ME Martino, Institute of Functional Genomics (IGFL), Lyon, France L Fasolato and B Cardazzo, University of Padova, Legnaro, Italy

ã 2016 Elsevier Ltd. All rights reserved.

Introduction Aeromonas and Laboratory Identification

The genus Aeromonas belongs to the family Aeromonas spp. can be easily isolated from clinical and envi- and comprises gram-negative, non-spore-forming, rod-shaped, ronmental samples. Several media are routinely used for Aero- facultative anaerobic that can be isolated from a very monas isolation, but their performances can vary according to wide spectrum of environmental niches. The history and the the nature of samples (food, clinical, or water) and some perception of Aeromonas by the scientific community have selective agents that can reduce the recovery of some species. evolved over 100 years, from its discovery in the late Aeromonas grow well on routine enteric isolation media nineteenth and early twentieth centuries until nowadays. Aero- (MacConkey, XLD, HE, SS, and DC media); however, the monads were first described as pathogens of poikilothermic lactose-negative isolates must be differentiated from com- animals. Today, they are recognized as causing severe illnesses monly isolated pathogens such as Salmonella and Shigella. in aquatic organisms (fish and other cold-blooded species) and The media that are frequently used for both qualitative and also as emerging pathogens associated with several human quantitative evaluations of Aeromonas spp. are listed in Table 2. infections and, in particular, as food-borne pathogens. In Microbiological methods are clearly needed for bacterial 2010, Janda and Abbott published an excellent review about isolation, but their use as species identification tools can be Aeromonas spp., providing a wide and comprehensive view of very challenging, especially for aeromonads. For instance, it the genus. However, many open questions regarding the ecol- can be difficult to separate A. veronii bv. sobria or A. caviae from ogy, pathogenicity, and of aeromonads were A. hydrophila or they may be confused with other genera, such present, and after 4 years, Aeromonas still represents a very as Vibrio and Plesiomonas. complex genus. In the last decade, DNA-based molecular methods have A distinctive characteristic of Aeromonas has always been its become more popular and widely acceptable for bacteria spe- controversial taxonomy. The complexity in identifying and cies identification due to their reproducibility, simplicity, and discriminating Aeromonas species relies on the extremely high high discriminatory power. Several molecular methods have intra- and interspecies genetic variability. The genus was first been applied for discriminating Aeromonas species. 16S rRNA discovered in 1891 and included in the family of Vibrionaceae gene sequencing represents the most commonly utilized together with Vibrio spp., Plesiomonas spp., and Photobacterium molecular technique for this purpose. However, it is now spp. This was due to the prevalence of these genera in the recognized to be problematic for bacterial characterization aquatic environments and the common phenotypic character- mainly because of its intragenomic heterogeneity. This sug- istics. The genus was officially created in 1943 and aeromonads gested that a single-gene-based identification approach may were roughly divided into two major groups, based upon not be appropriate for characterizing Aeromonas spp. As a con- growth characteristics and other biochemical features. The sequence, the multilocus sequence typing (MLST) approach mesophilic group, named A. hydrophila, consisted of motile became the new trend in the last 10 years. From 2011, three isolates that grew well at 35–37 C and were associated with MLST schemes were published for Aeromonas spp. demonstrat- a variety of human infections. The psychrophilic group, ing the validity of this technique in discriminating aeromonads referred to as A. salmonicida, included nonmotile strains that at species level. Moreover, the first Aeromonas MLST online had optimal growth temperatures of 22–25 C and caused database was opened (www.pubmlst.org/aeromonas) and is diseases in fish. From the mid-1970s until nowadays, an enor- now available for collecting and sharing information about mous explosion in the number of proposed species has been Aeromonas strains from different laboratories all over the seen, and the list of species assigned to the genus is constantly world. changing. This is mainly due to two reasons: (1) the general and recent tendency to propose new species based upon single strains, especially in the last 5 years, and (2) the invalidity of Aeromonas in the Environment some species names or the use of heterotypic synonyms of previously published species. Aeromonas are described as ubiquitous bacteria. They can be To date, there are 27 valid published species names among isolated not only from a variety of aquatic environments and Aeromonas spp. included in the List of Prokaryotic names with from different terrestrial ecosystems, such as food, inverte- Standing in Nomenclature (Table 1), but the second edition of brates, plants, and slurry and fecal contents of farm animals, Bergey’s Manual of Systematic Bacteriology (Bergey’s) recognizes but also as a digestive tract symbiont of fish, leeches, and bats. far fewer. The genome sequences of 46 Aeromonas strains, Initially, three Aeromonas genomospecies (A. hydrophila, A. including both draft and complete genomes, are now available caviae, and A. veronii) were considered to be related to the vast in GenBank. majority to human infections, while A. salmonicida has been

Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00013-1 61 62 Aeromonas

Table 1 List of the valid and proposed species in the genus Aeromonas in Water Aeromonas The main reservoir of the genus Aeromonas has always been the Synonym aquatic environment, with isolates from rivers, lakes, ponds, Species Year of proposal (year of proposal) seawater (estuaries), drinking water, groundwater, wastewater, and sewage in various stages of treatments. A. hydrophila 1943 Many studies have demonstrated the ability of Aeromonas to A. salmonicida 1953 survive and grow in drinking water supplies. The bacterium can A. sobria 1981 A. media 1983 resist to water treatment strategies such as rapid/slow sand A. caviae 1984 A. punctata (1957) filtration, hyperchlorination/direct filtration, and the use of A. veronii 1988 A. ichthiosmia (1991), granular activated carbon. Studies indicated that after disinfec- À A. culicicola (2002) tion with 1 mg l 1 of chlorine, 10% of the pipes had aeromo- A. eucrenophila 1988 nads and that A. hydrophila in biofilms could survive up to À A. schubertii 1989 0.6 mg l 1 of monochloramine, which could remove E. coli A. enteropelogenes 1991 A. trota (1992) biofilms. Some studies reported that the presence of Aeromonas A. allosaccharophila 1992 in drinking water could lead to septicemia in immunocompro- A. jandaei 1992 mised persons, although no link has been demonstrated so far. A. encheleia 1995 Due to the prevalence of Aeromonas in drinking water, the A. bestiarum 1996 A. popoffii 1997 onset of new resistance mechanisms, and the presence of sev- A. simiae 2004 eral virulence factors, aeromonads are included in the ‘Con- A. molluscorum 2004 taminant Candidate List’ by the Environmental Protection A. bivalvium 2007 Agency. The World Health Organization lists Aeromonas in A. aquariorum 2008 the third edition of Guidelines for Drinking-water Quality. A. tecta 2008 On the basis of the Consumer Confidence Report Rule, public A. diversa 2010 water systems are required to report unregulated contaminants, A. fluvialis 2010 such as Aeromonas, when detected. Moreover, the presence of A. piscicola 2010 aeromonads in water supplies poses risk factors for the trans- A. sanarellii 2010 mission of these bacteria to food products such as ready-to-eat A. taiwanensis 2010 A. rivuli 2011 vegetables. Decontamination with a lactic acid solution and A. australiensis 2013 not chlorine seems to show the highest potential to reduce A. cavernicolaa 2013 Aeromonas spp. and to guarantee prolonged shelf lives of fresh-cut vegetables. aNot yet included in the species with standing in nomenclature.

Aeromonas in Animals included as the predominant species in fish and water samples. Animals represent a very frequent reservoir for the transmis- However, A. hydrophila and A. veronii have been also recognized sion of Aeromonas species in the environment. Aeromonads are as involved in fish diseases, resulting in enormous economic implicated in infections of both aquatic and terrestrial organ- losses. Some studies have also identified the presence of less isms. A. salmonicida causes fish furunculosis, especially in frequently encountered species in environmental samples, salmonids, and the disease has several presentations, from an such as A. schubertii in organic vegetables. However, although acute form characterized by septicemia with hemorrhages at Aeromonas are still described as ubiquitous, the preferential the bases of fins, inappetence, and melanosis to a chronic association and adaptation between particular species and variety in older fish, consisting of lethargy, slight exopht- defined habitats have been recently highlighted. Two main halmia, and hemorrhaging in muscle and internal organs. A. different habitats were identified for Aeromonas species: aquatic hydrophila and A. veronii cause similar diseases, including hem- (fish and water) and terrestrial (mainly food and human cases orrhagic septicemia in carp, perch, catfish, and salmon; red of disease). Aeromonas were first described as water bacteria, sore disease in bass and carp; and ulcerative infections in and the use of water on foods and irrigation and in human catfish, cod, carp, and goby. Aeromonas have been implicated consumption could have easily contributed to their wide dis- in several infectious processes; in seals, they can also cause ‘red persal. The differentiation of species to a particular habitat leg’ disease in frogs, ulcerative stomatitis in snakes and lizards, might be the result of their adaptation over time. Species septicemia in dogs and septic arthritis in calves, and seminal such A. hydrophila, A. salmonicida, A. veronii, A. bestiarum, vesiculitis in bulls. A. sobria, and A. allosaccharophila are commonly isolated from the aquatic environment, while species such A. caviae, A. media, A. enteropelogenes, A. jandaei, and A. schubertii are described as Aeromonas in Food ‘terrestrial’ (mainly associated with ready-to-eat food and human diseases). Unfortunately, limited data exist on the dis- In the last 15 years, many studies were conducted to determine tributions of newly described species (such as A. rivuli, A. both the frequency and the concentration of Aeromonas spp. in taiwanensis, A. sanarellii, A. australiensis, and A. cavernicola)in food products (Table 2) from supermarkets and retail stores, the environment outside their initial taxonomic description. and it has been observed that aeromonads are inhabitants of Aeromonas 63

Table 2 Isolation and characterization of Aeromonas spp. in food

Approach Medium Matrix N samples Species identificationa

Qualitativeb ADA, mBIBG Retail foods: vegetables, meat and 68 130 isolates and meat products, seafood • 73 fish (A. hydrophila 59%; A. caviae 12%) quantitative • 41 vegetables (A. caviae complex 71%; A. hydrophila 7%; A. bestiarum 5%) • 16 meat and poultry (A. hydrophila 37%; A. caviae 12%; A. veronii biovar sobria 19%) Ryan, SAA Ready-to-eat foods: vegetables, 320 51 isolates cheeses, meat products, and ice A. hydrophila 53%, A. caviae 45%, A. sobria 2% creams Ryan Minimally processed vegetables 26 46 isolates A. hydrophila group 72%, A. caviae group 28% Agar overlay Ready-to-eat foods: meat, milk, fish 557 74 isolates method in BBGS Swab samples A. hydrophila 43%, A. bestiarum 3%, A. caviae 12%, A. media 1%, A. eucrenophila 3%, A. sobria 5%, A. veronii bv. sobria 12%, A. veronii bv veronii 4%, A. jandaei 5%, unidentified 11% Qualitative SAA Organic vegetables 86 33 isolates A. schubertii 55%, A. trota 15%, A. hydrophila 15%, A. caviae 9%, A. veronii biovar veronii 6% BAA Frozen–thawed fish 250 82 Isolates A. salmonicida 63%, Aeromonas bestiarum 20%, A. veronii bv. Sobria 5%, A. hydrophila 2%, Aeromonas encheleia 4%, others 6% GSP, Ryan, TCBS, Raw fish 84 134 isolates EA, SCA (without A. hydrophila 68%, A. caviae 26%, A. sobria 6% ampicillin) ASA, ADA Meat, seafood, dairy products, 389 72 isolates vegetables, beverage, and rice A. sobria 47%, A. hydrophila 53% ASDAB Freshwater food fishes (healthy and 53 103 isolates diseased) A. hydrophila 48%, A. sobria 15%, A. caviae 15%, A. jandaei 11%, A. veronii 5%, A. schubertii 3%, and A. trota 3% Blood agar, Commercial sick chickens 2000 11 isolates MacConkey agar A. hydrophila 100% ASDAB Fish and fishery products 73 91 isolates (freshwater and marine fish and A. hydrophila 19%, A. sobria 13%, A. caviae 7%, shellfish) A. jandaei 4%, A. trota 8%, A. schubertii 5%

ADA, ampicillin-dextrin agar; ASA, Aeromonas-selective agar; ASDAB, Aeromonas Starch DNA Agar Base; BAA, blood agar with ampicillin; BBGS, bile salts–brilliant green starch agar; EA, enterohemolysin agar; GSP, Pseudomonas–Aeromonas-selective; mBIBG, modified bile salts–Irgasan–brilliant green agar; Ryan, Aeromonas medium base; SAA, starch ampicillin agar; SCA, standard count agar; TCBS, thiosulfate–citrate–bile salts–sucrose agar (vibrio-selective) aPercentage of species among isolates. bMainly APW (alkaline peptone water) enrichment.

most types of food, from seafood to vegetables, meats (lamb, (Table 2). Aeromonas is frequently found in vegetables, espe- veal, pork, chicken, and ground beef), and dairy products. cially in ready-to-eat salads that are usually consumed without Their presence in foods often leads to spoilage reactions, washing. The type of vegetables seems to influence the Aero- but in some products, such as milk, they can reach high con- monas growth rate (more than the type of the atmosphere À centrations (up to 108 CFU ml 1) without any detectable present), with more rapid growth occurring on shredded organoleptic changes. Since their main reservoir is the aquatic endive and lettuce than on sprouts or grated carrots. A work environment, they have been isolated from several seafood conducted on RTE at the University ‘Federico II’ of Naples on species and the most common Aeromonas species found were 320 food products revealed the presence of Aeromonas in 46% A. salmonicida, A. bestiarum, A. veronii, and A. encheleia. Their of samples, mostly vegetables (45% lettuce, 40% endive, and frequent presence in these food matrices represents again a 15% rocket), but also on dairy products (45% ricotta cheese) potential risk seen in the actual trend of eating raw seafood. and meat (25% salami and raw ham) (Table 2). A. hydrophila Stratev and colleagues recently published a detailed review was the most common species isolated from food of animal about the prevalence of Aeromonas spp. in food, but the final origin, while A. caviae was mostly found in vegetables. Initial À species description was clearly affected by the methods of counts in food ranged from <102 to >105 CFU g 1 at 5 C, isolation and identification (biochemical vs. biomolecular) and after 7 days at refrigeration temperature, Aeromonas 64 Aeromonas concentration increased one to three log as most aeromonads England and Wales, Aeromonas bacteremia is a voluntarily are psychrotolerant. In the majority of the studies, the isolates reportable condition and 82 cases of Aeromonas bacteremia were recovered after enrichments techniques, indicating that were recorded in 2004. Based upon these data, it has been Aeromonas concentrations were relatively low. However, calculated that the incidence of Aeromonas septicemia in enrichment is suggested for processed food, since food- England/Wales and the United States is 1.5 per million. To preserving methods affect the recovery of Aeromonas, while date, the exact incidence of Aeromonas infections on a global for raw foods, the quantitative methods could provide a view basis is unknown as many cases may be asymptomatic or not of contamination. reported. Aeromonas importance in food bacteriology is due to their It has been observed that Aeromonas species implicated as strong adaptive capacity, their high lipolytic and proteolytic causes of human colonizations and infections are not restricted action, and their surviving ability at wide ranges of tempera- to a single genomospecies, and it seems that an association tures and pH that make this genus able to grow on any food between some Aeromonas species and their effects on humans matrix. Moreover, some strains produce thermostable toxins exists. Among the 27 species identified to date, A. hydrophila, A. and can survive in some processed food. Aeromonas strains can caviae, A. veronii, and A. jandaei are most commonly associated be recovered from foods stored at À20 C for considerable with humans and account for more than 85% of all clinical periods (years) and it has been demonstrated that A. hydrophila isolates. resists to 5% NaCl at specific temperatures. Their capacity to While Aeromonas was originally thought to be an opportu- grow at low pH values or high NaCl concentrations may rep- nistic pathogen in immune-compromised humans, an increas- resent a risk in ready-to-eat products where acidifications tech- ing number of cases of Aeromonas-associated intestinal and niques are used for food conservation. Acetic, lactic, tartaric, extraintestinal disease documented worldwide seem to suggest citric, sulfuric, and hydrochloric acids are effective at restricting it is an emerging human pathogen irrespective of the host’s growth, and polyphosphates can also control their growth in immune status. A recent study reports 91 cases of bacteremia certain foods. Overall, Aeromonas grow anaerobically as they caused by Aeromonas spp. recorded in a computerized database do aerobically, their growth under modified atmospheres of a regional hospital in southern Taiwan and confirms this depends on the nature and number of competing microbiota, bacterium as a nosocomial pathogen. To date, it is described as and the use of modified atmospheres to extend shelf lives of bona fide enteropathogen, but it is not universally accepted as packaged meats and fresh vegetables may enable aeromonads a pathogen bacterium. The proof that establishes Aeromonas to grow to high populations. However, Aeromonas spp. are as a true pathogen is lacking, and this is mainly due to the readily killed by heat treatment or irradiation, but they are failure to identify a single clonally related outbreak of disease resistant to chlorination processes and to multiple antibiotics. and to detect an immune-specific response in human serum. However, Aeromonas spp. have been isolated from several cases of human infections and are described as responsible of several Aeromonas in Human Health intestinal and extraintestinal diseases and syndromes. Aeromo- From 1954, when Aeromonas was first associated with the death nas are mainly the cause of gastrointestinal syndromes, but of a 40-year-old-woman in Jamaica, to the present date, the they have been also described as causing other types of role of this bacterium in human colonization and infection is infections. still much debated. Although Aeromonas does not belong to the human enteric microbiota, it has been demonstrated that it is Aeromonas in gastroenteritis present in 1% of the adult people and this value increases to The gastrointestinal tract is the most common site from which 3% in warm periods and up to 30% in developing nations. aeromonads are recovered. The colonization of the human Since Aeromonas spp. are ubiquitous bacteria, the associa- gastrointestinal tract by aeromonads is most likely a result of tion with humans is easily established. They are mainly the consumption of food and drinking water containing Aero- acquired via contact with contaminated drinking water or monas spp. In recent years, the incidence of gastroenteritis due through the ingestion of foods that are naturally exposed to to Aeromonas spp. has increased significantly, affecting all age aeromonads through irrigation processes or other ‘farm-to- groups in both industrialized and developing nations. In table’ operations. Also, raw seafood represents a common industrialized countries, the frequency of Aeromonas in stool way of contamination. Bivalves such as oysters and mussels samples has been reported to be between 2.2% and 10%. The are naturally bathed in estuary waters containing these results recently obtained by Global Enteric Multicenter Study bacteria, and through their filter-feeding process, they actually to identify the etiology and population-based burden of pedi- concentrate these bacteria within their meats. It has been also atric diarrheal disease in developing countries report Aeromo- reported that recreational activities such as boating, fishing, nas as a common cause of diarrhea in children younger than 5 and diving can lead to infection, although no reliable data years in Pakistan and in Bangladesh. are available. Aeromonas infections of the gastrointestinal tract can lead to Janda and Abbott listed a detailed survey of the incidence of five different settings, from nondescript enteritis to more severe Aeromonas infections all over the world, based on available forms accompanied by bloody stools or chronic intestinal data. In 1988, California reported that the incidence of syndrome, traveler’s diarrhea, or even cholera-like disease. Aeromonas infections was 10.6 per million. In 2006, 99 Aero- The most common setting is the secretory enteritis, which has monas infections were reported in 70 hospitals in France; this been reported to account for up to 89% of all cases of Aero- represents a prevalence of 1.62 infections per million, a value monas gastroenteritis. It includes fever and abdominal pain and much lower than that reported in the Californian study. In in some cases also vomiting. The dysenteric form is more rare, Aeromonas 65 accounting for up to 22% of Aeromonas gastroenteritis. More- and pancreatic systems. In Southeast Asia, Aeromonas is the third over, there are several complications associated to Aeromonas most common gram-negative cause of peritonitis. The peritoni- gastroenteritis; they mainly include segmental colitis or the tis caused by Aeromonas results mainly from extensions of infec- hemolytic uremic syndrome. tions from the biliary or gastrointestinal tract. However, the Despite all these data, Aeromonas is not officially considered source of infections is unclear in most cases, with few medical a gastrointestinal pathogen, as a real proof of pathogenicity histories suggesting an environmental origin. still lacks. To date, there is no animal model that can faithfully Aeromonas have been associated with respiratory tract infec- reproduce the Aeromonas-associated diarrheal syndrome, tions, not only mainly pneumonia but also with cases of although many attempts have been made. However, several empyema. The main cause was the presence of near-drowning studies reported Aeromonas as the sole pathogen present in events involving seawater and other massive aquatic exposures. patients affected by gastroenteritis, but it was also found in Again, the presence of underlying syndromes has often been the stools of 1–4% of asymptomatic individuals. Thus, their reported role in gastroenteritis is still problematic. Finally, Aeromonas species have been occasionally impli- A hypothesis that seems to be the most reliable so far cated in eye and urogenital tract infections. suggests the possibility that the pathogenicity of Aeromonas spp. relies on the presence of specific virulence factors in the genome and of particular conditions in hosts that favor the onset of the disease (i.e., immunocompromised patients and Pathogenicity persons with hematologic cancers, tumors of the gastrointesti- nal tractor, and other underlying pathological anomalies of the As already discussed, it is presently unclear whether aeromo- alimentary canal). nads can be considered proper pathogens. An animal model of infection is lacking and the attempts made to reproduce the Other infections illnesses were unsuccessful. In addition, the microbial factors Aeromonas spp. are also associated with a variety of skin and soft responsible for the onset of the diseases are still unknown and tissue infections, mainly as a consequence of direct contact with their identification is even more clouded by the widespread contaminated water and traumatic injuries. In terms of inci- presence of genes potentially implicated in microbial infec- dence, wound infections are much less frequent than gastroen- tions throughout the genomes of most Aeromonas species. teritis and, while the overall incidence of Aeromonas infections in To shed light onto the role of Aeromonas in gastroenteritis, United States was 10 per million (when reported), wound the use of putative gene markers for pathogenicity has been infections were estimated to be 0.7 per million. The manifesta- widely applied to characterize strains from different food ori- tions range from mild infections of the subcutaneous tissues gins (Table 2), but their presence is still only indicative of (cellulitis), which represent the most common symptom, to potential virulence. serious conditions affecting deeper tissues (necrotizing fasciitis The species involved in the vast majority of systemic infec- and myonecrosis). Aeromonas infection was also associated with tions in humans are A. hydrophila, A. caviae, and A. veronii; the use of medicinal leech therapy that mainly causes cellulitis. however, recent environmental studies extended the knowl- Aeromonas species were recognized as important pathogens in edge of ‘human-related’ Aeromonas to other taxa, including A. natural disaster situations; they were isolated in high concentra- jandaei and A. enteropelogenes. Moreover, an ecological and À tions (106–107 CFU ml 1) after both the hurricane Katrina in genetic link has been found between species isolated from New Orleans and the tsunami in Thailand in 2004. food matrices and human cases of diseases. Thus, if initially Another disease form associated with Aeromonas infections the main cause of Aeromonas infection was considered to be is septicemia. The vast majority of cases are seen in persons just the aquatic environment, now, the connection between who are severely immunocompromised or have underlying human infections and the ingestion of contaminated food complications such as diabetes mellitus, renal problems, car- seems to become a more common scenario. The adaptation diac anomalies, and other hematologic conditions. However, to specific habitats may suggest that the infectious process some cases of septicemia caused by Aeromonas in healthy per- involves, at least in part, selection of species (or strains) with sons have been described. The most common symptoms certain characteristics that favor infections. However, this has include fever, jaundice, abdominal pain, septic shock and not been demonstrated so far. One of the problematic issues in dyspnea. Aeromonas septicemia is mostly caused by traumas understanding the pathogenicity of Aeromonas concerns the and direct contact with microorganisms through wounds, and, fact that this genus produces an impressive array of virulence in some instances, it was associated with leech therapy. As a factors and also the lack of consensus on standardization of matter of fact, leeches harbor aeromonads symbiotically and terminology regarding these factors between different research their use in therapeutic procedures may cause infections. Cur- groups. Aeromonas spp. produce several extracellular products rently, it is not possible to clinically distinguish Aeromonas that fall into several broad categories, including cytolytic toxins bacteremia from those caused by other gram-negative bacteria with hemolytic activity, cytotonic enterotoxins, hemolysins, such as Escherichia coli or Klebsiella pneumonia. However, a lipases, proteases, leukocidins, phospholipases, fimbriae or peculiar indicator of Aeromonas infection is the presence of adhesins, and the capacity to form capsules. ecthyma gangrenosum-like lesions in the form of petechiae Several classes of genes have been identified that play impor- or bullae. tant roles in the colonization of the leech digestive tract, includ- Aeromonads are also recognized as causing intra-abdominal ing bacterial cell surface modifications, regulatory factors, diseases such as peritonitis and infections of the hepatobiliary nutritional elements, and genes involved in the secretion system 66 Aeromonas

(SS) (such as T2SS and T3SS). Aeromonas spp. produce a wide TagA has been described as a new virulence factor found in range of proteases, which cause tissue damage and aid in estab- an A. hydrophila isolate from diarrhea and only present in lishing an infection by overcoming host defenses and by pro- pathogens as E. coli O157:H7 and V. cholerae ; its role seems viding nutrients for cell proliferation. Lipases secreted by to be related to the inhibition of the classical complement- Aeromonas may also constitute virulence factors by interacting mediated lysis of the erythrocytes but, even in V. cholera, its with human leukocytes or by affecting several immune func- function in pathogenesis is speculative. tions through fatty acids generated by lipolytic activity. Two There are a large number of unresolved questions regarding factors thought to play intimate roles especially in the coloniza- the role of the potential virulence factors in Aeromonas infec- tion of gastrointestinal tract are flagella and pili. Aeromonas tions. Some genes, such as act, are also found in species that are produces two types of flagella, a constitutively expressed polar infrequently associated with human diseases (A. bestiarum). flagellum (Pof) and multiple inducible lateral flagella (Laf), Moreover, research on Aeromonas pathogenicity demonstrated which are, respectively, involved in the initial attachment of the enormous complexity of the situation involving polygenic bacteria to the gastrointestinal epithelium and in cell adherence, expression in both the pathogen and the host. Thus, there is long-term colonization, and biofilm formation. Biofilm devel- still much to be learned about Aeromonas virulence determi- opment may also be regulated by quorum sensing that appears nants and how they combine to result in the virulent subsets to act in concert with T3SS to regulate the expression of the within each Aeromonas species that causes disease. At present, Aeromonas enterotoxins, as its production increases when bacte- it is not possible to identify the disease-causing strains be- rial cell density increases. The cytotonic enterotoxin Act/Asa is a cause of the incomplete understanding of Aeromonas virulence pore-forming toxin, also known as aerolysin AerA: it was origi- mechanisms. nally recovered from a diarrheal isolate of A. hydrophila and was subsequently determined to possess a variety of biological activ- ities, including hemolysis, cytotoxicity, and enterotoxicity, and to cause lethality in mice. While it is clear that Act induces Aeromonas and Antimicrobial Susceptibility extensive host cell signaling (it stimulates proinflammatory responses by increased cytokine production through elevated A particularly interesting area that is receiving more attention tumor necrosis factor, IL-1b and IL-6 levels), it is unknown in the last years is the susceptibility of Aeromonas to antimicro- how the toxin exerts the effects. Another well-characterized bial agents. The studies regarding this topic reported data on toxin (AHH1) belongs to the family of b-hemolysins and has a the three major species associated with human disease, A. high sequence homology to the HlyA hemolysin of Vibrio cho- hydrophila, A. caviae, and A. veronii, so we do not know yet if lerae. These toxins are also named Act- and aerolysin-like mole- the available information is also valid for the other species. cules and are enterotoxigenic cytolysins. Many Aeromonas strains The first studies recording the antibiotic susceptibility of possess a surface layer (S-layer), which resists complement- Aeromonas were conducted between the mid-1980s and mid- mediated killing of the organism by impeding complement 1990s. Inducible chromosomal b-lactamases are still the activation. It seems that the set of bacterial virulence factors major resistance mechanism for most aeromonads, although and host responses that eventually lead to Aeromonas-associated their resistance to other antibiotics is dramatically increasing. diseases are ill-defined. Regarding gastrointestinal diseases, aero- Upon characterization of the antimicrobial resistance of 94 monads can apparently produce diarrhea by elaboration of Aeromonas isolates from warm- and cold-water ornamental enterotoxigenic molecules and/or by invasion of the gastroin- fish species using microarray analysis and conventional testinal epithelium. At least two cytotonic toxins have been PCR, a surprisingly high level of antimicrobial tolerance was identified: a heat-labile cytotonic enterotoxin (Alt) and a heat- identified in the strains tested. Half of the Aeromonas spp. stable cytotonic enterotoxin (Ast). Invasins have also been isolates were tolerant to more than 15 antibiotics, represent- reported, but they are difficult to detect in vitro; some studies ing seven or more different classes of antimicrobials. suggested that only a fraction of Aeromonas strains are invasive The quinolone and fluoroquinolone resistance gene was and the degree of invasion is considerably less than the observed detected at high frequency, although it has been reported for classic enteropathogens, such as E. coli and Yersinia enteroco- that Aeromonas strains are almost universally susceptible to litica. The gene encoding enolase was also found in A. hydrophila fluoroquinolones. Resistance has been also observed to car- strains recovered from stools. Enolase is a glycolytic enzyme bapenems, imipenem, chloramphenicol, and florfenicol. whose surface expression was shown to be important in the Moreover, tetracyclines were particularly widespread across pathogenesis of Streptococcus pyogenes-associated rheumatic all screened isolates. fever. It has been suggested that the surface expression of enolase The discovery of multidrug resistance in strains isolated occurs only in gram-positive bacteria, while other researchers from wild shellfish in the Adriatic Sea suggests an involvement have demonstrated the ability of this protein to bind human of Aeromonas in the dissemination of antibiotic resistance in the plasminogen, potentially indicating an important role during environment and in seafood. The susceptibility status of Aero- Aeromonas infections. The T3SS includes several factors with monas isolates for therapeutically active drugs appears to be multiple biological functions, and the gene AexT, a homologue independent of species designation. While some species-specific of Pseudomonas aeruginosa T3SS-secreted ExoT/S, was detected in susceptibility differences have been found, these results should some Aeromonas isolates, but no information is available on its be considered preliminary at present. Moreover, no connection role in bacterial virulence using in vivo models. Another well- between a specific resistance pattern and the origin of isolation known T3SS gene is ascV that codes for an inner-membrane has been identified so far. Certainly, more studies need to be component of the T3SS channel. performed in this area. Aeromonas 67

Conclusions Holmes P, Niccolls LM, and Sartory DP (1996) The ecology of mesophilic Aeromonas in the aquatic environment. In: Austin B, Altwegg M, Gosling PJ, and Joseph S (eds.) The genus Aeromonas, pp. 127–150. West Sussex, England: After more than a century from its discovery, the Aeromonas Wiley. genus is still intricate. Indeed, great improvements have been Hussain IA, Jeyasekaran G, Shakila RJ, Raj KT, and Jeevithan E (2013) Prevalence of made in the last years, as molecular genetics have led to consid- hemolytic and enterotoxigenic Aeromonas spp. in healthy and diseased freshwater erable advantages in the taxonomic determination of these bac- food fishes as assessed by multiplex PCR. American Journal of Advanced Food Science and Technology 1: 70–85. teria, which was one of the most controversial issue of the genus. Isonhood JH and Drake M (2002) Aeromonas species in foods. Journal of Food Moreover, the ecology and the mechanisms of environmental Protection 65: 575–582. adaptation have been tackled, identifying a genetic adaptation Janda JM and Abbott SL (1996) Human pathogens. In: Austin B, Altwegg M, process of Aeromonas species toward specific habitats. However, Gosling PJ, and Joseph S (eds.) The genus Aeromonas, pp. 151–173. West Sussex, the image of Aeromonas as a human pathogen is still blurred, as England: Wiley. Janda JM and Abbott SL (2010) The genus Aeromonas: taxonomy, pathogenicity, and no evidences of a clear association exist. There is still much to be infection. Clinical Microbiology Reviews 23: 35–73. learnt about Aeromonas pathogenicity and virulence determi- McMahon MAS and Wilson IG (2001) The occurrence of enteric pathogens and nants and how they combine to result in disease, but the advent Aeromonas species in organic vegetables. International Journal of Food of next-generation techniques and the possibility to analyze and Microbiology 70: 155–162. Neyts K, Huys G, Uyttendaele M, Swings J, and Debevere J (2000) Incidence and combine massive amounts of data make us believe it is likely to identification of mesophilic Aeromonas spp. from retail food. Letters in Applied happen. Microbiology 31: 359–363. Villari P, Crispino M, Montuori P, and Stanzione S (2000) Prevalence and molecular characterisation of Aeromonas spp. in ready-to-eat foods in Italy. Journal of Food See also: Chilled Foods: Modified Atmosphere Packaging; Fish: Fish Protection 63: 1754–1757. in the Human Diet; Spoilage: Bacterial Spoilage. Relevant Websites

Further Reading http://www.bacterio.cict.fr – List of the prokaryotic names with standing in nomenclature. Cristi L, Galindo A, and Chopra K (2007) Aeromonas and Plesiomonas species. http://www.epa.gov/safewater/ucmr/data_aeromonas.html – United States In: Doyle MP and Beuchat LR (eds.) Food microbiology fundamentals and frontiers, Environmental Protection Agency – Aeromonas detection. 3rd ed., pp. 381–400. Washington, DC: ASM Press. http://www.the-icsp.org – International Committee on Systematics of Prokaryotes. Das A, Sindhuja ME, Rathore A, et al. (2013) Diagnosis of virulent strains of motile http://permanent.access.gpo.gov/lps21800/www.epa.gov/safewater/ccl/cclfs.html – Aeromonas from commercial food. International Journal of Current Microbiology Drinking Water Contaminant Candidate List. and Applied Sciences 2: 300–306. http://www.pubmlst.org/aeromonas – Aeromonas MLST Database. Figueras MJ (2005) Clinical relevance of Aeromonas sM503. Reviews in Medical http://www.who.int/water_sanitation_health/dwq/guidelines/en/index.html – WHO Microbiology. 16: 145–153. Guidelines for drinking-water quality.