CC BY-NC-ND
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Journal of Microbiology, Immunology and Infection (2014) xx, 1-7
ORIGINAL ARTICLE
Aeromonas stool isolates from individuals with or without diarrhea in southern Taiwan: Predominance of Aeromonas veronii
Po-Lin Chen a b, Pei-Jane Tsaic d, Chang-ShiChen e, Ying-Chuan Lu f, Hung-Mo Chen f, Nan-Yao Lee a, Ching-ChiLee a, Chia-Wen Lia, Ming-ChiLia, Chi-Jung Wu g **, Wen-Chien Ko a h *
a Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan b Graduate Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan c Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan
d Research Center of Infectious Disease and Signaling, National Cheng Kung University, Tainan, Taiwan e Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, Taiwan f Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan g National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan
h Department of Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan Received 12 June 2014; received in revised form 4 August 2014; accepted 7 August 2014 Available online ■ ■ ■
KEYWORDS
Aeromonas veronii; Cytotoxicity; Diarrhea; Taiwan
Background: Although aeromonads are important pathogens causing invasive infections in southern Taiwan, Aeromonas-associated intestinal infections have been rarely mentioned. Purpose: The aim of this study was to understand the frequency of isolation and clinical significance of aeromonads recovered from adult stool samples in southern Taiwan. Methods: During a 15-month study period, 514 adults with diarrhea and 167 asymptomatic controls were prospectively screened for the presence of aeromonads in stools. The identity of Aeromonas species was determined by the rpoD sequencing. Clinical information was retrieved from medical records, and in vitro cytotoxicity assay and polymerase chain reaction detection of putative virulent genes were performed.
* Corresponding author. Department of Internal Medicine, National Cheng Kung University Hospital, Number 138, Sheng Li Road, 70403 Tainan, Taiwan.
** Corresponding author. National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Number 367, Sheng Li Road, 704 Tainan, Taiwan.
E-mail addresses: wu.chijung@msa.hinet.net (C.-J. Wu), winston3415@gmail.com (W.-C. Ko).
http://dx.doi.org/10.1016/j.jmii.2014.08.007
1684-1182/Copyright © 2014, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. All rights reserved.
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Results: Thirteen (2.5 %) of 514 diarrheal patients and six (3.6%) of 167 asymptomatic controls had Aeromonas isolates in their stools. Of 11 diarrheal patients with available clinical information, Aeromonas veronii, the predominant species, was noted in six patients, and another potential enteropathogen was present in four patients. The cytotoxicity of A. veronii isolates to the HT-29 cell line was more potent in the isolates from diarrheal patients than those from asymptomatic controls (p = 0.015). The cytotoxicity of A. veronii isolates was more potent than that of A. caviae from symptomatic patients (p = 0.001). Putative virulence markers, including AHCYTONE, ascV, ascF-ascG, and aexT, were detected exclusively in A. veronii. The presence of the ascV gene was associated with cytotoxicity in A. veronii isolates. All Aeromonas isolates were susceptible to varied antimicrobial agents, except ampicillin/sulbactam. Conclusion: A. veronii is the predominant species in stools from individuals with or without diarrhea in southern Taiwan.
Copyright © 2014, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction
Aeromonas species are important endemic pathogens in southern Taiwan, in which a variety of human Aeromonas infections, such as bacteremia, biliary tract infection, soft tissue infection, and pneumonia, have been reported.1-4 In contrast, Aeromonas-associated intestinal infections in this area were rarely mentioned. Although Aeromonas-associated intestinal infections have been reported and discussed in the literature,5-8 there are still several controversies about its role as an enteropathogen. The arguments about aeromonads as a true enter-opathogen, summarized in a review by von Graevenitz,9 come from several lines of evidence: (1) failure to identify a single clonally-related outbreak of diarrhea caused by these pathogens, even though they are ubiquitous in environments; (2) the lack of proven experimental pathogenicity for humans; and (3) frequently a self-limited clinical course of Aeromonas-associated diarrhea. However, with the advance of molecular techniques, more Aeromonas species are considered the causes of diarrhea, and the number of enterotoxins responsible for enteritis has been identified increasingly.10 At least infraspecific subsets of Aeromonas strains with a particular array of enterotoxin genes are suggested to be potential enteropathogens.9
Several virulence factors have been considered to play an important role in causing gastrointestinal infections, such as type IV pili,11 heat-labile enterotoxins (Act and Alt), heat-stable enterotoxin (Ast), a variety of hemolysins (AerA, HlyA, Ahh1, and Asa1), and type III secretion system (TTSS).10-14 Some clinical studies have found that the cytotoxin was more frequently present in Aeromonas isolates from diarrheal patients than in the carriers.15 However, controversy remains because some reports showed contradictory results.16
In Taiwan, diarrheal patients are often screened by clinical microbiology laboratories for several bacterial pathogens, such as Campylobacter, Shigella, or Salmonella species, but not for Aeromonas species. Therefore, our study aim was to study the frequency of isolation and clinical significance of aeromonads in fecal specimens, as
well as their antimicrobial susceptibility, cytotoxicity, and virulence factors among fecal Aeromonas isolates obtained from diarrheal and asymptomatic patients.
Materials and methods
Cultivation and identification of Aeromonas isolates
This prospective study was conducted at the National Cheng Kung University Hospital, a medical center in southern Taiwan between September 2010 and December 2011. The stool samples from symptomatic patients for microbiological cultures of Salmonella, Shigella, or Vibrio species, were screened for Aeromonas species using the Aeromonas Selective Medium LabM 167 (Lab M; Lab M Ltd, Lancashire, UK) in the microbiological laboratory. Patients undergoing health examinations in the study hospital were recruited for participation (as the control group), and their stool samples were screened for aeromonads using the method described above.
The genus Aeromonas was identified as previously described.17 Aeromonas isolates were stored at -70°C until use. Final species identification was made through the partial sequences of rpoD.18 The reference strains for rpoD sequencing (GenBank accession no.) included Aeromonas dhakensis (Aeromonas aquariorum MDC47, FJ936132.1), Aeromonas veronii ATCC 9071T (FN773340.1), Aeromonas caviae ATCC 13136T (FN773319.1), and Aeromonas sanar-ellii A2-67T (FJ472929.1).
Antimicrobial susceptibility
The performance procedures for antimicrobial susceptibility by the disk diffusion method and the interpretative criteria were interpreted following the Clinical and Laboratory Standards Institute recommendations for Aeromonas species.19 The antimicrobial agents tested included amox-icillin/sulbactam, cefuroxime, ceftriaxone, cefepime, lev-ofloxacin, and imipenem.
Aeromonas-associated diarrhea
Detection of putative virulence factors
All isolates were studied by polymerase chain reaction to identify the genes encoding putative virulence factors: cytolytic enterotoxin (AHCYTOEN), aerolysin (aerA), hemolysin (hlyA), heat-labile enterotoxin (alt), heat-stable enterotoxin (ast), and three components of the TTSS—ascV, aexT, and ascF-ascG—as described elsewhere.20
Cytotoxicity of Aeromonas isolates
Fecal Aeromonas isolates were tested for cytotoxicity to the human colon carcinoma cell line, HT-29, according to a previously described method.10 A 0.1% Triton X-100 solution was used as a positive control and serum-free RPMI (Roswell Park Memorial Institute) medium (GIBCO, Grand Island, NY, USA) as a negative control. The cytotoxic activity was expressed as the mean of triplicate measurements of released leukocyte lactate dehydrogenase levels, compared with that of Triton X-100 solution (defined as 100% of cytotoxicity).
Patient information
Symptomatic patients with Aeromonas species in their stools were investigated for their clinical presentations, if informed consent was obtained. The information reviewed included demographic data, food and occupation exposure history, and clinical presentations. The study was ethically approved by the Institutional Review Board of the study hospital (IRB no. ER-99-086).
Statistical analysis
Categorical variables were compared using the Chi-square test or Fisher's exact test, if the expected counts were less than 5. The median cytotoxicity was compared using the Mann—Whitney test and plotted using GraphPad Prism, version 5.01 (GraphPad Software Inc., La Jolla, CA, USA). The Cochran—Armitage trending statistic test was performed to assess the relationship between the incidence of Aeromonas-associated diarrhea and age.
Results
During the study period, stool samples were collected from 514 adults (>18 years old) with diarrhea in the study hospital. In addition, 167 stool specimens were obtained from individuals undergoing health examinations during the study period. Of 514 patients with diarrhea, 13 (2.5%) had Aeromonas isolates in unformed stools. In contrast, 6 (3.6%) of 167 asymptomatic persons had Aeromonas in their stools. A total of 19 Aeromonas stool isolates were identified as A. veronii (10, 52.6%), A. caviae (7, 36.8%), A. sanarelli (1, 5.3%), and A. dhakensis (1, 5.3%), based on the matched rpoD sequences. The distribution of Aeromonas species in patients with and without diarrhea was similar (p = 0.72). The Aeromonas isolation rates among stool samples in different age stratification are given in Fig. 1. Our data showed a linear trend of the proportion of Aeromonas
Figure 1. Isolation rates of Aeromonas species in the stools from individuals with and without diarrhea in different age groups. The p value of the trend test for diarrhea (■) and no diarrhea (□) is 0.07 and 0.41, respectively.
species recovered from individuals with diarrhea increasing with age (p = 0.07). Ten of 13 diarrheal episodes occurred during the summer season (April—September) in southern Taiwan.
Informed consent was obtained from 11 of 13 symptomatic patients, and their clinical features are summarized in Table 1. In addition to diarrhea, their clinical presentations included abdominal cramping pain (3 patients), nausea/vomiting (1 patient), dysentery (1 patient), and fever (1 patient). Copathogens, including Clostridium difficile [2 patients with fecal growth of C. difficile and 1patient with fecal C. difficile toxin as revealed by: PREMIER ™ TOXINS A&B (Meridian Bioscience, Inc., Cincinnati, Ohio, USA), and Vibrio parahaemolyticus (1 patient), were noted. Eleven Aeromonas isolates were identified as A. veronii (6 patients), A. caviae (3 patients), A. dhakensis (1 patient), and A. sanarellii (1 patient). Their median age was 73 (range, 35—87) years. The underlying diseases of 11 patients included liver cirrhosis (2 patients), malignancy (2 patients), diabetes mellitus (2 patients), and Crohn's disease (1 patient). One patient had a history of eating lettuce with salad prior to the illness. Nine of 11 patients were admitted for medical care, and eight were treated by antibiotics. Although diarrhea lasting for more than 1 month was noted in two patients, enteritis symptoms resolved within 2 weeks in eight patients. Of note, a patient from whose stool A. veronii and C. difficile toxins were detected, developed severe colitis and hypotension requiring vasopressor support.
The identified virulence genes, including—AHCYTOEN, ascF-ascG, ascV, aexT, and hlyA—in Aeromonas stool isolates from patients with diarrhea are summarized in Table 1. Three genes, aerA, ast, and alt, were not detected. Of note, in five clinical A. veronii isolates, some putative virulence markers, including AHCYTOEN (4 isolates), ascF-ascG (3 isolates), ascV (3 isolates), aexT (1 isolate), and hlyA (1 isolate), were identified. All 19 isolates were susceptible to cefuroxime, ceftriaxone, cefepime, levofloxacin, and imipenem (Table 2). In contrast, the majority (94.7%) of isolates were not susceptible to ampicillin/sulbactam.
The cytotoxicity of Aeromonas isolates from the individuals with and without diarrhea, as assessed in the HT-
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Table 1 Clinical features of 11 patients with Aeromonas-associated diarrhea
Characteristics
No. of patients (%)
Age (median, range), y 73, 35-87
Sex, male 8 (72.7)
Underlying disease
Diabetes mellitus 2 (18.2)
Malignancy 2 (18.2)
Liver cirrhosis 2 (18.2)
HIV/AIDS 1 (9.1)
Crohn's disease 1 (9.1)
Treatment for diarrhea
Admission 9 (81.8)
Antibiotic treatment 8 (72.7)
Clinical presentations
Leukocytosis 5 (45.5)
Fever 3 (27.3)
Diarrhea >2 wk 2 (18.2)
Aeromonas species
A. veronii 6 (54.6)
A. caviae 3 (27.3)
A. sanarellii 1 (9.1)
A. dhakensis 1 (9.1)
Virulence genes
AHCYTOEN 4 (36.4)
ascF-ascG 4 (36.4)
ascV 3 (27.3)
aexT 1 (9.1)
hlyA 1 (9.1)
Virulent strainsa 5 (45.5)
Concurrent enteropathogens
Clostridium difficileb 3 (27.3)
Vibrio parahaemolyticus 1 (9.1)
a Defined as cytotoxicity > 50%, based on the released leukocyte lactate dehydrogenase level, as compared with that of Triton X-100 solution, which is defined as 100% of cytotoxicity in the HT-29 cell line.
b Clostridium difficile toxin A/B or toxigenic C. difficile was detected in stool.
29 cell line, is shown in Fig. 2. Five of 11 patients with diarrhea were infected with Aeromonas isolates with a cytotoxicity level of >50%. The median values of cytotox-icity levels for fecal A. veronii isolates from diarrheal
Figure 2. The medians with interquartiles of cytotoxicity for fecal Aeromonas veronii and Aeromonas caviae isolates from individuals with and without diarrhea.
patients were higher than those from asymptomatic controls (63.1% vs. 21.9%, p = 0.015). Nonetheless, the cytotoxicity of A. caviae isolates obtained from individuals with and without diarrhea was similar (10.7% vs. 7.30%, p = 0.46). However, cytotoxicity was more evident in fecal A. veronii than in A. caviae isolates from symptomatic patients (63.1% vs. 10.7%, p = 0.001). With regard to the correlation and virulence factors, the cytotoxicity was more evident in ascV+ A. veronii isolates (73.0% vs. 26.2%, p = 0.008).
Two A. veronii isolates from the patients with concomitant V. parahaemolyticus or C. difficile toxin in feces expressed high cytotoxicity levels (92.4% and 91.5%, respectively). In contrast, low cytotoxicity levels (15.9% and 21.7%, respectively) were present in an A. veronii isolate from a patient with healthcare-associated diarrhea, and an A. caviae isolate from a patient with AIDS and diarrhea. Although C. difficile was isolated from the stool samples of both patients, the toxigenic status of C. difficile isolates was not determined.
Table 2 In vitro susceptibility of Aeromonas stool isolates to six antimicrobial agents
Drugs A. veronii (n = 10) A. caviae (n = 7) Aeromonas isolates (n = 19)
S I R S I R S
Ampicillin/sulbactam 0 1 (10) 9 (90) 0 1 (14.3) 6 (85.7) 1 (5.3)
Cefuroxime 10 (100) 0 0 7 (100) 0 0 19 (100)
Ceftriaxone 10 (100) 0 0 7 (100) 0 0 19 (100)
Cefepime 10 (100) 0 0 7 (100) 0 0 19 (100)
Levofloxacin 10 (100) 0 0 7 (100) 0 0 19 (100)
Imipenem 10 (100) 0 0 7 (100) 0 0 19 (100)
I = intermediate; R = resistant; S = susceptible.
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Discussion
In Taiwan, invasive Aeromonas infections were more often reported than Aeromonas-associated diarrhea. In the present study, the isolation rate in clinical stool samples from diarrheal patients was 2.5%, which is comparable with the isolation rates reported in Spain (2%), Sweden (2%), Israel (2%), Switzerland (4.8%), and Japan (5.6%),6'21e24 and is similar to that in our asymptomatic controls (4.2%) and healthy individuals in Tokyo (3.8%, 74/1958 patients).24 However, the isolation rates varied widely in different countries. For example, up to 52.4% of diarrheal infants and 8.7% in controls were reported in Peru.25
Similar to our previous observation of seasonal preference of Aeromonas bacteremia,1'26 the predominance of our cases of Aeromonas-associated diarrhea was evident in warm seasons, which is probably related to the proliferation of aeromonads in water systems in higher ambient temperatures.27 Although the trend test was not significant, the elderly have an increased risk of acquiring Aero-monas-associated diarrhea in this study. Similar findings of Aeromonas bacteremia have been reported.28 Greater susceptibility to Aeromonas infections in the elderly may be related to concurrent chronic underlying diseases and waning host immunity.28,29 Therefore, physicians should consider Aeromonas spp. as one of the possible enter-opathogens causing gastrointestinal infections in the susceptible population.
The result that A. veronii was the predominant species in adults was not in accordance with other reports. In northern Taiwan, Aeromonas hydrophila has been discovered in 2.5% of 2150 diarrheal stool samples from children.30 In a review article, A. caviae was referred to be the most common species, followed by A. hydrophila and Aer-omonas veronii biovar sobria.9 Among the etiologies of travelers' diarrhea among Finnish tourists traveling to Morocco, A. veronii biovar. sobria was the major species.31 These varied results may be related to heterogeneous hosts, geographic locations, season of collections, and different culture media used.5 Of note, A. sanarellii was isolated from a female presenting to the emergency department with diarrhea as well as urinary tract infection. She recovered after taking oral cephalexin (for 3 days), which was not in vitro active against A. sanarellii, for urinary tract infection. A. sanarellii has been first identified in Taiwan and associated with clinical wounds in humans.3,32 Although further clinical evidence is required to confirm the enteropathogenicity of A. sanarellii in humans, this is the first report to describe the isolation of A. sanarellii from human stools.
Although many cases of diarrheal disease due to Aeromonas were mild, 82% (9/11 patients) of our cases were admitted and 73% (8/11 patients) received antibiotics, indicating severe illness in certain cases. Despite the fact that the role of antimicrobial therapy remains controversial for Aeromonas-associated diarrhea, most Aeromonas isolates were susceptible to broad-spectrum beta-lactams and fluoroquinolone. However, the clinical use of cephalothin, ampicillin, or ampicillin/sulbactam for Aeromonas-associ-ated diarrhea will be discouraged because of antimicrobial resistance.20,33
Our study indicated that cytotoxicity levels were significantly higher in A. veronii isolates from diarrheal patients, in accordance with several published studies in which a significant association was found between cyto-toxicity in cell lines and clinical diarrheal disease.34'35 For example, in an Iranian study, cytotoxicity was present in 67.9% of A. hydrophila isolates from diarrheal patients, in contrast to 22.7% in asymptomatic persons (p < 0.05).34 Moreover, the ascV gene encoding the TTSS has been considered an indicator of virulence in Aeromonas,36,37 which was further supported by our finding that cytotox-icity was more evident in ascV+ isolates than in ascV~ isolates.
Although there were similar Aeromonas isolation rates from the stools of individuals with and without diarrhea, the microbiological characteristic suggests that A. veronii is a potent enteropathogen. Cytotoxicity was present in clinical A. veronii isolates, which carry an array of genes encoding virulence factors. Of note, a significant proportion of A. veronii isolates carried genes encoding a cytotoxin, AHCYTONE, and the components of TTSS (ascV and ascF-ascG). Moreover, the variable cytotoxicity among A. veronii isolates indicates that the colonization of nonpathogenic or low-level pathogenic Aeromonas isolates in the intestinal tracts of humans is possible. In contrast, the pathologic role of A. caviae in enteritis, at least in our study, is equivocal owing to the absence of known virulence factors and cytotoxicity in clinical isolates.
Gastroenteritis due to coinfections with Aeromonas and other enteropathogens have been rarely reported. A Spanish study of the role of Aeromonas species in travelers' diarrhea found that three (16.7%) of 18 patients with Aer-omonas infections had other enteropathogens, such as Shigella sonnei, Giardia lamblia, or Salmonella typhimu-rium.6 Moreover, four (36.4%) of our 11 patients with Aer-omonas-associated diarrhea had another enteropathogen, either C. difficile or V. parahaemolyticus, in their stools. However, the enteropathogenicity or toxigenic status of the two C. difficile isolates was not confirmed. These results of the presence of concurrent enteropathogens and similar isolation rates of Aeromonas species from stool samples of individuals with and without diarrhea, not surprisingly, raise some concern regarding the enteropathogenicity of Aeromonas species in humans, a point of controversy raised by von Graevenitz.9 However, many experts still include Aeromonas species in the list of enteropathogens of travelers' diarrhea,38 acute dysentery,39 or chronic diarrhea.40
In conclusion, A. veronii is the predominant species among fecal Aeromonas isolates, and its cytotoxicity was more evident in the isolates from diarrheal patients than from asymptomatic controls.
Conflicts of interests
The authors report no conflicts of interests.
Acknowledgments
This study was supported by the grants from the National Cheng Kung University Hospital (NCKUH-10003006, NCKUH-
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10205011, and NCKUH-10307014), National Science Council (NSC 102-2314-B-006-055), National Health Research Institutes (ID-099-PP-17), Ministry of Health and Welfare (M0HW103-TDU-B-211-113002), Taiwan. We are grateful to Prof. Chung-Yi Li and Jia-Lin Wu from the Research Center of Clinical Medicine, National Cheng Kung University Hospital, for providing statistical consulting services.
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