Plesiomonas shigelloides infection, Ecuador, 2004-2008.
Subject: Diarrhea (Development and progression)
Diarrhea (Health aspects)
Infection (Development and progression)
Infection (Health aspects)
Medical research (Health aspects)
Medicine, Experimental (Health aspects)
Authors: Escobar, Juan C.
Bhavnani, Darlene
Trueba, Gabriel
Ponce, Karina
Cevallos, William
Eisenberg, Joseph
Pub Date: 02/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Feb, 2012 Source Volume: 18 Source Issue: 2
Product: Product Code: 8000200 Medical Research; 9105220 Health Research Programs; 8000240 Epilepsy & Muscle Disease R&D NAICS Code: 54171 Research and Development in the Physical, Engineering, and Life Sciences; 92312 Administration of Public Health Programs
Geographic: Geographic Scope: Ecuador Geographic Code: 3ECUD Ecuador
Accession Number: 281175416
Full Text: Plesiomonas shigelloides (family Enterobacteriaceae) has been implicated in gastroenteritis outbreaks in travelers to tropical regions and in persons who have ingested contaminated food or water (1-3). For persons native to tropical regions, however, case-control studies have found little or no association between P. shigelloides infection and diarrhea (4-6). Although these studies have been conducted in areas where mixed infections are generally common, to our knowledge, none examined coinfections. We assessed the pathogenicity of P. shigelloides in the context of co-infections and across all age groups in a province in northwestern Ecuador.

The Study

During 2004-2008, serial case-control studies were conducted in 22 remote communities in Esmeraldas Province, Ecuador. Complete study design and laboratory procedures for pathogen detection have been described (7). Briefly, each community was visited 4-6 times on a rotating basis; each visit lasted for 15 days, during which all cases of diarrhea were identified by a visit to each household every morning. Household residents with cases had [greater than or equal to] 3 loose stools in a 24-hour period, and controls had no symptoms of diarrhea during the past 6 days. Fecal samples were collected from 3 healthy controls per person with diarrhea. These samples were plated on selective agar media, and 5 lactose-fermenting colonies were screened by PCR for enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, and enteroinvasive E. coli (EIEC). Lactose-negative isolates that were identified as either Shigella spp. or E. coli were also screened by PCR for the same molecular marker used for EIEC. All non-lactose-fermenting pathogens, including P. shigelloides, were biochemically identified by API 20E system (bioMerieux, Marey l'Etoile, France). Because shigellae are phylogenetically similar to E. coli pathotypes, we combined data from persons infected with E. coli and those infected with shigellae in our analysis. We tested for Giardia lamblia by using an ELISA kit (RIDASCREEN Giardia; R-Biopharm, Darmstadt, Germany), and rotavirus was detected with an enzyme immunoassay kit (RIDA Quick Rotavirus; R-Biopharm). We chose a molecular method for detecting E. coli pathotypes because they cannot be differentiated solely on the basis of biochemical tests; the metabolic homogeneity of P. shigelloides, however, makes this organism easily and clearly identifiable by biochemical test. Similarly, immunologic methods used for Giardia sp. and rotavirus detection are specific and sensitive enough to accurately detect these pathogens, and use of molecular methods would be justified only for deeper analysis. Institutional review board committees at the University of California, Berkeley; University of Michigan; Trinity College; and Universidad San Francisco de Quito approved all protocols.

During March 2004-March 2008, a total of 2,936 fecal samples were collected from persons of all ages (168 [6%] were <1 year of age, 597 [20%] were 1-4 years, 753 [26%] were 5-12 years, 1,362 [46%] were [greater than or equal to] 13 years, and 56 [2%] were missing a birth date), corresponding to 775 cases and 2,161 controls. P. shigelloides was isolated in 253 (8.6%) samples. This number exceeded isolation rates for all of the pathogens analyzed except G. lamblia, which was present in 701 (23.9%) samples. Rotavirus was detected in 225 (7.7%) samples and EIEC/shigellae in 188 (6.4%) samples. P. shigelloides was detected in 11.4% of case-patients with diarrhea (case prevalence), which is more than the 7.2% estimated in the community (weighted control prevalence; Figure). However, once we stratified by persons infected only with P. shigelloides and those infected with P. shigelloides plus [greater than or equal to] 1 of the other marker pathogens for which we tested, single infections with P. shigelloides were almost equally prevalent in the case-patients and in the community; in contrast, coinfections with P. shigelloides and other pathogens were more frequent in persons with diarrhea (Figure).

To determine whether P. shigelloides infection was associated with diarrhea, we estimated risk ratios (RRs) and bootstrapped 95% CIs for single and co-infection exposures (Table). A single infection with P. shigelloides was not associated with diarrhea (RR 1.5, 95% CI 0.9-2.2). Persons co-infected with P. shigelloides and another pathogen, however, had almost 6x the risk for diarrhea than those with no infection (RR 5.6, 95% CI 3.5-9.3) and simultaneous occurrence of P. shigelloides and rotavirus increased the risk for diarrhea to 16.2 (Table). We found no evidence for confounding of the association between P. shigelloides and diarrhea by co-infecting pathogens ([RR.sub.crude] = [RR.sub.MH-pooled]; where [RR.sub.crude] is the unadjusted RR and [RR.sub.MH-pooled] is the pooled Mantel-Haenszel RR estimate). However, we found some evidence for confounding by age of P. shigelloides co-infection ([RR.sub.MH-pooled] 4.2 [95% CI 2.1-8.1], compared with the crude estimate of 5.6) but no evidence for confounding by age for single infection with P. shigelloides.



A single infection with P. shigelloides resulted in a moderately increased risk for diarrheal disease, which suggests that this microorganism plays a minor role as a pathogen. This result agrees with findings of previous studies (4,8,9). Analysis of the co-infections, however, suggests that P. shigelloides may be pathogenic in the presence of another pathogen. Specifically, co-infections of P. shigelloides with either rotavirus or pathogenic E. coli (including shigellae) were 16.2x (95% CI 5.5-62.3) and 13.8x (95% CI 3.3-69.3) more likely to result in diarrhea, respectively. We cannot, therefore, rule out the pathogenic capacity of P. shigelloides even though single infection may not be sufficient to cause disease.

This co-infection analysis might be limited by the number of pathogens considered (Giardia sp., rotavirus, pathogenic E. coli, and shigellae). However, the high isolation rates suggest we are detecting the major pathogens in the region. Other pathogens that may be useful to consider, given their attention in the literature, include Entaemobae histolytica and Cryptosporidium spp.

Although we found nothing in the literature that addresses the role of co-infection in the pathogenicity of P. shigelloides, co-infection with enteric pathogens is a well-known phenomenon, especially in tropical regions (6). Co-infection with ETEC and enteropathogenic E. coli increases virulence (10). Other studies have shown that the severity of disease is increased when rotavirus infections occur alongside another infection with another enteric pathogen (11).

P. shigelloides may take advantage of the disruption of the normal gut microbiota and gut physiology because of the concurrent presence of other pathogens, establishing a pathology in the human gut. For example, diarrhea caused by enterotoxins produced by pathogens, such as ETEC, and Vibrio cholerae (12), may remove normal gut microbiota, enabling P. shigelloides to establish an infection. The disruption of gut microbiota that facilitates gut colonization has been demonstrated in murine models infected with Citrobacter rodentium and Salmonella enterica serovar Typhimurium (13).

Most medical literature considers infectious diarrhea as a monopathogenic phenomenon (12,14). In the data presented here, the crude risk ratio suggests that P. shigelloides is pathogenic. When looking at single infections, we found no evidence that P. shigelloides is pathogenic. When looking at co-infection data, however, we found associations between infection and diarrhea. Our findings suggest that multipathogenic infections may play a role in the pathogenesis of infectious diarrhea.

This study was supported by the US National Institute of Allergy and Infectious Disease, grant no. RO1-AI050038.

Dr Escobar is a graduate student at Universidad San Francisco de Quito, Quito, Ecuador. His research interests are molecular epidemiology of enteric pathogens and co-infections that cause diarrheal diseases.


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Author affiliations: University of Michigan, Ann Arbor, Michigan, USA (J. Eisenberg, D. Bhavnani); and Universidad San Francisco de Quito, Quito, Ecuador (G. Trueba, W. Cevallos, K. Ponce, J.-C. Escobar)


Address for correspondence: Joseph Eisenberg, University of Michigan, 1415 Washington Heights, Ann Arbor, MI 48109, USA; email: jnse@
Table. RRs and bootstrapped 95% CIs for single infections and co-
infections with Plesiomonas shigelloides, Ecuador, 2004-2008 *

                 [RR.sub.Single       [RR.sub.Co-      [RR.sub.Crude]
Co-infection    P.shig] (95% CI)  Infection] (95% CI)     (95% CI)

Any pathogen     1.5 (0.9-2.2)       5.6 (3.5-9.3)     2.6 (1.9-3.5)
Rotavirus        1.5 (0.9-2.2)      16.2 (5.5-62.3)    1.7 (1.1-2.5)
Giardia sp.      1.5 (0.9-2.2)       2.1 (1.0-3.9)     1.5 (1.0-2.2)
Escherichia      1.5 (0.9-2.2)      13.8 (3.3-69.3)    1.6 (1.1-2.4)
colil shigellae

                [RR.sub.MH-Pooled]  Wald test for
Co-infection         (95% CI)       heterogeneity  p value

Any pathogen      2.7 (1.9-3.6)         32.1       <0.001
Rotavirus         1.9 (1.2-2.9)         61.8       <0.001
Giardia sp.       1.6 (1.1-2.3)          1.3         0.2
Escherichia       1.7 (1.1-2.6)         32.8       <0.001

* RR, risk ratio. [RR.sub.crude] = the unadjusted RR and
[RR.sub.HH-pooled] is the pooled Mantel-Haenszel RR ratio estimate.
The Wald test assesses whether the strata [RR.sub.Single Pshig] and
[] differ. Because of the clustered study design
and the unequal sampling probabilities of controls, we chose not to
use logistic regression models. Instead, we applied a nonparametric
approach by using sampling weights to estimate RRs, as one would
for a cohort study. We bootstrapped 1,000 samples from the original
dataset, and with each new sample, we estimated the RR associated
with single infection and co-infection. The lower 0.025 and upper
0.975 percentiles of the bootstrap distribution are reported as 95%
CIs. Statistical analyses were conducted by using R version 2.11.1
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