates. The experiment was performed with 14 different Arcobacter-like species that belonged to 5 out of 7 different genera according to the new classification of Perez-Catalunya et al. 2 shown in Table 1. Most bacterial isolates used in this study were previously isolated in IRTA (Sant Carles de la Rapita, Spain) and Universitat Rovira i Virgili (Reus, Spain) from shellfish or their water environment; type strains have been included for comparative purposes ( Table 1). Out of 14 Arcobacter-like species used in this study, 13 species were isolated previously from Alfacs Bay and shellfish exposed to Poble Nou Channel Water and one Arcobacter-like Type strain was purchased from Spanish Culture Collection, CECT ( Preparation of oyster matrices for media supplementation. Two types of oyster matrices were prepared as the organic nutrient source and were supplemented into the conventional broth media, as explained below. Oyster homogenate (OH). Two kilograms (2 kg) of depurated oysters (Crassostrea gigas) produced locally in the Ebro Delta bays (Tarragona, Spain) were purchased from a commercial store. The oysters (n = 30) were of commercial size, ranging from 65 to 97 cm in shell length. They were rinsed and cleaned externally before opening. The oyster flesh (299 g) with its inter-valval liquid was collected in a sterile beaker. Then the samples were homogenized with a sterile kitchen blender. The homogenized oyster flesh was used in the preparation of the different kinds of broth used in the experiments.
Lyophilized oyster (LO). The Pacific oysters (C. gigas) were obtained from Ebro Delta, Spain and dry mass of lyophilized oysters, was prepared by freeze drying flesh and inter-valval liquid of oysters in a lyophiliser. The dry mass was then ground to a fine powder. The fine powder obtained after grinding was used as a supplement in the different experimental broth media.
Media preparation. Broth media preparation. Five groups of different broth media were prepared. These groups were based on using three conventional broth media with or without oyster supplementation (OH or LO) and prepared using different salt compositions (NaCl/ Artificial seawater [ASW]) and strengths (50% ASW or 100% ASW). The pH and salinity of each broth was measured. The experiment was done in duplicate.  Table 2).

Enrichment broth media containing lyophilized oyster:
3a. Conventional broth media supplemented with lyophilized oyster: This group of media was prepared by supplementing 12 g/L Arcobacter broth or 18.7 g/L marine broth i.e. half amount of that given by manufacturer instruction for both conventional broth media, with 5 g/L lyophilized oyster. Arcobacter broth supplemented with LO (ABLO) were also prepared with 2.5% NaCl or 50% ASW or 100% ASW. This group contained five types of broth media (Broth# 16-20 of Table 2). 3b. Lyophilized oyster broth media: This group of media consisted of four types of broth (Broth# 21-24 of Table 2) by adding 10 g/L of lyophilized oyster powder in to either distilled water or distilled water containing 2.5% NaCl or 50% or 100% ASW. www.nature.com/scientificreports/ Experimental procedures. Inoculum preparation. Fresh colonies of the 14 Arcobacter-like species grown on marine and blood agar mentioned above (Table 1) were transferred to sterile marine broth and tryptic soy broth (TSB) tubes, respectively. The tubes were vortexed for 10 s and incubated in aerobic condition at 30 °C for 24 h to get the uniform fresh culture of the colonies in a liquid medium. The optical density at 420 nm was adjusted, with sterile marine broth, to OD value 0.1 for all the strains used in the study (Table 1).

Broth inoculation and incubation:
. From the inoculum, 0.1 ml was transferred to the 9.9 ml pre-labelled broth tubes of different enrichment broth, vortexed and incubated in aerobic condition at 30 °C. The OD 420nm was measured for each strain at 0 h, 24 h and 48 h in different broth media (Table 2). Sterile broth of each formulation was used as control for the OD reading.
Inoculation on solid media:. After the incubation for 48 h, the growth of all the strains in the 24 types of enrichment broth was determined by culturing on marine agar, 100 µl of each enrichment broth. The plates were then incubated in aerobic condition at 30 °C for 48 h. Purity of the cultures was assessed, and relative growth was recorded as number of colonies following a scale from 0 to 4 (0 = No growth; 1 = 1-30 colonies; 2 = 31-150 colonies; 3 = 151-300 colonies; 4 = > 300 colonies).
Statistical analysis. The growth results are expressed in the terms of optical density (OD 420nm ) for each strain tested in each enrichment broth used in this study. Values of the mean and standard deviation were calculated for duplicate OD 420nm values. Statistical analysis was performed in SPSS 21.0 (IBM SPSS Statistics, SPSS Inc., USA). Analysis of variance (ANOVA) and differences among the mean values were tested by Tukey's test. Significant difference was accepted for p < 0.05.