Isolation of enterotoxigenic, hemolytic, and antibiotic-resistant Aeromonas hydrophila strains from infected fish in Bangladesh.

Strains of Aeromonas hydrophila isolated from skin infections of common freshwater fish in Bangladesh were tested for enterotoxin production, hemolysin production, and any correlation between these two activities. We also tested the resistance patterns of A. hydrophila to different drugs, especially in relation to ampicillin. The A. hydrophila strains produced an enterotoxin that was related to their beta-hemolytic activities. Production of beta-hemolysin may thus be an indicator of enterotoxicity. As 50% of the strains of A. hydrophila were found to be susceptible to 12.5 micrograms of ampicillin per ml, media containing this antibiotic may not be suitable for their isolation.     

Production of cholera-like enterotoxin by Aeromonas hydrophila

A total of 249 strains of mesophilic Aeromonas including 179 A. hydrophila and 70 A. caviae were tested for production of cholera-like enterotoxin by reversed passive latex agglutination (RPLA) assay. A cholera-like enterotoxin neutralized with cholera antitoxin was demonstrated in the culture filtrates from eight (4.5%) of the 179 A. hydrophila strains, while none of A. caviae strains revealed the enterotoxin production in the test. Production of the cholera-like enterotoxin in the eight strains of A. hydrophila was also confirmed by enzyme-linked immunosorbent assay (ELISA).     

Production of exotoxins by Aeromonas spp. at 5 degrees C

The ability of 60 strains of Aeromonas to produce enterotoxin and haemolysin after cultivation at 5 degrees C for 7-10 d was investigated. The strains were isolated from lamb meat, offal, carcasses and faeces, and had previously been tested for their ability to produce these exotoxins at 37 degrees C. The results showed that some strains of Aeromonas hydrophila and A. sobria were capable of producing enterotoxin and haemolysin at 5 degrees C, but none of the A. caviae strains tested produced these two factors. Of the 30 A. hydrophila strains investigated 25 and 27 were enterotoxigenic and haemolytic respectively. Likewise, of the 24 A. sobria strains investigated 16 and 18 were enterotoxigenic and haemolytic respectively. The results indicate that certain strains of Aeromonas species, in particular A. hydrophila and A. sobria, are of potential public health significance in meats stored at refrigeration temperature.     

Production of exotoxins by Aeromonas spp. at 5°C

The ability of 60 strains of Aeromonas to produce enterotoxin and haemolysin after cultivation at 5°C for 7–10 d was investigated. The strains were isolated from lamb meat, offal, carcasses and faeces, and had previously been tested for their ability to produce these exotoxins at 37°C. The results showed that some strains of Aeromonas hydrophila and A. sobria were capable of producing enterotoxin and haemolysin at 5°C, but none of the A. caviae strains tested produced these two factors. Of the 30 A. hydrophila strains investigated 25 and 27 were enterotoxigenic and haemolytic respectively. Likewise, of the 24 A. sobria strains investigated 16 and 18 were enterotoxigenic and haemolytic respectively. The results indicate that certain strains of Aeromonas species, in particular A. hydrophila and A. sobria , are of potential public health significance in meats stored at refrigeration temperature.     

Incidence of aeromonads in samples from an abattoir processing lambs

Two hundred and thirty three samples of lamb carcasses, livers, kidneys and faeces, collected at a local abattoir, were examined to determine the incidence of motile Aeromonas spp. Wash water from the abattoir was also tested. Direct plating on starch ampicillin agar and enrichment in alkaline peptone water were used. The incidence of aeromonads was low. They were detected only after enrichment in 5/47 faecal samples and 11/50 carcass samples. The 41 strains of Aeromonas isolated were identified to species level and 93% of them were able to grow at 5 degrees C. The ability to produce both haemolysin and enterotoxin was species-related and was more common in Aeromonas hydrophila and A. sobria strains than in A. caviae strains.     

Sequence analysis of amplified DNA fragments containing the region encoding the putative lipase substrate-binding domain and genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

Transposition of ampicillin resistance to an enterotoxin plasmid in an Escherichia coli strain of human origin.

We examined a strain of Escherichia coli, serotype O159.H34, of human origin which produced heat-stable and heat-labile enterotoxins, was resistant to ampicillin, and produced colicin. By conjugation and transformation experiments plasmids coding for enterotoxin production (Ent), enterotoxin production and ampicillin resistance (Ap-Ent), ampicillin resistance (Ap), and colicin production were isolated. Both the Ent and Ap-Ent plasmids were autotransferring and belonged to the F-incompatibility complex. However, the Apr Ent+ transconjugants showed differences in their levels of resistance and in their abilities to propagate F-specific phages and to transfer resistance. The results suggested there was transposition from the small Ap plasmid to the Ent plasmid. The Ap-Ent plasmids were larger than the enterotoxin factor and when treated with restriction endonuclease BamHI showed an additional fragment not present in the enterotoxin plasmid. The insertion of ampicillin resistance probably occurred at different sites on the enterotoxin plasmid, resulting in the observed variation in phenotype.     

Experimental evidence for enteropathogenicity in Aeromonas veronii

Eleven ornithine-positive strains of Aeromonas (9 A. veronii and 2 provisionally classified as Aeromonas species ornithine positive) were tested for ability to cause fluid accumulation in the rabbit ileal loop. All eight beta-hemolytic strains caused fluid accumulation. Gel diffusion analysis revealed that the A. veronii beta-hemolysin was serologically related to the A. hydrophila beta-hemolysin, a known enterotoxic molecule. The biological activity of the A. veronii hemolysin was neutralized by antiserum to A. hydrophila hemolysin. One of three strains that were not beta-hemolytic caused fluid accumulation but only when the ileal loops were inoculated with live cultures. These results suggest that A. veronii is a potential enteropathogen that can cause diarrhea by means of a cell-freed enterotoxin (beta-hemolysin) or by a second mechanism that requires the presence of whole cells.     

Plasmids coding for heat-labile enterotoxin production isolated from Escherichia coli O78: comparison of properties.

Nineteen enterotoxigenic Escherichia coli strains of serogroup O78, isolated in different geographical areas from humans with diarrheal diseases, were tested for their ability to transfer enterotoxin production. All of the strains originally produced heat-labile enterotoxin, and 16 also produced heat-stable enterotoxin and colonization factor antigen I. Plasmids coding for the production of heat-labile enterotoxin only were transferred from 13 strains. Some properties of these plasmids were compared. All were fi+, but they could be divided into three groups on the basis of their incompatibility reactions, ability to restrict E. coli K-12 phages, and size. The three heat-labile enterotoxin plasmids isolated from African strains all belonged to one enterotoxin plasmid group. The heat-labile enterotoxin plasmids from the Asian strains were divided into two groups, those from serotype O78.H11 differing from those from serotype O78.H12.     

Serogroups, K1 antigen, and antimicrobial resistance patterns of Aeromonas spp. strains isolated from different sources in Mexico

A total of 221 strains of Aeromonas species isolated in Mexico from clinical (161), environmental (40), and food (20) samples were identified using the automated system bioMérieux-Vitek. Antisera for serogroups O1 to 044 were tested using the Shimada and Sakazaki scheme. The K1 antigen was examined using as antiserum the O7:K1C of Escherichia coli. Besides, we studied the antimicrobial patterns according to Vitek AutoMicrobic system. Among the 161 clinical strains 60% were identified as A. hydrophila, 20.4% as A. caviae, and 19.25% as A. veronii biovar sobria. Only A. hydrophila and A. veronii biovar sobria were found in food (55 and 90% respectively) and environmental sources (45 and 10% respectively). Using "O" antisera, only 42.5% (94/221) of the strains were serologically identified, 55% (121/221) were non-typable, and 2.5% (6/221) were rough strains. Twenty-two different serogroups were found, O14, O16, O19, O22, and O34 represented 60% of the serotyped strains. More than 50% of Aeromonas strain examined (112/221) expressed K1 encapsulating antigen; this characteristic was predominant among Aeromonas strains of clinical origin. Resistance to ampicillin/sulbactam and cephazolin was detected in 100 and 67% of Aeromonas strain tested for their susceptibility to antibiotics. In conclusion, antibiotic-resistant Aeromonas species that possess the K1 encapsulating antigen and represent serogroups associated with clinical syndrome in man are not uncommon among Aeromonas strains isolated from clinical, food and environmental sources in Mexico.     

湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究

为了调查引起鱼类运动型气单胞菌败血症(俗称暴发病)的嗜水气单胞菌的遗传多样性和毒力特征, 阐明其流行规律, 研究于2006-2009年度从湖北省内3个不同地区的6个发病鱼塘中分离了30株嗜水气单胞菌, 其中20株为临床株(分离自血液、肝脏、肾脏或腹水), 6株为肠道株, 4株为池水株。基于所有菌株gyrB基因序列, 构建了系统发育树; 通过ERIC (Enterobacterial repetitive intergenic consensus, 肠道细菌基因间重复序列)指纹图谱进行菌株的遗传分型; 用PCR方法检测了7个毒力基因在菌株中的分布模式。这7个基因包括气溶素(aerA)、溶血素(hlyA)、热不稳定性细胞兴奋性肠毒素(alt)、热稳定性细胞兴奋性肠毒素(ast)、弹性蛋白酶(ahpB)、脂酶(lip)和鞭毛基因(fla)。此外, 以斑马鱼为感染对象, 通过腹腔注射测定了15株代表菌株的毒力。结果表明: 不同来源的20株临床株、1株肠道株和3株池水株具有相同的遗传特性, 体现为在系统树上聚为一枝, 序列相似性为100%, 具有相同的ERIC指纹图谱, 毒力基因分布模式为: aerA+hlyA+alt+ast+ahpB+lip+fla+, 且均为强毒株(LD50 9.74104cfu/尾)。与临床株相比, 其余5株肠道株和1株池水株或具有不同的ERIC指纹图谱或具有不同的毒力基因分布模式, 显示出了遗传多样性, 且毒力均弱于临床株(LD501.01106cfu/尾)。这说明在一定时间、一定区域内, 作为暴发病病原的嗜水气单胞菌为同一克隆系在流行, 不存在明显的变异或遗传多样性。此结果有助于阐明嗜水气单胞菌引起的暴发病的流行规律, 制定相应的防御措施。多种毒力基因在致病性菌株中的联合流行为发病机理的解析奠定了基础。此外, 鉴于毒力基因谱与致病性之间的相关性, 表明毒力基因可作为标记基因, 用于致病性菌株的检测。       

Enterotoxigenic aeromonads on retail lamb meat and offal

Enrichment in alkaline peptone water was compared with the direct plating method for the isolation of Aeromonas spp. from lamb meat and offal samples. The enrichment method significantly increased the isolation rate of aeromonads. Motile Aeromonas species (A. hydrophila, A. sobria and A. caviae) were present in all kinds of samples investigated. Seventy-three Aeromonas strains isolated in this survey were characterized to species level and examined for their ability to produce virulence factors. Strains identified as A. sobria were the strongest producers of haemolysin and enterotoxin, whereas A. caviae strains were consistently non-haemolytic and non-enterotoxigenic. Thus it is likely that lamb meat and offal are potentially significant sources of virulent Aeromonas species and may play an important role in the aetiology of Aeromonas-associated gastro-enteritis.     

Enterotoxigenic aeromonads on retail lamb meat and offal

Enrichment in alkaline peptone water was compared with the direct plating method for the isolation of Aeromonas spp. from lamb meat and offal samples. The enrichment method significantly increased the isolation rate of aeromonads. Motile Aeromonas species ( A. hydrophila, A. sobria and A. caviae ) were present in all kinds of samples investigated. Seventy-three Aeromonas strains isolated in this survey were characterized to species level and examined for their ability to produce virulence factors. Strains identified as A. sobria were the strongest producers of haemolysin and enterotoxin, whereas A. caviae strains were consistently non-haemolytic and non-enterotoxigenic. Thus it is likely that lamb meat and offal are potentially significant sources of virulent Aeromonas species and may play an important role in the aetiology of Aeromonas -associated gastro-enteritis.     

Antimicrobial activity among Pseudomonas and related strains of mineral water origin

Siderophore production in 382 Pseudomonas and related strains of mineral water origin were screened and the antimicrobial activities of 158 of these tested against nine target organisms of health significance. Presence of siderophores could be detected in 54·4% and the majority of strains tested (91·2%) inhibited at least one of the nine target strains. Staphylococcus, Escherichia coli and Aeromonas hydrophila were particularly sensitive. Addition of iron eliminated the inhibitory activity in 96·7% of cases ; the antagonistic effect should be largely determined by siderophore-mediated competition for iron. Most of the inhibitory strains produced siderophores, whereas the non-inhibitory strains did not. Few strains also produced bacteriocins showing activity against Pseudomonas aeruginosa and Aer. hydrophila. Strains isolated from mineral water have a broad antibacterial potential.     

Virulence profiles and other biological characters in water isolated Aeromonas hydrophila

Thirty water isolates of A. hydrophila were tested for potential virulence profiles, antibiotic resistance and Bacteriocin-Like Substances (BLS) production. Cytotoxic activity was present in all strains tested, 87% were hemolytic and 70% adhesive. Lysine decarboxylase reactions (LDC) positivity was correlated with virulence factors: 100% versus cytotoxicity, 84% versus adherence, 76% versus hemolytic activity. The correlation was also present in the LDC-negative strains. Hemolytic and cytotoxic activities were frequently associated: high cytotoxicity, corresponding to high hemolytic activity and vice versa. The in vitro susceptibility of A. hydrophila to 28 antibacterial agents showed that cefotaxime was the most active beta-lactam antibiotic, and Cefuroxime inhibited 90% of the strains. Isolates were resistant to Penicillin G, Ampicillin, Carbenicillin, Amoxicillin, Cephalotin and Cefaclor. Tetracycline, Chloramphenicol, Nitrofurantoine, the quinolones and the aminoglycosides (except Streptomycin) were consistently active. BLS production never emerged against closely-related microorganisms. On the contrary A. hydrophila presented a heteroinhibitory activity against non-taxonomically related genera such as Listeria spp. (L. seeligeri NCTC 11856, L. welshimeri NCTC 11857, L. ivanovii NCTC 11846) and S. aureus ATCC 25923. Although a large number of strains showed virulence determinants together with other biological characters such as antibiotic resistance and BLS production, it was not possible to relate these factors to the observed plasmids.     

Distribution and characterization of hemolytic, and enteropathogenic motile Aeromonas in aquatic environment

A year-long survey on the distribution of motile Aeromonas species in the surface waters of riverine and marine environments was conducted. The filtered membranes were directly placed onto the modified Pril-xylose-ampicillin agar for the enumeration of Aeromonas species. High counts of motile aromonads were found in riverine stations and this bacterial population was also observed in significant quantities in polluted marine samples. In the identification of 2,444 isolates, three species of motile Aeromonas were observed. A. caviae (43%) was prevalent followed by A. sobria (35%) and A. hydrophila (20%). A. hydrophila was high in clean riverine samples, A. sobria was predominantly isolated from a stagnant water sampling area, and A. caviae was distributed more in marine samples. Statistical analyses suggested that the densities of Aeromonas were related to the cumulative effect of various physicochemical parameters rather than to a single factor. Among the species of Aeromonas, A. hydrophila, and A. sobria were highly hemolytic whereas only 11% of A. caviae were observed to lyse sheep erythrocytes. Suckling-mouse assay was performed to elucidate the enterotoxicity of motile aeromonads and 21% of the tested strains (one A. caviae strain) were found to produce enterotoxin.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.     

The Biochemical Activity of Enterotoxin and Non-Enterotoxin Producing Staphylococci

One hundred and sixty-nine staphylococcal strains of human origin have been tested for production of enterotoxin A, B or Ci, coagulase activity, DNase activity, typical growth on ETGP-agar, hemolysin production and the breakdown of mannitol under aerobic conditions. Very good correlation was observed between enterotoxin production and coagulase activity, in that 82 % of the enterotoxin producing strains also synthesized coagulase. The correlation between DNase activity and positive reaction in mannitol to enterotoxin production was also good (80 % of the enterotoxic strains produced both DNase and aerobic acid from mannitol). Of the enterotoxin producing strains 66 % hemolysed bovine erythrocytes and 61 % were ETGP-positive. However, the frequency of hemolysing respectively ETGP-positive but non-enterotoxin producing strains was very high, viz. 46 % respectively 32 %. It is concluded that enterotoxin production can not to a satisfactory degree of security be predicted by means of the other biochemical characters.     

Prevalence, species differentiation, haemolytic activity, and antibiotic susceptibility of aeromonads in untreated well water

The use of untreated water for drinking and other activities have been associated with intestinal and extraintestinal infections in humans due to Aeromonas species. In the present study aeromonads were isolated from 48.7% of 1,000 water samples obtained from wells and other miscellaneous sources. Aeromonas species were detected in 45% of samples tested in spring, 34.5% in summer, 48% in autumn and 60% of samples tested in winter. Speciation of 382 strains resulted in 225 (59%) being A. hydrophila, 103 (27%) A. caviae, 42 (11%) A. sobria and 11 (3%) atypical aeromonads. Of 171 Aeromonas strains tested for their haemolytic activity, 53%, 49%, 40% and 37% were positive in this assay using human, horse, sheep and camel erythrocytes respectively. The results obtained indicate that potentially enteropathogenic Aeromonas species are commonly present in untreated drinking water obtained from wells in Libya (this may also apply to other neighbouring countries) which may pose a health problem to users of such water supplies. In addition, ceftriaxone and ciprofloxacin are suitable drugs that can be used in the treatment of Aeromonas-associated infections, particularly in the immunocompromised, resulting from contact with untreated sources of water.     

Distribution of virulence genes in Aeromonas spp. isolated from Sardinian waters and from patients with diarrhoea

AIMS: To characterize 46 isolates of different Aeromonas spp. strains (26 Aeromonas hydrophila, 13 Aeromonas sobria and 7 Aeromonas salmonicida) isolated from coastal water and clinical sources in Sardinia, Italy. METHODS AND RESULTS: The isolates were analysed for the production of the following virulence properties: slime, haemolysin, gelatinase and protease production, and adhesion to eucaryotic epithelial cells. The presence of known virulence genes: A. hydrophila cytolytic enterotoxin gene AHCYTOEN; type IV pilus gene Tap; Bundle forming pilus genes BfpA and BfpG were investigated using polymerase chain reaction (PCR). In addition the enterobacterial repetitive intergenic consensus sequences (ERIC)-PCR fingerprinting was used to further differentiate the strains. CONCLUSIONS: This study confirms the presence of virulent Aeromonas strains in the Mediterranean sea. The study also found a greater prevalence of haemolysin, protease and gelatinase production, as well as a higher adhesion capacity, among strains isolated from patients with diarrhoea. SIGNIFICANCE AND IMAPCT OF THE STUDY: This is the first time that Aeromonads have been isolated and characterized from Sardinian waters and from patients with diarrhoea in Sardinia. This study adds to our knowledge of the ecology of this micro-organism and may in the future help prevent infections both in fish and in humans.