Marine microorganisms: the world also changes - …formatex.info/microbiology3/book/1285-1292.pdf · Marine microorganisms: the world also changes Pilar González-Párraga, Alberto

Marine microorganisms: the world also changes

Pilar González-Párraga, Alberto Cuesta, J. Meseguer and Mª Ángeles Esteban

Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain

In recent years, the increasing trend of pollution has been extended to the marine environment, although it has been one of natural places more stable for years, thanks to the great development of marine aquaculture worldwide at present is being severely affected. The indiscriminate use of antimicrobial compounds begins to promote an important change in the microflora of both marine and inland waters. Microorganisms previously known as non-pathogenic or even producers of antimicrobial compounds, which had been previously described as commensally or asymptomatic organisms, begin now to be described as opportunistic pathogens, being this character often associated to stress conditions. This chapter will describe the major groups of marine microorganisms previously described as asymptomatic and members of the external bacterial microflora of fish. Within these organisms is distinguished between two groups, those who were known as asymptomatic, in which recently began to appear potentially pathogenic species or even strains and a second group consisting of bacterial type previously described as pathogenic which start having antimicrobial resistance known or permitted in the actual aquaculture practices.

Keywords: Marine; drugs; resistance; bacteria.

Introduction

The marine environment hosts a large biodiversity and is the mainstay of economic activities with a long tradition in our society. Despite its importance as a source of food-related resources, energy or medicine, we know very little about the species of microorganisms living and functioning in aquatic ecosystems. This aquatic ecosystem includes bacteria involved in the processing of organic matter and those who live as parasites or symbionts. Many microorganisms in marine medium often produce antibacterial substances that allow the ecological stability of ecosystem [1]. These bacteria-bacteria interactions are a mechanism to keep the microhabitat to certain species of microorganisms [2]. Most of these studies have addressed the effect of other pathogenic bacteria in fish and mollusks [1]. However, these mechanisms for regulating the growth of other bacteria appear to be inhibited in situations of stress such as changes in water temperature [3]. Aquaculture is an important contribution to the nutrition of many communities around the world, but overfishing, introduction of exotic or alien species to the environment habitat or environmental degradation and water pollution have contributed to destruction and alteration of the ecosystem. One of the most serious problems facing the ocean is the pollutants from land-based sources or activities, as well as marine industrial activities such as aquaculture. The extensive freshwater or marine farming systems result in overcrowding. This increase in biomass is limited by the capacity of the marine ecosystem which brings a decline in growth, increased mortality and increased diseases. The microbiological activity is by far the most important factor influencing fish quality [4]. However, the growth and even the survival of the aquaculture industry are threatened by uncontrolled microbial diseases that cause extensive losses [5]. In this chapter we will describe some of the major bacterial species that are becoming responsible for important economic losses in the aquaculture industry in contrast to the previously well-known pathogens. Microorganisms usually described as normal in the surface microflora of fish have recently been described as opportunistic pathogens. In many cases, strains have been also described with antibiotic resistance capacity and even the inhibitory compounds produced by other bacteria of the microflora.

Microorganism distribution in marine water

Microorganisms are extremely important for marine life because they are essential in the destruction of organic matter. In the field of marine microbiology, the marine microbiologist CE Zobell conducted studies to demonstrate the essential role of bacteria in the cycle of living matter in seawater. In general, the higher amounts of bacteria are commonly found in the sea surface, in the highlights zone and together with the phytoplankton, and decreasing with depth. The distribution of bacteria in the seas tends to be parallel to the plankton [6]. In the sediments there are high counts of bacteria and fungi, which play an important role in the remineralization of organic matter and feeding of the deep sea fauna. Researchers believe that there are thousands of other undiscovered marine bacteria because the list of species of marine bacteria isolated to date, does not reflect the diversity of forms present in the sea. Some of these marine bacteria present great difficulties in identification or taxonomy because of its difficult isolation and culture since many of them have special needs for growth or are only able to grow associated with other organisms [3]. There are not marine

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bacteria that are harmful and dangerous to humans. But instead, a number of pathogenic microbes can be washed out to sea by sewage from landfills and sewers, or rivers and adapt to the marine environments. This ability to adapt is linked to the problem that some marine organisms feed by filtration of these contaminated waters, and therefore pathogenic bacteria for humans. In general, populations of marine bacteria can not survive the strong environmental changes, such as heat, or changes in their physical and chemical conditions. Normally their living conditions are more limited and lack of resting states in their life cycle. However, the ability of this marine bacterium to grow at relatively high salt concentration (up to 10%) is noteworthy. The distribution of bacteria in seawater is determined by a series of physicochemical and biological factors, and apparently there are two fundamental causes of this distribution, the amount of available decaying organic matter and the density of planktonic organisms within the waters. Repeated observations support the conclusion that the abundance of bacteria depends on the amount of plankton organisms, which represent the main source of food for bacteria and natural bases which are linked. It has been found that the distribution of bacteria in the marine environment is extremely abundant in the coastal region and decreases dramatically as the coast is becoming more remote. In areas near the coast most of the bacteria keep similarity with the land while in remote areas all organisms are halophilic and psychrophilic although there are also barophiles. Its categorisation in terms of temperature is more complicated [7]. The maximal cell densities is achieved between the growth temperatures of 10 and 15º C (at all salinities examined) and the poor growth yields above 25º C suggest a classification within the psychrotolerant organisms [7]. The bacteria most frequently isolated from the marine environment are gram negative, mobile, while in sediments are usually gram positive. Regarding the vertical distribution, the largest number of organisms found in the superficial zone, where many groups of bacteria and the number is decreasing to the great depths, but increased again in the sediment where we found up to 420 million bacteria per gram of mud from the sea.

Effects of aquaculture on the environment

The marine environment has been one of the best maintained over time. However, environmental problems derived from aquaculture, such as overfishing, introduction of new species with different microflora brought from other countries, the excess of biomass and waste generated, as well as the indiscriminate use of antimicrobial compounds begins to produce a major change in the microflora of both marine and inland waters. In example, pathogenic microorganisms such as Pseudomonas, both the man and aquatic organisms, begin to be dangerous unexplored [3]. Antibiotics are used by farmers both to prevent infection by pathogens as well as to treat the fish affected for them [8, 9]. The Common European Directives (2003/99/EC) are one example of regulations dealing with this issue. However, the use of antibiotics in aquaculture is limited, because the marine pathogens have also shown resistance mechanisms to these antibiotics [10]. An alternative method to control various microbiological problems that occur in the farms would be the addition of pure cultures of bacteria producing inhibitory substances for the pathogenic ones [10, 11, 12]. As occurs in clinic, the origin of the antibiotic resistance genes has long been a mystery [13]. The widespread of these genes is usually recognized by scientists and the public owing to the overuse of antibiotic drugs [14]. There are some increasing evidences that antibiotic resistance genes in pathogens have an environmental origin [13, 14]. The NDM-1 encoding plasmids also harbour genes conferring resistance to almost all antibiotics. These data infer that marine originated β-lactamases may stringently be regulated as a protective mechanism against β-lactam molecule secreted by antibiotic producers that share the same ecological niche [14]. The field of marine microbiology is still evolving and one can expect significant progress on the problems of pollution of sea water such as bacterial degradation of oil that is under investigation nowadays. To counter these problems every day becomes more significant in the seas.

Description of the main microorganisms affecting aquaculture

As we have mentioned above, below we will described some of the bacterial genera associated with fish farming that begin to describe how new pathogens or antibiotic resistance all display did not have before following the classification described by Bergey bacteriological (Manual of Systematic Bacteriology). There are pathogens found among all the domains of life, the Archaea, Eucarya and Bacteria [15]. In this chapter we will do reference to only pathogen genus or strains included in domain Bacteria, Class Betaproteobacteria (Alcaligenes sp and Comamonas sp), Class Gammaproteobacteria (Halomonas sp, Pseudomonas sp, Psychrobacter sp, Alteromonas sp, Pseudoalteromonas sp, Shewanella sp, Vibrio sp, Photobacterium sp, Aeromonas sp and Providencia sp), Class Bacilli (Staphylococcus sp), Class Flavobacteria (Tenacibaculum sp) and Class Sphingobacteria (Sphingobacterium sp).

Class Betaproteobacteria: Genus Alcaligenes

Alcaligenes is a gram-negative, rod-shaped or globular, motile obligate aerobe, heterotrophic nitrifier that is commonly found in the environment [16, 17]. Genus Alcaligenes is part of natural nonpathogenic bacterial microbiota and had been isolated from water as well as in the mussel samples [18]. Alcaligenes faecalis is not described as a microorganism

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that causes disease in aquaculture, although a subspecie A. faecalis subsp homari was transferred to the genus Halomonas as H. aquamarina, which produce disease in crustaceans such as lobster, being isolated from the hemolymph and inducing a softening of the shells [19, 20]. Recent researches have reported on a novel substance with immunostimulating activity which is present in culture supernatants of the Alcaligenes faecalis FY-3 strain [17]. As far as sensitivity to antibiotics, Alcaligenes faecalis is sensitive to antibiotics like meropenem and imipenem [21]. Other antimicrobials have moderate activity (ceftazidime, cefoperazone, minocycline, piperacilline and colistine), while trimethoprim-sulfamethoxazole was not active against A. faecalis and other drugs like ciprofloxacin, norfloxacin, aztreonam, gentamicin and amikacin were not active against most strains of this species [22, 23].

Genus Comamonas

Comamonas is an aerobic gram-negative bacillus that grows forming pink-pigmented colonies, that is motile, nonglucose fermenting, nonspore forming. Comamonas are commonly found in soil, plants, water saprophytes, and also in humidifier reservoir water. Although they are usually considered a commensal, some species of Comamonas are potentially pathogenic but has not been well studied [24]. Comamonas testosteroni, classified as Pseudomonas testosteroni until 1987, has been scarcely implicated as causative of human infections in sites such as the abdomen, bloodstream, central nervous system, and urinary tract [25]. These bacteria are usually associated with treatments with antibiotics. There are data in the literature that report the case of bacteremia in humans associated with exposure to tropical fish [26]. Within this genus enclosed a lot of recently described species, some as Comamonas koreensis [27] or Comamonas nitrativorans [28] isolated from waste treatment plants or sewage. A recent study identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity to the Pasteurella multocida glycosaminoglycan synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule [29]. Differences have been reported in the antibiotic susceptibilities of different isolates of C. testosteroni. Isolates from blood were susceptible to tetracycline, trimethoprim/sulfamethoxazole, penicillins and cephalosporins with mixed

susceptibility patterns to aminoglycosides and quinolones while isolates from intra-abdominal infections showed uniform susceptibility to aminoglycosides. In general, all of the isolates were resistant to ampicillin and some isolates are resistant to aminoglycosides and susceptible to quinolones and ceftazidime [30].

Class Gammaproteobacteria: Genus Halomonas

Halomonas are gram-negative rods, nonfermentative, moderately halophiles, requiring high NaCl for growth in a variety of temperature and pH conditions. In addition, several members of the Halomonas group are capable to partially oxidize sulfur compounds such as thiosulfate and sulfide into tetrathionate under halo-alkaline conditions [31]. The family Halomonadaceae forms a separate phylogenetic lineage within the Gammaproteobacteria according to 16S rRNA gene sequence analysis and is made up mostly of halophilic bacteria [32, 33]. The genus Halomonas is not monophyletic and comprises two clearly separated phylogenetic groups that now contain larger numbers of species [33]. Some species belonging to this genus have been reclassified and included in genus Cobetia or Chromohalobacter [33]. The genus Halomonas is ubiquitous and most of the Halomonas species are a constituent of a bacterial group that is very abundant in marine environments [34]. It comprises a set of species that live in high salinity conditions [35] such as fermented and salty foods [36]. In the case of marine organisms, representatives of the genus Halomonas have been isolated and identified such as the specie H. venusta found in the surface of the seaweed Ulva pertusa, Laminaria japonica, Gracilaria textorii and Polysiphonia urceolate [37]. The halophilic proteobacterial genera Halomonas sp and Salinivibrio sp dominated the branchiopod genus Artemia (brine shrimp) microbiota in both two different stages in development: in nauplii and in the intestine of adult animals [38]. Autochthonous microbial communities can also act as probiotics providing digestive enzymes such as Halomonas sp, which is known to produce extracellular a-amylase and may have high lipolytic and DNase activity, moderate proteolytic, xylanolytic and inulinolytic activity [38]. Various studies have demonstrated the exopolysaccharide production properties of Halomonas species [34] but only one Halomonas strains have been associated with larval scallop mortality. It seems that Halomonas pathology appears to be directly related to poor husbandry, suggesting that sources of bacterial infection are broodstock, algal cultures, and incoming seawater, as previously stated by Elston [34]. Halomonas venusta is even known for a human infection caused from the bite of a fish [39]. In general, bacterial infections that involve a biofilm are not susceptible to the antibacterial treatments. In particular, Halomonas sp is resistant to tetracycline and florfenicol during scallop cultivation. The routine antibiotic treatment or UV sterilisation as a preventive measure in scallop hatcheries could also be effective to control planktonic cell numbers of genus Halomonas before bacterial adhesion on scallop shells, thus reducing the potential to form biofilms [34].

Genus Pseudomonas

Strains of genus Pseudomonas are curved rods, gram negative, motile with one or more polar flagellums. This genus is in general catalase and oxidase positive, strictly aerobic but some strains can use nitrate in anaerobic conditions. It is

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common the presence of plasmids and do not form spores. Some species synthesize an exopolysaccharide capsule that facilitates cell adhesion, biofilm formation and protects against phagocytosis, antibody or complement binding, thus increasing its pathogenicity [40]. Pseudomonas are characterized by an enormous metabolic capacity which is reflected by their capability to adapt to diverse environments, such as terrestrial and marine, degrade compounds and synthesize great variety of molecules. Pseudomonas is the most common genera in crustaceans, marine fish and bivalves [41]. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants [42]. Plasmids and bacteriophages are important contributors to the genetic diversity found in Pseudomonas sp [42]. Pseudomonas aeruginosa participates in infections in immunocompromised patiens and has emerged as a model organism for biofilm studies. The group of bacteria related to the genus Pseudomonas is very broad and includes species pathogenic for humans and plants commonly found in fresh altered water. In the case of marine organisms species of the genus Pseudomonas have been isolated and identified from the microbiota of farmed fish such as rainbow trout (Oncorhynchus mykiss) [43], perch (Perca fluviatilis) [44] and rohu (Labeo rohita) [45]. There are also data that report the role as a fish pathogen of some species such as P. fluorescens [46], P. anguilliseptica, which has been identified as the cause of mortalities in Japanese eel (Anguilla japonica) and European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus) [47, 48, 49]. P. plecoglossicida causes hemorrhagic ascites in freshwater fishes of Japan [50] and P. luteola causes mortalities in rainbow trout [51, 52] and there are other diseases in fish that have not been determined the Pseudomonas species that has caused them [53]. Korkea-Aho et al [54] have described that Pseudomonas M174 strain is a potential probiotic against Flavobacterium psychrophilum and has several modes of action. An additional property of Pseudomonas sp is their resistance to many antibiotics [55]. Pseudomonas aeruginosa is responsible for a high percentage of nosocomial infections. The difficulty in its treatment by antibiotics arises from its capability of nearly expressing all mechanisms of antibiotic resistance; hence, it is considered a multidrug-resistant (MDR) organism [56]. Many compounds have been identified as efflux pump inhibitors (EPIs) when used as adjuvants or in combination with the effective antibiotics [56]. Certain parameters are essential for choosing between the general EPI that can inhibit the action of one transporter that expels various antibiotics in one bacterial species or a specific EPI that inhibits the pumping of one antibiotic family in various bacteria [56].

Genus Psychrobacter

Psychrobacter are psychrophilic and halophilic microorganisms, aerobic, stationary and catalase and oxidase positive, also show no pigment [57]. The optimum growth temperature is about 20º C but authors have described Psychrobacter strains which can growth about -10º C until 26º C [58]. The Psychrobacter strains have diverse representatives of the permafrost community, should carry traits that has allowed them to adapt to these conditions [58]. Changes in membrane composition and exopolysaccharides are the results of growing in presence and absence of 5% NaCl [58]. The role of the genus Psychrobacter as a pathogen is unclear and reported only one species, Psychrobacter immobilis, which was isolated from rainbow trout showing a natural infection [59]. Under certain experimental conditions, infection with Psychrobacter immobilis failed to produce mortalities although external symptoms, such as darkening of the skin, pale gills and abnormal swimming and other internal symptoms such as dilation of vascular structures, infiltration of mononuclear cells in the liver, degeneration of gills, vascular congestion, etc. were described [59]. The microorganism P. marincola is described as new specie of the genus Psychrobacter by Romanenko et al [57], which was isolated from seawater samples as well as tissues of a tunicate. Resistance to antibiotics of Psychobacter is beginning to be described. Some authors have described that this sensitivity or resistance depends on the particular strain and the growth temperature [58]. Sulfonamide resistance was reported for the first time by Byrne-Bailey et al [60] and a Psychrobacter psychrophilus strain has been described like resistant to tetracycline and streptomycin was isolated from subsoil sediment sampled from the coast of the Eastern-Siberian Sea. The genes conferring antibiotic resistance were localized on a plasmid [61].

Genus Alteromonas

Alteromonas is a genus of gram-negative aquatic bacterium with curved rods and motile by means of a single polar flagellum; require a seawater base for growth. Alteromonadaceae family includes genera such as Alteromonas, Pseudoalteromonas, Marinobacter and Shewanella which include marine bacteria. Encompassed many of the bacteria in the genus Alteromonas previously have been relocated to the genus Pseudoalteromonas after phylogenetic analysis of 16S rRNA, as in the case of Alteromonas elyakovii [62] is currently identified as Pseudoalteromonas elyakovii. Pseudoalteromonas genus has species associated with marine organisms, but only some of them are related to disease. This is the case of certain crustaceans and mollusks [63]. In an epidemiological study, representatives of four species of Pseudoalteromonas were isolated from the internal organs of European sea bass (Dicentrarchus labrax) and seabream [64]. In addition, this genus proved to be the prevalent microorganisms isolated from sea bass showing clinical symptoms and gold without it. However, in experiments to test the possible pathogenicity of these species could be demonstrated that only one of the species tested, identified as P. undine, was weakly virulent to sea bass.



The genus Alteromonas is resistant to different antimicrobial agents such as ampicilline, kanamycin, rhodamine 6G, crystal violet, malachite green and sodium dodecyl sulphate [65]. Other studies provide further evidence that environmental, particularly aquatic bacterium Aeromonas sp contains functional resistance genes with the potential to impact on the effectiveness of clinically important antibiotics [66].

Genus Shewanella

This is a genus of gram negative and facultatively anaerobic rods. It is a saprophytic marine organism which is often isolated from spoiling fish. Shewanella is the sole genus included in the Shewanellaceae family of marine bacteria. Shewanella sp are Proteobacteria than shared taxonomic category with Salmonella or Campylobacter. This genus belongs to the class of the Gammaproteobacteria and the order of the Alteromonadales. Shewanella putrefaciens, previously classified as Achromobacter putrefaciens, Alteromonas putrefaciens and Pseudomonas putrefaciens, is a species widely distributed in nature and a well-known agent of fish spoilage. One species, Shewanella algae, has been linked to the production of a tetrodotoxin [67]. The genus Shewanella comprises species which are widely distributed in aquatic environments. Shewanella species have been isolated from marine fish [68, 69], freshwater fish [44] and molluscs [70]. Some Shewanella species such as S. marisflavi [71] and S. alga [72] and freshwater as S. putrefaciens have been described as pathogens of marine organisms. There is information about the role that some strains of these species plays as antimicrobial against certain fish pathogens and improve the tolerance of golden stress induced by culture at high densities [73]. Furthermore, Shewanella putrefaciens, is not only a member of the microbial association found in fish from temperate waters, but they also contribute significantly to the spoilage of fish stored under different conditions. Thus, Shewanella sp were found to be the specific spoilage organism in fish from Mediterranean Sea (temperate waters) stored aerobically in ice [74]. Genes that confer resistance to antibiotics are commonly found in plasmids. Transferable qnr genes are usually carried by large conjugative plasmids that often encode extended spectrum lactamases (ESBLs) or AmpC-type -lactamases. The qnr genes were shown to be located in the vicinity of intact, antibiotic resistance determinant-containing class 1 integrons. Transfer of plasmid-borne qnr was shown to occur by conjugation. Chromosome borne qnr-type genes were discovered in environmental bacteria such as Photobacterium profundum, Vibrionaceae and Shewanella algae [75].

Genus Vibrio

Microorganisms belonging to the genus Vibrio are gram-negative curved bacilli, facultative anaerobes, oxidase and catalase positive, non spore with a polar flagellum which gives them great mobility. They are aquatic bacteria, most are halophilic and any of them may also live in alkaline environments. Many species of this genus are pathogens, symbionts or parasitic [76]. Infections caused by these organisms are usually associated with ingestion of raw shellfish or exposure of wounds to sea water [76]. Within this genus there are species such as Vibrio alginolyticus, V. chagasii, V. gigantis, V. harveyi, V. rumoriensis and V. tubiashii isolated from marine organisms [77]. V. alginolyticus has been associated with vibriosis, which has produced gold and mortalities in cultured sea bass in the Mediterranean countries [78, 79]. It is a bacterial specie that shows an enormous intraspecific variability and its pathogenicity appears to be specific to a strain of V. alginolyticus in particular. In fact, it seems that the pathogenesis of V. alginolyticus is the sum of the concentrated action of multiple virulence factors. Certain experiments showed the virulence of V. alginolyticus was correlated to that of the collagenase-SSCP type, which demonstrated that some strains were low-virulent isolates, while different strains were highly virulent [80]. Vibrio chagasii, phylogenetically related to V. splendidus and other species such as V. lentus, V. pomeroyi and V. kanaloae, has been associated with pathological symptoms of certain marine species [81]. It was described as a new species of the genus Vibrio by Thompson et al [82], being isolated from turbot larvae (Psetta maxima) and cultured rotifers. Likewise, this species has been identified as one of the most abundant taxa present in the mucus of the coral Mussismilia hispida [83]. Vibrio gigantis was isolated and identified for the first time as a new specie of the genus Vibrio by Le Roux et al [84] from marine organisms such as sea cucumber (Apostichopus japonicus) [85]. V. harveyi is one of the genus Vibrio species most frequently isolated [86], and has been associated with high losses in cultured shellfish and opportunistic diseases in marine fish [87]. The administration of V. harveyi on diet causes stress after 48 h in seabream. Results from our group support the idea that this bacterium alters immune system parameters at the cellular level, reducing the phagocytic ability and respiratory burst of head-kidney leukocytes. For the same case humoral immunity was not affected (unpublished data). Zorrilla et al [88] have demonstrated the important role that extracellular products (ECPs) play in the pathogenesis of this microorganism, which has also been seen in the case of shrimp and Atlantic salmon (Salmo salar) [89]. In this sense, Rico et al [90] have seen that there are differences between their patterns of proteins in the ECPs between virulent and avirulent strains of V. harveyi isolated from diseased senegalese sole (Solea senegalensis). Vibrio rumoiensis corresponds to a psychrophilic microorganism isolated and identified from a fish processing plant, which shows a extremely high catalase activity [91]. V. tubiashii was initially described as a pathogen of larval and juvenile mollusks such as oysters (Ostrea edulis) and clams (Ruditapes philippinarum) [92]. Recently, returned to isolate looking like a resurgence of this species as a pathogen of these organisms. This microorganism is capable of producing

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some ECPs that are closely related to its virulence [93]. The pathogenicity of this species to induce disease in certain fish, such as rainbow trout [94] has also been reported and their description among the dominant members of the intestinal microbiota of larval seabream and sea bass [95]. Some Vibrio species isolated from Italian aquaculture showed resistance to ampicillin, carbenicillin, kanamycin, cefalothin, while they were sensitive to chloramphenicol, nitrofurantoin and tobramycin; the sulfadiazine-trimethoprim association was completely ineffective [96]. The treated effluent systems are reservoirs for various antibiotic resistance genes and Vibrio species isolated from wastewater final effluents showed resistances against erythromycin (100%), chloramphenicol (100%), nitrofurantoin, cefuroxime and cephalothin (90-95%) [97].

Genus Photobacterium

Microorganisms included in the genus Photobacterium are gram-negative rod-shaped bacteria, halophilic, facultatively anaerobic, that are common in the marine environment and on the surfaces and in the intestinal contents of marine animals. Some species are bioluminescent and are found as symbionts in specialized luminous organs of fish. P. phosphoreum contains in its genome a lux gene that codes for the enzyme luciferase. This enzyme transforms chemical energy into light energy. Luciferase is a heterodimer with alpha and beta subunits. These two subunits are coded by luxA and luxB respectively [98]. Photobacterium includes species that are pathogenic for various farmed fish. P. damselae subsp piscicida [99] previously named Pasteurella piscicida, which has been described as the aetiological agent of pasteurellosis in marine fish. Adherence by a polysaccharide capsule and extracellular products are an important first step in the pathogenesis of this disease, P. damselae subsp damselae [100] and species that have a symbiotic relationship with fish [101]. Other members of this genus have been described as members of the intestinal microbiota of Nephrops norvegicus [102], Salmo salar [103] and certain corals [104]. Photobacterium damsela subsp piscicida isolated from different geographical areas, found that the majority (93%) of the isolates were resistant to erythromycin, and also, in a lower percentage (less than 10%), to amoxicillin, ampicillin, florfenicol and trimethoprim-sulfamethoxazole [96]. In particular, European P. piscicida isolates were sensitive to kanamycin, florfenicol and trimethoprim- sulfamethoxazole while the Japanese strains are more resistant [96].

Genus Aeromonas

The genus Aeromonas comprises gram negative rod, facultative anaerobic, non-sporulating, motile with a single polar flagellum and grow in the temperature range of mesophilic [40]. Individuals are ubiquitous in fresh and salt water. The genus Aeromonas belongs to the Vibrionaceae family, and has many similarities to the family Enterobacteriaceae. The genus is divided into two groups: the group of psychrophilic Aeromonas which consists of a single species, A. salmonicida and mesophilic Aeromonas group is formed by the species A. hydrophila, A. caviae, A. veronii subsp. sober, A. jandaei and A. veronii subsp. schubertii. Aeromonas hydrophila behaves as an opportunistic pathogen in both aquatic and host environments. It can cause hemorrhagic septicemia, resulting in fin and tail rot and epizootic ulcerative syndrome in juvenile and mature fish or intestinal and wound infection in human [105]. Usually, the identified species of this genus such as Aeromonas bivalvium, Aeromonas hydrophila and Aeromonas salmonicida subsp. salmonicida are associated with marine organisms. The first one was firstly described as a new species of the genus Aeromonas by Minana-Galbis et al [106], following the identification of a strain isolated from bivalve mollusks. However, it has not been described as fish pathogens so far. Aeromonas hydrophila is a ubiquitous organism found in the microbiota of different fish and other aquatic organisms. It can cause disease under stress conditions and is the causative agent of the disease known as hemorrhagic septicemia, or ulcerative disease [107]. This is a microorganism implicated in several epizootic outbreaks in aquaculture [108], some of them affecting sea bass [109]. Fish infected with this bacterium have different symptoms that can range from a lack of appetite, abnormal swimming, pale gills and skin ulcerations [110]. In Atlantic salmon, A. salmonicida causes the disease known as furunculosis and it causes lesions in the dermis, leading to ulcers, added to its ability to penetrate the tissues and organs [20]. It has been isolated from various fish such as salmon, perch, carp, different types flounder, eel, catfish, etc. [111], but has virtually no impact on the golden crops and there is a reference to an infection by A. salmonicida achromogenes in sea bass in Turkey [112]. In particular, Aeromonas is one of the major causes of bacterial infections affecting tilapia [113]. The pathogenicity of A. hydrophila depends on the production of potential virulence factors, such as exoproteases and exotoxin [105]. Application of antibiotics and chemical drugs is a conventional method to control this disease, but generally results in the constant emergence of "superbugs" and chemical accumulation in the food chain [105]. Pathogenic strains of Aeromonas veronii resistant to multiple antibiotics were isolated from A. ocellatus individuals showing signs of infectious abdominal dropsy. The moribund fish showed haemorrhage in all internal organs, and pure cultures could be obtained from the abdominal fluid [114].



Genus Providencia

Bacteria of the genus Providencia are rods, gram-negative opportunistic pathogens that have been isolated from a wide variety of environments and organisms ranging from humans to insects to sea turtles and shark mouths [115]. Members of the genus have repeatedly been found in association with humans, insects and many other vertebrate and invertebrate animals in both pathogenic and non-pathogenic contexts [116]. Numerous studies surveying bacteria associated with insects although it is unclear whether these and other associations have meant the bacteria were acting as pathogens or were simply present in the insects’ environment [115]. As regards aquaculture, Providencia rettgeri has been associated with mass mortalities of carp (Hypophthalmichthys molitrix) in Israel [117]. The organism was isolated from internal organs and skin ulcers in fish affected of severe sepsis. Subsequently, also it was described its presence in the feces of sea turtles (Caretta caretta) [118]. In this study, antibiotic resistant bacteria reflect marine contamination by polluted effluents and C. caretta is considered a bioindicator which can be used as a monitor for pollution [118]. One Providencia stuartii strain was been described like resistant to all -lactams including carbapenems [119]. They encoded a novel metallo--lactamase which was inhibited by EDTA and hydrolyzed penicillins, cephalosporins, and carbapenems [119].

Class Bacilli: Genus Staphylococcus

Staphylococci are gram positive bacterial parasites, coccoid, facultative anaerobes that form irregular colonies. They often cause fever or septicemia. Most species of this genus are described as pathogens for humans and animals and there are two distinct groups: coagulase-positive (Staphylococcus aureus) or coagulase-negative (S. epidermidis, S. haemolyticus, S. simularas, S. hominis, S. saprophyticus, S. capitis and S. warner) [120]. Among them, S. warneri has been detected in diseased freshwater fish [121], as well as in facilities that processed fish products and foods derived from marine products. Signs associated with the pathology produced by this species in fish correspond with exophthalmia, ulcerated fins and there was also isolated from internal organs [121].

Genus Tenacibaculum

Tenacibaculum means ‘rod-shaped bacterium that adheres to surfaces, and all reported species within the genus Tenacibaculum have been isolated from surfaces of marine organisms [122]. The individuals of this genus are gram-negative, oxidase- and catalase-positive. Cells are rods during exponential growth and spherical cells occur in stationary phase. Colonies are bright yellow and flexirubin-type pigment is absent. They are mesophilic with optimal growth around 25–37º C and at pH 6–9. They were capable of hydrolysing several polymeric compounds as casein, collagen, hydroxyethyl cellulose, starch, barley -glucan and pullulan [122]. The genus contains the former Flexibacter maritimus, which has been reclassified to Tenacibaculum maritimum, the type species of the genus [123]. Tenacibaculum mesophilum has been isolated from marine organisms such as sponges and macroalgae and identified as new specie in 2001 [123]. It has been isolated from biofilms and marine organisms since then [124]. Although it has been isolated from disease marine organisms, it has demonstrated its pathogenic potential in experimental infections made with the clam larvae Venerupis pollastre [125]. Avendaño-Herrera et al [3] have tested the in vitro susceptibility of isolates of Tenacibaculum maritimum from fish farms to chemotherapeutic agents used for the treatment of bacterial diseases in fish. Their results indicated that all strains assayed were resistant to oxolinic acid, enrofloxacin and flumequine but susceptible to amoxicillin, nitrofurantoin, florfenicol, oxytetracycline and trimethoprim-sulphamethoxazole [3].

Class Sphingobacteria: Genus Sphingobacterium

The genus Sphingobacterium is gram-negative, strictly aerobic, chemoorganotrophic, catalase, oxidase positive, non-motile, non-spore-forming, non-flagellated, rod-shaped bacterial [46]. Some strains makes colonies grown on triptone soy agar (TSA) are yellow, circular and convex with entire margins. They can growth at 10-45º C, pH 6–9 and 0–5 % NaCl [126]. In the field of aquaculture, Sphingobacterium representatives of the genus have been identified as symbionts microorganisms isolated from the outer membrane of Chinese salmon eggs (Oncorhynchus tshawytscha) but failed to determine the pathogenic nature [127]. When analyzing the effect of certain antimicrobial compounds have on these bacteria can be seen that show sensitivity to the new fluoroquinolones sparfloxacin and levofloxacin, as well as trimethoprim-sulfamethoxazole and ciprofloxacin [128]. However, there are strains resistant to imipenem [128] although its mechanism of resistance has not been elucidated.

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Conclusion

Little is known of marine organisms and much less than the interactions between them. In this chapter we have collected a few examples of bacteria that begin to develop resistance to antibiotics. Many of them are derived from terrestrial bacteria pathogenic or nonpathogenic adapting to life at sea and other marine which acquire resistance with plasmids. Thus, microorganism’s one point considered sensitive over time become resistant to various antimicrobial compounds to adapt to the environment. These microbial habitat changes are due primarily to human activity that takes place exclusively in areas near the coasts. However, we must rely on men to take social awareness and learn to care for the ocean, minimizing such changes and, moreover, we can think that the sea is able to undertake these changes as part of the process of evolution.

Acknowledgements. The authors want to thank the regional Fundación Séneca (04538/GERM/06). A. Cuesta thanks the Ministry of Education and Science (Spain) his contract Ramón y Cajal.

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Marine microorganisms: the world also changes - …formatex.info/microbiology3/book/1285-1292.pdf · Marine microorganisms: the world also changes Pilar González-Párraga, Alberto