1

Molecular characterization of mycobiota in four different

drinking water sources in Jeddah City (Saudi Arabia)

Rukaia M. Gashgari1, Hesham M. Elhariry

2 and Youssuf A. Gherbawy

*2,3

1Girls college, King Abdulaziz University, Jeddah , Saudi Arabia

2Biological sciences department, Faculty of science, Taif University, Taif, Saudi

Arabia

3Botany Department, Faculty of Science, South Valley University, Qena, Egypt

Running title (Drinking water mycobiota …..)

*The Corresponding Author

Prof. Dr. Youssuf A. Gherbawy

Biological sciences department,

Faculty of science,

Taif University,

Taif, P.O. Box 888

Saudi Arabia

Youssufgherbawy@yahoo.com

Tele 00966553993906

2

Abstract

The study included collection of water samples (150 samples) from different

sources of treated water (30 samples) and tap water in some hospitals (30 samples from

cold water tap and 30 samples from hot water tap) and private houses (60 samples)

belonging to each water network in Jeddah City (Saudi Arabia). From these sources, fungi

were isolated and identified using the traditional methods. Moreover, the molecular genetic

techniques including PCR amplification and sequencing of ITSs of unculturable fungi were

also considered. All of sequenced strains were identified by similarity searches with ITS

sequences in EMBL/GenBank. All strains that are defined in water samples of the

distribution stations and private homes have been defined by traditional methods and has

not been appreciated strains unable to grow (unculturable) on synthetic media.

Acremonium strictum was detected as unculturable strain in hospital cold water. In hospital

hot water, three fungal species have been identified by the traditional methods, which were

also defined using the methods of molecular diagnostics (partial sequencing of ITS gene).

In addition, 6 other species including Acremonium strictum, Cladosporium

cladosporioides, Rhizopus azygosporus, Penicillium citrinum, Penicillium oxalicum and

Trichoderma viride were identified using the molecular method. It can be concluded that,

drinking water in Jeddah city contain many different fungi species that do not affect the

quality of drinking water. However, some of these strains cause disease in humans,

produce mycotoxins, or may cause allergic diseases. The study also emphasized the need

for application of molecular diagnostic techniques as rapid and accurate methods for

detecting the presence of fungi and to ensure the quality and safety of the water.

Keywords: DGGE, Hospital, ITS, PCR

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Introduction

Limited attention has been given to the presence of fungi in the aquatic environment

(Standard Method 9610, 1995; Dogget, 2000; Arvanitidou et al., 2002; Hageskal et al., 2006;

Hageskal et al., 2007; Pereira et al., 2009). Further research is therefore needed in this area

since waterborne fungi could also be associated with a variety of health related effects, taste

and odour problems and contamination in food and beverages (Dogget, 2000). A study of

fungal diversity and significance in the two USA distribution systems has also recently been

conducted (Kelley et al., 2003), involving samples taken from several points throughout the

systems. This included an examination of the ability of selected fungi to grow in water and to

produce metabolites of concern to human health, and also looked at the effectiveness of

different control measures. Kinsey et al. (1999) have summarized many of the main

considerations in sampling fungi from water. Hageskal et al. (2006) reported a wide

occurrence of fungi in Norwegian drinking water and concluded that the mycobiota of water

should be considered when the microbiological safety and quality of drinking water are

assessed. Several studies have focused on analysing hospital water systems with respect to the

presence of fungi (Arvanitidou et al., 2000, Anaissie et al., 2001, 2003, Warris et al.,

2001,2003; Panagopoulou et al., 2002, Hapcioglu et al., 2005, Kanzler et al., 2007, Kennedy

and Williams, 2007, Pires-Gonçalves et al., 2008,). The results indicate that hospital and

private home water may contain a wide diversity of fungi, including potential pathogens. The

hypothesis is that fungi in water are aerosolized into air when water passes installations, such

as taps and showers, and thus introduced to severely immunocompromised patients with a

high risk of fungal infections. Several microorganisms can cause taste and odor problems in

drinking water, and such sensoric problems have been connected to the presence of fungi.

Several fungal species, such as, e.g. Chaetomium globosum (Kikuchi et al. 1981), have been

found to produce geosmin, a compound often associated with earthy odor and taste in

drinking water (Paterson et al. 2007). In addition, fungi may produce a variety of other

4

compounds with distinctive off-flavours and tastes (Ezeonu et al., 1994, Kaminski et al.,

1974, Mattheis and Roberts, 1992, Montiel et al., 1999 and Nyström et al., 1992).

With the continued development of genomic techniques, it is now possible to detect

and analyse fungal DNA in environmental samples (Bridge et al., 1998). These techniques

are based on the polymerase chain reaction (PCR) (Edel, 1998). A widely used approach is

to conduct PCR using oligonucleotide primers specific for the conserved flanking regions of

the internally transcriber spacers (ITS) of the fungal rRNA gene (White et al., 1990). The

ITS primers are regarded as universal for fungi because rRNA genes appear to be present in

most fungi (Gardes and Bruns, 1993). The technique has the potential for very sensitive

detection of fungi in environmental samples. However, with environmental samples there is

a need for caution as a range of factors can bias the DNA amplification, such as achieving

initial DNA extractions for only a subset of the total species present, the presence of

compounds inhibitory to amplification, obtaining primerprimer annealment, or the formation

of chimeric co-amplified sequences (Wang and Wang, 1997; Hastings, 1999; Head, 1999).

As PCR techniques continue to become more widely used, further development will

undoubtedly resolve many of these potential problems (Viaud et al., 2000). It is usual to

incorporate further analysis of the PCR product, and this essentially involves either

sequence analysis, restriction fragment analysis, or the use of taxon specific probes or

primers (Bridge et al., 1998). In many cases the techniques target specific fungal taxa but

some, although originally designed for bacteriological studies (examples in Hurst et al.,

1997; Edwards, 1999), are targeted much more broadly and are being adapted for analyzing

community structures in mixed fungal populations. Denaturing gradient gel electrophoresis

DGGE involves running PCR product (using ITS primers) through a gel containing a

gradient of denaturants; the sequence of the bases in the DNA determines at what point on

the gel’s gradient the strands denature and hinder further migration, so that on visualization

the resulting bands reflect the diversity of fungi present in the original sample. DGGE is

5

now beginning to be used for fungal populations (Kowalchuk et al., 1997; Vainio and

Hantula, 2000).

This study was designed to achieve the following objectives: Applying new rapid

molecular technique for detecting the fungal contaminants in water supply sources in Jeddah

city, recognize the ability of these modern procedures for detecting the unculturable fungi in

water sources.

Materials and Methods

Water samples

The sampling protocol of Hageskal et al (2006) was applied in this study. Briefly,

about 150 water samples were collected from 10 water supplies. Each water supply

was

sampled three times. Sample points were included treated water at the treatment plants and

hot- and cold-water taps in hospitals and private homes attached to

each supply network.

Isolation of fungi

The isolation procedure of Hageskal et al (2006) was used in the present study.

Briefly, 100-ml water samples were filtered through membrane filters. These filters were

incubated on dichloran-18% glycerol agar (Hocking and Pitt 1980) in darkness at 20°C ±

1°C for up to 2 weeks. The number of colonies was determined and expressed as the number

of CFU per 100-ml water sample.

The isolation frequency (percent) for each species was calculated

by dividing the

number of positive samples by the total number of samples.

Concentrations of each species were expressed as minimum and maximum numbers

of CFU per 100 ml.

For isolation of pure single colonies, positive cultures were subcultured

on potato

dextrose agar (Oxoid, Basingstoke, United Kingdom) or malt extract agar (Oxoid) and

incubated at 25°C ± 1°C for 7 days. For further work, representative isolates were stored on

potato dextrose agar slants at 4°C.

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Identification of fungi

Classic identification

For identification, the fungi were subcultured on suitable agar medium as described

by Samson et al. (2004). Whenever possible, the fungi were phenotypically identified to the

species level

by macroscopic and microscopic characteristics by employing

light

microscopy (de Hoog, 2000; Domsch, 1980; Ellis, 1988;1989; Klich 2002, Pitt, 1979 and

1988; Ramires, 1982. Raper and Fennel. 1977. Samson and Frisvad. 2004. Samson, et al.

2004. Samson and Pitt. 1990. Schwab and Straus. 2004). Slide preparations were stained

with lactofuchsin, with or without alcohol, in lactic acid or in distilled water.

To ensure correct identification of closely related species, some Penicillium species,

in

addition to morphological identification, were confirmed by detection

of indole

metabolites by a filter paper method (Lund, 1995.). Briefly, a piece of filter paper dipped in

Ehrlich reagent (Samson, et al. 2004) was placed on an agar plug with mycelia. In the case

of indole metabolite production, a violet ring will appeared within 10 min.

Rapid molecular identification

DNA extraction

The genomic DNA of culturable fungi isolates was extracted using the CTAB-

method described by Ausubel et al. (1987). For direct extraction of DNA from water

samples, 1 liter of water sample filtered through a Nuclepore polycarbonate filter, 47 mm in

diameter, with a pore size of 0.45 µm (Whatman, Clifton, NJ). With sterile forceps, the

membrane was carefully separated from the pad/base and placed into an empty, sterile Petri

dish. Using flame-sterilized scissors, the membrane was cut into small pieces (about 1 cm2).

DNA extraction was achieved using the Watermaster-DNAPurification Kit (Epicenter-

Biotechnologies, WI, USA), which is referenced for DNA extraction from water matrices

since it reduces possible PCR inhibition by natural organic matter (Pereira et al., 2010).

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Determine fungi population profiles using denaturing gradient gel electrophoresis

Amplification of the internal transcribed spacer (ITS) gene was used for the

comparison of different culture media in terms of diversity coverage and population

dynamics in time (Pereira et al., 2010). Nested-PCR was used to improve the amplification

of fungi with low number of representatives in the sample. The first step was carried out

using primers ITS1 and ITS4 (White et al., 1990), without GC-clamp to facilitate

amplification from whole genomic DNA. The resulting almost complete ITS fragments

were then used as template in the second step, where a 40 bp GC-clamp (Muyzer et al.,

1993) was incorporated to the ITS5 primer (White et al., 1990). The PCR mixture consisted

of 1X ThermoPol buffer, 0.5 μM of each primer, 0.2 mM of dNTPs and 1.25 U of Taq

DNA polymerase. The PCR protocol included a 7 min initial denaturation at 95 °C, 35

cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by 5 min of final

extension.

The PCR products were analyzed by DGGE that was performed using DCode

System DGGE Apparatus (Bio-Rad, Hercules, USA), following the manufacturer’s

instructions (Novinscak et al., 2008). The denaturing gradient was performed 25%–75% in

a gel of 6% Acrylamide/Bis 40% after 16 h at 80 V in 1X TAE buffer. Gels were then

stained for 10 min in ethidium bromide and destained in distilled water for 10 min. Bands

of interest were excised from the gels and placed in 30 μl of 10 mM Tris overnight at 4 ºC.

9 μl of the Tris/band solution was used for sequencing with the Big Dye terminator

sequencing kit v. 3.1 (Applied Biosystems, Foster City, USA) and primer R1378 on a

3130xl ABI Analyser (Applied Biosystems). All of sequenced strains were identified by

similarity searches with ITS sequences in EMBL/GenBank.

Results and Discussion

In the present study, 150 water samples were collected from 50 points in the drinking

water distribution system of Jeddah city, kingdom of Saudi Arabia. Samples were tested for

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the presence of fungi. The highest isolation frequency of fungi (90%) was recorded for water

samples collected from the private home (Table 1). The isolation frequency in cold and hot

water obtained from hospitals were 70 and 76.7%, respectively. On the other hand, the lowest

isolation frequency of fungi (40%) was obtained by the water samples collected from drinking

water treatment plants. These results indicated that, the best quality of drinking water, from

the mycobiologist point of view, was recorded in treatment plants. This quality was

deteriorated from the starting point to the end points of distribution system. This finding was

in agreement with the finding of Hageskal et al., (2006). They demonstrated that, a wide

variety of fungi species is present in all parts of the water distribution systems in Norway.

Moreover, piped drinking water can offer a transmission route for fungi.

Fungi numbers were also determined in all positive samples. Averages of fungi

numbers in the positive samples of each tested points were statistically analyzed and

illustrated in Figure 1. The lowest obtained numbers of fungi were found in the treatment

water (TW). These numbers were significantly (p<0.5) increased in hospital cold water

(HCW). More significant increase was also recorded when the private home water (PHW)

was investigated. These results demonstrated that, not only isolation frequency (previously

discussed) but also the mycobiota load was increased through the drinking water distribution

system of Jeddah city. On the other hand, no significant (p>0.05) difference in the mycobiota

load was obtained between TW and the hospital hot water (HHW). Comparing with PHW,

HCW and HHW had low fungi number. This may be due to some additional treatments such

as UV-treatment or ozonation that might be process to improve water quality. Also, heating of

water might be lead to reduce the total number of fungi.

Occurrence of fungi species in the different four tested-points were tested in the

positive samples (Table 2). In the treated water (TW), six species were identified by the

classic methods. These species were Alternaria alternate, Acremonium strictum, Mucor

plumbeus, Penicillium montanense, Penicillium melinii and. The most frequently fungi

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species occurred in TW were Trichoderma viride (66.6 %) followed by Alternaria alternate

and Acremonium strictum (50 %), whereas the lowest frequently occurred species was

Penicillium melinii (25 %). Pereira et al., 2009 detected genera Aspergillus, Cladosporium,

Penicillium, and Candida in three different drinking water sources in Portugal.

In the hospital cold water (HCW), 13 different fungal strains were identified by the

classical methods. By applying the molecular method an addition species, Acremonium

strictum, was identified. This means that, Acremonium strictum could be characterized as an

unculturable strain. In general, Penicillium montanense was the dominant fungi strain in

HCW followed by Aspergillus fumigates (Table 2).

In the hospital hot water (HHW), only three different fungi species were classically

identified (Table 2). In addition to these three species, six additional species were also

identified by the molecular method. In addition to Acremonium strictum, the species

Cladosporium cladosporioides, Rhizopus azygosporus, Penicillium citrinum, Penicillium

oxalicum and Trichoderma viride were also identified as unculturable strains in HHW. In a

recent study of Pereira et al. (2010) Acremonium sp. was identified as unculturable strain in

spring water. The highest occurrence percentage (19 %) was recorded by Alternaria alternate

and Penicillium montanense in HHW. These two species were identified by both classic and

molecular method (Table 2).

In private home water (PHW), 16 different fungi species were identified by the two

examined procedure (classic and molecular methods). This means that, unculturable fungi

species were not detected by the molecular method applied in this work (Table 2).

Trichoderma viride was the dominant fungi (35 %) in PHW followed by Alternaria alternate

and Aspergillus niger (27.8 %).

Generally, most filamentous fungi identified are known to be widely distributed in

nature and could therefore present no harmful impact in terms of water quality. However,

among the isolated fungi, there are potentially pathogenic, allergenic, and toxigenic species.

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For instance, Aspergillus fumigates was recovered from 19 % of the positive tested samples in

general. This species was recovered from 26.1 % and 24.1 % from HCW and PHW,

respectively (Table 2). The occurrence of potentially pathogenic species, such as A.

fumigatus, in drinking water has lead to speculations whether hospital water systems may

serve as a transmission route for fungal infections. Several studies have focused on analyzing

hospital water systems with respect to the presence of fungi (Anaissie et al., 2003; Kanzler et

al. 2007; Kennedy & Williams 2007; Pires-Goncalves et al. 2008). The results indicate that

hospital water may contain a wide diversity of fungi, including potential pathogens. The

hypothesis is that fungi in water are aerosolized into air when water passes installations, such

as taps and showers, and thus introduced to severely immunocompromised patients with a

high risk of fungal infections.

Penicillium species have been frequently recovered from water in the various studies

performed. Several of the species in both genus Penicillium and Aspergillus are known to

produce mycotoxins in other substrates, such as food and beverages (Pitt & Hocking 1999).

However, mycotoxins produced in water will of course be extremely diluted, and are perhaps

of minor concern (Hageskal et al., 2009).

On the other hand, several microorganisms can cause taste and odor troubles in

drinking water. These sensory problems have been connected to the presence of fungi. In this

respect, Chaetomium globosum, have been found in PHW in the present study. This fungus is

known as a geosmin producer, a compound often associated with earthy odor and taste in

drinking water (Paterson et al. 2007).

Generally, the total positive samples were 110 from total of 150 examined water

samples from the different four sampling points. It can be concluded that, there are 18

different fungi species distributed in the drinking water network. From these species 6, 14 and

16 species were found in TW, HCW and HHW, respectively. This result indicated that

number of species The dominant fungi species in the drinking water distribution systems in

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general was Trichoderma viride (29.1 %) followed by Penicillium montanense (25.5 %) and

Alternaria alternate (25.5 %) (Fig. 2). These species were found in all sampling points by

different recovery percentage within each water type. This finding is in agreement with those

mentioned by Hageskal et al. (2006). They stated that Trichoderma viride was found to be the

most dominant species in Norwegian drinking water.

Conclusions

The general result of the present study demonstrated that, a wide diversity of fungi

species has been isolated from drinking water. Also, the molecular techniques used in this

study established an alternative quick procedure for determine the mycobiota in water supply

sources.

Acknowledgment

This work was supported by a grant (Contract No. 18-012-430) from King Abdulaziz

University, Kingdom of Saudi Arabia. So, authors are very grateful for the deanship of

scientific research at king Abdulaziz University for the financial support.

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Table 1. Distribution and isolation frequency of fungi at sample points of drinking water

network in Jeddah city

Sample point No. of

tested

points

No. of

samples

No. of

Positive

samples

Isolation

frequency

Treatment Plants 10 30 12 40

Hospital

Cold-water tap 10 30 23 76.7

Hot-water tap

10 30 21 70

Private home 20 60 54 90

19

Table 2. Occurrence of fungi species in the positive samples collected from different points

in the drinking water distribution system of Jeddah city.

Fungi species

Occurrence in positive samples (%) Species

concentration

Min-Max

range

(CFU/100mL)

Accession

Numbers TW

n=12

HCW

n=23

HHW

n=21

PHW

n=54

Total

positive

samples

n= 110

Alternaria alternate 6 (50) 3 (13) 4 (19) 15 (27.8) 28 (25.5) 4 – 15 FR717899

Aspergillus niger ---- 5 (21.7) 2 (9.5) 15 (27.8) 22 (20) 2-18 FR717900

Aspergillus fumigates ---- 6 (26.1) ---- 13 (24.1) 19 (17.3) 1-8 FR717901

Acremonium strictum 6 (50) 1 (4.3)* 1 (4.8)* 11 (20.4) 19 (17.3) 4 – 32 FR717902

Chaetomium globosum ---- ---- ---- 12 (22.2) 12 (10.9) 3 – 12 FR717903

Cladosporium cladosporioides ---- 3 (13) 1 (4.8)* 12 (22.2) 16 (14.5) 6 – 21 FR717904

Cladosporium herbarum ---- 4 (17.4) ---- 15 (27.8) 19 (17.3) 2 – 4 FR717905

Fusarium dimerum ---- 1 (4.3) ---- 11 (20.4) 12 (10.9) 8 – 43 FR717906

Rhizopus azygosporus 2 (8.7) 1 (4.8)* 11 (20.4) 14 (12.7) 1 – 3 FR717915

Mucor plumbeus 2 (16.7) ---- ---- ---- 2 (1.8) 2 – 5 FR717907

Penicillium brevicompactum ---- 2 (8.7) ---- 12 (22.2) 14 (12.7) 3 – 8 FR717908

Penicillium citrinum ---- 3 (13) 2 (9.5)* ---- 5 (4.5) 1 – 4 FR717909

Penicillium glabrum ---- ---- ---- 11 (20.4) 11 (10) 0 – 2 FR717910

Penicillium montanense 4 (33.3) 7 (30.4) 4 (19) 13 (24.1) 28 (25.5) 2 – 5 FR717916

Penicillium melinii 3 (25) ---- ---- 13 (24.1) 16 (14.5) 0 – 2 FR717911

Penicillium olsonii ---- 1 (4.3) ---- 11 (20.4) 12 (10.9) 0 – 2 FR717912

Penicillium oxalicum ---- 1 (4.3) 1 (4.8)* 12 (22.2) 14 (12.7) 2 – 6 FR717913

Trichoderma viride 8 (66.6) 3 (13) 2 (9.5)* 19 (35.2) 32 (29.1) 6 – 22 FR717914

* species which did not able to phenotypically identify but could be identified by partial

sequence of ITS gene as a rapid molecular technique.

20

TW HCW HHW PHW -- -- --

0

500

1000

1500

2000

c c

a

bF

ungi num

bers

(C

FU

/ 1

00 m

L w

ate

r)

Water source

Figure 1: Average of total fungi number in the positive samples of treatment water (TW, n=

12), hospital cold water (HCW n= 23), hospital hot water (HHW n= 21) and private home

water (PHW n= 54) in drinking water distribution system of Jeddah city. Error bars represent

the standard deviations. Columns with the same letter are not significantly different.

21

Figure 2: Occurrence percentage of the aquatic fungi species in drinking water distribution

system of Jeddah city.

Altern

aria

alte

rnat

e

Asper

gillu

s nige

r

Asper

gillu

s fu

mig

ates

Acrem

onium

stri

ctum

Cha

etom

ium

globo

sum

Cla

dosp

orium

cla

dosp

orioid

es

Cla

dosp

orium

her

baru

m

Fusar

ium

dim

erum

Rhizo

pus az

ygos

poru

s

Muc

or p

lum

beus

Penicilli

um b

revico

mpa

ctum

Penicilli

um citr

inum

Penicilli

um g

labr

um

Penicilli

um m

onta

nens

e

Penicilli

um m

elin

ii

Penicilli

um o

lson

ii

Penicilli

um o

xalic

um

Tricho

derm

a virid

e

0

5

10

15

20

25

30

% O

ccuure

nce in p

ositiv

e s

am

ple

s (

n=

11

0)