1
RNA-colony blot hybridization method for enumeration 1
of culturable Vibrio cholerae and Vibrio mimicus 2
Running Title: Enumeration of V. cholerae and V. mimicus by RNA colony blot 3
hybridization 4
5
Christopher J. Grim1,2
, Young-Gun Zo3, Nur A. Hasan
2,4, Afsar Ali
5, Wasimul B. Chowdhury
4, 6
Atiqul Islam4, Mohammed H. Rashid
5, Munirul Alam
4, J. Glenn Morris, Jr.
5, Anwar Huq
1,2 and 7
Rita R. Colwell1,2
* 8
9
1University of Maryland Institute for Advanced Computer Studies, University of Maryland, 10
College Park, Maryland 11
2Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland 12
3Department of Environmental Science, Kangwon National University, Chuncheon 200-701, 13
Republic of Korea 14
4International Center for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh 15
5Emerging Pathogens Institute, University of Florida, Gainesville, FL 32610 16
17
18
* Corresponding author, Mailing address: 3103 Biomolecular Sciences Building, no. 296, 19
College Park, MD 20742. Phone: (301) 405-9550. Fax: (301) 314-6654. E-mail: 20
[email protected]. 21
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02007-08 AEM Accepts, published online ahead of print on 26 June 2009
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Abstract 1
A species-specific RNA colony blot hybridization protocol was developed for enumeration of 2
culturable V. cholerae and V. mimicus in environmental water samples. Bacterial colonies on 3
selective or non-selective plates were lysed by SDS and the lysates were immobilized on nylon 4
membranes. A fluorescence-labeled oligonucleotide probe targeting a phylogenetic signature 5
sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules 6
immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all 7
colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of 8
the probe for the targeted species. For visible colonies of 10 non-target species, specificity of the 9
probe was calculated to be 90% because of a weak positive signal produced by Grimontia 10
(Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay 11
were evaluated, using lake water samples amended with a bioluminescent V. cholerae strain no 12
false negative or false positive was found, indicating 100% sensitivity and specificity for 13
culturable bacterial populations in freshwater samples, when G. hollisae was not present. When 14
the protocol was applied to laboratory microcosms containing V. cholerae, attached to live 15
copepods, copepods were found to carry approximately 10,000 to 50,000 CFUs of V. cholerae 16
per individual copepod. The protocol was also used to analyze pond water samples collected in a 17
cholera-endemic area of Bangladesh over a nine month period. Water samples collected from 18
six ponds demonstrated a peak in abundance of total culturable V. cholerae 1-2 months prior to 19
an observed increase in pathogenic V. cholerae and in clinical cases recorded by the area health 20
clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of 21
V. cholerae in the environment. The RNA blot hybridization protocol also can be applied to 22
detection of other gram-negative bacteria, for taxon-specific enumeration. 23
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Introduction 2
Vibrio cholerae is autochthonous to the aquatic environment; yet, some strains produce 3
enterotoxins and are capable of causing epidemics of the human disease, cholera. Strains of V. 4
cholerae are classified by their O-antigen, with over 210 serogroups recognized to date. Seven 5
cholera pandemics have occurred since 1832: while microbiologic data on the earlier pandemics 6
are not available, the last two are known to have been caused by strains within serogroup O1, 7
with the major pathogenic factor being production of cholera toxin. The genes encoding for 8
cholera toxin and other pathogenic factors have been shown to reside in a mobile genetic 9
element of phage origin, designated CTXΦ (22). 10
Standard microbiologic methods for isolation of V. cholerae present in natural waters rely 11
primarily on a method originally developed for clinical diagnosis, namely enrichment in alkaline 12
peptone water, followed by subculture on selective media, and confirmation using selected 13
biochemical and immunological tests (9). The alkaline nature of the enrichment broth allows 14
differential multiplication of Vibrio species, but renders this method inappropriate for 15
enumeration. PCR methods and oligonucleotide hybridization have been used in detecting and 16
enumerating toxigenic V. cholerae (3, 13, 14, 16, 17, 23). These methods typically rely on 17
amplification of or hybridization to pathogenic markers, such as O1/O139 wbe, tcpA, and ctxA 18
DNA sequences. 19
However, occasional localized outbreaks of cholera have been caused by non-O1/non-20
O139 V. cholerae, which may be toxigenic or non-toxigenic. And, conversely, many 21
environmental V. cholerae O1 strains isolated from endemic areas do not harbor ctx genes (11). 22
It has also been shown that CTXΦ is capable of lysogenic conversion of strains that are CTXΦ- 23
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(22). Additionally, the cholera toxin (CTX) prophage has also been detected in clinical strains of 1
V. mimicus and V. mimicus has been proposed as a natural reservoir for CTXΦ (2). Furthermore, 2
ecological studies of V. cholerae often are hampered by the fact that toxigenic strains represent 3
only a small percent of the total V. cholerae population in the environment, especially in cholera 4
non-endemic areas. These facts underline a need for a method of detection of the total number of 5
V. cholerae present in environmental samples. 6
The many copies of 16S rRNA molecules in each V. cholerae cell offer appropriate 7
targets for species-specific enumeration. In this study, the probe Vchomim1276, previously 8
described by Heidelberg (4-6), was employed in an RNA colony blot hybridization protocol. 9
The specificity and sensitivity of the probe was tested using type strains, and environmental and 10
clinical isolates. The method was evaluated using laboratory microcosms to which cells of V. 11
cholerae were added, and the protocol was used to enumerate V. cholerae from samples 12
collected from ponds in a cholera endemic region of Bangladesh.13
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Materials and Methods 2
Bacterial strains. Bacterial strains used in this study are listed in Table 1. Sources for 3
strains included the American Type Culture Collection (ATCC, Manassas, VA) and clinical and 4
environmental isolates from the United States, Mexico, Bangladesh, England, and Ecuador. 5
Unless specified, all strains were grown on LB-20 agar, Luria-Bertani agar, amended with NaCl 6
to a final concentration of 2%. The bacterial cultures were maintained at -80°C in LB broth to 7
which 25% glycerol had been added, and on LB-20 agar slants at room temperature. 8
V. cholerae-specific probe. We examined 207 16S rRNA gene sequences deposited in 9
GenBank of Vibrio or Photobacterium origin to determine V. cholerae specific sequence motifs 10
in 16S rRNA genes (25). However, a stretch of parsimony informative sites between 16S rRNA 11
gene sequences of V. cholerae and V. mimicus was not found. A previously described 16S rRNA 12
gene probe, Vchomim1276 was selected as the oligonucleotide probe, specific for V. cholerae 13
and V. mimicus (7, 8). The probe, ACT TTG TGA GAT TCG CTC CAC CTC G (Tm = 72°C), 14
was 5’ end-labeled with either fluorescein or Cy3 at the time of oligomer manufacturing by 15
Sigma-Genosys (St. Louis, MO). 16
Colony blot lift. Strains were inoculated onto LB-20 agar and grown overnight at room 17
temperature to a colony size no larger than 5 mm in diameter. A previously reported RNA 18
colony blot lift method (10) was employed to liberate, denature, and immobilize nucleic acid 19
onto nylon or nitrocellulose membranes (GE Osmonics). Briefly, colony containing agar plates 20
were overlaid with membranes and transfer of colony materials was allowed to proceed for at 21
least 15 min. Membranes were transferred colony side up onto filter papers wetted with 10% 22
SDS and incubated for 5-10 min. Membranes were then transferred to filter paper wetted with 23
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3X SSC, pre-warmed to 65°C, and incubated at 65°C for 10-15 min. Membranes were dried at 1
37°C for 10 min, and baked at 70°C for 15 min. Methylene blue was used for qualitative 2
examination of RNA and/or DNA immobilized onto membranes. 3
RNA colony blot hybridization. All solutions used for RNA colony blot lift and 4
hybridization were treated with diethyl pyrocarbonate (DEPC) to eliminate RNase 5
contamination. Colony blots were washed in 3X SSC, 0.1% SDS, three times at room 6
temperature for 10 min each, and once at 65°C for 2 hr, to prevent cellular debris from 7
interfering with hybridization. Membranes were prehybridized at 60ºC for 30 min in 8
hybridization solution base (10 ml per 100 cm2 membrane; 0.9 M NaCl, 50 mM sodium 9
phosphate/pH 7.0, 5 mM EDTA, 0.5% SDS in DEPC-treated water). Hybridizations were 10
performed overnight (approximately 16 hr) at 60ºC, using hybridization solution base (10 ml per 11
100 cm2 membrane) containing fluorescently labeled Vchomim1276 probe (80 ng/ml). After 12
hybridization, membranes were washed in washing solution, 1% SDS, 1X SSC in DEPC-treated 13
water, at the hybridization temperature, for 30 min. Membranes were wrapped in plastic wrap or 14
placed in a hybridization bag and visualized using a Typhoon 9410 variable mode imager (GE 15
Healthcare, Piscataway, NJ), in the fluorescence mode. Alternatively, colony blots hybridized 16
with Cy3- or fluorescein-labeled Vchomim1276 were visualized with a Dark Reader hand lamp 17
(Clare Chemical; Dolores, CO). 18
Estimation of sensitivity and specificity of RNA colony blot hybridization. The 19
sensitivity and specificity of the Vchomim1276 probe, under conditions employed for RNA 20
colony blot hybridization, were estimated using colony blots containing pure cultures of V. 21
cholerae and sister species. According to their statistical definition (1, 20), sensitivity was 22
determined using the equation, Sensitivity = # true positives / (# true positives + # false 23
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negatives). Specificity was determined using the equation, Specificity = # true negatives / (# true 1
negatives + # false positives). Extensive testing was also previously performed for the 2
specificity of probe Vchomim1276 (4). 3
Spiking experiments. For evaluation of sensitivity and specificity of the overall 4
hybridization assay, a freshwater sample with known abundance of V. cholerae was employed. 5
Aliquots from fresh (Lake Artemesia, MD) water samples were amended with luminescent V. 6
cholerae non-O1/non-O139 strain CB99-18 that had been isolated from Chesapeake Bay, MD 7
(15). Appropriate dilutions were made to obtain approximately 150 total colonies, including 8
approximately 40 V. cholerae colonies, per spread plate using heterotrophic plate count agar or 9
LB-20 agar. Colony lift and RNA colony blot hybridization were performed as described above, 10
and prevalence of Vchomim1276-positive colonies was compared with expected prevalence of 11
the amended culture, based on luminescence of the V. cholerae CB99-18 strain. Thus, we were 12
able to confirm that the lake water did not contain luminescent bacteria. 13
Copepod microcosm. Microcosms containing copepods collected from Baltimore Harbor, 14
MD during winter months were incubated with cells of V. cholerae CB99-18 at room 15
temperature for 18 hr. The copepods in the microcosms were collected using a nylon mesh (64 16
μm) plankton net, washed to remove unattached bacteria, homogenized, and plated for 17
enumeration of attached V. cholerae. Plates were incubated at room temperature for 1-2 days, 18
after which RNA colony blots were prepared using Cy3-labeled Vchomim1276. 19
Field trial. The probe, Vchomim1276, and the RNA colony blot hybridization protocol 20
were employed to enumerate V. cholerae from water samples collected from a cholera-endemic 21
area, Mathbaria, in Bangladesh. It is located adjacent to the Bay of Bengal, and approximately 22
400 km southwest of Dhaka, the capital city of Bangladesh. In this study, samples from 23
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Mathbaria were collected biweekly from July, 2006 to March, 2007 from six man-made ponds 1
that are heavily used as a source for drinking water and for other domestic purposes. A major 2
river, Baleshwar, flows along the western boundary of Mathbaria, the other side of which is the 3
tropical mangrove forest of the ‘Sundarbans’. RNA colony blot lifts from L agar spread plates of 4
unconcentrated water samples were prepared and hybridized using the probe, Vchomim1276. A 5
total of 108 samples were tested. In parallel, colony blots from the same water samples, using 6
the same media, were assayed for culturable toxigenic V. cholerae using an alkaline phosphatase 7
labeled DNA probe, as described previously (24). Total bacteria were determined for each 8
sample using the fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI), and epifluorescent 9
microscopy (18). 10
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Results 2
Development of the RNA colony blot hybridization method. In developing the RNA 3
colony blot hybridization method specific for V. cholerae, four methods of colony blot lift, two 4
targeting DNA (GE Osmonics, membrane manufacturer) (24) and two targeting RNA (10, 12) 5
were evaluated. To assess the amount of nucleic acid immobilized onto membranes using each 6
method, the blots were stained with methylene blue (Fig. 1A). All four methods were effective 7
in liberating, denaturing, and immobilizing nucleic acid to the nylon membrane. The nylon 8
membranes were then cut into quarters and hybridized with Cy3-labeled Vchomim1276 probe. 9
The method of Ivanov and Gigova (10), targeting RNAs on the blot, proved far superior to the 10
other three methods in terms of hybridization signal (Fig. 1B). A modified method was 11
developed, which included pre-hybridization washing and the use of DEPC-treated solutions, 12
and the full method is presented in the Materials and Methods section. 13
Evaluation of RNA colony blot hybridization. The probe Vchomim1276, applied in the 14
RNA colony blot hybridization, was evaluated for accuracy in detecting colonies of the targeted 15
species. For qualitative variables, such as species identification, accuracy of the probe can be 16
evaluated in terms of sensitivity ( proportion of positives correctly identified by the test) and 17
specificity (proportion of negatives correctly identified by the test). According to the results of 18
application of the probe in RNA-colony lift hybridization (Table 1), the probe had 100% 19
sensitivity. However, specificity was 90% because of a weak positive signal produced by G.. 20
hollisae (21), under the conditions used in the RNA colony blot hybridization (Fig. 2). Sequence 21
comparison on the probe target site revealed that the cross reactivity is due to 16S rRNA 22
sequence similarity in the target site rather than non-specific binding of the probe to other 23
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cellular components. The cross-reaction is easily distinguished from that of V. cholerae or V. 1
mimicus (Fig. 2) by comparing the intensity of the signals. Signal intensity statistics can be used 2
to develop separation of the weak false reactivity displayed by G. hollisae, i.e., >25% of the 3
signal strength of a positive control signal, to resolve false positives. 4
To evaluate accuracy of the overall assay of the RNA colony blot hybridization protocol, 5
experiments were performed whereby natural water samples were amended with laboratory 6
cultures of V. cholerae. The RNA colony blot method was able to identify and enumerate V. 7
cholerae from two different types of environmental samples. Figure 3A is a digital fluorescence 8
image of a colony blot in which luminescent V. cholerae CB99-18 was added to Lake Artemesia, 9
MD pond water and spread plated, using 10-fold serial dilutions. A total of 58 V. cholerae 10
colonies were enumerated on this membrane by the probe. A highly luminescent strain of V. 11
cholerae, CB99-18, was used as the surrogate to validate blot results. Plate counts done prior to 12
RNA colony blot lift and hybridization, yielded a total of 187 colonies, 58 of which were 13
luminescent, indicating that the probe hybridization method detected V. cholerae CB99-18 cells 14
without false positives or false negatives, yielding 100% sensitivity and 100% specificity for 15
aquatic bacteria in the lake water when G. hollisae is absent. 16
Additionally, the RNA colony blot hybridization protocol was used to identify V. 17
cholerae isolates from a complex community of surface attached bacteria on copepods (Fig. 3B). 18
These experiments indicate that individual copepods can carry 1 – 5 × 104 cells of V. cholerae, 19
with an attachment rate of 0.3 -0.05% after overnight co-cultivation. As expected, the negative 20
control, copepods without V. cholerae added to the microcosm, did not produce culturable V. 21
cholerae signals by the RNA colony blot hybridization protocol since the copepods were 22
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collected during a time when the species is most likely in a viable but non-culturable (VBNC) 1
state. 2
Application to population dynamics of V. cholerae. To assess the practicality of the RNA 3
colony blot lift and hybridization method, water samples collected from a cholera endemic area 4
were tested. Fig. 4 shows results for two of the positive blots prepared using water collected 5
from Mathbaria province, Bangladesh (site 1 and site 2). Survey of V. cholerae in freshwater 6
ponds in Bangladesh over a nine month period demonstrated an abundance of planktonic cells, as 7
high as 104 cfu ml
-1, 1-2 months prior to an environmental peak in pathogenic V. cholerae 8
isolation and typical seasonal epidemics (Oct-Nov and April-May) (Fig. 5). In fact, the trend is a 9
reduction in the number of V. cholerae in the water immediately prior to the onset of an outbreak 10
suggesting that the cases may have a longer than expected incubation period. In contrast to this 11
trend, the total bacterial abundance, given by DAPI total direct count, remains relatively constant 12
at approximately 106 cells/ml. Interestingly, at the timepoints during which typical seasonal 13
epidemics occur, the number of ctxA-positive colonies are higher than the species-positive 14
colonies, suggesting that either free ctx phage can be detected or that ctx phage has infected other 15
species. At all other times, the number of ctxA-positive colonies is less than the number of V. 16
cholerae species colonies, as expected. 17
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Discussion 1
In this study, we developed an RNA colony blot hybridization method using a 2
fluorescently labeled oligonucleotide probe specific for V. cholerae and V. mimicus (4). A 3
simple RNA colony blot method, originally reported in 1986 (10), proved superior to others 4
tested and was modified for use in this study. A commercially available fluorochrome label was 5
preferred over other labels because of laboratory personnel safety, operational regulations, 6
availability, and/or economic reasons. By targeting RNA, instead of DNA, the fluorochrome 7
signal was boosted sufficiently for reliable detection. The probe is unable to resolve V. cholerae 8
from V. mimicus, since the 16S rRNA sequences from V. cholerae and V. mimicus differ by only 9
~6 of 1456 nucleotides (19). However, inclusion of V. mimicus in the V. cholerae RNA colony 10
blot hybridization method was deemed acceptable, since V. mimicus has been proposed to be a 11
reservoir for CTX Φ (2). Also, these two species can be separated, when necessary, by using 12
TCBS agar for the spread plates, since V. mimicus typically produces green, sucrose non-13
fermenting colonies and V. cholerae yellow, sucrose-fermenting colonies. 14
Results from experiments, in which V. cholerae was added to samples and in field trials, 15
demonstrate that the RNA colony blot hybridization method can be used to detect and enumerate 16
V. cholerae in different types of environmental water samples. For samples containing or 17
expected to contain high numbers of V. cholerae, colony blot lifts from spread plates are 18
satisfactory for enumeration of V. cholerae. In this case, there is no need for an enrichment step 19
or use of selective media. The method can be adapted based on expected density of cells of V. 20
cholerae. For samples in which the expected density of V. cholerae is low, such as water 21
samples collected during winter months, filtration can be used to concentrate samples directly 22
onto membranes. In this case, several sample volumes should be filtered to yield well separated 23
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colonies of sufficient size to give a reliable signal (~2-3 mm in diameter). Larger membranes 1
(137 mm) can be used to sample even larger volumes of water (100 to 200-ml), when the number 2
of V. cholerae cells is extremely low. When filtration is used, a selective medium such as TCBS 3
should be used to inhibit growth of competitor cells/colonies. 4
The method can also be adapted for isolation of the organism. If the colony blot lift is 5
made from a spread-plate, the master plate can be stored at 15°C, until the hybridization is 6
complete, at which time the positive signals can be correlated to colonies and subsequently 7
subcultured. If the colony blot is made by filtering a water sample directly onto the membrane 8
(and then overlaying onto an agar plate), subsequent growth on the membrane can be replica-9
plated onto a fresh agar plate prior to colony lysis. Again, positive signals can be correlated with 10
colonies, subcultured and confirmed. For this purpose, this method is preferred to traditional 11
enrichment/selective medium isolation methods, because the necessity for extensive and 12
laborious biochemical tests can be avoided. 13
14
Acknowledgement: This research was supported by the NIH research grant 1RO1A13912901 15
under collaborative agreements between the Johns Hopkins Bloomberg School of Public Health 16
and the ICDDR,B. Dr. C. Grim was supported by the IC Postdoctoral Fellowship Program 17
(NGA grant# HM15820612010). Young-Gun Zo was supported by the 2nd phase Brain Korea 18
21 Project in 2008. 19
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References 1
1. Altman, D. G., and J. M. Bland. 1994. Statistical notes: diagnostic tests 1: sensitivity 2
and specificity. BMJ 308. 3
2. Boyd, E. F., K. E. Moyer, L. Shi, and M. K. Waldor. 2000. Infectious CTXΦ and the 4
vibrio pathogenicity island prophage in Vibrio mimicus: evidence for recent horizontal 5
transfer between V. mimicus and V. cholerae. Infection and Immunity 68:1507-1513. 6
3. Goel, A. K., S. Ponmariappan, D. V. Kamboj, and L. Singh. 2007. Single multiplex 7
polymerase chain reaction for environmental surveillance of toxigenic-pathogenic O1 and 8
non-O1 Vibrio cholerae. Folia Microbiol (Praha) 52:81-5. 9
4. Heidelberg, J. 1997. Seasonal abundance of bacterioplankton populations of Bacteria, 10
gamma proteobacteria, Vibrio/Photobacterium, Vibrio vulnificus, Vibrio cholerae/Vibrio 11
mimicus, and Vibrio cincinnatiensis associated with zooplankton in the Choptank River, 12
Maryland. Ph.D. thesis. University of Maryland College Park. 13
5. Heidelberg, J., K. Heidelberg, and R. Colwell. 2002. Bacteria of the gamma-subclass 14
Proteobacteria associated with zooplankton in Chesapeake Bay. Appl Environ Microbiol 15
68:5498-5507. 16
6. Heidelberg, J., K. Heidelberg, and R. Colwell. 2002. Seasonality of Chesapeake Bay 17
bacterioplankton species. Appl Environ Microbiol 68:5488-5497. 18
7. Heidelberg, J. F., K. B. Heidelberg, and R. R. Colwell. 2002. Bacteria of the gamma-19
subclass Proteobacteria associated with zooplankton in Chesapeake Bay. Appl Environ 20
Microbiol 68:5498-507. 21
8. Heidelberg, J. F., K. B. Heidelberg, and R. R. Colwell. 2002. Seasonality of 22
Chesapeake Bay bacterioplankton species. Appl Environ Microbiol 68:5488-97. 23
on May 26, 2018 by guest
http://aem.asm
.org/D
ownloaded from
15
9. Huq, A., Grim, C., Colwell, R.R., and Nair, G.B. 2006. Detection, isolation, and 1
identification of Vibrio cholerae from the environment. In R. Coico, Kowalik, T., 2
Quarles, J., Stevenson, B., Taylor, R. (ed.), Current Protocols in Microbiology. John 3
Wiley & Sons, Inc. 4
10. Ivanov, I., and L. Gigova. 1986. RNA colony hybridization method. Gene 46:287-90. 5
11. Kaper, J. B., J. G. Morris, Jr., and M. M. Levine. 1995. Cholera. Clin Microbiol Rev 6
8:48-86. 7
12. Kaufmann, P., A. Pfefferkorn, M. Teuber, and L. Meile. 1997. Identification and 8
quantification of Bifidobacterium species isolated from food with genus-specific 16S 9
rRNA-targeted probes by colony hybridization and PCR. Appl Environ Microbiol 10
63:1268-73. 11
13. Khuntia, H. K., B. B. Pal, and G. P. Chhotray. 2008. Quadriplex PCR for 12
simultaneous detection of serotype (O1 and/or O139), biotype, toxigenic potential and 13
Central regulating factor of V. cholerae. J Clin Microbiol. 14
14. Koch, W. H., W. L. Payne, B. A. Wentz, and T. A. Cebula. 1993. Rapid polymerase 15
chain reaction method for detection of Vibrio cholerae in foods. Appl Environ Microbiol 16
59:556-60. 17
15. Louis, V. R., E. Russek-Cohen, N. Choopun, I. N. Rivera, B. Gangle, S. C. Jiang, A. 18
Rubin, J. A. Patz, A. Huq, and R. R. Colwell. 2003. Predictability of Vibrio cholerae in 19
Chesapeake Bay. Appl Environ Microbiol 69:2773-85. 20
16. Mantri, C. K., S. S. Mohapatra, T. Ramamurthy, R. Ghosh, R. R. Colwell, and D. V. 21
Singh. 2006. Septaplex PCR assay for rapid identification of Vibrio cholerae including 22
detection of virulence and int SXT genes. FEMS Microbiol Lett 265:208-14. 23
on May 26, 2018 by guest
http://aem.asm
.org/D
ownloaded from
16
17. Mendes, C. L., F. G. Abath, and N. C. Leal. 2008. Development of a multiplex single-1
tube nested PCR (MSTNPCR) assay for Vibrio cholerae O1 detection. J Microbiol 2
Methods 72:191-6. 3
18. Porter, K. G., and Y. S. Feig. 1980. The use of DAPI for identifying and counting 4
aquatic microflora. Limnol. and Oceanog. 25:943-948. 5
19. Ruimy, R., V. Breittmayer, P. Elbaze, B. Lafay, O. Boussemart, M. Gauthier, and R. 6
Christen. 1994. Phylogenetic analysis and assessment of the genera Vibrio, 7
photobacterium, Aeromonas and Plesiomonas deduced from small subunit rRna 8
sequencer. Int. J. Syst. Bact. 44:416-426. 9
20. Sokal, R. R., and F. J. Rohlf. 1995. Biometry: the principles and practices of statistics in 10
biological research, 3rd ed. W.H. Freeman and Co., New York, NY. 11
21. Thompson, F. L., B. Hoste, K. Vandemeulebroecke, and J. Swings. 2003. 12
Reclassification of Vibrio hollisae as Grimontia hollisae gen. nov., comb. nov. Int J Syst 13
Evol Microbiol 53:1615-1617. 14
22. Waldor, M. K., and J. J. Mekalanos. 1996. Lysogenic conversion by a filamentous 15
phage encoding cholera toxin. Science 272:1910-1914. 16
23. Wright, A., Y. Guo, J. Johnson, J. Nataro, and J. J. Morris. 1992. Development and 17
testing of a nonradioactive DNA oligonicleotide probe that is specific for Vibrio cholerae 18
cholera toxin. J Clin Microbiol 30:2302-2306. 19
24. Wright, A. C., Y. Guo, J. A. Johnson, J. P. Nataro, and J. G. Morris, Jr. 1992. 20
Development and testing of a nonradioactive DNA oligonucleotide probe that is specific 21
for Vibrio cholerae cholera toxin. J Clin Microbiol 30:2302-6. 22
on May 26, 2018 by guest
http://aem.asm
.org/D
ownloaded from
17
25. Zo, Y. G., and R. R. Colwell. 2008. A simple binomial test for estimating sequencing 1
errors in public repository 16S rRNA sequences. J Microbiol Methods 72:166-79. 2
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Figure Legends: 23
24
Figure 1 A. Methylene blue staining of RNA colony blot lift on nylon membranes prepared 25
using spread plates of V. cholerae CB99-18 on LB-20 agar. B. Digital fluorescence image of 26
colony blot from A, cut into quarters (for optimization experiments) and hybridized with theCy3-27
labeled probe, Vchomim1276. 28
Figure 2. A. schematic representation and B. Digital fluorescence image of RNA colony blot 29
hybridization with Cy3-labeled Vchomim1276 to determine specificity of probe. 30
Figure 3. Digital fluorescence images of RNA colony blots, to which V. cholerae CB99-18 had 31
been added, hybridized with Cy3-labeled probe Vchomim1276. A = pond water, Lake Artemisia, 32
MD, and B = copepod microcosm. 33
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Figure 4. Representative RNA colony blots hybridized with probe Vchomim1276. Blots were 1
prepared from spread plates of 100µL of water and/or dilution. Blots shown were prepared using 2
100µL water sample from pond 3, July, 2006, Round # 60 (A) and pond 1, Jan, 2007, Round#73 3
(B) Mathbaria, Bangladesh. 4
Figure 5. Seasonality and abundance of pathogenic V. cholerae determined by conventional 5
culture (solid line) and molecular methods (targeting ctxA) and total V. cholerae by RNA colony 6
blot, compared to total bacterial counts (DAPI) in coastal areas of Mathbaria, Bangladesh. 7
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Table 1. Bacterial strains used to evaluate the RNA colony blot hybridization method employing 1
probe Vchomim1276. 2
Strain Hybridization
V. cholerae O1 classical ATCC14035 +
V. cholerae O1 classical ATCC11623 +
V. cholerae O139 AI1877 +
V. cholerae O139 EM-0208 +
V. cholerae O1 El Tor N16961 +
V. cholerae O1 El Tor EB-0184 +
V. cholerae non-O1/non-O139 CB98-203 +
V. cholerae non-O1/non-O139 CB99-18 +
V. cholerae non-O1/non-O139 EC1 +
V. cholerae non-O1/non-O139 UM4089 +
V. cholerae non-O1/non-O139 TMA21 +
V. cholerae non-O1/non-O139 EB-0172 +
V. cholerae non-O1/non-O139 EM-0232 +
V. mimicus ATCC33563 +
V. mimicus UM4198 +
Aeromonas caviae ATCC15468 -
Escherichia coli K12 -
V. salmonicida ATCC43839 -
V. vulnificus ATCC27562 -
V. orientalis ATCC33934 -
V. splendidus ATCC33125 -
V. furnissii ATCC35016 -
V. anguillarum ATCC19264 -
Grimontia (Vibrio) hollisae ATCC33564 -*
V. fluvialis CB99-14 -
*weak cross-reactivity; V. hollisae was reclassified as Grimontia hollisae by Thompsion et al. 3
(21) based on 16S rRNA-based genetic distance from representative Vibrio species. 4
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1
Figure 1. A. Methylene blue staining of RNA colony blot lift on nylon membranes prepared 2
using spread plates of V. cholerae CB99-18 on LB-20 agar. B. Digital fluorescence image of 3
colony blot from A, cut into quarters (for optimization experiments) and hybridized with theCy3-4
labeled probe, Vchomim1276. Inserted line is for orientation purposes only. 5
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1
Figure 2. A. schematic representation and B. Digital fluorescence image of RNA colony blot 2
hybridization with Cy3-labeled Vchomim1276 to determine specificity of probe. A, clinical V. 3
cholerae O1; B, environmental V. cholerae O1; C, clinical V. mimicus; D, environmental V. 4
mimicus; E, V. anguillarum; F, V. fluvialis; G, G. hollisae; H, V. vulnificus; I, Aeromonas caviae; 5
J, E. coli. 6
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A B
1
Figure 3. Digital fluorescence images of RNA colony blots, to which V. cholerae CB99-18 had 2
been added, hybridized with Cy3-labeled probe Vchomim1276. A, pond water, Lake Artemisia, 3
MD; and B, copepod microcosm. 4
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1
Figure 4. Representative RNA colony blots hybridized with probe Vchomim1276. Blots were 2
prepared from spread plates of 100µL of water and/or dilution. Blots shown were prepared using 3
100µL water sample from A, pond 3, July, 2006, Round # 60; and B, pond 1, Jan, 2007, 4
Round#73; Mathbaria, Bangladesh. 5
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1
2
3
Figure 5. Seasonality and abundance of pathogenic V. cholerae determined by conventional 4
culture (solid line) and molecular methods (targeting ctxA) and culturable V. cholerae by RNA 5
colony blot, compared to total bacterial counts (DAPI) in coastal areas of Mathbaria, 6
Bangladesh. 7
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