AEM Accepted Manuscript Posted Online 2 January 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.03540-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Vibrio cholerae non-O1/non-O139 carrying multiple virulence factors and V. cholerae O1 in the Chesapeake Bay, Maryland Running title: Pathogenic V. cholerae in the Chesapeake Bay Daniela Ceccarelli1, Arlene Chen1, Nur A Hasan1,2,3, Shah M. Rashed1,4, Anwar Huq1,5, Rita R. Colwell1,2,3,5,6* 1

Maryland Pathogen Research Institute, University of Maryland, College Park, MD, USA;

2

CosmosID Inc., College Park, MD, USA;

3

University of Maryland Institute for Advanced Computer Studies, University of Maryland,

College Park, MD, USA; 4

International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka,

Bangladesh; 5

Maryland Institute of Applied Environmental Health, University of Maryland, College Park,

MD, USA; 6

Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD,

USA;

*Corresponding Author: Prof. Rita R. Colwell Center of Bioinformatics and Computational Biology University of Maryland Institute of Advanced Computer Studies University of Maryland, College Park, MD 20742, USA. Tel. (+1) 301 405 9550; Fax (+1) 301 314 6654; [email protected]

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1

Abstract

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V. cholerae non-O1/non-O139 inhabits estuarine and coastal waters globally but its clinical

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significance has not been sufficiently investigated, despite the fact that it has been associated

4

with septicemia and gastroenteritis. Emergence of virulent V. cholerae non-O1/non-O139 is

5

consistent with recognition of new pathogenic variants worldwide. Oyster, sediment, and water

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samples were collected during a Vibrio surveillance program carried out in 2009-2012 in the

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Chesapeake Bay, Maryland (USA). V. cholerae O1 was detected by direct fluorescent antibody

8

(DFA) but not successfully cultured, whereas 395 isolates of V. cholerae non-O1/non-O139 were

9

confirmed by multiplex PCR and serology. Only a few of the V. cholerae non-O1/non-O139

10

isolates were resistant to ampicillin and/or penicillin. Most were sensitive to all antibiotics tested

11

and 77-90% carried hemolysin hlyAET, actin cross-linking repeats in toxin rtxA, haemagglutinin

12

protease hap, and type 6 secretion system. Ca. 19 to 21% of the isolates encoded neuraminidase

13

nanH and/or heat-stable enterotoxin NAG-ST and only 5% contained type 3 secretion system.

14

None of the V. cholerae non-O1/non-O139 isolates contained VPI associated genes. However,

15

ctxA, ace, or zot were present in nine isolates. Fifty-five different genotypes carried up to 12

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virulence factors, independent of source of isolation, and represent the first report of both

17

antibiotic susceptibility and virulence associated with V. cholerae non-O1/non-O139 from the

18

Chesapeake Bay. Since these results confirm the presence of potentially pathogenic V. cholerae

19

non-O1/non-O139, monitoring for total V. cholerae, regardless of serotype, should be done

20

within the context of public health.

21

2

22

Introduction

23

Vibrio cholerae, a water-borne bacterial pathogen, is an autochthonous inhabitant of

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riverine and estuarine aquatic environments. There are more than 200 serogroups, based on O

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antigenic characters, but only serogroups O1 and O139 have been associated with epidemic

26

cholera and both are considered a major public health threat for developing countries (1).

27

Developed countries today rarely witness cholera cases caused by the epidemic strains of

28

V. cholerae and outbreaks are typically travel-associated (2). Yet, infections other than cholera

29

can be caused by the non-epidemic V. cholerae serogroups that, collectively, are referred to as

30

non-O1/non-O139 and are generally acquired through raw or undercooked seafood consumption.

31

V. cholerae non-O1/non-O139 infections are continuously reported worldwide (3, 4),

32

emphasizing their clinical significance. Although V. cholerae non-O1/non-O139 strains

33

generally do not produce cholera toxin, other virulence factors contribute to their pathogenicity,

34

including hemolysin hlyA (5), protease hapA (6), cytotoxic actin cross-linking repeats in toxin

35

rtxA (7), sialidase nanH (8), heat stable toxin NAG-ST (9), a type 6 secretion system (T6SS)

36

(10), and a type 3 secretion system (T3SS) (11). Occasionally, cholera toxin ctxA and toxin-

37

coregulated-pilus-associated genes tcpA and tcpI are reported to be present in V. cholerae non-

38

O1/non-O139 isolates (12, 13).

39

The CDC reported in 2010-2011 that V. cholerae O75 caused sporadic cholera cases traced

40

to contaminated shellfish consumption in the US Gulf Coast (14), and in 2011-2012 that

41

toxigenic V. cholerae O141 infections in New Jersey and Arizona were likely associated with

42

raw clam consumption and unsafe drinking water (2). In Maryland, vibriosis is mainly associated

43

with V. parahaemolyticus and V. vulnificus, but 5-10% of all cases yearly are caused by V.

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cholerae non-O1/non-O139 (15) and increasing so over the last decade (16). According to CDC

3

45

guidelines, oral rehydration is the therapy of choice for mild V. cholerae non-O1/non-O139

46

infections, whereas severe infections and septicemia should be treated with ciprofloxacin and/or

47

third-generation cephalosporins (ceftazidime and ceftriaxone) (17).

48

The Chesapeake Bay is the largest estuary in the Unites States and has been the subject of

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many microbiological studies over the last forty years. Occurrence of V. cholerae in the

50

Chesapeake Bay was first documented in the late 70s, when both V. cholerae non-O1/non-O139

51

(18) and non-toxigenic V. cholerae O1 (19) were isolated in different locations of the Bay.

52

Ecological surveys and genetic diversity analysis of V. cholerae were subsequently undertaken

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(20, 21), showing V. cholerae to be a naturally occurring component of estuarine and marine

54

coastal microbiota.

55

As reported by Baquero et al., “the study of antibiotic resistance in indigenous water

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organisms is important, as it might indicate the extent of alteration of water ecosystems by

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human action” (22). The Chesapeake Bay is characterized by high recreational use, heavy

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commercial fishing, and wastewater overflows from treatment plants. This composite aquatic

59

environment makes the Bay a potential bioreactor for genetic exchange among bacteria subjected

60

to antibiotic treatment (agricultural operations, poultry farms, isolates of human origin) and

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autochthonous microorganisms, enhancing the spread of drug resistance in aquatic environments.

62

V. cholerae isolated from seawater has been shown to be antibiotic resistant worldwide (23-25)

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but no information is available about V. cholerae populations of the Chesapeake Bay.

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The reported increase in V. cholerae non-O1/non-O139 cases in Maryland (16) demands

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better understanding of both antibiotic resistance and pathogenic properties of these bacteria,

66

considering no such data are available for environmental V. cholerae in the Chesapeake Bay. The

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aim of this study, therefore, was to undertake extensive analysis of virulence determinants and

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antibiotic resistance patterns of V. cholerae isolates collected during a 43 month surveillance

69

carried out in the Chesapeake Bay.

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Material and methods

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Sample collection, processing, and strain isolation

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From February, 2009, to August, 2012, oyster, sediment, and water samples were collected

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from the Chester River (CR) and Tangier Sound (TS), Chesapeake Bay, Maryland, USA. The

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two sampling sites were chosen based on ecological and environmental conditions: CR station,

76

located at the mouth of the Chester river, is representative of the upper Chesapeake Bay (39º 05'

77

09” N; 076º 09' 49” W), whereas the TS station is located in the lower Chesapeake Bay

78

(3810.976 N; 7557.901 W). Sampling was performed twice per month during summer (June to

79

August) and once per month the rest of the year (September to May). At each site, 12 liters of

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epipelagic water (whole water, plankton free water (PFW), and plankton fraction), 20-25 oysters,

81

and 80-100g of sediment were collected and V. cholerae isolated using alkaline peptone water

82

enrichment according to standard protocols (26). Briefly, three volumes of water (1L, 100ml,

83

10ml), homogenized oysters (10g, 1g, 0.1g), and sediment samples each added to 10X alkaline

84

peptone water were incubated statically at 35°C for 16-18 hours. 1 ml of each O/N enrichment

85

was used for DNA extraction by boiling (27) and tested by multiplex PCR for ctxA and toxigenic

86

V. cholerae O1 and O139 (28). A loopful of pellicle from each overnight enrichment was

87

streaked onto selective media, CHROMagar™ Vibrio (CHROMagar, USA), and Thiosulfate

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citrate bile salts sucrose (TCBS) agar (Difco, USA), and incubated overnight at 37°C.

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Presumptive V. cholerae colonies were subcultured onto LB agar and multiplex toxR PCR was

90

used to confirm V. cholerae (Table 1). Antisera kits for O1 (Vibrio cholerae Antiserum Poly,

5

91

Difco, USA) and O139 (O139 “Bengal”, Hardy Diagnostics, USA) V. cholerae were used to

92

determine serotype by slide agglutination, per manufacturer instructions. Serotyping was

93

confirmed by multiplex PCR, as previously described (Table 1). Bacterial isolates were stored at

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-80°C in LB broth containing 50% (vol/vol) glycerol.

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Direct fluorescent antibody assay-Direct viable count (DFA-DVC)

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Direct fluorescent antibody (DFA) detection of V. cholerae O1 and O139 was performed

97

using V. cholerae serogroup O1 (Cholera DFA) or O139 (Bengal DFA) kits (New Horizons,

98

MD). Briefly, 1 ml water and plankton samples were incubated overnight at 30°C with 0.002%

99

nalidixic acid and 0.025% yeast extract; samples were fixed with 2% formaldehyde, and stored at

100

room temperature until processed (26). Samples were processed according to the DFA O1 kit

101

instructions and slides were examined by epifluorescence microscope (Carl Zeiss AxioScope).

102

Antibiotic susceptibility

103

Antibiotic susceptibility was determined by disk diffusion on Muller-Hinton Agar (BD,

104

USA), according to Clinical and Laboratory Standards Institute guidelines for V. cholerae (29)

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and Enterobacteriaceae (30). Escherichia coli ATCC 25922 was used as quality control strain.

106

All strains were tested for resistance to: Ampicillin (AM-10µg), Ciprofloxacin (CIP-5µg),

107

Chloramphenicol (C-30µg), Erythromycin (E-15µg), Kanamycin (K-30µg), Nalidixic Acid (NA-

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30µg),

109

Sulfamethoxazole-Trimethoprim (SXT-23.75/1.25µg), and Tetracycline (T-30µg). Ampicillin

110

resistant strains were also screened for monobactams, carbapenems, second-, third- and fourth-

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generation cephalosporins: Cefotaxime (CTX-30µg), Ceftazidime (CAZ-30µg), Ceftriaxone

112

(CRO-30µg), Cefoxitin (FOX-30µg), Cefepim (FEP-30µg), Imipenem (IPM-10µg), and

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Aztreonam (ATM-30µg). MIC for strains showing intermediate susceptibility to erythromycin

Penicillin

(P-10µg),

Spectinomycin

6

(SPT-100µg),

Streptomycin

(S-10µg),

114

was determined using Ery EM256 (0.016-256 µg/ml) E-test strips (bioMerieux-USA), according

115

to manufacturer’s instructions.

116

DNA extraction and PCR amplification

117

Genomic DNA was extracted by boiling protocol according to Ausubel et al. (27). PCR

118

was performed in 25 μl reaction mix containing 12.5 μl of GoTaq Master Mix polymerase

119

(Promega) and 50ng/μl DNA. Gene targets and oligonucleotides sequences are listed in Table 1.

120

For thermal cycling conditions see specific references (Table 1). PCR amplicons were confirmed

121

by sequencing done at Eurofins Genomics (USA), and Invitrogen Vector NTI® software was

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used to compare DNA sequences against the GenBank Nucleotide database. Reference strains V.

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cholerae O1 N16961, INDRE 91/1 and O395, V. cholerae non-O1/non-O139 RC385, RC66, and

124

AM-19226, and V. cholerae O139 MO10 were included as positive and negative controls where

125

appropriate. Data presented in Table 4 are results of at least two independent experiments. DNA

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sequences were determined by Eurofins Genomics (Huntsville, USA). Simpson's Index of

127

Diversity was used to calculate sample diversity.

128 129

Results

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Detection and isolation of V. cholerae

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Altogether, 111 rounds of sampling took place at the Chester River (CR, n=54) site and

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Tangier Sound (TS, n=57) site between February, 2009, and August, 2012. Chester River

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samples were more frequently positive for V. cholerae than Tangier Sound, with 63% (34/ 54)

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positive rounds compared to 31% (17/54), respectively. Water temperature in the Chesapeake

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Bay displayed annual seasonal patterns with dramatic changes over the study period at both sites,

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from a low of 0.5°C in January to a maximum of 30.14°C in July. Seasonal fluctuation of salinity

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in the upper and mid-Chesapeake Bay is shown in Figure 1. Salinity ranged between 3 and 12.5

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ppt in Chester River, and 7.3 and 19.1 ppt in Tangier Sound.

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Combined sampling at both sites yielded 395 V. cholerae isolates by enrichment method

140

(Table 2) and all were confirmed V. cholerae non-O1/non-O139 by multiplex PCR and serology.

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From the CR site, a total of 312 V. cholerae isolates were isolated, half of which were from

142

water samples. V. cholerae was also isolated from oysters (six isolates from two rounds) and

143

sediment (twenty-one isolates from seven rounds) predominantly during the summer months of

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2011 and 2012 (Figure 1). Over the entire 43 month study at the CR site, the yield of V. cholerae

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isolates for the summer months (June to August) in 2010 was approximately 60% less than in

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2011 (40 and 139 isolates, respectively) and, interestingly, the proportion of positive samples

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dropped by approximately one-tenth in the summer of 2012 (15 isolates from 6 samplings). The

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peak of V. cholerae isolation in the summer of 2011 was strongly correlated (R2 = 0.9) with

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lower salinity (3.9 to 6.4 ppt), compared with higher salinity in 2011 (8 to 9.5 ppt) and 2012 (8.2

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to 11.1). V. cholerae isolation was episodic in Tangier Sound the entire sampling time, with 83

151

isolates from the three water fractions and none from oysters or sediment (Table 2). The largest

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number of isolates was obtained during February to May, 2010, and these were mainly from

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water but there was no recurrent seasonal pattern detected during the 43 month study (Figure 1).

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V. cholerae O1 and O139 were not isolated in culture from any of the samples collected in

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this study, and their presence was never detected by multiplex PCR (28) directly from O/N

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enrichment samples. Since V. cholerae can be present in the natural environment in a viable but

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non-culturable state, a direct fluorescent antibody (DFA) method was employed to detect V.

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cholerae O1 and O139 in all of the plankton and PFW samples. V. cholerae O139 was negative

159

for all samples tested during the entire surveillance. V. cholerae O1 was positive for plankton

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and/or PFW samples collected from both Chester River and Tangier Sound, but the results

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showed a scattering of V. cholerae O1 presence during the entire sampling (Table 3). All

162

samples collected during 2011 were negative for V. cholerae O1. Overall, 32 of 111 sampling

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rounds (29%) were positive for V. cholerae O1 from plankton and/or PFW samples. The average

164

V. cholerae O1 cell counts ranged from 8 to 45 x 103 cells/ml, and 5 to 10 x 103 cells/ml for

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plankton and PFW samples, respectively (Table 3). The percent positive rounds per year varied

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from 6.3 to 42.6%. The proportion of positive samples was higher for water samples than for

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plankton in the Chester River, a result reverse to that for Tangier Sound, where V. cholerae O1

168

positive samples were detected mainly in plankton samples. Furthermore, an increased frequency

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of V. cholerae O1 positive samples was observed during the summer months (May to August) of

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2010 and 2012 in Tangier Sound.

171 172

V. cholerae non-O1/non-O139 antibiotic resistance

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Antimicrobial susceptibility testing was carried out using disk diffusion assay for 11

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antibiotics (Figure 2) on a selection of the V. cholerae non-O1/non-O139 isolates obtained in this

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study (n=307), and selected for analysis according to site and date of isolation as representative

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(~78% of the total set of isolates). No significant difference was observed between isolates from

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the Chester River and the Tangier Sound. Nor was there a significant difference according to

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sample type (oysters, plankton, water, or sediment). Multidrug resistant isolates were not

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detected. All of the V. cholerae isolates were sensitive to chloramphenicol, ciprofloxacin,

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kanamycin, nalidixic acid, spectinomycin, streptomycin, sulfamethoxazole-trimethoprim and

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tetracycline. Of the 307 V. cholerae environmental isolates, 13% showed resistance to one or two

182

of the antibiotics tested: 20 to both ampicillin and penicillin (oysters, water, and PFW); 17 to

9

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penicillin (plankton and water); one to penicillin and erythromycin (water sample from Tangier

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Sound); and one to ampicillin (oysters in the Chester River). Intermediate resistance was

185

detected for kanamycin (11%), spectinomycin (7%) and streptomycin (8%). Interestingly, 71%

186

of all the isolates showed intermediate susceptibility to erythromycin. MIC was determined for a

187

selected set of strains showing intermediate resistance (n=110), and all showed an MIC of ≤4

188

µg/ml.

189

Twenty-one ampicillin resistant isolates were also tested for resistance to monobactam,

190

carbapenem, and second-, third- and fourth- generation cephalosporins. Two isolates from water

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samples collected from the Chester River were resistant to ceftriaxone, whereas three isolates

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from water samples collected in Tangier Sound were resistant to aztreonam. None of the AmpC

193

(MOX, CMY, FOX, LAT, ACC, MIR, DHA) and β-lactamase (blaOXA, blaSHV, blaCTX, blaTEM,

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blaIMP) gene determinants were detected in the Chesapeake Bay V. cholerae isolates, nor were

195

class 1 integrons or SXT/R391 ICE integrases (data not shown).

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Distribution of virulence factors among V. cholerae non-O1/non-O139

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Virulence factors detected in the V. cholerae isolates obtained in the study are listed in

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Table 4. Almost all of the V. cholerae isolates carried the following virulence factors: El Tor

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variant hemolysin hlyAET (83%), haemagglutinin protease hap (83.3%), actin cross-linking

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repeats in toxin rtxA (77.7%), and T6SS vasAKH (77.7-90.4%). Ca. 19.7% of the isolates

202

encoded neuraminidase nanH. The heat-stable toxin NAG-ST, encoded by stn (confirmed by

203

amplicon sequencing, data not shown), was found in 86 of the isolates (21.8%) from both

204

sampling sites. Only 5% of the isolates carried T3SS (vcsC/vcsV/vcsN/vspD). Genes ctxA, ace,

205

and/or zot were absent in all but nine of the V. cholerae non-O1/non-O139 (0.3-1%) from both

10

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the Chester River and Tangier Sound. None carried any of the toxin-coregulated pilus genes

207

(tcpA, tcpI, and tcpH).

208

Fifty-five profiles were obtained, showing up to 12 different virulence associated genes in

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the V. cholerae isolates from both sampling sites. Representative genotypes are shown in Table

210

5. Fourteen of the isolates carried no virulence associated factors, and 24 of the isolates each had

211

unique profiles. In general, 83 of the V. cholerae isolates from Tangier Sound showed greater

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variability, with 35 different virulence profiles (D=0.90), compared with 45 profiles observed in

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312 of the V. cholerae isolates from the Chester River (D=0.81). Analysis of the different water

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fractions and samples, showed greatest variation in virulence among V. cholerae isolates from

215

water samples (D=0.88) and lowest among V. cholerae isolates from sediment (D=0.60).

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The most frequent genotype detected (143 out of 395 isolates) was hlyA hap rtxA vasA

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vasK vasH. Seven variants of this profile, lacking rtxA and/or hap, with stn/sto and/or nanH were

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observed among 130 isolates (Table 5). Nine isolates were characterized by ctxA, ace or zot and

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virulence genes (hlyA, hap, stn/sto, rtxA, nanH, vasA, vasK, or vasH), but not the TCP-related

220

genes. These were mainly unique virulence profiles (Table 5). Twenty-one isolates had both type

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3 (vcsN, vcsV, vcsC, and vspD) and type 6 (vasA, vasK, and vasH) secretion systems, in different

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combinations with hlyA, hap, rtxA, and stn/sto (Table 5).

223 224

Discussion

225

The present study was aimed at gaining an understanding of the presence, virulence and

226

antibiotic resistance profiles of V. cholerae in the Chesapeake Bay. These bacteria are widely

227

distributed in the aquatic environment and are readily isolated into culture whereas V. cholerae

228

O1 is difficult to isolate even in cholera-endemic areas (31). However, V. cholerae O1 was

11

229

detected using DFA, as has been reported by previous investigators carrying such studies in the

230

Bay (20, 21). Attempts to isolate V. cholerae O1 into culture were not successful. It is concluded

231

that V. cholerae O1 is present in the Chesapeake Bay and detected by DFA, but in very low

232

numbers. The limit of detection might also depend on V. cholerae O1 being outnumbered by

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non-O1/non-O139 V. cholerae as well as the former being in a viable but non-culturable

234

(VBNC) state (32); both hypothesis are consistent with previous findings in both cholera

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endemic and non-endemic areas of the world (31, 33).

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Our results indicate, both in terms of percentage of positive rounds of sampling and number

237

of isolates, that V. cholerae is detected more frequently at the northern site (CR) than at the

238

southern site (TS) in the Chesapeake Bay, likely linked to the lower salinity registered in the

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Chester River, compared to Tangier Sound where the highest salinity points were registered over

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the entire study period. These findings are in agreement with previous studies conducted in the

241

Chesapeake Bay (20, 21) indicating that a salinity range between 4 and 14 ppt is optimal for V.

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cholerae.

243

Bacteria resistant to antibiotics have been reported to be present in aquatic environments

244

(22). Intensive use of antibiotics in medicine and in animal farming has been suggested to be the

245

source of such resistance (34, 35) and V. cholerae isolated from seawater have been shown to be

246

antibiotic resistant (23, 24). The data presented here provide the first report of antimicrobial

247

susceptibility for V. cholerae non-O1/non-O139 from the Chesapeake Bay. A very narrow

248

resistance profile was found with neither transfer of resistance from industrial or clinical strains

249

nor intrinsic “resistome” of naturally occurring isolates being significant for this V. cholerae

250

population.

12

251

Compared to other Vibrio spp. isolated from the Chesapeake Bay (36), V. cholerae non-

252

O1/non-O139 isolates in this study showed lower resistance to ampicillin and penicillin (9-20%)

253

than V. parahaemolyticus (53-68%), and intermediate resistance to streptomycin, compared to V.

254

vulnificus (36). Penicillin resistance, almost ubiquitous in both clinical and environmental V.

255

cholerae worldwide (23, 25), is likely associated with mutations of the penicillin binding

256

proteins 1 and/or 2, as observed for several sequenced V. cholerae strains (i.e. N16961, MJ-1236,

257

MO10). Ampicillin resistance has been reported for clinical V. cholerae non-O1/non-O139 and

258

V. parahaemolyticus isolated in Maryland as early as 1984 (37). Our data suggest that

259

mechanisms other than AmpC and β-lactamase genes may be responsible for ampicillin

260

resistance, such as variation in cellular impermeability or efflux pump activity, but this will

261

require further investigation to resolve.

262

Erythromycin is frequently used as a growth promoter in food animal production (38) and

263

the possible release of this antibiotic with wastewater into the Bay may explain the widespread

264

intermediate resistance observed in the V. cholerae non-O1/non-O139 isolates obtained in this

265

study. No data are available for erythromycin resistance for V. parahaemolyticus and V.

266

vulnificus from the Chesapeake Bay. Although antibiotic treatment with third-generation

267

cephalosporin is recommended only for severe infections and septicemia (17), the detection of

268

ceftriaxone resistant isolates of V. cholerae non-O1/non-O139 should raise concern, but

269

reassuringly all isolates were susceptible to ciprofloxacin, also recommended for clinical

270

treatment by the CDC (17).

271

Toxigenic V. cholerae non-O1/non-O139 strains appear to be more frequently isolated

272

worldwide, from both clinical and environmental samples, and are reported to be highly diverse

273

(39, 40). Variability amongst the isolates of this study was observed, with fifty-five profiles

13

274

comprising up to 12 virulence factors (Table 5). From the sequenced genomes of non-O1/non-

275

O139 V. cholerae available to date it is clear that these strains are genetically divergent from

276

each other and from V. cholerae O1 and O139 strains. Most of the virulence genes (nanH, hlyA,

277

hap, rtxA, stn) can be located on both chromosomes and also can be associated with

278

pathogenicity islands (T3SS) or encoded by different gene clusters on two separate chromosomes

279

(T6SS). This creates a number of different gene combinations that are virtually impossible to

280

predict and cannot be explained by acquisition of multiple clustered genes in one transfer event

281

without further analysis.

282

Genetic diversity was not associated with sampling location and identical profiles were

283

observed for isolates from both sampling sites. Some virulence genotypes were associated with

284

strains isolated at the same time of sampling. Rigorous phylogenetic analysis of the isolates was

285

not done and further investigation in this direction is required. Nevertheless, the same virulence

286

genotypes may represent a clonal population of V. cholerae. Interestingly, no association was

287

observed between isolates and location. Furthermore, the genetic profiles within a given

288

sampling round varied among the isolates. For example, 34 and 11 isolates were isolated from

289

sampling rounds CR037 and CR023, respectively, but these isolates for both of the sampling

290

rounds yielded five different genotypes, whereas sampling rounds CR021 and TS017 yielded 13

291

and 16 isolates with eight different genotypes recorded.

292

Genes ctxA, ace or zot were present in only nine isolates and these were from both the

293

Chester River and Tangier Sound. Other investigators have reported that environmental V.

294

cholerae non-O1/non-O139 generally do not produce cholera toxin (40, 41), even though V.

295

cholerae O1 and O139 isolated from the aquatic environment have been found to produce toxin

296

(12). Transfer of cholera toxin genes to non-O1/non-O139 strains in the aquatic environment can

14

297

be mediated by generalized transduction via CTXΦ ,(42), and environmental vibriophages have

298

been demonstrated to transfer toxin genes from CTXΦ positive strains to environmental non-

299

O1⁄non-O139 V. cholerae isolates (43). V. cholerae O1 isolated in the Chesapeake Bay (19) and

300

detected in this study by DFA, can perhaps be considered a source of toxin genes.

301

Previous studies have shown that non-toxigenic V. cholerae non-O1/non-O139 have been

302

associated with disease (7). Genes hlyA and hapA code for a hemolysin that exhibits vacuolating

303

activity (5) and a protease that affects epithelial tight junction-associated proteins (6),

304

respectively. In some cases, these factors are accompanied by rtxA cytotoxic activity, causing

305

mammalian cells to detach and round up (7). In our analysis, hlyA, hap, and rtxA were common

306

virulence factors, with a frequency similar to results of environmental surveillance in Argentina

307

(15), Iceland (19), Italy (23), Bangladesh (20), and China (13). Almost ubiquitous in this study

308

was the V. cholerae type 6 secretion system, with 76% of the isolates encoding all three genes

309

(vasAKH). The gene variability observed for T3SS and T6SS might be a consequence of gene

310

absence or amplification failure due to single nucleotide polymorphisms (SNPs). It has been

311

reported that T6SS contributes to pathogenesis in humans and to fitness for the bacterium,

312

protecting V. cholerae against other Gram-negative bacteria both in the human intestine and in

313

the environment (10).

314

Almost a quarter of the isolates of this study encoded the non-agglutinating heat stable

315

toxin NAG-ST and/or the sialidase nanH (8). Our findings differ from the relatively rare

316

detection of these two putative virulence factors reported for environmental isolates worldwide

317

(13, 44). Given the role of NAG-ST in severe diarrheal disease in human volunteers reported by

318

Morris et al. (9), the widespread distribution of this pathogenic factor in V. cholerae non-

319

O1/non-O139 in the Chesapeake Bay should be noted by public health authorities.

15

320

The observation that only 5% of V. cholerae non-O1/non-O139, mostly from the Chester

321

River, possessed a type 3 secretion system similar to the T3SS2 of V. parahaemolyticus is very

322

interesting. T3SS was found in three isolates with a composite genotype of hlyA stn/sto hap rtxA

323

nanH vcsC vspD vcsN vcsV vasA vasK vasH. T3SS-dependent virulence has been demonstrated

324

in the infant rabbit model, where V. cholerae non-O1/non-O139 was able to colonize the

325

intestine, induce pathological changes, and elicit diarrhea (11). T3SS was documented in V.

326

cholerae O75, a strain isolated from oysters harvested from Apalachicola Bay (Florida) and

327

responsible for a local cholera outbreak in 2010 (45).

328

Six V. cholerae non-O1/non-O139 were isolated from oysters in June and August, 2011,

329

when water temperatures were elevated and bacterial concentrations were usually high. Their

330

virulence genotype was found to be T6SS, hlyA and hapA and, in some cases, accompanied by

331

rtxA and/or nanH. The combined action of these virulence factors in V. cholerae non-O1/non-

332

O139 can be interpreted as enabling the bacterium to induce acute gastroenteritis if present in

333

raw or undercooked seafood that is consumed.

334

In summary, based on the V. cholerae non-O1/non-O139 virulence determinant and

335

antibiotic resistance profiles for isolates from the Chesapeake Bay, we confirm the presence of

336

potentially pathogenic forms of V. cholerae non-O1/non-O139 and support the view that

337

estuarine and marine bacteria comprise a significant reservoir of virulence and fitness genes.

338

These findings reinforce the connection between environmental reservoir and human infection

339

and confirm the value of monitoring V. cholerae within the context of public health.

340

16

341

Acknowledgments

342

This research was supported by National Science Foundation Grant No. 0813066 and

343

National Institutes of Health Grant No. 2RO1A1039129-11A2. The funders had no role in study

344

design, data collection and analysis, decision to publish or preparation of the manuscript.

345

We are grateful to Mitch Tarnowski and David White (Department of Natural Resources,

346

USA) and Kathy Brohawn, Sarah Harvey, Rusty McKay, and Steve Hiner (Maryland

347

Department of the Environment, USA) for their valuable and much appreciated assistance during

348

sampling.

349

17

350

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351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

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45. Haley BJ, Choi SY, Grim CJ, Onifade TJ, Cinar HN, Tall BD, Taviani E, Hasan NA, Abdullah AH, Carter L, Sahu SN, Kothary MH, Chen A, Baker R, Hutchinson R, Blackmore C, Cebula TA, Huq A, Colwell RR. 2014. Genomic and phenotypic characterization of Vibrio cholerae non-O1 isolates from a US Gulf Coast cholera outbreak. PLoS ONE 9:e86264. 46. Bauer A, Rørvik LM. 2007. A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. Lett. Appl. Microbiol. 45:371-375. 47. Vora GJ, Meador CE, Bird MM, Bopp CA, Andreadis JD, Stenger DA. 2005. Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp. Proc. Natl. Acad. Sci. U. S. A. 102:19109-19114. 48. Rivera ING, Chun J, Huq A, Sack RB, Colwell RR. 2001. Genotypes associated with virulence in environmental isolates of Vibrio cholerae. Appl Environ Microbiol 67:24212429. 49. Kumar P, Peter WA, Thomas S. 2010. Rapid detection of virulence-associated genes in environmental strains of Vibrio cholerae by multiplex PCR. Curr. Microbiol. 60:199-202. 50. O'Shea YA, Reen FJ, Quirke AM, Boyd EF. 2004. Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles. J. Clin. Microbiol. 42:4657-4671.

506 507

21

Table 1. Sequences and references for the oligonucleotides used in this study.

Gene

Primer

Sequence (5’-3’)

toxR

UtoxF

GASTTTGTTTGGCGYGARCAAGGTT

vctoxR

GGTTAGCAACGATGCGTAAG

640

(46)

vptoxR

GGTTCAACGATTGCGTCAGAAG

297

(46)

vvtoxR

AACGGAACTTAGACTCCGAC

435

(46)

O1F2-1

GTTTCACTGAACAGATGGG

192

(28)

O1R2-2

GGTCATCTGTAAGTACAAC

O139F2

AGCCTCTTTATTACGGGTGG

O139R2

GTCAAACCCGATCGTAAAGG

VCT1

ACAGAGTGAGTACTTTGACC

VCT2

ATACCATCCATATATTTGGGAG

ctxB-F

ATGCACATGGAACACCTCAAAATATTACTG

ctxB-R

TCCTCAGGGTATCCTTCATCCTTTCAATC

132-F

TAGCCTTAGTTCTCAGCAGGCA

951-R

GGCAATAGTGTCGAGCTCGTTA

O1-rfb

O139-rfb

ctxA

ctxB

tcpI

Amplicon (bp)

Reference (46)

(28) 449

(28) (28)

308

22

(28) (28)

231

(47) (47)

862

(48) (48)

tcpA

tcpH/A

hlyA

stn/sto

zot

ace

72-F

CACGATAAGAAAACCGGTCAAGAG

481 (El Tor)

(48)

477-R

CGAAAGCACCTTCTTTCACGTTG

620 (Classical)

(48)

647-R

TTACCAAATGCAACGCCGAATG

tcA-F

ATGCAATTATTAAAACAGCTTTTTAAG

tcA-R

TTAGCTGTTACCAAATGCAACAG

tcpH-1

AGCCGCCTAGATAGTCTGTG

tcpA-4

TCGCCTCCAATAATCCGAC

489-F

GGCAAACAGCGAAACAAATACC

481 (El Tor)

(48)

744-F

GAGCCGGCATTCATCTGAAT

738/727 (Classical)

(48)

1184-R

CTCAGCGGGCTAATACGGTTTA

67-F

TCGCATTTAGCCAAACAGTAGAAA

194-R

GCTGGATTGCAACATATTTCGC

225-F

TCGCTTAACGATGGCGCGTTTT

1129-R

AAC CCC GTT TCA CTT CTA CCC A

Ace-F

TGATGGCTTTACGTGGCTTGTGATC

Ace-R

GCCTGTTGGATAAGCGGATAGATGG

(48) 627 (Atypical)

(49) (49)

1289

(50) (50)

23

(48) 172

(48) (48)

947

(48) (48)

134

(44) (44)

hap

rtxA

nanH

vcsC

vcsV

Hap-F

ACGTTAGTGCCCATGAGGTC

351

Hap-R

ACGGCAAACACTTCAAAACC

Rtx-F

CTGAATATGAGTGGGTGACTTACG

Rtx-R

GTGTATTGTTCGATATCCGCTACG

nanH-F

CTTCCTCCAATACGGTTCTTGTCTCTTATGC

nanH-R

TTCGGCTACCATCGGCAACTTGTATC

vcsC2-F

GGAAAGATCTATGCGTCGACGTTACCGATGCTATGGG

vcsC2-R

CATATGGAATTCCCGGGATCCATGCTCTAGAAGTCGGTTGTTTCGGTAA

vcsV2-F

ATGCAGATCTTTTGGCTCACTTGATGG

vcsV2-R

ATGCGTCGACGCCACATCATTGCTTGC

(44) (44)

417

(44) (44)

314

(26) (26)

535

(26) (26)

742

(26) (26)

GGATCCCGGGAATTCCATATGCGTCGACAGTTGAGCCAAT vcsN

vcsN2-F

484

(26)

TCCATT

vspD

vasH

vcsN2-R

CGGGGTACCATGCTCTAGACGACCAAACGAGATAAT

vspD-F

ATCGTCTAGAACTCGAAGAGCAGAAAAAAGC

vspD-R

ATCGGTCGACCTTCCCGCTTTTGATGAAAT

vasH-857F

GTGGCACGCTATTTCTGGAT

(26) 422

(26) 385

24

(26)

(12)

vasA

vasK

vasH-1242R

TTTCAGCTCACGCACATTTC

vasA-104F

GTACGACCGATCCTGACGTT

vasA-446R

ATCTGAATGGTCGTGGCTTC

vasK-1851F

GCGTCAAATTCAGGAAGAGC

vasK-2250R

CTGTCCCAGAACCCAACTGT

(12) 342

(12) (12)

399

(12) (12)

508 509

25

Table 2. V. cholerae non-O1/non-O139 isolated by enrichment from the Chester River and Tangier Sound between February, 2009, and August, 2012. Chester River

Tangier Sound

Sediment

21

0

Oyster

6

0

Water

174

60

Plankton Free Water

56

9

Plankton

55

14

Total (395)

312

83

510 511

26

Table 3. Number of V. cholerae O1 positive rounds detected by DFA and average V. cholerae O1 cell counts by year for the entire studya.

Tangier Sound Total

Chester River

No. of positive rounds

No. of positive rounds cells/ml (x 103)

Year rounds

cells/ml (x 103)

Total rounds

(% of total rounds)

(% of total rounds)

P

PFW

P

PFW

P

PFW

P

PFW

2009

n =13

1 (7.7)

3 (23.1)

45.00

8.33

n = 14

5 (35.7)

6 (42.6)

8

8.75

2010

n = 15

5 (33.3)

5 (33.3)

11

25

n = 16

5 (31.3)

1 (6.3)

20

5

2012

n = 11

3 (27.3)

4 (36.4)

15

6.25

n = 11

4 (36.4)

2 (18.2)

16.25

10

512 513

P, plankton; PFW, plankton free water.

514

a

DFA analysis was negative for V. cholerae O1 in all rounds during 2011.

515 516

27

Table 4. Virulence gene profiles of 395 V. cholerae non-O1/non-O139 isolates. No. of positive isolates (%) Gene

Chester River (n = 312)

Tangier Sound (n = 83)

Total (n = 395)

ctxA

4 (1.3)

0

4 (1)

ctxB

0

0

0

tcpAa

0

0

0

tcpI

0

0

0

tcpH/A

0

0

0

ace

1 (0.3)

0

1 (0.2)

zot

2 (0.6)

2 (2.4)

4 (1)

hlyAETb

279 (89.4)

49 (59)

328 (83)

stn/sto

80 (25.6)

6 (7.2)

86 (21.8)

hap

280 (89.7)

49 (59)

329 (83.3)

rtxA

251 (80.4)

56 (67.5)

307 (77.7)

nanH

63 (20.2)

15 (18.1)

78 (19.7)

vscC

19 (6.1)

3 (3.6)

22 (5.6)

vspD

18 (5.8)

3 (3.6)

21 (5.3)

vscN

20 (6.4)

3 (3.6)

23 (5.8)

vscV

18 (5.8)

3 (3.6)

21 (5.3)

vasA

284 (91)

73 (88)

357 (90.4)

vasK

283 (90.7)

67 (80.7)

350 (88.6)

T3SS

T6SS

28

vasH

268 (85.9)

39 (47)

307 (77.7)

517 518

a

tcpA classical, El Tor and atypical alleles were investigated (see Table 1);

519

b

hlyAClassical allele negative for all isolates.

520

29

Table 5. Representative virulence genotypes of V. cholerae non-O1/non-O139. Genotype

Number of isolates

Source

hlyA hap rtxA vasA vasK vasH

143 (CR, TS)

W, PFW, P, S, O

hlyA stn/sto hap rtxA vasA vasK vasH

41 (CR)

W, PFW, P, S, O

hlyA hap rtxA nanH vasA vasK vasH

24 (CR, TS)

W, PFW, P, O

hlyA hap vasA vasK vasH

21 (CR, TS)

W, PFW, P, S, O

hlyA stn/sto hap rtxA nanH vasA vasK vasH

16 (CR, TS)

W, PFW, O

hlyA hap rtxA nanH vcsC vspD vcsN vcsV vasA vasK vasH

12 (CR, TS)

W, PFW, P

hlyA stn/sto hap vasA vasK vasH

9 (CR)

W, PFW, P, S

hlyA hap nanH vasA vasK vasH

5 (CR, TS)

W, PFW

hlyA hap rtxA vasA vasK

5 (CR)

PFW, P

hlyA hap rtxA vcsC vspD vcsN vcsV vasA vasK vasH

3 (CR, TS)

W, PFW, P

hlyA stn/sto rtxA nanH vasA vasK vasH

3 (CR)

W

hlyA stn/sto hap rtxA nanH vcsC vspD vcsN vcsV vasA vasK vasH

3 (CR)

W, P

hlyA stn/sto hap rtxA vcsC vspD vcsN vcsV vasA vasK vasH

2 (TS, CR)

W

ctxA hlyA hap rtxA vasA vasK vasH

2 (CR)

PFW, S

30

521

zot hlyA hap vasA vasK vasH

1 (CR)

W

ctxA hlyA stn/sto nanH vasA vasK vasH

1 (CR)

W

ace hlyA hap rtxA nanH vasA vasK vasH

1 (CR)

P

ctxA hlyA hap rtxA nanH vasA vasK vasH

1 (CR)

W

zot hlyA hap rtxA nanH vasA vasK vasH

1 (CR)

W

zot hlyA rtxA vasA vasK vasH

1 (TS)

W

zot rtxA vasA vasK

1 (TS)

W

CR, Chester River; TS, Tangier Sound; O, oyster; S, sediment; W, water; P, plankton; PFW, plankton free water.

522

31

523

Figure legends

524 525

Figure 1. Isolation of V. cholerae by enrichment over the course of the 43-month study in the

526

Chester River (top) and Tangier Sound (bottom). Water temperature in degrees C (black dots,

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right hand Y-axis) and V. cholerae detection (left hand Y-axis) in sediment (S, light blue bar),

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oyster (O, purple bar), plankton fraction (P, green bar), water (W, red bar), and plankton-free

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water (PFW, blue bar).

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Figure 2. Percentage of antibiotic resistant environmental V. cholerae non-O1/non-O139.

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R, resistant; I, intermediate; S, sensitive. AM, Ampicillin; CIP, Ciprofloxacin; C,

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Chloramphenicol; E, Erythromycin; K, Kanamycin; NA, Nalidixic Acid; P, Penicillin; SPT,

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Spectinomycin; S, Streptomycin; SXT, Sulfamethoxazole-Trimethoprim; T, Tetracycline.

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