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Aeromonas caviae septicemia in immunocompetent gastrointestinal carriers.

Aeromonas species are Gram-negative, motile, facultative anaerobic, rod shaped, oxidase positive bacteria of the family Aeromonadaceae [1]. Aeromonas species are environmental bacteria which are widely distributed in aquatic environment (both fresh and saltwater), soil and agricultural products [1]. Aeromonads cause acute diarrheal disease of short duration or chronic loose stools in children, the elderly, or the immunocompromised, and they have been implicated as a cause of travelers' diarrhea.

The significance of Aeromonas species as causative agent of human diarrhea has been well established [2]. Incidence of diarrheal disease caused by Aeromonas species is higher in developing countries than to developed countries [3] and extra-intestinal infections caused by these organisms are being recognized with increasing frequency [4]. These extra-intestinal infections include occasional primary infections in normal hosts (like cystitis and wound infections) as well as severe infections (like septicemia, peritonitis, endocarditis, osteomyelitis, meningitis, necrotizing fasciitis etc.) in immuno-compromised patients [1]. There are presently 17 species in the genera Aeromonas. At least 10 of these have been identified in human diseases, but important species are Aeromonas hydrophila (A. hydrophila), A. caviae and A. veronii biovar sobria [5]. Most invasive infections are caused by A. hydrophila in patients with compromised immune systems, usually in association with malignancies or liver cirrhosis [6].

Here we are reporting three cases of septicemia with asymptomatic gastrointestinal carriage of A. caviae (having substantial degree of invasiveness), in immunocompetent patients.

Case Report

We have isolated three stains of A. caviae from the blood of patients (residents of Lucknow, U.P., India) with community acquired bloodstream infections in the year 2008 (since Jan 2008 to June 2008) from patients getting admitted to Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, UP, a tertiary care hospital based in the largest state of north India. All the patients were apparently healthy before the onset of symptoms of septicemia and had no recent history of diarrhea, previous hospitalization, peritonitis, endocarditis, osteomyelitis, meningitis or necrotizing fasciitis.

Stool samples (3 samples, 24 hours apart) from each patient were also collected. Stool samples were cultured directly on MacConkey agar, Ampicillin sheep blood agar, and Xylose deoxycholate citrate agar. Simultaneous enrichment was also done in Gram-negative broth and alkaline peptone water, incubated overnight at 37[degrees]C and then sub cultured on the above mentioned media. Stool samples from two of the patients showed heavy growth of the A. caviae (as confirmed by the standard biochemical tests) on the primary isolation medium [7]. While from one patient's stool it was grown only after enrichment.

Antimicrobial susceptibility testing was performed using Mueller-Hinton agar (Oxoid, UK) by the disc diffusion method according to Clinical and Laboratory Standards Institute (CLSI) recommendations [8]. A. caviae isolates of each patient from both sources (blood and stool) showed similar antibiotic susceptibilities. All the six isolates were susceptible to Gentamicin, Amikacin and Imipenem; Resistant to Amoxycillin, Cotrimoxazole, Cephalothin and Cefazolin; 4 isolates were sensitive to Cefotaxime (2 isolates from one patients being resistant), while only 2 isolates (from one patient) were sensitive to Ciprofloxacin.

To test invasiveness of the strains Hep-2 cells were grown in minimal Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine and 20 mM HEPES (Hi Media). These cells were grown at 37[degrees]C in a humidified atmosphere under 5% C[O.sub.2]. Twenty hours prior to the infection, approximately 4 x 105 cells were seeded per well of 6-well tissue culture plates (BD Falcon, U.S.).

Bacteria were grown in BHI broth at 28[degrees]C for 12 hours and added to the cell monolayer at a multiplicity of infection (MOI) of 50:1. To determine the invasiveness of bacteria, at 3 hours of infection, Hep-2 cells were washed thrice with Phosphate Buffer Saline (PBS) and incubated further for 90 min in MEM containing 100 [micro]L/mL of gentamicin. The numbers of intracellular bacteria were determined by lysing the cells in PBS containing 0.1% digitonin and plating on trypticase soy agar (TSA). Cell invasion tests were found positive in all the isolate, 0.069% to 0.098% (mean being 0.073%) of the inoculated bacteria have invaded the Hep-2 cells. This cellline invasion test was performed in Department of Biochemistry, Lucknow University, Lucknow, UP, India.

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Slow intravenous infusion of Amikacin in a dose of 7.5 mg/kg body weight was given to the each patient every 12 hours for a period of 10 days and the patients were treated successfully. Follow-up cultures of blood and stool taken on 7th and 10th day were negative for A. caviae. Patients were asymptomatic after the antibiotic course. HIV ELISA was performed and was found negative in all the three patients.

Discussion

Aeromonas spp. cause cellulitis or wound infections following traumatic injury in an aqueous environment. They also cause various infections associated with hospitalization such as rare urinary tract infections, surgical wound infections, meningitis, peritonitis, endocarditis, or other serious infections [1,7]. Major predisposing conditions for Aeromonas infections include cirrhosis or other hepatic disease, hematologic malignancies and hepatobiliary diseases [9]. Aeromonas bacteremia is a rare disease usually seen in patients with multiple medical problems and in immunocompromised hosts, especially those with malignant or hepatobiliary diseases [6]. Sepsis may also occur as a result of contamination of wounds from fresh water or soil sources [1] or with the use of medicinal leeches [1]. The most common species involved in Aeromonas septicemia is A. hydrophila (just as in case of diarrhea) [6]. Although, from China bacteremia caused by A. caviae has been reported among patients with underlying illnesses like liver cirrhosis, malignancy and hepatobiliary diseases [9]; no case of A. caviae septicemia is reported from India.

We have found cases of septicemia with asymptomatic gastrointestinal carriage of A. caviae in immunocompetent patients having no history of diarrhea or previous hospitalization. These strains of A. caviae having considerable degree of invasiveness might have invaded the gastrointestinal tract and gained assess to the bloodstream (just as in case of Salmonella typhi, Yersinia enterocolitica, Shigella dysenteriae etc).

Asymptomatic gastrointestinal carriage of invasive A. caviae strains is an unusual finding and is of epidemiological importance as several areas of India have very high rates of Aeromonas induced acute diarrhea/gastroenteritis (up to 13%) [3], which may lead to asymptomatic gastro-intestinal carriage later on. Finding of these cases unleash a possibility of asymptomatic gastrointestinal carriage of such invasive strains of A. caviae in a very large population of India, which needs to be evaluated further in India as well as other countries having high rates of Aeromonas induced acute diarrhea/gastroenteritis.

Acknowledgments

Mayank Dwivedi acknowledges the financial assistance from the Scientific and Industrial Research, New Delhi, India. Amit Prasad acknowledges the financial assistance from the University Grant Commission, New Delhi, India and Council for Scientific and Industrial Research, New Delhi, India, respectively. We also acknowledge Mr. Nalin Sajwan for his scientific assistance.

References

[1.] Janda J.M., Abbott S.L. Evolving concepts regarding the genus Aeromonas: an expanding Panorama of species, disease presentations, and unanswered questions. Clin Infect Dis 1998;27:332-44.

[2.] von Graevenitz A. The role of Aeromonas in diarrhea: a review. Infection 2007;35:59-64.

[3.] Alavandi S.V., Ananthan S. Biochemical characteristics, serogroups, and virulence factors of aeromonas species isolated from cases of diarrhoea and domestic water samples in Chennai. Indian J Med Microbiol 2003;21:233-8.

[4.] Janda J.M., Abbott S.L., Khashe S., et al. Further studies on biochemical characteristics and serologic properties of the genus Aeromonas. J Clin Microbiol 1996;34:1930-3.

[5.] Chang B.J., Janda J.M. Aeromonas, 10th ed. London: Hodder Arnold; 2006.

[6.] Ko W.C., Chuang Y.C. Aeromonas bacteremia: review of 59 episodes. Clin Infect Dis 1995;20:1298-304.

[7.] Al-Benwan K., Abbott S., Janda J.M., et al. Cystitis caused by Aeromonas caviae. J Clin Microbiol 2007;45:2348-50.

[8.] Clinical and Laboratory Standards Institute. Performance standard for antimicrobial disk susceptibility testing. Approved standard. 9th ed. M2-A9 v, no. 1. Clinical and Laboratory Standards Institute, Wayne, Pa, 2006.

[9.] Wu C.J., Wu J.J., Yan J.J., et al. Clinical significance and distribution of putative virulence markers of 116 consecutive clinical Aeromonas isolates in southern Taiwan. J Infect 2007;54: 151-8.

M. Dwivedi (1), A. Mishra (2), A. Prasad (1), A. Azim (3), R.K. Singh (3), A.K. Baronia (3), K.N. Prasad (1) and U.N. Dwivedi (2)

(1) Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences; (2) Department of Biochemistry, Lucknow University; (3) Department of Critical Care Medicine, Sanjay Gandhi Post Graduate Institute of Medical Sciences; Lucknow, India

Received on 8 July 2008; revised 24 November 2008.

Address for correspondence: Dr. K.N. Prasad. Professor, Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India-226014. E-mail: knprasad@sgpgi.ac.in. Telephone No: 941501158.
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Author:Dwivedi, M.; Mishra, A.; Prasad, A.; Azim, A.; Singh, R.K.; Baronia, A.K.; Prasad, K.N.; Dwivedi, U.
Publication:The Brazilian Journal of Infectious Diseases
Article Type:Report
Geographic Code:3BRAZ
Date:Dec 1, 2008
Words:1470
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