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Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
Risk Assessment of Heterotrophic Bacteria from
Bottled Mineral Water Consumed in Syria
Nazih Daood
Department of Botany, Faculty of Sciences, Tishreen University, Syria
Received 18/09/2008
Accepted 12/05/2009
ABSTRACT
Four hundred and thirty samples of bottled mineral water belonging to ten
different local and imported brands collected from the Syrian market have
been analyzed bacteriologically. Heterotrophic plate count (HPC) was
determined using R2A agar culture medium and incubating at 22οC for 5–7
days. Pseudomonads and Aeromonads members were selectively investigated
and identified biochemically.
Around 70 % of the imported water samples exhibited HPC counts more
than 100 cfu/ml versus 13 % for local ones. Risk assessment of the
heterotrophic bacteria revealed that 53.49% of the strains showed resistance to
one or two of the twenty antibiotics tested and the highest resistance was found
against nalidixic acid, novobiocin, ampicillin, streptomycin, imipenem and
ampicillin/sulbactam. The majority of Pseudomonas spp. and Aeromonas spp.
strains were found to be resistant to nalidixic acid, trimethoprim/
sulfamethoxazole, ampicillin, and novobiocin. Pseudomonas spp. showed also
high resistance to tetracycline, imipenem, ceftazidime, amikacin, erythromycin
and carbencillin. The most effective antibiotics against Pseudomonas spp. were
ciprofloxacin, gentamicin, aztreonam, colistin, kanamycin and ceftriaxone.
However, the following antibiotics showed complete activity against Aeromonas
spp. amikacin, ceftazidime, ceftriaxone, ciprofloxacin and gentamicin. The
complete activity was also seen for aztreonam, chloramphenicol and gentamicin
against other HPC bacteria. Strains with multiple antibiotic resistance (MAR)
represented 60.18% of all isolates and the most resistant organism belonged to
the genus Pseudomonas followed by Aeromonas strains. The high load of
heterotrophic bacteria with relatively high counts of opportunists such as
Pseudomonads and Aeromonads in the studied mineral bottled water
represents hygienic and quality challenge for this ready-to-consume
commodity. Moreover, multiple antibiotic resistance detected among these
bacteria might pose health significances, at least to some defined sensitive
individuals, and should be considered properly.
Keywords: Bottled mineral water; Risk assessment; Heterotrophic
Pseudomonas; Aeromonas; Antibiotic Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
ﺓﺄﺒﻌﻤﻟﺍ
ﺔﻴﻨﺩﻌ ﻩﺎﻴﻤﻟﺍ ﻲﻓ ﺔﻴﺫﻐﺘﻟﺍ ﺔﻴﺭﻴﻏ ﻡﻴﺜﺍﺭﺠﻟﺍ ﺓﺭﻭﻁﺨ ﺭﻴﺩﻘﺘ
ﻲﻓ ﺔﻜﻠﻬﺘﺴﻤﻟﺍ
ﺩﺅﺍﺩ ﻪـﻴﺯﻨ
ﺔﻴﺭﻭﺴ ـ
ﻥﻴﺭﺸﺘ ﺔﻌﻤﺎﺠ ـ ﻡﻭﻠﻌﻟﺍ ﺔﻴﻠﻜ ـ ﺕﺎﺒﻨﻟﺍ
ﻡﻠﻋ ﻡﺴﻗ
/ ﻉﺍﺩﻴﻹﺍ ﺦـﻴﺭﺎﺘ
/ ﻲﻓ ﺭـﺸﻨﻠﻟ لﺒﻗ
ﺔـﻴﻨﺩﻌﻤﻟﺍ ﻩﺎـﻴﻤﻟﺍ ﻥﻤ ﺓﺩﺭﻭﺘﺴﻤﻭ
ﺔﻴﻠﺤﻤ ﺔﻔﻠﺘﺨﻤ ﺕﺎﻜﺭﺎﻤ ﺭﺸﻋ ﻰﻟﺇ ﺩﻭﻌﺘ ﺔﻨﻴﻋ ﻥﻭﺜﻼﺜﻭ ﻤﻌﺒﺭﺃ
ﻡﺍﺩﺨﺘـﺴ ﺎﺒ ﻱﺭﺘﺒ ﻕﺎﺒﻁﺃ ﻲﻓ ﺔﻴﺫﻐﺘﻟﺍ ﺔﻴﺭﻴﻏ ﻡﻴﺜﺍﺭﺠﻟﺍ
ﺕﻟﺯﻋ ﻴﻤﻭﺜﺭﺠ ﺕﻠﻠﺤﻭ ﺔﻴﺭﻭﺴﻟﺍ ﻕﺍﻭﺴﻷﺍ ﻥﻤ ﺓﺄﺒﻌﻤﻟﺍ
ﻑـﺌﺍﻭﺯﻟﺍ ﺩﺍﺭـﻓﺃ ﻥـﻋ ﻱﺭـﺤﺘﻟﺍ ﻡـﺘ ﻡﺎـﻴﺃ
7 5 ﺓﺩـﻤ 22
ﺔـﺠﺭﺩﻟﺍ ﺩﻨﻋ ﻥﻀﺤﻟﺍﻭ R2A ﺕﺒﻨﺘﺴﻤﻟﺍ
ﺔﻠﻜﺸﻤ ﺓﺩﺤﻭ 100 ﻥﻤ ﺭﺜﻜﺃ ﺔﻴﺫﻐﺘﻟﺍ ﺔﻴﺭﻴﻏ
ﺓﺩﺭﻭﺘﺴﻤﻟﺍ ﻩﺎﻴﻤﻟﺍ
ﻥﻤ 70 ﺕﺭﻬﻅﺃ
ﺕﺩـﺒﺃ ﺔـﻴﺫﻐﺘﻟﺍ ﺔـﻴﺭﻴﻏ ﻡﻴﺜﺍﺭﺠﻟﺍ ﺓﺭﻭﻁﺨ ﺭﻴﺩﻘﺘ ﺔﻴﻠﺤﻤﻟﺍ
لﺒﺎﻘﻤ ﺩﺤﺍﻭﻟﺍ ﺭﺘﻴﻠﻠﻤﻟﺍ ﻲﻓ
ﺔـﻤﻭﺎﻘﻤﻟﺍﻭ ﺓﺭـﺒﺘﺨﻤﻟﺍ ﻥﻴﺭﺸـﻌﻟﺍ ﺔـﻴﻭﻴﺤﻟﺍ ﺕﺍﺩﺎﺼﻟﺍ ﻥﻤ ﻥﻴﻨﺜﺃ ﻭﺃ ﺩﺤﺍﻭﻟ ﺔﻤﻭﺎﻘﻤ ﺕﻻﻼﺴﻟﺍ %
ﻥﻤ 53.5
، ﻙﻴﺴﻜﻴﺩ ﻟﺎﻨ
ﻴ ﺩﻀ ﺕﻨﺎﻜ ﻯﻭﺼﻘﻟﺍ
ﻙﻴﺴـﻜﻴﺩﻴﻟﺎﻨ
ﻠﻟ ﹰ ـﻤﻭﺎﻘﻤ
ﺱﺎـﻨﻭﻤﻭﺭﻴﻷﺍﻭ ﻑـﺌﺍﻭﺯﻟﺍ ﻉﺍﻭـﻨﺃ ﻥـﻤ ـﻅﻋﻷﺍ
ﻡﺴﻘﻟﺍ ﻥﺎﻜ ﻡﺎﺘﻜﺎﺒﻟﻭﺴ
ﺔـﻌﻔﺘﺭﻤ ﺔﻤﻭﺎﻘﻤ ﹰﺎﻀﻴﺃ ﻑﺌﺍﻭﺯﻟﺍ ﻉﺍﻭﻨﺃ ﺕﺭﻬﻅﺃ ﻥﻴﺴﻭﻴﺒﻭﻓﻭﻨ
ﻉﺍﻭﻨﺃ ﺩﻀ ﺔﻴﻠﻋﺎﻓ ﺭﺜﻜﻷﺍ
ﻉﺍﻭـﻨﺃ ﺩـﻀ ﺔ
ﺓﺩﺩﻌﺘﻤﻟﺍ ﺔﻤﻭﺎﻘﻤﻟﺍ ﺕﺍﺫ ﺕﻻﻼﺴﻟﺍ ﺕﻠﻜﺸ ﻯﺭﺨﻷﺍ
ﺱﻨﺠ ﻰﻟﺇ ﺩﻭﻌﺘ ﺔﻤﻭﺎﻘﻤ ﺭﺜﻜﻷﺍ ﺔﻴﺤﻟﺍ ﺕﺎﻨﺌﺎﻜﻟﺍ ﺕﻨﺎﻜ
ﻉﻭﻤﺠﻤ %
ﻥﻤ 60.2 ﺔﻴﻭﻴﺤﻟﺍ ﺕﺍﺩﺎﺼﻠﻟ
ﺓﺭـﻴﺒﻜﻟﺍ ﺩﺍﺩـﻋﻷﺍﻭ ﺔـﻴﺫﻐﺘﻟﺍ ﺔﻴﺭﻴﻏ ﻡﻴﺜﺍﺭﺠﻟ ﺍﻥﻤ ﻲﻟﺎﻌﻟﺍ ﻯﻭﺘﺤﻤﻟﺍ ﻥﺇ ﺱﺎﻨﻭﻤﻭﺭﻴﻷﺍ
ﺕﻻﻼﺴ ﺎﻬﻴﻠﺘ ﻑﺌﺍﻭﺯﻟﺍ
ﹰﺎ ﺩﻴﺤﺘ لﺜﻤﺘ ﺔﺴﻭﺭﺩﻤﻟﺍ ﺓﺄﺒﻌﻤﻟﺍ ﺔﻴﻨﺩﻌﻤﻟﺍ ﻩﺎﻴﻤﻟﺍ ﻲﻓ ﺕﺎﺴﺎﻨﻭﻤﻭﺭﻴﻷﺍﻭ ﻑﺌﺍﻭﺯﻟﺎﻜ ﺔﻴﺯﺎﻬﺘﻨﻻﺍ ﻡﻴﺜﺍﺭﺠﻟﺍ ﻥﻤ ﹰﺎﻴﺒﺴﻨ
ﺕﺍﺩﺎﺼـﻠﻟ ﺓﺩﺩـﻌﺘﻤﻟﺍ ﺔـﻤﻭﺎﻘﻤﻟﺍ
ﻰـﻟﺇ ﻑﻀﺃ ﺓﺭﺸﺎﺒﻤ
ﺔﻜﻠﻬﺘﺴﻤﻟﺍ ﺔﻋﺎﻀﺒﻟﺍ ﻩﺫﻬﻟ ﹰ ﻴﻋﻭﻨﻭ
ﻥﻴﺴﺎﺴـﺤﻟﺍ ﺩﺍﺭـﻓﻷﺍ ﺽﻌﺒ ﺩﻨﻋ لﻗﻷﺍ ﻰﻠﻋ ﺔﻴﺤﺼ ﺔﻴﻤﻫﺃ ﺕﺍﺫ ﻥﻭﻜﺘ ﻥﺃ ﻥﻜﻤﻴ ﺜﺍﺭﺠﻟﺍ
ﻩﺫﻫ ﺩﻨﻋ ﺔﻔﺸﺘﻜﻤﻟﺍﻭ
ﻥﻤ ﺩﻴﺯﻤﺒ لﻤﺎﻌﺘ ﻥﺃ ﺏﺠﻴﻭ
، ﺔـﻴﺫﻐﺘﻟﺍ ﺔـﻴﺭﻴﻏ ﻡﻴﺜﺍﺭـﺠ ، ﺓﺭﻭـﻁﺨﻟﺍ ﺭﻴﺩﻘﺘ ، ﺓﺄﺒﻌﻤ ﺔﻴﻨﺩﻌﻤ ﻩﺎﻴﻤ : ﺤﺎﺘﻔﻤﻟﺍ
.ﺕﺍﺩﺎﺼ ﺔﻤﻭﺎﻘﻤ ، Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
I- Introduction
There has been a dramatic increase in the consumption of bottled drinking water, especially, natural mineral water in Syria. Although the growing of tourism industry in Syria can be considered as a main cause, the increase in demand worldwide has been attributed to many factors. The public's concern over increased water pollution and the so common belief among people that bottled water is superior to municipal water in that it contains no microorganisms (Papapetropoulou et al., 1997; Hunter, 1993), and fashioned trends towards the consumption of designer and trademarked water (Hunter, 1994). In addition, bottled water is often recommended for patients with immune-system deficiencies as well as marketed as ideal for infant nutrition and reconstitution of foods (Warburton, 1993). Objection to the local water supplies for offensive taste and odor as well as fluoride, chlorine and other additives might be considered as additional factors (Warburton et al., 1992; Tamagnini and Gonzalez., 1997; Bharath et al., 2003). Because the emergent mineral water is an oligotrophic environment, their content of viable bacterial cell is as low as 10 cfu ml-1 (Ferreira et al., 1994; Leclerc, 1994). This low count of autochthonous organisms are of little concern to the healthy consumer. However, as contamination may be induced during bottling process, potential pathogens may persist. Therefore, because there is no permitted disinfection or sterilization process of commercially available mineral waters for removal of microorganisms, the presence of such pathogens may be of great public health concern. Because Pseudomonads constitute the main part of these both naturally occurring and contaminating bacteria (Rosenberg and Hernandez- Duquino, 1988; Guillot and Leclerc, 1993) as well as their role in opportunistic infections (Rusin et al., 1997; Kudinha et al., 2000), the assessment of the health risk from these organisms after bottling continues to be of high interest for both of microbiologists and health workers. Aeromonas spp. are widespread in surface waters. Clinically, they are incriminated as a causes of diarrhea, peritonitis, endocarditis, meningitis, septicemia, urinary tract and wound infections mostly associated with consumption of contaminated drinking water (Pin et al., 1997; Rusin et al., 1997; Kudinha et al., 2000). Therefore, as for Psd.aeruginosa, their occurrence in mineral water is considered as quality indicator. Drinking water has been suggested as an important Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
source of human infections caused by the members of Aeromonas (Burke et al., 1984; Havelaar et al., 1990). From health risk point of view, the great distribution of Pseudomonas spp. and Aeromonas spp. as the main components of heterotrophic bacteria in bottled mineral water and their increased resistance to clinically available antimicrobial agents has been considered as health concern particularly to immunocompromised patient and because there is the risk of transferring the resistance to other bacteria present in the human body and some pathogenic ones (Rosenberg and Hernandez- Duquino, 1988; Massa et al., 1995; Mary et al., 2000). By taking into account that there are many local and imported brands of bottled mineral water in the Syrian market, and there is no documented and reliable information on the microbiological quality of these water products and consequently their hygienic safety for consumptions, the increased consumption of such waters raises the question as to whether they are hygienically safe. Therefore, the aim of the study is to provide an adequate information on the microbiological quality of these water products and an assessment of their health risks through investigation of the antibiotic resistance among Pseudomonads spp., Aeromonads spp. and other heterotrophic bacteria isolated from it. II- Materials and methods
Collection of samples. A total of 160 bottles of natural Syrian mineral water as well as 270 bottles of imported bottled natural mineral water were directly collected from the retail outlets in Syria throughout the years 2006 and 2007 . The studied local water are Syrian in origin and produced (bottled) locally. Mineral water bottles were stored at room temperature (20 to 22°C) prior to investigation. Bottles were vigorously agitated before analysis. All analysis tests were performed in laboratories of Tishreen University. Bacteriological analysis: The samples were analyzed to investigate the members of Psuodomonads and Aeromonads as well as total heterotrophic test. For heterotrophic plate counts (HPC), different volumes of undiluted water samples were plated on Tryptic Soy Agar TSA (Oxoide ltd) and R2A medium (Reasoner and Geldreich, 1985) and incubated at 22° C for 5–7 days. For Pseudomons spp. and Aeromonas spp., membrane filtration technique was applied. Volumes up to 1L of sample were filtrated using cellulose membrane filter of 0.45 µm pores (Millipore Ltd) and filters were Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
transported onto selective culture media and incubated up to 72 hr at 30°C. Pseudomonas Agar Base (Oxoide Ltd) supplemented with 200 mg/l cetrimide and 15 mg/l nalidixic acid was used for detecting of Pseudomons aeruginosa. Other Psuodomonads members were presumptively isolated by using pseudomonas isolation agar (Hi media Ltd) as selective culture medium. Selective isolation of Aeromonas hydrophila was performed by using Pril-ampicillin-dextrin-ethanol [PADE] agar containing 15-30 µg ampicillin (Imziln et al., 1997) . For isolation of the other presumptive members of Aeromonas spp., filters were transferred to Glutamate Starch Phenol Red (GSP) agar plates (Merck Ltd) supplemented with 100 000 U l-1 of penicillin G (Sigma Chemical). Yellow colonies were counted and submitted to further identification tests as mentioned below. Identification and classification. For identification purposes, all suspected colonies either from heterotrophic plate count (HPC) or membrane filters were subcultured on Tryptic Soy Agar for 24 hr at 30° C. Each strain was tested by Gram stain, for oxidase production and glucose assimilation; further identification was carried out with the API 20 NE identification system for nonfermenters (Bio Mereux ltd). All tests were carried out at 22°C. Further confirmation tests were carried out to identify the isolates of Aeromonas spp. and Pseudomonas spp. ; these tests are: the oxidation/fermentation (O/F), sensitivity to vibriostatic agent O/129, gas production from glucose, H2S production from cysteine, esculin hydrolysis, motility, growth at 41.5° C , characteristic growth on King's A' agar, growth on acetamide broth, denitrification, arginine dihydrolase, and gelatin liquefaction (Popoff, 1984; Carnahan et al., 1991; Murray et al., 1999). Antibacterial susceptibility testing. For all strains isolated, antibiotic sensitivity was tested by the agar disk diffusion method (Bauer et al., 1966). Bacterial strains were suspended in sterile 0.85% saline and the cell density was adjusted to match the turbidity of McFarland No. 2 standard, diluted 1:20, and swabbed on Mueller- Hinton agar (Hi media Ltd) using sterile cotton swabs. The antibiotics and the concentrations used were as follows: amikacin (AMK: 30 µg), ampicillin (AMP: 10 µg), ampicillin-sulbactam (SAM: 10/10 µg), aztreonam (AZT: 30 µg), carbencillin (CAB: 100 µg), ceftazidime (CFZ: 30 µg), ceftriaxone (CRO: 30 µg), chloramphenicol (CHL: 30 µg), ciprofloxacin (CIP: 5 mg), colistin (COL: 10 µg), erythromycin (ERY: 15 µg), gentamicin (GEN: 10 µg), imipenem (IMP: 10 µg), Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
kanamycin (KAN: 30 µg), nalidixic acid (NAL: 30 µg), novobiocin (NOV: 30 µg), streptomycin (STR: 10 µg), tetracycline (TET: 30 µg), tobramycin (TOB: 10 µg), trimethoprim/sulfamethoxazole (SXT: 1.25/23.75 µg). Plates were incubated at 30 ºC for 24–48 h. Results of susceptibility were reported as sensitive, intermediate or resistant strain upon the size of the inhibition zone around each disk and information supplied by the antibiotics manufacturing company (Hi Media ltd) and referenced criteria (NCCLS, 1999). Multiple antibiotic resistance (MAR) indexing of each isolate was carried out by dividing the number of antibiotics to which the isolate was resistant to the total number of antibiotics to which the isolate was III- Results
A total of 216 bacterial isolates were obtained and subjected to susceptibility tests. These isolates can be divided into three categories: Pseudomonads, Aeromonads, and other heterotrophic bacteria (HPC) as shown in Table 1. The count of viable heterotrophic bacteria as measured on R2A ranged from 10 to 5.6.105 cfu ml-1 for imported water and from 0 to 8.7.102 cfu ml-1 with only two samples exceeded 103 cfu ml-1 for local water. It is clear that the level of occurrence of Pseudomonas spp. is somewhat related to HPC counts while this is not the case for Aeromonas spp. any way. So, samples with more than 10000 cfu ml-1 were found to be crowded with Pseudomonas members. As shown in Table 1, there were 22 (13.75%) positive local samples for occurrence of Pseudomonas spp. Of these, Pseudomonas spp. were recovered in only 10 (6.25%) samples with tested volumes less than 250 ml. In contrast, the imported samples showed as high as 81 (30%) positive samples and 59 (21.85%) of them were less than 250 ml. On the other hand, Aeromonas spp. showed high frequency of isolation and occurred in 42 (26.25%) and 118 (43.70%) samples of local and imported waters respectively.
Table 1. Percentages of positive samples of bottled mineral waters for presence
of Pseudomonas spp. and Aeromonas spp. as related to HPC counts.
No and (%) of samples positives when
Bacteria
HPC as cfu*/ml is:
0-102 102–103 103–104
Pseudomonas spp. 5 (3.12) 15 (9.37) 2 (1.25) Aeromonas spp. 19 (11.87) 21 (13.12) 2 (1.25) Pseudomonas spp. 8 (2.96) 18 (6.67) 24 (8.89) 31 (11.48) Imported
Aeromonas spp. 28 (10.37) 30 (11.11) 27 (10) 33 (12.22) * cfu: colony forming unite Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
Of 87 strains isolated of Pseudomonads, 23 (26.44%) were tentatively identified as Psd.aeruginosa followed by Pseudomonas fluorescens (16.09%), and the rest i.e. (58.61%) of strains distributed to other Pseudomonas spp. as illustrated in Table 2.
Table 2. Total number and (%) of the identified species of Pseudomonads and
Aeromonads isolated from bottled mineral waters.
Number and (% ) of strains identified
Bacteria
Mineral water
Local Imported
Pseudomonas spp. Psd. aeruginosa Psd. fluorescens Psd. alcaligenes Psd. putida Psd. vesiculares Psd. stutzeri Psd. picketti Psd. diminuta Psd. paucimobilis Other Pseudomonas Aeromonas spp. Aer hydrophila 7 Aer .caviae Aer .sorbia Other Aeromonas 92 141 233
Aeromonas hydrophila has been found to be predominant species among the isolated strains of Aeromonas spp.; So, of 60 strains, 19(31.66%) were identified as Aer.hydrophila followed by other species (Table 2). In addition, there were 86 isolates of heterotrophic bacteria other than Pseudomonads and Aeromonads. This separation was tentatively achieved by subculturing each HPC colony on Pseudomonads and Aeromonads selective culture media mentioned above. Furthermore, each colony was subjected to the main biochemical tests for identification of Pseudomonads and Aeromonads. Consequently, any colony failed to meet these criteria was considered as HPC isolate. As shown in Table 2, the identified strains belonged to genus Pseudomonads were represented by nine species. However, there were difficulties to classify some of the suspected strains of Pseudomonas spp. and hence cited as other Pseudomonads. As it can be seen from Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
Table 2, the obvious difference between the two sources of water in term of species distribution was significant. Notably, the imported water has showed higher counts of Pseudomonas spp. and subsequently Psd.aeruginosa compared with local one. However, this is not the status of Aeromonas spp. distribution since Aeromonas hydrophila were observed with almost similar percentages of occurrence for local and imported water. Results of the antibiotic sensitivity testing are presented in table 3 and figure 1. At the level of species isolated, and as related to antibiotic sensitivity profile, no significant differences were found between the two sources of water. Figure 1. Percentage of antibiotic resistance among Pseudomonas spp.,
Aeromonas spp., and other heterotrophic bacteria isolated from
bottled mineral water.
AMK: amikacin 30 µg, AMP: ampicillin 10 µg, SAM: ampicillin-sulbactam 10/10 µg, AZT: aztreonam 30 µg, CAB: carbencillin 100 µg, CFZ: ceftazidime 30 µg, CRO: ceftriaxone 30 µg, CHL: chloramphenicol 30 µg, CIP: ciprofloxacin 5 mg, COL: colistin 10 µg, ERY: erythromycin 15 µg, GEN: gentamicin 10 µg, IMP: imipenem 10 µg, KAN: kanamycin 30 µg, NAL: nalidixic acid 30 µg, NOV: novobiocin 30 µg, STR: streptomycin 10 µg, TET: tetracycline 30 µg, TOB: tobramycin 10 µg, SXT: trimethoprim/sulfamethoxazole 1.25/23.75 µg. Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
Most of the heterotrophic bacteria (HPC) isolated from the bottled mineral water were resistant to nalidixic acid, novobiocin, and ampicillin. Complete sensitivity were recorded towards aztreonam, chloramphenicol, and gentamicin. Intermediate resistances were more often observed against the following antibiotics: streptomycin, nalidixic acid, chloramphenicol, tetracycline, ampicillin and novobiocin. (Table 3, figure 1) For Pseudomonas spp., the highest frequency of resistance was to nalidixic acid (76.92) followed by trimethoprim/sulfamethoxazol (68.96%) and ampicillin (67.95%). (Table 3).
Table 3. Percentage of antibiotic resistance among Pseudomonas spp.,
Aeromonas spp., and other heterotrophic bacteria isolated from
bottled mineral water.
Percentage of sensitive, intermediate resistant and resistant strains of :
Pseudomonas spp.
Aeromonas spp.
Other HPC bacteria
Antibiotic
(n*= 78)
Amikacin
Ampicillin
Aztreonam
Ceftazidime
Ceftriaxone
Chloramphenicol 62.83 21.79 15.38 71.67 13.33
Colistin
Gentamicin
Imipenem
Kanamycin
Nalidixic acid
26.67 11.66 61.67 10.47 18.60 Novobiocin
43.59 35.90 20.51 26.67 10 63.33 47.67 19.77 21.80 14.10 64.10 56.67 8.33 35 70.94 13.95 Tobramycin
n*= number of strains isolated. #: S= sensitive, ##:I= intermediate resistant, ###:R= resistant. Six antibiotics have showed noticeable activity against Pseudomonas spp. These antibiotics and their sensitivity percentages are ciprofloxacin (97.44%), gentamicin (91.03%), aztreonam (88.46%), colistin (87.18%), kanamycin (87.18%) and ceftriaxone (84.62%). The most effective antibiotics against Pseudomonas spp. were ciprofloxacin, gentamicin, aztreonam, colistin, kanamycin and Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
ceftriaxone with sensitivity levels ranging from 97.44% for ciprofloxacin to 84.62% for ceftriaxone. Intermediate resistance were exclusively found with the antibiotics streptomycin, chloramphenicol, nalidixic acid and tetracycline. Overall, although there were some exceptions, all strains of Pseudomonas spp. have showed relatively high level of resistance to antibiotic tested since a 77.14% of strains were resistant to three or more of twenty antibiotics tested (Table 4). All isolates of Aeromonas spp. were found to be susceptible to amikacin, ceftazidime, ceftriaxone, ciprofloxacin and gentamicin.
Sensitivity was almost complete to aztreonam, carbencillin, and tobramycin since resistance rate did not exceed 10% of the tested strains. Highest resistance level were detected for streptomycin, nalidixic acid, and ampicillin. In addition to high resistance to ampicillin and ampicillin/ sulbactam, Aer.hydrophila strains have showed high resistance levels towards erythromycin, imipenem, streptomycin and novobiocin. Strains resistant to more than two antibiotics were considered as multiple antibiotic resistant (MAR). Accordingly, as high as 77.14%, 60%, and 46.51% of the isolates of Pseudomonas spp., Aeromonas spp. and HPC respectively in our investigation were in that category. As indicated previously, multiple antibiotic resistance (MAR) indexing gives some information about the size of resistance for individual isolate. In this study, a value greater than 0.15 indicates that the isolate is multiple antibiotic resistant. The MAR index values and the percentages of MAR strains of all bacterial strains studied are shown in Table 4. Single resistance (i.e.: resistance to one antibiotic) occurred in only 8.57 % of pseudomonas spp. strains, and 14.28 % were also resistant to two drugs. Single resistance was found in 18.33% of the Aeromonas spp. strains, 21.67% had double resistance. However, single and double resistance among HPC bacteria were observably increased to 23.25 and 30.23 respectively. Thereby, MAR strains were less common among other HPC than that of Pseudomonas spp. and Aeromonas spp. Accordingly, the MAR strains of Pseudomonas spp. had resistance indices ranging from 0.15 to 0.85 as compared with 0.15 to 0.65 for Aeromonas spp. strains. However, HPC strains had low MAR indices and the range was 0.15 - 0.45. Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
The most occurred profiles of resistance among Pseudomonas spp. strains were the combined resistance to nalidixic acid, with tetracycline, ampicillin,amikacin and erythromycin; nalidixic acid, with carbencillin, ampicillin, ceftazidime, imipenem and trim/sulfamethoxazol. In case of Aeromonas spp., the resistance profile most recorded was the combined resistance to Streptomycin with nalidixic acid, ampicillin/ sulbactam and erythromycin or novobiocin.
Table 4. Multiple antibiotic resistance (MAR) index and the percentages of
MAR strains of Pseudomonas spp., Aeromonas spp., and
heterotrophic bacteria (HPC) from bottled mineral water.
Number and (%) of resistant and MAR strains.
index antibiotics Pseudomonas spp., Aeromonas, spp.,
20 (23.25) 37 (17.13) 26 (30.23) 49 (22.68) 11 (12.79) 17 (7.87) IV- Discussion
In general, the viable counts of heterotrophic bacteria has been suggested as satisfactory indicator of the overall quality of bottled mineral water production (Ferreira et al., 1994). However, in many countries, and as quality parameters demanded of bottled mineral waters, mineral water must be free of Pseudomonas aeruginosa in any 250-ml sample (European Community, 1980).This because the opportunistic pathogen Psd. aeruginosa is currently considered a Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
primary infectious agent implicated in foodborne and waterborne diseases (Warburton, 1993; Morais et al., 1997). The significance of Pseudomonas spp. and related species in bottled mineral waters is related to their capability of multiplying abundantly in such low- nutrient water (Gonzalez et al., 1987; Mavridou et al., 1994; Tsai and Yu, 1997) and surviving with somewhat constant numbers for at least 6 months after bottling (Hunter, 1993; Leclerc and Moreau, 2002; Daood, 2008). It is also worthy of noting that, Psd. aeruginosa has been previously suggested as a surrogate indicator for the presence of other opportunistic pathogens (Geldreich, 1992). Accordingly, and since Pseudomonas spp. were discovered in high numbers in volumes of 250 ml, this puts as total as 59 (21.85%) samples of imported studied waters as unsatisfactory. Fortunately, this rate decreased to 10 (6.25%) samples of local waters. As Pseudomonas spp. have occurred in only 13.75% and 30% of domestic and imported water samples respectively (Table 1), our results about the predomination of Pseudomonas species are not fully in agreement with those obtained by many authors. In 1988, Rosenberg and Hemandez- Duquino showed that various Pseudomonas spp. presented in 45% of the samples from 87 different carbonated and noncarbonated mineral waters purchased in Germany (Rosenberg and Hemandez- Duquino, 1988). This proportion was decreased to 30% in a study performed by (Mavridou, 1992). Likewise, Guillot and Leclerc reported that 40% of the isolates originated from mineral water were identified as Pseudomonas species (Guillot and Leclerc, 1993). Pseudomonas spp. are also abundantly isolated from most of the mineral water samples analyzed by (Manaia et al., 1990). All Pseudomonal species identified in this study (Table 2) and our previous study (Daood, 2008) have been frequently isolated by others. Psd. aeruginosa , Psd. Stutzeri. Psd fluorescens and Psd. putida have been commonly isolated from drinking bottled water (Hernandez- Duquino and Rosenberg, 1987) and bottled mineral water (Rosenberg and Hernandez-Duquino, 1988; Venieri et al., 2006) In their study on five brands of French mineral water, Mary et al., (2000) have identified the following pseudomonal species: Psd. maltophilia, Psd. fluorescens,and Psd. alcaligenes. More recently, the species Psd. aeruginosa, Psd. testoalcaligenes, Psd. maltophilia, Psd. diminuto, Psd. fluorescens, and Psd. vesicularis were isolated from domestic bottled water in Greece with being Psd. aeruginosa is the most isolated microorganisms (Venieri et al., 2006). Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
Although there are numerous reports that have documented, at least under some circumstances, the incidence of diarrheal disease after ingestion of drinking water contaminated with Aeromonas spp., the health significance of Aeromonas spp. in water is not fully understood (Borchardt et al., 2003; WHO, 2003). However, there is a worldwide distribution of Aeromonas spp. in bottled drinking water (Slade and Falah, 1986; Manaia et al., 1990; Villari et al., 2002) and approved ability of growth in it (WHO, 2003). It is common to isolate Aeromonas spp. from both drinking and mineral water as well as food and environmental samples (Araujo et al., 1991; Hanninen and Siitonen, 1995; Sen and Rodgers, 2004; Daood, 2008). Pavlov et al (2004) have reported that Aeromonas spp. constituted 18.10% of the isolated bacteria. Contrary, Aeromonas spp. have predominantly found in 26.25% and 43.70% in samples of local and imported waters. (Table 1) Aeromonas hydrophila has been frequently isolated; for examples, it constituted 0.3%, 3.3% and 3.11% of isolates from bottled water (Venieri et al., 2006), municipally treated tap water (Emekdas et al., 2006) and bottled mineral water (Daood, 2008) respectively. In this study, this proportion was notably increased to 8.15% of all bacterial isolates. Because it is not possible to recognize the potential pathogenity of its components, HPC test is still somewhat ambiguous term and consequently it is not recommended to rely on to assess the health significance and risks associated with drinking water (Edberg and Allen, 2004; Allen et al., 2004). Furthermore, there is no clear-cut evidence to link the gastroenteritis with heterotrophic bacteria ingested by healthy people (Rusin et al., 1997; Colford et al., 2002). Under these circumstances, the antimicrobial sensitivity profile of the heterotrophic bacteria constituted the HPC and particularly the members of Pseudomonas and Aeromonas seems to be, at least currently, reasonable measurement in this situation. Collectively, the incidence of antibiotic resistance among aquatic bacteria is a growing area of global public health concern (Pavlov et al., 2004; Shrivastava et al., 2004; Hernandez-Duquino and Rosenberg, 1987; Schwartz et al., 2003; Venieri et al., 2006). The problem will become worse with increased levels of multiresistances. The antibiotic resistance among heterotrophic bacteria isolated from the bottled drinking water has been well studied. Collectively, high resistance levels have been reported among the majority of HPC towards penicillin and ampicillen (Pavlov et al 2004), ampicillin, nalidixic acid and novobiocin (Jeena et al., 2006), ampicillin and Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
nalidixic (Mary et al., 2000; Papandreou et al., 2000), and ampicillin, colistin, and chloramphenicol (Messi et al., 2005). Resistance to antibiotics such tetracycline, streptomycin, were less common with percentages ranged from 20% to 40% (Jeena et al., 2006). On the other hand, the majority of such studies have showed the somewhat complete susceptibility of HPC to ciprofloxacin and to less extent to gentamicin (Pavlov et al., 2004; Jeena et al., 2006; Mary et al., 2000; Papandreou et al., 2000). However, in mineral water, one study has recorded high resistance rates among HPC to quinolones and fluoroquinolones represented by nalidixic acid and ciprofloxacin respectively (Guyard et al., 1999). Although there is some agreement, the resistance levels of HPC reported above were higher than those encountered in the present investigation. High sensitivity levels were recorded most of the tested twenty antibiotics. For instance, in addition to absolute activity showed by two antibiotics: azterionam and gentamicin, the sensitivity levels ranged between 83.72% to 96.51% for eleven antibiotics. (Table 3). As such, this result might be considered as a unique in such studies. Clinical strains of Pseudomonas aeruginosa have been frequently found to be resistant to the following antimicrobials: ceftazidime, piperacillin/tazobactam, imipenem, aztreonam, amikacin, tobramycin, gentamicin, and ciprofloxacin (Pitten et al., 2001; Wang et al., 2006). Fortunately, in the present study, some of the above antibiotics have showed considerable activity against isolates of Pseudomonas. Namely, ciprofloxacin, gentamicin, and aztreonam were the most effective antibiotics against Pseudomonas spp. and tobramycin has also appeared to be relatively effective. However, of the twenty antibiotics tested in the present study, ten had no significant inhibition of Pseudomonas spp. Thus, as much as 60% to 76% of the tested isolates were resistant towards the following antibiotics: carbencillin, erythromycin, amikacin, ceftazidime, imipenem, tetracycline, novobiocin, ampicillin, trimethoprim/sulfamethoxazole, and nalidixic acid. It is noticeable in the present study, a part of their innate resistance to primary antibiotics such as penicillin, tetracycline, and erythromycin, to find some of such that non-clinical Pseudomonas aeruginosa isolates being resistant to what so called anti-pseudomonal antibiotics (i.e.: imipenem, amikacin, ceftazidime, and carbencillin). The obvious exception is colistin that has anti-pseudomonal activity and has been used previously for treatment of pneumonia caused by multidrug resistant Pseudomonas aeruginosa (Levin et al., Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
1999). In the present study, this antibiotic has showed observable activity toward both Pseudomonas spp. and Aeromonas spp. The results of complete or high susceptibility rates of Aeromonas spp. isolates towards amikacin, aztreonam, ceftriaxone, gentamicin and ciprofloxacin are in agreement with those obtained by other studies on drinking water and other sources (Emekdas et al., 2006; Motyl et al., 1985; Reinhard and George,1985; Bizani and Brandelli, 2001). Comparatively, the high level of resistance towards ampicillin/sulbactam showed by our Aeromonas spp. isolates (40% resistant) were lower than that found among food and clinical ones recorded by Palu et al., (2006) who reported that 73.5% of isolates were resistant to that antibiotic. However, the high resistance to this β- lactamic antibiotics (ampicillin 60% and ampicillin/sulbactam 40%) might be explained by natural ability of Aeromonas spp. to produce β- lactamase (Fosse et al., 2003). High susceptibility to trimethoprim/sulfamethoxazole is in agreement with that has been recorded among freshwater Aeromonas spp. (Miranda and Castillo, 1998). However, resistance to tetracyclines and trimethoprim/sulfamethoxazole has been described for some isolates of Aeromonas spp. (Ko et al., 1996; Vila et al., 2003). In recent study, food strains of Aeromonas spp. have showed resistance to ampicillin/sulbactam, cefoxitin and tetracycline while clinical strains were found resistance to ampicillin/sulbactam, cefotaxime, ceftazidime, cefoxitin, sulfamethoxazole/trimethoprim, chloramphenicol and tetracycline (Palu et al., 2006). Our Aer.hydrophila strains were found highly resistant to ampicillin and ampicillin/sulbactam, erythromycin, imipenem, streptomycin and novobiocin. These results are partially in agreement with that recorded by Imziln et al., (1996) on wastewater strains and by Ilhan et al., (2006) on veterinary strains of Aer. hydrophila. In the latter study, high resistance were recorded to penicillin G, erythromycin and gentamicin while these strains were found susceptible to the other antibiotics with amoxicillin and enrofloxacin being the most effective. Multiple resistance to antibiotics have been frequently recorded among both of environmental and non-environmental isolates of Pseudomonas spp. and Aeromonas spp. Multiresistant Aeromonas spp. have been isolated from clinical and food samples (Radu et al., 2003; Palu et al., 2006). Furthermore, the resistance to chloramphenicol, sulfamethoxazole/trimethoprim and tetracycline has been recently suggested to be mediated by plasmids (Schmidt et al., 2001; Casas et al., 2005; Palu et al., 2006). Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
It is not uncommon to find multiresistant HPC strains of mineral water origin, for instances, Messi et al., (2005) have found that 55% of HPC strains were MAR whereas as high as 80% of these isolates were resistant to one or more antibiotics. More recently, Jeena et al., (2006) have reported that 45% of HPC strains showed multiple resistance. The very related result recorded in the present study (i.e.: 46.5% of HPC strains were MAR) suggests to the relative uniformity of the HPC components and their response to antibiotics in different studies on mineral waters. However, the high values of MAR for Pseudomonas spp. (77.14%) and Aeromonas spp. (60%) recorded in the current study could have two significant perspectives; first is the obvious contribution of such opportunists in elevation of antibiotic resistance of HPC when they are involved in it. Second is a health consideration resulted from the presence of these MAR opportunists in bottled mineral water that should be considered whenever the sanitary quality of mineral water is assessed. Given increased findings about the possibility of conjugational and transformational transfer of resistance elements to indigenous flora of aquatic, terrestrial environments and biofilms (Witte, 2000; Lilley and Bailey, 2002), the concern is coming from the potential possibility of transfer the resistance factors namely plasmids to the normal human susceptible microflora. This will create real hazards in patients on antibiotic as well as the possibility of emerging more virulent pathogens. Given that, as reported by Boronin, (1992), plasmids are ubiquitous in Pseudomonas spp., where in one study for example, plasmids were detected in 46% of Pseudomonads isolates (Messi et al., 2005) as well as the ability of such bacteria to transfer them to other ones, their occurrence in drinking water might be considered with particular health significance. So, although there is no clear cut off evidence of responsibility of these plasmids in huge prevalence of more resistant bacteria, the presence of such plasmid-harboring bacteria in drinking water must be taken seriously. V- Conclusion
In conclusion, the increases in antibiotic resistance in bottled water heterotrophic bacteria (HPC) and particularly among the members of Pseudomonas and somewhat Aeromonas is of clinical concern, both because this kind of water is ready to consume commodity, and because some of these bacteria can act as real pathogens and induce infections in humans. Fortunately, in this study, many antibiotics are still active against opportunists such as Aeromonas spp. and Pseudomonas spp. Overall, irrespective of some exceptions, the other heterotrophic bacteria (HPC) and Aeromonas spp. have showed more Damascus University Journal for BASIC SCIENCES Vol. 25, No 1, 2009
susceptibility to available antibiotics. Moreover, some of the antibiotics have maintain their complete activity in these situations. It has been observed that might be somewhat potential relationship between the load of heterotrophic bacteria and the level of antibiotic resistance. So, in our study, increased HPCs had mostly led to increased resistance to antibiotics among more virulent bacteria like Pseudomona spp. and Aeromonas spp. It is recommended, upon the results obtained, to consider the risk of presence of such resistant bacteria in bottled mineral water in the Syrian market. Partially, this may be achieved by routine monitoring and applying the international criteria for bottling and importing of these goods. Daood - Risk Assessment of Heterotrophic Bacteria from Bottled Mineral Water …
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