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Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China
YJINF3433_proof ■ 19 November 2014 ■ 1/9 Journal of Infection (2014) xx, 1e9
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www.elsevierhealth.com/journals/jinf
Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China Q3
Fengjuan Li a,b,f, Wenqing Wang c,f, Zhaoqin Zhu d,f, Aiping Chen e,f, Pengcheng Du a,b, Ruibai Wang a,b, Haili Chen d, Yunwen Hu d, Jie Li a,b, Biao Kan a,b,*, Duochun Wang a,b,* a
National Institute for Communicable Disease Control and Prevention, China CDC /State Key Laboratory for Infectious Disease Prevention and Control, Beijing, China b Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China c CDC of Pudong New Area, Shanghai, China d Shanghai Public Health Clinical Center, Fudan University, Shanghai, China e CDC of Fujian Province, Fuzhou, China Accepted 8 November 2014 Available online - - -
KEYWORDS Distribution; Virulence-associated genes; Antimicrobial resistance; Aeromonas isolates; China
Summary Objectives: To determine the prevalence of Aeromonas infections in diarrheal patients, the distribution of virulence-associated genes and antibiotic resistance among different Aeromonas species in China. Methods: We conducted continual active surveillance aimed on Aeromonas from diarrheal patients and aquatic samples. Aeromonas strains were identified by biochemical tests, further confirmed to species level by a multilocus phylogenetic analysis. Potential virulence genes were detected by PCR. Antibiotics susceptibility testing was carried based on the minimal inhibitory concentration. Results: From 5069 samples (stool specimens, n Z 4529; water samples, n Z 540) in China, 257 Aeromonas isolates [stools, n Z 193 (4.3%); water, n Z 64 (11.9%)] were identified by biochemical tests. The most common species from stools and water were Aeromonas veronii (42.5%) and Aeromonas caviae (37.5%), respectively. Distribution of five potential genes were significantly different between stool and water samples, two genes (ast and alt) were higher in stool than in water samples (P < 0.01). Meanwhile, three species (A. veronii, A. caviae and
* Corresponding authors. National Institute for Communicable Disease Control and Prevention, China CDC /State Key Laboratory for Infectious Disease Prevention and Control, Beijing, China. Q1 E-mail addresses: [email protected] (B. Kan), [email protected] (D. Wang). f Equal contributors. http://dx.doi.org/10.1016/j.jinf.2014.11.004 0163-4453/ª 2014 Published by Elsevier Ltd on behalf of The British Infection Association. Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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F. Li et al. Aeromonas aquariorum) account for the six most prevalent combination patterns of potential genes. Furthermore, strains resistant to nine antibiotics was markedly higher in strains isolated from water than those from stools (P 0.003); in contrast, resistance to only two antibiotics was higher in strains isolated from stools compared to those from water. In addition, strains containing multiple antibiotic resistance (MAR) from stools (8.6%; 16/187) and water (30.2%; 19/63) were resistant to ten or more antibiotics. Conclusion: Our study highlights the multiple factors involved in the pathogenesis of Aeromonas and reveals that environmental Aeromonas has acquired a wide range of MAR compared to those from clinical sources. ª 2014 Published by Elsevier Ltd on behalf of The British Infection Association.
Aeromonas species are Gram-negative, waterborne organisms that are often found to be environmental and food contaminants. This genus includes the causative agents of infectious diseases, the most common of which are diarrhea, bacteremia and localized soft-tissue infections.1 The gastrointestinal tract is by far the most common anatomic site from which Aeromonas spp. are recovered.1 Twenty-six species of Aeromonas have been identified.2 The Aeromonas species that are most frequently associated with diarrhea in humans are Aeromonas hydrophila, Aeromonas veronii and Aeromonas caviae,3 but their distribution varies according to geography. A recent report showed that many Aeromonas aquariorum isolates exist in a wide range of clinical and environmental water samples, but this species is often misidentified as A. hydrophila due to the use of traditional biochemical methods.4 Diarrheal diseases remain a leading cause of morbidity and mortality in China: more than 70 million cases are reported annually with an incidence of over 60/10 million,5 and only approximately 25% of all the reported cases were lab-confirmed.6 Few studies have been conducted on non-lab-confirmed pathogens in China. Moreover, because Aeromonas infections are not required reporting in China and because most studies have been limited to a few diarrheic bacteria,7 little attention has been focused on the prevalence of Aeromonas infections in diarrheal patients. The mechanism of Aeromonas pathogenesis is complex and not well understood. Current theory suggests that the virulence of Aeromonas species may be multifactorial. Several virulence factors have been identified in Aeromonas, including toxins (cytotoxic and cytotonic), proteases, hemolysins, lipases, adhesions, agglutinins, pili, enterotoxins, various enzymes, and outer membrane arrays. In 2004, genes for a type III secretion system (TTSS) were identified in this genus.8,9 Therefore, the detection of virulence genes in Aeromonas is essential for determining the potential pathogenicity of the organism; however, the difference in virulence-associated genes between diarrheal patients and environmental water, as well as the distribution of virulence-associated genes among different species, is unclear. Futhermore, the abuse of broad-spectrum antibiotics in clinical settings lead to an increase in antibiotic resistance among disease-causing Aeromonas species, high level of resistance to commonly used antibiotics, such as ampicillin, tetracycline and ciprofloxacin, have been emerged in both
clinical and environmental Aeromonas isolates.10,11 This causes bacteria face strong selection pressure to acquire resistance through a variety of mechanisms, such as genetic mutation and horizontal transfer of resistance genes. The antibiotic resistance of Aeromonas spp. isolated from diarrheal patients in China, and the extent of antibiotic resistance in Aeromonas from environmental water samples, have remained uninvestigated. Hence, we conducted active surveillance of Aeromonas, with a total of 257 Aeromonas isolates identified from 2010 to 2012 from diarrheal patients and environmental water in China. Our aim was to compare the distribution of Aeromonas species between diarrheal patients and environmental water and characterize the distribution of virulenceassociated genes and antimicrobial resistance profile among Aeromonas species from those two sources.
Materials and methods Active surveillance of Aeromonas Stool specimens were collected from diarrheal patients admitted to sixteen hospitals in the city of Shanghai, China, from June 25, 2010 to Dec. 31, 2012. Patients age from 5 month to 82 years (mean age 42.1 19.2). Specimens were collected by rectal swabs in CaryeBlair transport media and then transported to the laboratories of the Pudong District Center for Disease Prevention and Control (CDC), Shanghai, and the Shanghai Public Health Clinical Center, Fudan University. For the isolation of Aeromonas, refer to a previous report.7 Aquatic samples were collected by a monthly active surveillance program from the Minjiang River, Fujian province, China, from June, 2007 to Nov., 2008, at thirty samples per month. Aquatic samples were collected in sterile bottles and then transported to the CDC laboratory of Fujian province; for the isolation of Aeromonas, refer to a previous report.7 All of the bacterial isolates were screened for the oxidase reaction, subjected to biochemical tests (BioMe ´rieux, France), and identified as belonging to the Aeromonas genus by PCR of the 16S rRNA gene and sequencing analysis employing nucleotide blast in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Specimens from diarrheal patients were also detected for the following enteropathogens: Shigella, Salmonella, Campylobacter spp., Vibrio spp., diarrheagenic Escherichia coli, and Yersinia by conventional methods.7 Viruses were detected by RTPCR as described previously.12
Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Characters of Aeromonas isolates in China 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
PCR template preparation
3
The genomic DNA from Aeromonas samples was extracted using a genomic DNA purification kit (Tiangen Biotech, Beijing, China) in accordance with the manufacturer’s instructions. Extracted DNA was dissolved in TE buffer and stored at 20 C until use as PCR templates.
azithromycin (AZI), nitrofurantoin (NIT), kanamycin sulfate (KAN), and sulfamethoxazole (SMZ). Antibiotic susceptibility testing was carried out in 96-well plates, and the protocol was designed based on the minimal inhibitory concentration (MIC). E. coli ATCC 25922 was used as the quality-control strain. Multiple antibiotic resistance (MAR) was defined as resistance to three or more antibiotics.
PCR amplification and sequencing
Statistical analysis
To identify the Aeromonas strains in this study at the species level, seven housekeeping genes were targeted for analysis: atpD, dnaX, dnaJ, gyrA, gyrB, recA, and rpoD.13 Eleven genes were selected as potential markers of virulence,9,14,15,16 including aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), hemolysin (hlyA), serine protease (ahp), phospholipase (lip), flagellin (fla), ascF-G, type III secretion dependent ADP-ribosylating toxins, and a laf gene encoding a lateral flagella. For PCR amplification, each reaction was performed in a final volume of 50 ml containing 25 ml of 2 Taq PCR MasterMix (Tiangen Biotech, Beijing, China), 1.5 ml 10 mM of each forward and reverse primer, 2 ml DNA template, and 20 ml ddH2O.The reaction mixture was subjected to denaturation at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 54 Ce63 C for 30 s and extension at 72 C for 1 min/kb. An extension step of 7 min at 72 C was carried out following the last cycle to ensure fulllength synthesis of the fragment. All PCR products of the seven housekeeping genes were commercially direct sequenced in both directions (TaKaRa, Dalian, China).
The data were analyzed by using SPSS version 17.0 (SPSS Inc.,Chicago, IL). X2 test or Fisher’s exact test were used to analyze the results. A P value of <0.05 was considered statistically significant, while a P value of <0.01 was considered highly significant.
Phylogenetic data analysis A phylogenetic tree was constructed by multilocus phylogenetic analysis (MLPA) of the concatenated 4705 bp sequence of the seven housekeeping gene fragments.13 Reference nucleotide sequences of those seven housekeeping genes were obtained from the GenBank database, which included the twenty-six available representative species, listed in Table S1. Comparison analyses of the sequences were conducted with BioEdit software (Ibis Biosciences, Carlsbad, CA, USA). Clustal-W was used to perform multiple alignments of the nucleotide sequences. The phylogenetic trees were generated with MEGA 5.0 software by the neighbor-joining method. Bootstrap values were calculated based on 1000 replicates.
Antibiotic susceptibility test All isolates were tested for susceptibility to twenty-four antibiotics, according to the standards outlined by the Clinical and Laboratory Standards Institute.17 The antimicrobial drugs tested were streptomycin (STR), polymyxin B (POL), erythrocin (ERY), cefalotin (CEO), gentamycin (GEN), amikacin (AMI), cefixime (CEF), cefuroxime (CFX), imipenem (IMI), amoxicillin clavulanic acid (AMO), levofloxacin (LEV), cefoxitin (CFT), ampicillin (AMP), ceftriaxone (CFN), nalidixic acid (NAL), ciprofloxacin (CIP), norfloxacin (NOR), chloramphenicol (CHL), tetracycline (TET), doxycycline (DOX),
Results Prevalence of Aeromonas and co-infection of active surveillance We collected 5069 specimens in our active surveillance, including 4529 stool specimens from diarrheal patients and 540 aquatic samples. By traditional biochemical tests: oxidase positive, have facultative anaerobic metabolism, resistance to O/129 (150 mg) (Oxoid), and API-20E system (BioMe ´rieux, France), 193 (4.3%) samples from diarrheal patients and 64 (11.9%) samples from the aquatic environment were identified as the genus level of Aeromonas strains. For diarrheal patients, Aeromonas was found in 55 patients who were positive for other known enteropathogens: 27 patients were co-infected with bacteria, including enteropathogenic E. coli (9 cases), Salmonella spp.,6 Shigella spp.,3 Vibrio parahemolyticus,3 Campylobacter jejuni,3 Yersinia pseudotuberculosis2 and Campylobacter coli,1 22 were co-infected with viruses, including Norovirus,8 Sapovirus,2 and Rotavirus,12 and 6 were infected with three pathogens (Table S2).
MLPA identification and distribution of Aeromonas species The sequencing analysis of the concatenated 4705 bp sequence of gyrB-rpoD-recA-dnaJ-gyrA-dnaX-atpD sequence found that all 257 strains belonged to ten different species (Fig. 1), with the top four most prevalent species being A. veronii (32.3%), A. caviae (28.4%), A. aquariorum (17.1%), and A. hydrophila (8.2%). Worthy of note, 5 strains could not be clustered into any of the 26 known species and might be new species. The distribution of Aeromonas species in clinical and environmental samples differed from each other (Table 1). In clinical isolates, the most prevalent species were A. veronii (42.5%), A. caviae (25.3%) and A. aquariorum (14.5%), whereas A. caviae (37.5%), A. aquariorum (25.0%), A. hydrophila (15.6%) and A. jandaei (15.6%) were the most common in aquatic water. The difference in prevalence between clinical and environment isolates was significant for the species A. veronii, A. aquariorum, A. hydrophila and A. jandaei (P < 0.05).
Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Figure 1 Neighbor-joining phylogenetic tree of Aeromonas isolates recovered from stools and water. The tree based on MLPA of the concatenated 4705 bp sequence of seven housekeeping genes (gyrB-rpoD-recA-dnaJ-gyrA-dnaX-atpD). Color tree lines and fonts represent species involved in this study, brackets indicated the numbers of identified strains in this study. Reference nucleotide sequences of twenty-six Aeromonas species (colored in black fronts) are available from the GenBank database, as listed in Table S1. Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Characters of Aeromonas isolates in China 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
Table 1
5
Distribution of Aeromonas spp. in stool and water samples.
Species
No. total strains (%)
No. stool strains (%)
No. water strains (%)
P Value
A. veronii A. caviae A. aquariorum A. hydrophila A. jandaei A. enteropelogenes A. media unknown A. salmonicida A. diversa A. allosaccharophila Total
83 (32.3) 73 (28.4) 44 (17.1) 21 (8.2) 10 (3.9) 9 (3.5) 6 (2.3) 5 (1.9) 4 (1.6) 1 (0.4) 1 (0.4) 257
82 (42.5) 49 (25.3) 28 (14.5) 11 (5.7) 0 9 (4.7) 6 (3.1) 3 (1.6) 4 (2.1) 0 1 (0.5) 193
1 24 16 10 10 0 0 2 0 1 0 64
0a 0.063 0.015a 0.005a 0a 0.118 0.341 0.336 0.575 0.25 1
a
(1.6) (37.5) (25.0) (15.6) (15.6)
(3.1) (1.6)
Statistically significant difference between stool and water strains.
Distribution of putative virulence genes among Aeromonas isolates
Combination patterns of putative virulence genes among Aeromonas species
We detected eleven potential virulence genes in the Aeromonas strains (Table 2). Of the strains, 85.9% carried Elastase, followed by fla (66.5%), act (64.5%) and Lipase (60.3%), and three genes (laf, aer and ascF-G) were present in approximately 10% of the isolates. The four most prevalent virulence genes from both stool and water isolates were Elastase, fla, act and Lipase, which ranged from 54.4% to 95.3%. The prevalence of five virulence genes (fla, Elastase, ast, lip, hlyA, alt and ahp) were significantly different between stool and water isolates (P < 0.05, Table 2), but only alt and ast were found to be more common in stool isolates than in water isolates. Eleven virulence genes varied significantly among the most prevalent species (Fig. 2), for example, ahp (61.6%), hlyA (72.7%) and ast (69.9%) were highly prevalent in A. caviae, A. aquariorum and A. veronii, respectively, but low in other species. Both Lipase (21.7%) and Elastase (73.5%) were uncommon in A. veronii, but highly prevalent in other species; some genes, such as laf, aer and ascF-G, were uncommon in all species.
Multiple virulence genes were common and varied among different Aeromonas species in this study. We found 147 combination patterns (PTs) of eleven examined putative virulence genes (Table S3). The six most prevalent PT (n > 5) were PT1 (Fla/Ela/ast/act, n Z 22), PT2 (fla/Lip/ Ela/act/ahp, n Z 15), PT3 (fla/ast/act, n Z 13) PT4 (fla/ Lip/Ela/hlyA/act, n Z 9), PT5 (Ela/ast/act, n Z 8) and PT6 (Fla/Lip/Ela/ahp, n Z 7). The other 141 PTs carried by eleven Aeromonas species and unknown species, and account for the remaining 183 stains. Notably, among the six most prevalent PTs, A. veronii carried three (PT1, 3 and 5), A. caviae carried two (PT2 and 7), while A. aquariorum comprised one (PT4) (Table S3).
Table 2
Antibiotic resistance patterns among Aeromonas isolates All 257 strains were susceptible to 19 of the 24 tested antibiotics with ranges over 50%e99.2%; susceptible rates of 10 antibiotics were over 90%. All strains were resistant
Distribution of virulence associate genes between stool and water Aeromonas.
Gene
Total
Patient
Environment
P Value
fla Elastase ast act lip hlyA alt ascF-G ahp laf aer
171 221 88 166 155 85 81 26 76 33 27
126 160 79 126 105 60 76 20 53 20 22
45 61 9 40 50 25 5 6 23 13 5
0.46 0.013a 0a 0.686 0.001a 0.24 0a 0.82 0.198 0.039a 0.417
a
(66.5%) (85.9%) (34.2%) (64.5%) (60.3%) (33.1%) (31.5%) (10.1%) (29.5%) (12.8%) (10.5%)
(65.3) (82.9) (40.9) (65.3) (54.4) (31.2) (39.4) (10.4) (27.5) (10.4) (11.4)
(70.3) (95.3) (14.1) (62.5) (78.1) (39.1) (7.8) (9.4) (36.0) (20.3) (7.8)
Statistical difference between stool and water isolates.
Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Figure 2 Distribution of virulence genes among the four most common species. The proportion of red shadow represents the distributed proportion of virulence gene within species.
to the twenty-four tested antimicrobial agents to different degrees (Fig. 3). The percentage of strains resistant to five antibiotics (AMP, AMO, ERY, NAL and CEO) was over 50% (55.3e89.1%). Notably, for nine antibiotics (POL, STR, CFN, SMZ, KAN, CFX, CEF, GEN, DOX), the resistant rates were significantly higher for isolates from water than for isolates from stools (P 0.003). In contrast, only two antibiotics (AMP and AMO) showed significantly higher resistant rates for isolates from stools than for isolates from water (Fig. 3). Among different species, the number of antibiotics to which the strain exhibited a resistant rate over 50% was as follows: 6 (A. aquariorum), 5 (A. hydrophila), 4 (A. caviae), 4 (A. veronii) and 6 (other species) (Table S4). Of the 257 strains, except for seven strains (six isolated from stools and one from water), the remaining 250 strains contained 176 multiple antibiotic resistance (MAR) patterns to twenty-four tested antimicrobial agents (Table S5), with the most MAR pattern including resistance to seventeen
antibiotics. There were 151 MAR patterns that were unique to only one strain. In total, 8.6% (16/187) of isolates from stools and 30.2% (19/63) of isolates from water were resistant to 10 or more antibiotics.
Discussion Our active surveillance indicated the wide-spread distribution of Aeromonas species in aquatic environments, which were the major sources of contamination in acquired Aeromonas infections.1 The percentage of Aeromonas-positive samples from diarrheal patients was 4.5%, which is similar to the rate in other countries.3,18 Here, we did not find proof of outbreaks caused by Aeromonas alone, so the role of Aeromonas as an etiologic agent of diarrhea is still to be a controversial issue,19 further epidemiology and etiology should provide support for this point of view. In fact, several sporadic cases3,20 and even small
Figure 3 Antibiotic resistance patterns results of all strains. Patient group represents resistance rate from patient higher than that of environment, environment group represents resistance rate from water higher than that of patient, colored fronts represent statistical difference between patient and water isolates. Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Characters of Aeromonas isolates in China 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
outbreaks21 have indicated that Aeromonas has the capacity to produce diarrhea. In addition, we found a certain proportion of co-infection, similar to the reports in India,22 Pakistan23 and Hong Kong.24 This might be an indication that co-infection causes diarrhea; for instance, the co-infection of Pseudomonas has been proved to cause diarrhea together with Aeromonas or aggravate the disease.25 Our MLPA results showed that the most prevalent species isolated from stools were A. veronii, A. caviae, and A. aquariorum, in contrast to A. caviae, A. aquariorum and A. hydrophila/A. jandaei in water. The results were somewhat different from diarrheal patients in Israel (A. caviae, A. veronii and A. taiwanensis)18 and Malaysia (A. caviae, A. hydrophila and an unknown group)26 and a report of traveler’s diarrhea (A. veronii, A. caviae, and A. jandaei/A. hydrophila).27 One reason for this difference might be different identification methods; for example, A. aquariorum was frequently isolated species in clinical and water samples but can be misidentified as A. hydrophila by phenotypic methods.4 Another reason may be the different geographical distribution.1 In addition, we also found five strains that could not be identified by MLPA. The identification of Aeromonas has long been thought of as complicated and changing. 16S rRNA gene sequencing has been a good technology to use to identify and distinguish bacteria28; however, the accuracy 16S rRNA is significantly lower when distinguishing bacteria at the species level.29 Previously, one or two housekeeping genes have been used to identify and distinguish Aeromonas, but this technique turned out to be inaccurate or unstable, especially when genetic recombination occurs more frequently30 or when horizontal gene transfer occurred.31 When a concatenated sequence of seven housekeeping genes fragments is used, no overlaps occur either intraspecies or inter-species, and the classification of all strains is in separate clusters.13 We collected and analyzed the seven housekeeping genes of each of the 26 known species from GenBank database and then chose them as references; this may ensure accuracy. However, we also found inconsistencies between MLPA and single housekeeping genes in a few strains (data not shown), which may be caused by mutation and deletion at bases within a single gene; thus, to identify Aeromonas more accurately, more conservative and species-specific gene(s) should be explored and combined with phenotypic characteristics. Nevertheless, full-genome sequencing and microarray analysis is a more promising method for solving Aeromonas taxonomy issues.32,33 Due to the complexity of the pathogenic mechanism of Aeromonas, a single virulence gene cannot explain the disease caused by Aeromonas.23 Flagella are thought to play intimate roles in colonization of the gastrointestinal tract; two types of flagella, polar flagellum (fla) and lateral flagella (laf ), were identified in Aeromonas.34 We found fla present in the majority of strains from both patients and water sources, while laf was relative lower, suggesting that flagella may play more of a role in the pathogenisis of diarrhea. In addition, the enterotoxin genes ast and alt were significantly higher among samples from stools
7 than in those form water. This agrees with the data presented in a previous study, in which mutants of ast and alt exhibited a significantly reduced capacity to evoke fluid secretion in mice compared to wild-type A. hydrophila.35 Diarrhea caused by alt- and ast-carrying Aeromonas is more serious,23 indicating that these two enterotoxins have a close correlation to diarrhea. We also found that the virulence genes varied among different species, which may suggest the pathogenesis of Aeromonas varies among different species.36 Furthermore, differential expression of genes might be an important factor in the pathogenesis of Aeromonas species.1 It should be noted that we found 147 combination patterns of putative virulence genes among the 257 strains, although three species, A. veronii, A. caviae and A. aquariorum account for the six most prevalent patterns, the major patterns were carried by eleven Aeromonas species and unknown species. Thus, we emphasizes that multiple factors must be involved in the complicated process of Aeromonas pathogenesis. In this study, nearly all strains were resistant to the twenty-four tested antibiotics to different degrees. For five antibiotics, the resistant rates were over 50%, indicating high resistance to antibiotics of Aeromonas strains from both diarrheal patients and aquatic sources. What is more, the most common species from both diarrheal patients and aquatic sources were resistant to the types of antibiotics for which resistant rates were over 50%. This result most likely reflects the existence of selective pressures in those environments due to the use of antimicrobial agents in the prevention and treatment of human diseases.2 Our results showed that, as with other intestinal pathogens, the antibiotic resistance of Aeromonas is due to selection pressure from facing various classes of antimicrobial agents. Although some drugs, such as amoxicillin clavulanic acid, ampicillin and erythromycin, were once effective against Aeromonas infection,37 our strains exhibited low levels of sensitivity to these drugs. Fortunately, there are still several antibiotics to which Aeromonas is still sensitive, such as gentamycin, amikacin, cefixime, cefuroxime, levofloxacin, ciprofloxacin, norfloxacin, chloramphenicol, doxycycline and nitrofurantoin, which can be used as preferred drugs for the treatment of diarrhea. We found that the antibiotic sensitivity may differ among the species of Aeromonas, which suggests that we should choose the drugs used based on the species causing the infection. Notably, both the number of antibiotics to which isolates were resistant and the multiple antibiotic resistance (MAR) rate were significantly higher in samples from water than in samples from stools, with the highest MAR, to seventeen antibiotics, seen in a strain originating from water. Although MAR strains have been widely reported,10,11 this results was still unexpected. This result indicates that the environmental Aeromonas strains have acquired a wide range of resistance to antibiotics and MAR compared to those from clinical sources. Highly resistant Aeromonas strains may differ by geographical region, but in any case, Aeromonas from the aquatic environment has endured a larger variety of selective pressure from antibiotics.1 Thus, it is urgent to rediscover and evaluate the pathogenicity of environmental Aeromonas.
Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Acknowledgments This work was supported by the Priority Project on Infectious Disease Control and Prevention (2008ZX10004-008, 201202006, 2012ZX10004215, 2012ZX10004-201 and 2013ZX10004221-004) from the Ministry of Health, China, Key discipline construction of health system in Pudong New Area, Shanghai, China (No. PWZxk2010-09).
17.
18.
19.
Appendix A. Supplementary data 20.
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2014.11.004.
References 1. Janda JM, Abbott SL. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 2010;23(1):35e73. 2. Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease. J Infect 2011;62(2):109e18. 3. Figueras MJ, Alperi A, Saavedra MJ, Ko WC, Gonzalo N, Navarro M, et al. Clinical relevance of the recently described species Aeromonas aquariorum. J Clin Microbiol 2009;47(11):3742e6. 4. Aravena-Roman M, Harnett GB, Riley TV, Inglis TJ, Chang BJ. Aeromonas aquariorum is widely distributed in clinical and environmental specimens and can be misidentified as Aeromonas hydrophila. J Clin Microbiol 2011;49(8):3006e8. 5. Liu HX, Zhang J. Analysis of reported infectious diarrhea (other than cholera, dysentery, typhoid and paratyphoid) in China in 2011. Zhonghua Yu Fang Yi Xue Za Zhi 2013;47(4):328e32. 6. Hao R, Li P, Wang Y, Qiu S, Wang L, Li Z, et al. Diversity of pathogens responsible for acute diarrheal disease in China. Clin Infect Dis 2013;57(12):1788e90. 7. Qu M, Deng Y, Zhang X, Liu G, Huang Y, Lin C, et al. Etiology of acute diarrhea due to enteropathogenic bacteria in Beijing, China. J Infect 2012;65(3):214e22. 8. Yu HB, Rao PS, Lee HC, Vilches S, Merino S, Tomas JM, et al. A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis. Infect Immun 2004;72(3):1248e56. 9. Chacon MR, Soler L, Groisman EA, Guarro J, Figueras MJ. Type III secretion system genes in clinical Aeromonas isolates. J Clin Microbiol 2004;42(3):1285e7. 10. Palu AP, Gomes LM, Miguel MA, Balassiano IT, Queiroz ML, Freitas-Almeida AC, et al. Antimicrobial resistance in food and clinical Aeromonas isolates. Food Microbiol 2006;23(5):504e9. 11. Carvalho MJ, Martinez-Murcia A, Esteves AC, Correia A, Saavedra MJ. Phylogenetic diversity, antibiotic resistance and virulence traits of Aeromonas spp. from untreated waters for human consumption. Int J Food Microbiol 2012;159(3):230e9. 12. Gao Y, Jin M, Cong X, Duan Z, Li HY, Guo X, et al. Clinical and molecular epidemiologic analyses of norovirus-associated sporadic gastroenteritis in adults from Beijing, China. J Med Virol 2011;83(6):1078e85. 13. Martinez-Murcia AJ, Monera A, Saavedra MJ, Oncina R, LopezAlvarez M, Lara E, et al. Multilocus phylogenetic analysis of the genus Aeromonas. Syst Appl Microbiol 2011;34(3):189e99. 14. Santos JA, Gonzalez CJ, Otero A, Garcia-Lopez ML. Hemolytic activity and siderophore production in different Aeromonas species isolated from fish. Appl Environ Microbiol 1999; 65(12):5612e4. 15. Sen K, Rodgers M. Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification. J Appl Microbiol 2004;97(5):1077e86. 16. Martinez O, Rodriguez-Calleja JM, Santos JA, Otero A, GarciaLopez ML. Foodborne and indicator bacteria in farmed
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enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity. Infect Immun 2002;70(4):1924e35. 36. Wu CJ, Wu JJ, Yan JJ, Lee HC, Lee NY, Chang CM, et al. Clinical significance and distribution of putative virulence markers of 116 consecutive clinical Aeromonas isolates in southern Taiwan. J Infect 2007;54(2):151e8.
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www.elsevierhealth.com/journals/jinf
Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China Q3
Fengjuan Li a,b,f, Wenqing Wang c,f, Zhaoqin Zhu d,f, Aiping Chen e,f, Pengcheng Du a,b, Ruibai Wang a,b, Haili Chen d, Yunwen Hu d, Jie Li a,b, Biao Kan a,b,*, Duochun Wang a,b,* a
National Institute for Communicable Disease Control and Prevention, China CDC /State Key Laboratory for Infectious Disease Prevention and Control, Beijing, China b Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China c CDC of Pudong New Area, Shanghai, China d Shanghai Public Health Clinical Center, Fudan University, Shanghai, China e CDC of Fujian Province, Fuzhou, China Accepted 8 November 2014 Available online - - -
KEYWORDS Distribution; Virulence-associated genes; Antimicrobial resistance; Aeromonas isolates; China
Summary Objectives: To determine the prevalence of Aeromonas infections in diarrheal patients, the distribution of virulence-associated genes and antibiotic resistance among different Aeromonas species in China. Methods: We conducted continual active surveillance aimed on Aeromonas from diarrheal patients and aquatic samples. Aeromonas strains were identified by biochemical tests, further confirmed to species level by a multilocus phylogenetic analysis. Potential virulence genes were detected by PCR. Antibiotics susceptibility testing was carried based on the minimal inhibitory concentration. Results: From 5069 samples (stool specimens, n Z 4529; water samples, n Z 540) in China, 257 Aeromonas isolates [stools, n Z 193 (4.3%); water, n Z 64 (11.9%)] were identified by biochemical tests. The most common species from stools and water were Aeromonas veronii (42.5%) and Aeromonas caviae (37.5%), respectively. Distribution of five potential genes were significantly different between stool and water samples, two genes (ast and alt) were higher in stool than in water samples (P < 0.01). Meanwhile, three species (A. veronii, A. caviae and
* Corresponding authors. National Institute for Communicable Disease Control and Prevention, China CDC /State Key Laboratory for Infectious Disease Prevention and Control, Beijing, China. Q1 E-mail addresses: [email protected] (B. Kan), [email protected] (D. Wang). f Equal contributors. http://dx.doi.org/10.1016/j.jinf.2014.11.004 0163-4453/ª 2014 Published by Elsevier Ltd on behalf of The British Infection Association. Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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F. Li et al. Aeromonas aquariorum) account for the six most prevalent combination patterns of potential genes. Furthermore, strains resistant to nine antibiotics was markedly higher in strains isolated from water than those from stools (P 0.003); in contrast, resistance to only two antibiotics was higher in strains isolated from stools compared to those from water. In addition, strains containing multiple antibiotic resistance (MAR) from stools (8.6%; 16/187) and water (30.2%; 19/63) were resistant to ten or more antibiotics. Conclusion: Our study highlights the multiple factors involved in the pathogenesis of Aeromonas and reveals that environmental Aeromonas has acquired a wide range of MAR compared to those from clinical sources. ª 2014 Published by Elsevier Ltd on behalf of The British Infection Association.
Aeromonas species are Gram-negative, waterborne organisms that are often found to be environmental and food contaminants. This genus includes the causative agents of infectious diseases, the most common of which are diarrhea, bacteremia and localized soft-tissue infections.1 The gastrointestinal tract is by far the most common anatomic site from which Aeromonas spp. are recovered.1 Twenty-six species of Aeromonas have been identified.2 The Aeromonas species that are most frequently associated with diarrhea in humans are Aeromonas hydrophila, Aeromonas veronii and Aeromonas caviae,3 but their distribution varies according to geography. A recent report showed that many Aeromonas aquariorum isolates exist in a wide range of clinical and environmental water samples, but this species is often misidentified as A. hydrophila due to the use of traditional biochemical methods.4 Diarrheal diseases remain a leading cause of morbidity and mortality in China: more than 70 million cases are reported annually with an incidence of over 60/10 million,5 and only approximately 25% of all the reported cases were lab-confirmed.6 Few studies have been conducted on non-lab-confirmed pathogens in China. Moreover, because Aeromonas infections are not required reporting in China and because most studies have been limited to a few diarrheic bacteria,7 little attention has been focused on the prevalence of Aeromonas infections in diarrheal patients. The mechanism of Aeromonas pathogenesis is complex and not well understood. Current theory suggests that the virulence of Aeromonas species may be multifactorial. Several virulence factors have been identified in Aeromonas, including toxins (cytotoxic and cytotonic), proteases, hemolysins, lipases, adhesions, agglutinins, pili, enterotoxins, various enzymes, and outer membrane arrays. In 2004, genes for a type III secretion system (TTSS) were identified in this genus.8,9 Therefore, the detection of virulence genes in Aeromonas is essential for determining the potential pathogenicity of the organism; however, the difference in virulence-associated genes between diarrheal patients and environmental water, as well as the distribution of virulence-associated genes among different species, is unclear. Futhermore, the abuse of broad-spectrum antibiotics in clinical settings lead to an increase in antibiotic resistance among disease-causing Aeromonas species, high level of resistance to commonly used antibiotics, such as ampicillin, tetracycline and ciprofloxacin, have been emerged in both
clinical and environmental Aeromonas isolates.10,11 This causes bacteria face strong selection pressure to acquire resistance through a variety of mechanisms, such as genetic mutation and horizontal transfer of resistance genes. The antibiotic resistance of Aeromonas spp. isolated from diarrheal patients in China, and the extent of antibiotic resistance in Aeromonas from environmental water samples, have remained uninvestigated. Hence, we conducted active surveillance of Aeromonas, with a total of 257 Aeromonas isolates identified from 2010 to 2012 from diarrheal patients and environmental water in China. Our aim was to compare the distribution of Aeromonas species between diarrheal patients and environmental water and characterize the distribution of virulenceassociated genes and antimicrobial resistance profile among Aeromonas species from those two sources.
Materials and methods Active surveillance of Aeromonas Stool specimens were collected from diarrheal patients admitted to sixteen hospitals in the city of Shanghai, China, from June 25, 2010 to Dec. 31, 2012. Patients age from 5 month to 82 years (mean age 42.1 19.2). Specimens were collected by rectal swabs in CaryeBlair transport media and then transported to the laboratories of the Pudong District Center for Disease Prevention and Control (CDC), Shanghai, and the Shanghai Public Health Clinical Center, Fudan University. For the isolation of Aeromonas, refer to a previous report.7 Aquatic samples were collected by a monthly active surveillance program from the Minjiang River, Fujian province, China, from June, 2007 to Nov., 2008, at thirty samples per month. Aquatic samples were collected in sterile bottles and then transported to the CDC laboratory of Fujian province; for the isolation of Aeromonas, refer to a previous report.7 All of the bacterial isolates were screened for the oxidase reaction, subjected to biochemical tests (BioMe ´rieux, France), and identified as belonging to the Aeromonas genus by PCR of the 16S rRNA gene and sequencing analysis employing nucleotide blast in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Specimens from diarrheal patients were also detected for the following enteropathogens: Shigella, Salmonella, Campylobacter spp., Vibrio spp., diarrheagenic Escherichia coli, and Yersinia by conventional methods.7 Viruses were detected by RTPCR as described previously.12
Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Characters of Aeromonas isolates in China 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
PCR template preparation
3
The genomic DNA from Aeromonas samples was extracted using a genomic DNA purification kit (Tiangen Biotech, Beijing, China) in accordance with the manufacturer’s instructions. Extracted DNA was dissolved in TE buffer and stored at 20 C until use as PCR templates.
azithromycin (AZI), nitrofurantoin (NIT), kanamycin sulfate (KAN), and sulfamethoxazole (SMZ). Antibiotic susceptibility testing was carried out in 96-well plates, and the protocol was designed based on the minimal inhibitory concentration (MIC). E. coli ATCC 25922 was used as the quality-control strain. Multiple antibiotic resistance (MAR) was defined as resistance to three or more antibiotics.
PCR amplification and sequencing
Statistical analysis
To identify the Aeromonas strains in this study at the species level, seven housekeeping genes were targeted for analysis: atpD, dnaX, dnaJ, gyrA, gyrB, recA, and rpoD.13 Eleven genes were selected as potential markers of virulence,9,14,15,16 including aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), hemolysin (hlyA), serine protease (ahp), phospholipase (lip), flagellin (fla), ascF-G, type III secretion dependent ADP-ribosylating toxins, and a laf gene encoding a lateral flagella. For PCR amplification, each reaction was performed in a final volume of 50 ml containing 25 ml of 2 Taq PCR MasterMix (Tiangen Biotech, Beijing, China), 1.5 ml 10 mM of each forward and reverse primer, 2 ml DNA template, and 20 ml ddH2O.The reaction mixture was subjected to denaturation at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 54 Ce63 C for 30 s and extension at 72 C for 1 min/kb. An extension step of 7 min at 72 C was carried out following the last cycle to ensure fulllength synthesis of the fragment. All PCR products of the seven housekeeping genes were commercially direct sequenced in both directions (TaKaRa, Dalian, China).
The data were analyzed by using SPSS version 17.0 (SPSS Inc.,Chicago, IL). X2 test or Fisher’s exact test were used to analyze the results. A P value of <0.05 was considered statistically significant, while a P value of <0.01 was considered highly significant.
Phylogenetic data analysis A phylogenetic tree was constructed by multilocus phylogenetic analysis (MLPA) of the concatenated 4705 bp sequence of the seven housekeeping gene fragments.13 Reference nucleotide sequences of those seven housekeeping genes were obtained from the GenBank database, which included the twenty-six available representative species, listed in Table S1. Comparison analyses of the sequences were conducted with BioEdit software (Ibis Biosciences, Carlsbad, CA, USA). Clustal-W was used to perform multiple alignments of the nucleotide sequences. The phylogenetic trees were generated with MEGA 5.0 software by the neighbor-joining method. Bootstrap values were calculated based on 1000 replicates.
Antibiotic susceptibility test All isolates were tested for susceptibility to twenty-four antibiotics, according to the standards outlined by the Clinical and Laboratory Standards Institute.17 The antimicrobial drugs tested were streptomycin (STR), polymyxin B (POL), erythrocin (ERY), cefalotin (CEO), gentamycin (GEN), amikacin (AMI), cefixime (CEF), cefuroxime (CFX), imipenem (IMI), amoxicillin clavulanic acid (AMO), levofloxacin (LEV), cefoxitin (CFT), ampicillin (AMP), ceftriaxone (CFN), nalidixic acid (NAL), ciprofloxacin (CIP), norfloxacin (NOR), chloramphenicol (CHL), tetracycline (TET), doxycycline (DOX),
Results Prevalence of Aeromonas and co-infection of active surveillance We collected 5069 specimens in our active surveillance, including 4529 stool specimens from diarrheal patients and 540 aquatic samples. By traditional biochemical tests: oxidase positive, have facultative anaerobic metabolism, resistance to O/129 (150 mg) (Oxoid), and API-20E system (BioMe ´rieux, France), 193 (4.3%) samples from diarrheal patients and 64 (11.9%) samples from the aquatic environment were identified as the genus level of Aeromonas strains. For diarrheal patients, Aeromonas was found in 55 patients who were positive for other known enteropathogens: 27 patients were co-infected with bacteria, including enteropathogenic E. coli (9 cases), Salmonella spp.,6 Shigella spp.,3 Vibrio parahemolyticus,3 Campylobacter jejuni,3 Yersinia pseudotuberculosis2 and Campylobacter coli,1 22 were co-infected with viruses, including Norovirus,8 Sapovirus,2 and Rotavirus,12 and 6 were infected with three pathogens (Table S2).
MLPA identification and distribution of Aeromonas species The sequencing analysis of the concatenated 4705 bp sequence of gyrB-rpoD-recA-dnaJ-gyrA-dnaX-atpD sequence found that all 257 strains belonged to ten different species (Fig. 1), with the top four most prevalent species being A. veronii (32.3%), A. caviae (28.4%), A. aquariorum (17.1%), and A. hydrophila (8.2%). Worthy of note, 5 strains could not be clustered into any of the 26 known species and might be new species. The distribution of Aeromonas species in clinical and environmental samples differed from each other (Table 1). In clinical isolates, the most prevalent species were A. veronii (42.5%), A. caviae (25.3%) and A. aquariorum (14.5%), whereas A. caviae (37.5%), A. aquariorum (25.0%), A. hydrophila (15.6%) and A. jandaei (15.6%) were the most common in aquatic water. The difference in prevalence between clinical and environment isolates was significant for the species A. veronii, A. aquariorum, A. hydrophila and A. jandaei (P < 0.05).
Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Figure 1 Neighbor-joining phylogenetic tree of Aeromonas isolates recovered from stools and water. The tree based on MLPA of the concatenated 4705 bp sequence of seven housekeeping genes (gyrB-rpoD-recA-dnaJ-gyrA-dnaX-atpD). Color tree lines and fonts represent species involved in this study, brackets indicated the numbers of identified strains in this study. Reference nucleotide sequences of twenty-six Aeromonas species (colored in black fronts) are available from the GenBank database, as listed in Table S1. Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Characters of Aeromonas isolates in China 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
Table 1
5
Distribution of Aeromonas spp. in stool and water samples.
Species
No. total strains (%)
No. stool strains (%)
No. water strains (%)
P Value
A. veronii A. caviae A. aquariorum A. hydrophila A. jandaei A. enteropelogenes A. media unknown A. salmonicida A. diversa A. allosaccharophila Total
83 (32.3) 73 (28.4) 44 (17.1) 21 (8.2) 10 (3.9) 9 (3.5) 6 (2.3) 5 (1.9) 4 (1.6) 1 (0.4) 1 (0.4) 257
82 (42.5) 49 (25.3) 28 (14.5) 11 (5.7) 0 9 (4.7) 6 (3.1) 3 (1.6) 4 (2.1) 0 1 (0.5) 193
1 24 16 10 10 0 0 2 0 1 0 64
0a 0.063 0.015a 0.005a 0a 0.118 0.341 0.336 0.575 0.25 1
a
(1.6) (37.5) (25.0) (15.6) (15.6)
(3.1) (1.6)
Statistically significant difference between stool and water strains.
Distribution of putative virulence genes among Aeromonas isolates
Combination patterns of putative virulence genes among Aeromonas species
We detected eleven potential virulence genes in the Aeromonas strains (Table 2). Of the strains, 85.9% carried Elastase, followed by fla (66.5%), act (64.5%) and Lipase (60.3%), and three genes (laf, aer and ascF-G) were present in approximately 10% of the isolates. The four most prevalent virulence genes from both stool and water isolates were Elastase, fla, act and Lipase, which ranged from 54.4% to 95.3%. The prevalence of five virulence genes (fla, Elastase, ast, lip, hlyA, alt and ahp) were significantly different between stool and water isolates (P < 0.05, Table 2), but only alt and ast were found to be more common in stool isolates than in water isolates. Eleven virulence genes varied significantly among the most prevalent species (Fig. 2), for example, ahp (61.6%), hlyA (72.7%) and ast (69.9%) were highly prevalent in A. caviae, A. aquariorum and A. veronii, respectively, but low in other species. Both Lipase (21.7%) and Elastase (73.5%) were uncommon in A. veronii, but highly prevalent in other species; some genes, such as laf, aer and ascF-G, were uncommon in all species.
Multiple virulence genes were common and varied among different Aeromonas species in this study. We found 147 combination patterns (PTs) of eleven examined putative virulence genes (Table S3). The six most prevalent PT (n > 5) were PT1 (Fla/Ela/ast/act, n Z 22), PT2 (fla/Lip/ Ela/act/ahp, n Z 15), PT3 (fla/ast/act, n Z 13) PT4 (fla/ Lip/Ela/hlyA/act, n Z 9), PT5 (Ela/ast/act, n Z 8) and PT6 (Fla/Lip/Ela/ahp, n Z 7). The other 141 PTs carried by eleven Aeromonas species and unknown species, and account for the remaining 183 stains. Notably, among the six most prevalent PTs, A. veronii carried three (PT1, 3 and 5), A. caviae carried two (PT2 and 7), while A. aquariorum comprised one (PT4) (Table S3).
Table 2
Antibiotic resistance patterns among Aeromonas isolates All 257 strains were susceptible to 19 of the 24 tested antibiotics with ranges over 50%e99.2%; susceptible rates of 10 antibiotics were over 90%. All strains were resistant
Distribution of virulence associate genes between stool and water Aeromonas.
Gene
Total
Patient
Environment
P Value
fla Elastase ast act lip hlyA alt ascF-G ahp laf aer
171 221 88 166 155 85 81 26 76 33 27
126 160 79 126 105 60 76 20 53 20 22
45 61 9 40 50 25 5 6 23 13 5
0.46 0.013a 0a 0.686 0.001a 0.24 0a 0.82 0.198 0.039a 0.417
a
(66.5%) (85.9%) (34.2%) (64.5%) (60.3%) (33.1%) (31.5%) (10.1%) (29.5%) (12.8%) (10.5%)
(65.3) (82.9) (40.9) (65.3) (54.4) (31.2) (39.4) (10.4) (27.5) (10.4) (11.4)
(70.3) (95.3) (14.1) (62.5) (78.1) (39.1) (7.8) (9.4) (36.0) (20.3) (7.8)
Statistical difference between stool and water isolates.
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Figure 2 Distribution of virulence genes among the four most common species. The proportion of red shadow represents the distributed proportion of virulence gene within species.
to the twenty-four tested antimicrobial agents to different degrees (Fig. 3). The percentage of strains resistant to five antibiotics (AMP, AMO, ERY, NAL and CEO) was over 50% (55.3e89.1%). Notably, for nine antibiotics (POL, STR, CFN, SMZ, KAN, CFX, CEF, GEN, DOX), the resistant rates were significantly higher for isolates from water than for isolates from stools (P 0.003). In contrast, only two antibiotics (AMP and AMO) showed significantly higher resistant rates for isolates from stools than for isolates from water (Fig. 3). Among different species, the number of antibiotics to which the strain exhibited a resistant rate over 50% was as follows: 6 (A. aquariorum), 5 (A. hydrophila), 4 (A. caviae), 4 (A. veronii) and 6 (other species) (Table S4). Of the 257 strains, except for seven strains (six isolated from stools and one from water), the remaining 250 strains contained 176 multiple antibiotic resistance (MAR) patterns to twenty-four tested antimicrobial agents (Table S5), with the most MAR pattern including resistance to seventeen
antibiotics. There were 151 MAR patterns that were unique to only one strain. In total, 8.6% (16/187) of isolates from stools and 30.2% (19/63) of isolates from water were resistant to 10 or more antibiotics.
Discussion Our active surveillance indicated the wide-spread distribution of Aeromonas species in aquatic environments, which were the major sources of contamination in acquired Aeromonas infections.1 The percentage of Aeromonas-positive samples from diarrheal patients was 4.5%, which is similar to the rate in other countries.3,18 Here, we did not find proof of outbreaks caused by Aeromonas alone, so the role of Aeromonas as an etiologic agent of diarrhea is still to be a controversial issue,19 further epidemiology and etiology should provide support for this point of view. In fact, several sporadic cases3,20 and even small
Figure 3 Antibiotic resistance patterns results of all strains. Patient group represents resistance rate from patient higher than that of environment, environment group represents resistance rate from water higher than that of patient, colored fronts represent statistical difference between patient and water isolates. Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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Characters of Aeromonas isolates in China 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
outbreaks21 have indicated that Aeromonas has the capacity to produce diarrhea. In addition, we found a certain proportion of co-infection, similar to the reports in India,22 Pakistan23 and Hong Kong.24 This might be an indication that co-infection causes diarrhea; for instance, the co-infection of Pseudomonas has been proved to cause diarrhea together with Aeromonas or aggravate the disease.25 Our MLPA results showed that the most prevalent species isolated from stools were A. veronii, A. caviae, and A. aquariorum, in contrast to A. caviae, A. aquariorum and A. hydrophila/A. jandaei in water. The results were somewhat different from diarrheal patients in Israel (A. caviae, A. veronii and A. taiwanensis)18 and Malaysia (A. caviae, A. hydrophila and an unknown group)26 and a report of traveler’s diarrhea (A. veronii, A. caviae, and A. jandaei/A. hydrophila).27 One reason for this difference might be different identification methods; for example, A. aquariorum was frequently isolated species in clinical and water samples but can be misidentified as A. hydrophila by phenotypic methods.4 Another reason may be the different geographical distribution.1 In addition, we also found five strains that could not be identified by MLPA. The identification of Aeromonas has long been thought of as complicated and changing. 16S rRNA gene sequencing has been a good technology to use to identify and distinguish bacteria28; however, the accuracy 16S rRNA is significantly lower when distinguishing bacteria at the species level.29 Previously, one or two housekeeping genes have been used to identify and distinguish Aeromonas, but this technique turned out to be inaccurate or unstable, especially when genetic recombination occurs more frequently30 or when horizontal gene transfer occurred.31 When a concatenated sequence of seven housekeeping genes fragments is used, no overlaps occur either intraspecies or inter-species, and the classification of all strains is in separate clusters.13 We collected and analyzed the seven housekeeping genes of each of the 26 known species from GenBank database and then chose them as references; this may ensure accuracy. However, we also found inconsistencies between MLPA and single housekeeping genes in a few strains (data not shown), which may be caused by mutation and deletion at bases within a single gene; thus, to identify Aeromonas more accurately, more conservative and species-specific gene(s) should be explored and combined with phenotypic characteristics. Nevertheless, full-genome sequencing and microarray analysis is a more promising method for solving Aeromonas taxonomy issues.32,33 Due to the complexity of the pathogenic mechanism of Aeromonas, a single virulence gene cannot explain the disease caused by Aeromonas.23 Flagella are thought to play intimate roles in colonization of the gastrointestinal tract; two types of flagella, polar flagellum (fla) and lateral flagella (laf ), were identified in Aeromonas.34 We found fla present in the majority of strains from both patients and water sources, while laf was relative lower, suggesting that flagella may play more of a role in the pathogenisis of diarrhea. In addition, the enterotoxin genes ast and alt were significantly higher among samples from stools
7 than in those form water. This agrees with the data presented in a previous study, in which mutants of ast and alt exhibited a significantly reduced capacity to evoke fluid secretion in mice compared to wild-type A. hydrophila.35 Diarrhea caused by alt- and ast-carrying Aeromonas is more serious,23 indicating that these two enterotoxins have a close correlation to diarrhea. We also found that the virulence genes varied among different species, which may suggest the pathogenesis of Aeromonas varies among different species.36 Furthermore, differential expression of genes might be an important factor in the pathogenesis of Aeromonas species.1 It should be noted that we found 147 combination patterns of putative virulence genes among the 257 strains, although three species, A. veronii, A. caviae and A. aquariorum account for the six most prevalent patterns, the major patterns were carried by eleven Aeromonas species and unknown species. Thus, we emphasizes that multiple factors must be involved in the complicated process of Aeromonas pathogenesis. In this study, nearly all strains were resistant to the twenty-four tested antibiotics to different degrees. For five antibiotics, the resistant rates were over 50%, indicating high resistance to antibiotics of Aeromonas strains from both diarrheal patients and aquatic sources. What is more, the most common species from both diarrheal patients and aquatic sources were resistant to the types of antibiotics for which resistant rates were over 50%. This result most likely reflects the existence of selective pressures in those environments due to the use of antimicrobial agents in the prevention and treatment of human diseases.2 Our results showed that, as with other intestinal pathogens, the antibiotic resistance of Aeromonas is due to selection pressure from facing various classes of antimicrobial agents. Although some drugs, such as amoxicillin clavulanic acid, ampicillin and erythromycin, were once effective against Aeromonas infection,37 our strains exhibited low levels of sensitivity to these drugs. Fortunately, there are still several antibiotics to which Aeromonas is still sensitive, such as gentamycin, amikacin, cefixime, cefuroxime, levofloxacin, ciprofloxacin, norfloxacin, chloramphenicol, doxycycline and nitrofurantoin, which can be used as preferred drugs for the treatment of diarrhea. We found that the antibiotic sensitivity may differ among the species of Aeromonas, which suggests that we should choose the drugs used based on the species causing the infection. Notably, both the number of antibiotics to which isolates were resistant and the multiple antibiotic resistance (MAR) rate were significantly higher in samples from water than in samples from stools, with the highest MAR, to seventeen antibiotics, seen in a strain originating from water. Although MAR strains have been widely reported,10,11 this results was still unexpected. This result indicates that the environmental Aeromonas strains have acquired a wide range of resistance to antibiotics and MAR compared to those from clinical sources. Highly resistant Aeromonas strains may differ by geographical region, but in any case, Aeromonas from the aquatic environment has endured a larger variety of selective pressure from antibiotics.1 Thus, it is urgent to rediscover and evaluate the pathogenicity of environmental Aeromonas.
Please cite this article in press as: Li F, et al., Distribution, virulence-associated genes and antimicrobial resistance of Aeromonas isolates from diarrheal patients and water, China, J Infect (2014), http://dx.doi.org/10.1016/j.jinf.2014.11.004
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F. Li et al.
Acknowledgments This work was supported by the Priority Project on Infectious Disease Control and Prevention (2008ZX10004-008, 201202006, 2012ZX10004215, 2012ZX10004-201 and 2013ZX10004221-004) from the Ministry of Health, China, Key discipline construction of health system in Pudong New Area, Shanghai, China (No. PWZxk2010-09).
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Appendix A. Supplementary data 20.
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2014.11.004.
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