Marly Paiva Nunes

Occurrence and characterization of Aeromonas species in pasteurized milk and white cheese in Rio de Janeiro, Brazil

A total of 35 samples (1000 ml each) of pasteurized milk and 25 samples (100 g each) of white cheese purchased at supermarkets in Rio de Janeiro were analyzed for the presence of Aeromonas . Strains of Aeromonas were isolated from 28.5% of pasteurized milk and 32% of white cheese samples. Standard Plate counts in the pasteurized milk samples ranged from 7.2 × 10* to 2.5 × 105 CFU/ml. Total and fecal coliform counts in white cheese samples ranged from 1.9 × 10* to 2.4 × 105 most probable number per g and 3.2 × 102 to 1.2 × 105 most probable number per g, respectively. It was possible to identify Aeromonas caviae (58.9%), Aeromonas hydrophila (12.8%), and Aeromonas schubertii (2.5%) among the cultures isolated from pasteurized milk samples. Twenty-five percent of the strains could only be classified as Aeromonas spp. In white cheese samples, unclassified strains were the most frequent isolates (61.5%) followed by A. hydrophila (26.9%), A. caviae (7.6%) and Aeromonas sobria (3.8%). Only strains of A. hydrophila and A. sobria showed high rate of positive results when tested for the production of hemolysin, cytotoxin, and staphylolytic activity. Heat-stable enterotoxin and autoagglutination test did not correlate as virulence factors. The presence of Aeromonas species in refrigerated food samples suggests that this microorganism could be a potential foodborne pathogen, and dairy products may represent an important vehicle of its transmission.

Occurrence of Plesiomonas shigelloides in water environments of Rio de Janeiro city

Fresh and salt water samples analyzed in Rio de Janeiro city showed the presence of Plesiomonas shigelloides. Forty-six strains were isolated from both environments. A high incidence of P. shigelloides was achieved in polluted fresh and salt waters as well as in samples from non-polluted streams. P. shigelloides isolates had biochemical characteristics similar to those already described in the literature. None of the isolates analyzed produced enterotoxin in the suckling mouse assay. Hemolytic activity against sheep and human type A erythrocytes was detected in the strains tested. The results of the antibiotic susceptibility tests indicated that all the isolates were susceptible to the cephalosporins, penicillins combined with a beta-lactamase inhibitor, aminoglycosides, imipenem, norfloxacin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. All the isolates were resistant to the penicillins.

Occurrence of Yersinia species in raw and pasteurized milk in Rio de Janeiro, Brazil

Thirty-seven (16.9%) of 219 raw milk samples and 38 (13.7%) of 280 pasteurized milk samples were positive for Yersinia sp. The isolates from raw milk samples include Yersinia enterocolitica (32.4%) comprising biotype 1 (0:5, 10.8%), and biotype 2 (0:10 K1, 1.6%); Yersinia intermedia (64.9%) comprising 0:18 (40.5%), 0:7,8 (8.1%), 0:16 (2,7%) and non-typable (13.5%) and Yersinia frederiksenii (0:22, 2.7%). The isolates from pasteurized milk samples include Y. enterocolitica (41.5%) comprising 0:5 (31.7%), 0:13 (2.4%), 0:7,8 (2.4%) and 0:16 (4.8%); Y. frederiksenii (56.1%) comprising 0:27 (7.3%), 0:25,35 (12.2%), non-typable (36.6%) and Y. intermedia (non-typable, 2.4%). Most Y. enterocolitica and about one third of non- Y. enterocolitica strains produce heat-stable toxin (ST). Antibiotic susceptibility, autoagglutination capacity and calcium-dependency of strains also were investigated.

Incidence of motile Aeromonas species in aquatic environments of Rio De Janeiro; Brazil

Fresh and salt water samples collected in Rio de Janeiro city were analysed for the presence of motile Aeromonas species. Twenty-six out of 50 aliquots analysed (52%) were positive for Aeromonas . One hundred strains were isolated from both environments ( A. caviae , 60%; A. veronii , 14%; A. hydrophila , 1%; A. sobria , 1%; and Aeromonas sp., 24%). Minimal tests such as oxidase, motility, sensitivity to the vibriostatic agent 2,4-diamino-6,7 - diisopropylpteridine, fermentation and gas from glucose, acetoin from glucose (Voges-Proskauer), lysine decarboxylase, ornithine decarboxylase, and esculin hydrolysis were sufficient to classify the majority Aeromonas strains into species. No Aeromonas was found in nonpolluted waters but, in contrast, both fresh and salt polluted waters showed a high incidence of isolates. Most of the Aeromonas strains analysed produced hemolysin and/or heat-stable enterotoxin. The latter was produced by 73% of the A. veronii isolates.

Aeromonas Hydrophila Isolated from Cases of Bovine Seminal Vesiculitis in South Brazil

Preliminary studies on pathologic conditions in the testis, epididymis, and the accessory sex glands of cattle have demonstrated that vesiculitis is the most common inflammatory condition in the genitals of Danish bulls.5 Occasionally, this condition has an acute onset that produces clinical signs and then becomes a chronic disease.18 Four-polled Hereford bulls approximatedly 1 year of age (1–2 years old) from a farm in southern Brazil were examined by rectal palpation. During this procedure, enlargement of the seminal vesicles was detected. The animals were slaughtered, and samples were collected for bacteriologic and histopathologic examination. Macroscopically, the seminal vesicles were enlarged and firm and contained foci of yellow exudate. The histopathologic examination revealed interstitial seminal vesiculitis with areas of lymphocytic and plasma cell infiltration surrounding alveoli containing suppurative exudate. Varying degrees of fibrosis were found in the glands. Samples of a selected group of organs were available for sectioning. For bacteriologic examination, fragments of seminal vesicles were inoculated asseptically onto 5% (w/v) sheep blood agar and incubated aerobically at 37 C for 24–48 hours. Microorganisms from all seminal vesicles were isolated in pure culture. The colonies were beta-hemolytic, smooth, small, and white and had characteristics of the genus Aeromonas (gram-negative rods, oxidase positive, fermentative metabolism, and resistant to vibriostatic agent 2,4,diamino6,7-diisopropylpteridine, O/129, 150 mg). According to the biochemical profile the Aeromonas isolates were classified as Aeromonas hydrophila (Table 1).14 There have been no previous reports of seminal vesiculitis in bulls associated with Aeromonas spp. However, in a previous report of the isolation of A. hydrophila from feces of healthy cattle, this species was suggested as an occasional host to Aeromonas.1 Aeromonas hydrophila isolated from the genital tract of cows was associated with abortion.28 Nonetheless, bovine seminal vesiculitis associated with other microrganisms such as Actinomyces pyogenes,4,5,8 Escherichia coli,3 Brucella abortus,24 Mycoplasma bovigenitalium,2,13 Haemophilus somnus,12 and Pseudomonas aeruginosa4 have also been described. Although, some authors consider it unlikely that any bac-

ANTI-BRUCELLA AGGLUTININS IN BATS AND “CALUTHRIX” MONKEYS

Anti-Brucella agglutinins were found in 5 of 53 (9.4%) vampire bats Desmodus rotundus, captured in the State of Bahia, Brasil. Two specimens of Diphylla ecaudata were negative. Fifty specimens of the small monkey, Callithrix penicillata, were also negative.

Bacteriocin-like substance of Aeromonas hydrophila.

The genus Aeromonas comprises Gram negative rods found mainly in aquatical environments that may infect humans and animals (JM Janda 1991 Clin Microbiol Rev 4: 397-410). In humans, some Aeromonas species have been associated with intestinal and extraintestinal infections and enterotoxins, cytotoxins as well as invasive mechanisms have been incriminated in the development of illness in the host (Janda loc. cit.). Bacteriocin-like substances (BLS) are protein compounds produced by some bacteria (G Ivanovics 1962 Bacteriol Rev 26: 108-118) showing antagonic activity against their own species (isoinhibitory activity IA) or other non-related species (heteroinhibitory activity HA). The use of the expression BLS is recommended to nominate bacterial products showing antagonic activity though not characterized (K Sandhu et al. 1983 J Clin Microbiol 17: 511-515). These substances have been widespread utilized in epidemiological studies as specific marker properties of bacteria, in the regulation of population dynamics in bacterial ecosystems and clinical treatment (V Fantinato & F Zelante 1991 Rev Microbiol 22: 49-51). As BLS have not been currently described in Aeromonas species, the purpose of this study was to investigate their production in strains isolated from animal, clinical and environmental sources. The assays for the production of BLS were performed according to Sandhu et al. (loc. cit.). The strains used as BLS producers and BLS indicators are listed in Table. Our results showed that among 32 Aeromonas strains, the BLS could be only demonstrated in a strain of A. hydrophila isolated from a water tank containing alligators. This strain demonstrated heteroinhibitory activity against four Staphylococcus aureus strains (one ATCC 6538 and three methicillin-resistant MRSA). The heteroinhibitory activity was demonstrated after an incubation of 48 hr at 37°C and not at 25°C, conditions also observed for the BLS production in Serratia, Pseudomonas, Leuconostoc and Enterococcus strains (JD Foulds & D Shemin 1969 J Bacteriol 99: 655-660, JRW Govan & G Harris 1985 J Clin Microbiol 22: 490-494, F Mathleu et al. 1993 J Appl Bacteriol 74: 372-379, F Villani 1993 J Appl Bacteriol 74: 380-387). The heteroinhibitory activity was demonstrated by inhibitory zones ranging from 17 to 27mm. JR Govan (1986 Scand J Infec Dis 49: 31-37) proposed that different inhibition zone diameters may depend on the potency either of BLS action or they may be correlated with the number of receptors for BLS in the bacterial surface. In spite of these results, further research is necessary to better understand the bacteriocin-like activity in Aeromonas species as well as to investigate the chemical nature of this substance and its pharmacological usefulness.

Behaviour of Aeromonas spp. after animal passage

The Aeromonas genus have been consideredas important infectious agents in humans and ani-mals (JM Janda 1991 Clin Microbiol Reviews 4:397-410). The biological characteristics such as,toxins production (e. g. hemolysins, cytotoxins andenterotoxins) and also cell-associated features, thatappear to play important role in infectious pro-cesses in humans and animals, have been studiedin the attempt of elucidate patogenicity of the dif-ferent Aeromonas species (Janda loc. cit.). Evi-dences indicate that the animal passage may influ-ence in the expression of biological characteris-tics in many organisms such as, Plesiomonasshigelloides (SC Sanyal et al. 1980 J Med Microbiol13: 401-409) and Vibrio cholera O1 biotype ElTor (A Tikoo et al. 1994 J Med Microbiol 40: 246-251). DV Singh and SC Sanyal (1992 J MedMicrobiol 37: 262-267) reported that passagethrough rabbit instestines may control the expres-sion of the genes responsible for toxins produc-tion in Aeromonas spp.In this communication we report on the alter-ation of the hemolytic and enterotoxigenic charac-ter and also surface characteristics in one strain ofAeromonas isolated from environment, after ani-mal passage by endovenous route.Four samples of Aeromonas were used in thisstudy, three from polluted estuary water ( A. caviae- 030, Aeromonas sp. - 057, A. trota - 058) and onefrom drain treatment station supplyed by FundacaoOswaldo Cruz, Rio de Janeiro (A. hydrophila -T336). Those strains were maintend in nutrient agar(NA) with 1% of NaCl (FW Hickman-Brenner etal. 1987 J Clin Microbiol 25: 900- 906) at roomtemperature.All samples were tested with regar to hemol-ysin production, through β hemolysis zones aroundthe colonies in rabbit blood agar with 5% (v/v) oferitrocytes and the haemolitic activity was analyzedwith the metodology described by M Cumberbatchet al. (1979 Infect Immun 23: 829-837). The suck-ling mouse test (WA Dean et al. 1972 Infect Dis125: 407), was used for enterotoxin detection, theautoagglutination capacity for self pelleting (SP)and for preciptation after boiling (PAB) as de-scribed by JM Janda et al. (1987 Infect Immun 55:3070-77). The hidrofobic profile by phase parti-tioning with hydrocarbon solvents described by MRosenburg et al. (1980 Fems Microbiol Lett 9: 29-33).Strains for animal experiments were cultivatedin 5ml of BHI (OXOID) at 28

Prevention of suicide phenomenon in aeromonads

Clinical isolates ofAeromonas (13Aeromonas caviae), 8Aeromonas hydrophila, 3Aeromonas spp., and 2Aeromonas media recovered from diarrheal feces of children were submitted to the suicide phenomenon test and investigated at intervals of 24 h for up to 120 h. The same isolates were also stimulated by repeated passages in broth for ten days before the test. After the bacterial stimulus, a decrease in the number of aeromonads with suicidal capacity was observed. The suicide phenomenon was expressed after 72 h of incubation in only some isolates. The results show that the suicide phenomenon can be prevented by stimulation of bacterial metabolism.