Marine Pollution Bulletin 79 (2014) 354–358

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Marine Pollution Bulletin

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Detection of Helicobacter pylori in the coastal waters of Georgia, PuertoRico and Trinidad

0025-326X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.marpolbul.2013.11.021

⇑ Corresponding author. Tel.: +1 478 445 0812; fax: +1 478 445 5290.E-mail address: Dave.bachoon@gcsu.edu (D.S. Bachoon).

Chelsea B. Holman a, D.S. Bachoon a,⇑, Ernesto Otero b, Adesh Ramsubhag c

a Department of Biological and Environmental Sciences, Georgia College and State University, Campus Box 81, Milledgeville, GA 31061-0490, USAb Department of Marine Sciences, University of Puerto Rico, Mayaguez Campus, P.O. Box 9000, Mayaguez 00681, Puerto Ricoc Department of Life Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobago

a r t i c l e i n f o

Keywords:Helicobacter pyloriFecal pollutionCaribbeanPathogensMarine pollution

a b s t r a c t

Fecal pollution in the coastal marine environments was assessed at eleven sampling locations along theGeorgia coast and Trinidad, and nine sites from Puerto-Rico. Membrane filtration (EPA method 1604 andmethod 1600) was utilized for Escherichia coli and enterococci enumeration at each location. Quantitativepolymerase chain reaction (qPCR) amplification of the 16S ribosomal RNA gene was used to determinethe presence of the Helicobacter pylori in marine samples. There was no significant correlation betweenthe levels of E. coli, enterococci and H. pylori in these water samples. H. pylori was detected at four ofthe 31 locations sampled; Oak Grove Island and Village Creek Landing in Georgia, Maracas river in Trin-idad, and Ceiba Creek in Puerto Rico. The study confirms the potential public health risk to humans due tothe widespread distribution of H. pylori in subtropical and tropical costal marine waters.

� 2013 Elsevier Ltd. All rights reserved.

Fecal contamination of recreational water can have a detrimen-tal effect on the environment, ecology and economy of a region andincrease the risk for transmission of human pathogens (Staleyet al., 2012; Savichtcheva and Okabe 2006). Fecal indicator bacteria(FIB), such as Escherichia coli and Enterococcus spp. are often usedas a proxy to predict the presence of human pathogens in water-bodies (Savichtcheva and Okabe 2006). The acceptable levels ofthese fecal indicators have been determined from epidemiologicalstudies (USEPA, 1986). Recent studies have shown unacceptablyhigh levels of fecal pollution in several coastal regions of theGeorgia, Puerto-Rico and Trinidad impacted by human fecal pollu-tion (Bachoon et al., 2010). Therefore, it is likely that these marinewaters containing human fecal pollution may harbor pathogenicmicroorganisms. However the dangers of pathogenic bacteria inmarine waters of the Caribbean Islands is currently unknown.

A common human pathogen is Helicobacter pylori which infectsmore than 50% of the world’s population and as much as 70% of thepopulation in some developing countries (Kim et al., 2011; Janzonet al., 2009; Giao et al., 2008). This gram-negative spiral shapedbacterium commonly infects the gastric mucosa of humans(Vale and Vitor 2010). The vast majority of H. pylori infectionsare asymptomatic and only a small percentage of cases progressto cancer. However, it is estimated that 70% of gastric cancers aredirectly caused by this organism (Kim et al., 2011). It is suspectedthat H. pylori is transmitted person to person through

contaminated food or water (Vale and Vitor 2010; Janzon et al.,2009; Giao et al., 2008; Sen et al., 2007; Gomes and De Martinis,2004). Several studies have indicated that drinking fecal contami-nated water might aid the transmission of H. pylori (Ghosh andBodhankar 2012; Vale and Vitor 2010; Linke et al., 2010) andaquatic environments may be a potential natural reservoir forthese bacteria (Twing et al., 2011; Sen et al., 2007).

Traditionally, H. pylori was difficult to detect in natural watersamples by culture-based methods. Today, there are numerousPCR-based methods available for detecting the presence of H. pyloriin water samples that have used to demonstrate the widespreadpresence of this bacterium in marine water from Italy andDelaware, as well as freshwater samples from the United States,Mexico, Japan, England, Sweden, and Germany (Twing et al.,2011; Agustí et al., 2010; Sen et al., 2007; Carbone et al., 2005;Gomes and De Martinis, 2004; Maxzari-Hiriart et al., 2001; Mackayet al., 1998; Hulten et al., 1996). However it appears that the detec-tion of this bacterium in water samples does not always correlatewith high levels of fecal indicator bacteria (E. coli or enterococci) orthe presence of human fecal bacteria (Twing et al., 2011; Gomesand De Martinis, 2004). This can be very problematic for publichealth officials that often attempt to predict the safety of water-bodies based on fecal indicator bacteria levels. Additional studiesare needed to assess the distribution and relationship of H. pylorito common fecal indicator bacteria levels in other marine systemsover broader geographical scales.

In this study, H. pylori 16S ribosomal RNA gene fragment wasused as a molecular marker for detection of the bacterium in

C.B. Holman et al. / Marine Pollution Bulletin 79 (2014) 354–358 355

quantitative polymerase chain analysis assay (Kobayashi et al.,2002). The aim of this study was to determine the presence of H.pylori in marine water samples from Georgia, Puerto Rico and Trin-idad in addition to determining whether traditional FIB enumera-tion can be used to predict the presence of H. pylori insubtropical to tropical marine waters.

Between 2009 and 2010 water samples were collected in tripli-cate in sterile whirlpack bags or polypropylene bottles from 31coastal sites from U.S. Georgia coast, Puerto Rico and Trinidad(Fig. 1) kept on ice and processed within 6 h. Sampling sites wereclassified as Urban/Suburban and Rural based on arbitrary knowl-edge of existing population density.

The samples were filtered using USEPA method 1604 (USEPA2000) and USEPA method 1600 (USEPA 2000). Triplicates of100 ml volume of water from each sample site were filteredthrough a 0.45-lm GN-6 gridded membrane filter (Pall Corpora-tion, Ann Arbor, MI) and then incubated on MI plates of the fluoro-gen, 4-methylumbelliferyl-b-D-galactopyranoside (MUGal), andchromogen, indoxyl-b-D-glucuronide (IBDG), at 35 ± 0.5 �C for24 h and on m-EI plates, membrane-enterococcus indoxyl-b-D-glu-coside agar at 41 ± 0.5 �C for 24 h. The MI plates were displayedunder ultra-violet light (366 nm), the fluorescent colonies werecounted, and the data was recorded as colony forming units(CFU) per 100 ml (Zimbro and Power 2003).

Fig. 1. Sampling locations in Georgia, Puerto Rico and Trinidad. Sampled locati

Water samples were filtered through, 0.22-lm-pore nitrocellu-lose membrane filter (Type GS, Millipore, Billerica, MA, USA). Thefilters were frozen and shipped on dry ice by overnight courier toMilledgeville, GA. Filters were processed with the MoBio Ultra-clean™ Soil DNA Kit (Carlsbad, CA, USA) using a modified protocol(Bachoon et al., 2010; Sherchan and Bachoon 2011). Extracted DNAwas quantified using a nanodrop ND-1000 spectrophotometer(Wilmington, DE).

H. pylori genomic DNA from ATCC� 700392D-5 was used as po-sitive controls in 16S ribosomal RNA gene qPCR assays. The prim-ers used were HP-F, 50-CTCATTGCGAAGGCGACCT-30, and HP-R,50-TCTAATCCTGTTTGCTCCCCA-30 (Kobayashi et al., 2002) and pro-duced a amplicon of 85-bp. The specificity of the qPCR assay wasoptimized against common fecal bacteria (E. coli strain B genomicDNA Sigma� D4889, Enterococcus faecalis ATCC 29212, Bacteroi-des dorei DSM 17855 (DSMZ, Germany) and Bifidobacteriumadolescentis genomic DNA ATCC� number 15703D™). After which,we used B. adolescentis genomic DNA ATCC� number 15703D™was used as a negative control. DNA extracts were amplified usingthe Bio-Rad CFX96 (Hercules, CA). Samples were run using opti-mized qPCR assays. Each qPCR contained a 25 ll volume with12.5 ll of iQ™ SYBR� Green 2x Supermix, 12 ll deionized H2O,250 nM of primer, 1 ll (�10 ng) of template DNA and 0.04 ll ofmagnesium under the following conditions: initial denaturing at

ons are referenced in Tables 1–3 respectively. Horizontal scale bar = 2 km.

356 C.B. Holman et al. / Marine Pollution Bulletin 79 (2014) 354–358

95 �C for 3 min; 40 cycles of 95 �C for 10 s, and 67 �C for 20 s. Serialdilutions of H. pylori DNA (ATCC� 700392D-5) was used to gener-ate all standard curves from 4.81 � 106 gene copies to 4.81 � 101

gene copies. The lower limit of detection was 25 gene copies. Thepresence of PCR inhibition was evaluated using exogenous Salmontestis DNA from Oncorhynchus keta (Sigmaò, St. Louis, MO) as aninternal control. The salmon testes DNA was diluted in sterile dis-tilled H2O to create a standard curve ranging from 0.0007 to 7 ng ofDNA and detected by qPCR in the DNA extracts (Morrison et al.,2008). Changes of less than two CT value were observed, whichindicates that the extracted DNA did not contain impurities thatsignificantly inhibited the PCR (Morrison et al., 2008; Bachoonet al., 2010). Pearson and Spearman regression analysis were con-ducted (StatistiXL) to evaluate covariation of E. coli counts and H.pylori 16S RNA gene copy number.

It is well documented that the coastal marine environment inthe U.S. and the Caribbean region is impacted with elevated levelsof fecal indicator bacteria such as enterococci and E. coli that oftencan be traced to human sources (Bachoon et al., 2010; Morrisonet al., 2008; King et al., 2007). E. coli was enumerated using MIplates as a measure of fecal pollution in marine environments fromGeorgia and the Caribbean. While there is no standard for E. colienumeration in marine water, the statistical threshold value(STV) for recreational use in freshwater samples ranges from 320to 410 CFU 100 ml�1 depending on potential gastrointestinal ill-ness rate per thousand individuals and the STV for enterococci inmarine waters is 110–130 CFU 100 ml�1 (USEPA 2012). MI platecounts indicated that none of the sites in Georgia exceeded theSTV value for E. coli or enterococci (Table 1). In contrast, Table 2indicates that all the sampled locations from Puerto Rico containedE. coli concentrations higher than then STV value. The highest levelof E. coli contamination was detected at the fishers association andcontained approximately 42 times greater levels than the EPAstandard and Ceiba Creek contained approximately 18 times great-er levels of E. coli than the EPA standards for freshwater (USEPA

Table 2Enumeration of E. coli and detection of H. pylori from marine sites in Puerto Rico.

Site number Classification * Site location

1 SU Boquilla2 SU Oro Creek3 U Yaguez river4 U Fishers association5 U Ceiba Creek6 U Sabalos Creek7 R Guanajibo river8 SU Joyuda lagoon9 SU Boquerón wetland cre

ND, not detected; SU, sub-urban; U, urban; R, rural.

Table 1Enumeration of E. coli and detection of H. pylori from marine sites along the Georgia coas

Site Classification* Location

1 R Oak grove island (2010)2 R Blythe island Regional park (2010)3 R Jekyll island Warf (2010)4 R Village Creek Landing (2010)5 R Dunbar Creek SS1 sea island road bridge (2010)6 R Julienton river A (2009)7 R Julienton river B (2009)9 R Julienton river C (2009)8 R Mud river A (2009)

10 R Mud river B (2009)11 R Shell Creek (2009)

ND, not detected; SU, sub-urban; U, urban; R, rural.

2012; USEPA 2004). E. coli and enterococci enumeration in Trinidadsamples indicated that two locations, Carli and Balandra Bays, ex-ceeded the EPA standards (Table 3). It is often assumed by regula-tory agencies that high levels of fecal bacteria such as E. coli andenterococci can act as a reliable indicator for the presence of path-ogenic bacteria in an environment; unfortunately this assumptionmay not always be valid (Twing et al., 2011; Voytek et al., 2005).E. coli and enterococci are common bacteria of the human gutbut they also common in the gut of other warm-blooded mammals,therefore detection of FIB alone does not necessarily indicate hu-man fecal pollution (Staley et al., 2012).

A qPCR assay that was reported to detect down to ten 16S rRNAgene copies of H. pylori (Kobayashi et al., 2002) was used and de-tected the presence of this bacterium in water samples from allof the regions sampled (Table 3). In rural Georgia locations, OakGrove Island and Village Creek Landing which had modest levelsof E. coli (�135 cfu/100 ml) and enterococci 81 CFU 100 ml�1 con-tained an average of approximately 2.41 � 102 gene copies H. pyloribut in Shell Creek that had higher levels of E. coli contamination H.pylori was undetectable (Table 1). Based on USEPA, standards forE. coli and enterococci in recreational water use all of the sites inGeorgia can be considered safe for primary contact (USEPA2011). Samples were collected from areas in Puerto Rico knownto have had exceedingly high levels of fecal indicator bacteria thatmaybe from human or bovine sources (Bonkosky et al., 2008;Bachoon et al., 2010; Sherchan et al., 2013). H. pylori was only de-tected at Ceiba Creek and not at the Fishers Association site thathad twice as much E. coli counts (Table 2). In Trinidad, Maracasriver was the only location that was positive for the presence ofH. pylori with a mean of 2.41 � 101 gene copies per 100 ml. How-ever, H. pylori was not detected (detection limit 40 gene copiesper 100 ml) in two sites which exceeded the EPA standard forE. coli in recreational water; Carli Bay and Balandra Bay (Table 3).This was surprising because a previous study had shown that CarliBay was impacted by human fecal pollution (Bachoon et al., 2010)

Mean MI plate(CFU 100 ml�1)

Mean 16S rRNA gene copynumber/100 ml

2594 ND4737 ND1804 ND9924 ND4229 264.03034 ND4028 ND1551 ND

ek 385 ND

t.

Mean MI plate(CFU 100 ml�1)

Mean m-EI plate(CFU 100 ml�1)

Mean 16S rRNA gene copynumber/100 ml

143 81 240.5196 87 ND

86 24 ND136 31 240.5

35 18 ND44 10 ND82 16 ND48 30 ND12 8 ND41 3 ND

204 78 ND

Table 3Enumeration of E. coli and detection of H. pylori from marine sites in Trinidad.

Site number Classification* Site location Mean MI plate (CFU 100 ml�1) Mean m-EI plate (CFU 100 ml�1) Mean 16S rRNA gene copy number/100 ml

1 R Maracas river 198 166 24.12 R Maracas bay 191 121 ND3 SU Chaguaruamas bay 184 9 ND4 R Las cuevas 162 15 ND5 R Carli bay 1680 129 ND6 R Quinam bay * 108 ND7 R Morne diablo bay * 36 ND8 R Toco bay 9 15 ND9 R Balandra bay 276 113 ND

10 R Salybia bay 56 147 ND11 R Salybia river 112 50 ND

ND, not detected; SU, sub-urban; U, urban; R, rural.*No data available.

C.B. Holman et al. / Marine Pollution Bulletin 79 (2014) 354–358 357

and it is well established that H. pylori is solely a human pathogen(Vale and Vitor 2010). Pearson regression analysis indicated a non-significant covariation between E. coli counts and qPCR results(r2 = 0.002; P = 0.803). Spearman regression was used due to thelarge proportion of negative PCR reactions and confirmed theabove results (rs = 0.286; P = 0.133). Similar observations of a lackof correlation between fecal indicator bacteria concentration andthe presence of H. pylori in estuarine and beach locations have beenreported (Twing et al., 2011). In contrast, there are studies thatindicate H. pylori does correlate to the extent of fecal contamina-tion (Queralt et al., 2005).

Studies indicate that inoculation of water with feces from indi-viduals infected with H. pylori may act as a means of person to per-son transmission for H. pylori infection (Vale and Vitor 2010;Janzon et al. 2009). The ability of H. pylori to change to VBNC formsalso allows the bacteria to persist in the environment for incre-ments longer than other fecal bacteria (Twing et al., 2011). Fortu-nately, the levels of H. pylori detected (240 gene copies) in all ofthe samples were considered low compared to the infectious dosefor H. pylori in human which can be 105 cells (Vale and Vitor 2010).Therefore, it is uncertain if the levels of H. pylori detected in Geor-gia, Puerto Rico and Trinidad are sufficient to transmit infection tohumans because all of the routes of transmission of H. pylori to hu-mans are still unknown and H. pylori eventually enter a coccoidnoninfectious state (Agustí et al. 2010). However further studieswould be necessary to determine the viability of cells detectedand the public health risk of possible transmission from such cells.

Acknowledgements

This project is supported by Georgia College and State Univer-sity and was funded by a research grant by the Puerto Rico SeaGrant Program (R-92-1-08).

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