Ecotoxicology and Environmental Safety 106 (2014) 188–194
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Prevalence of antibiotic resistance in bacteria isolated from drinking well water available in Guinea-Bissau (West Africa) A. Machado a,b,n, A.A. Bordalo a,b a Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences (ICBAS-UP), University of Porto, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal b Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR ), University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal
art ic l e i nf o
a b s t r a c t
Article history: Received 4 November 2013 Received in revised form 21 April 2014 Accepted 23 April 2014 Available online 20 May 2014
The dissemination of antibiotic-resistant bacteria and the spread of antibiotic resistance genes are a major public health concern worldwide, being even proposed as emerging contaminants. The aquatic environment is a recognized reservoir of antibiotic resistant bacteria, and antibiotic resistance genes have been recently detected in drinking water. In this study, the water quality and the prevalence of antibiotic resistance of heterotrophic culturable bacteria were characterized seasonally in wells that serve the population of Guinea-Bissau (West Africa) as the sole source of water for drinking and other domestic proposes. The results revealed that well water was unfit for human consumption independently of the season, owing to high acidity and heavy fecal contamination. Moreover, potentially pathogenic bacteria, which showed resistance to the most prescribed antibiotics in Guinea-Bissau, were isolated from well water, posing an additional health risk. Our results suggest that well water not only fosters the transmission of potential pathogenic bacteria, but also represents an important reservoir for the proliferation of antibiotic resistant bacteria, that can aggravate the potential to cause disease in a very vulnerable population that has no other alternative but to consume such water. & 2014 Elsevier Inc. All rights reserved.
Keywords: Hand-dug well Water quality Antibiotic resistance Drinking water Africa
1. Introduction Water is indispensable for the maintenance of living organisms and ecosystem health, but 783 million people worldwide continued to use unimproved sources to meet their drinking water needs (WHO and UNICEF, 2010). Because of its importance to human existence and public health, much of the concern about drinking water has focused on microbial water quality, and the occurrence of pathogens (Szewzyk et al., 2000; Berry et al., 2006). The spread of antibiotic resistance bacteria and antibiotic resistance genes in the environment has been the subject of growing concern. Antibiotic resistance is naturally present in the environment, since most antibiotics used for treating infections have an environmental origin, being produced by microorganisms (Martínez, 2008). Indeed, natural environments like the aquatic one represents a reservoir of antibiotic resistance contributing for the spread and evolution of antibiotic resistance genes (e.g. Baquero et al., 2008; Martínez, 2008; Falcone-Dias et al., 2012). As the human population grow, anthropogenic pressures increase, including the use of antibiotics in medicine, agriculture, and n Corresponding author at: Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal. E-mail address: [email protected] (A. Machado).
http://dx.doi.org/10.1016/j.ecoenv.2014.04.037 0147-6513/& 2014 Elsevier Inc. All rights reserved.
veterinary. In this way, the increased prevalence of antibiotic resistance genes observed in the aquatic environment potentially favor further recombination and horizontal gene transfer (e.g. Baquero et al., 2008; Martínez, 2008; Zhang and Zhang, 2009; Lupo et al., 2012; Farkas et al., 2013). Presently, the presence of antibiotic resistance genes have been confirmed in different environments being recognized as a new emerging contaminant in the environment (Wells et al., 2007; Zou et al., 2011). Recent studies emphasized the presence of antibiotic resistance bacteria and antibiotic resistant genes in drinking water (e.g. Schwartz et al., 2003; Faria et al., 2009; Xi et al., 2009; VazMoreira et al., 2011; Falcone-Dias et al., 2012; Farkas et al., 2013; Shi et al., 2013), a potential risk to human and animals. Due to the potential direct transmission of antibiotic resistant pathogens to humans that may decrease the efficiency of the antibiotic therapy, and/or promote the transmission of antibiotic resistance genes to commensal or opportunist bacteria, drinking water may represent an important source of antibiotic resistance (Schwartz et al., 2003). This issue takes on special relevance in developing countries where the access to potable water is limited, and drinking water may be retrieved directly from contaminated environmental sources, and used without any treatment. The WHO and UNICEF (2010) reported that 37 percent of the people without access to improved sources of drinking water live in sub-Saharan Africa. In Guinea-Bissau, a West Africa country of
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1.6 million inhabitants living under extreme poverty, water supply and sanitation cover less than half of the population (INEC, 2004), and only 13.6 percent of the population has access to potentially potable water and no wastewater treatment is available (Bordalo and Savva-Bordalo, 2012). The population retrieves water for daily use, including drinking from open/unprotected shallow hand-dug wells, 80 percent of which are contaminated with fecal materials and have an acid pH (Bordalo and Savva-Bordalo, 2007). Traditionally, the population defecates on the wild, and the distance of the few existing latrines is often insufficient to avoid contamination of the water source with human-pathogenic microorganisms increasing the rise of contamination. In addition, animals such as cows, goats, chickens and pigs wander freely enabling the rise of contamination. Cholera periodically ravages the country, and during the 2008 epidemic 14228 cases of cholera were diagnosed, and 225 people died (Luquero et al., 2011). A 2012 outbreak yielded 3280 cases and 22 deaths (A.A. Bordalo, unpublished). Although some recent studies concerning water quality in Africa are available (e.g. Abdelrahman and Eltahir, 2011; Chigor et al., 2012), few were undertaken on shallow well water (Bordalo and Savva-Bordalo, 2007; Pritchard et al., 2007), and even fewer dealt with antibiotic resistance (e.g. Inyang, 2009; Emmanuel et al., 2011; Odeyemi et al., 2012). This study was designed to help respond to the need of knowledge concerning the water quality and antimicrobial resistance patterns in different environments towards the understanding of the associated human health risk and to develop global strategies to fight against infectious diseases. In this way, we investigated the water quality and antibiotic resistance susceptibility pattern of culturable bacteria from wells, during the dry and wet season, that serve the population as the sole source of water for drinking and other domestic uses.
2. Material and methods 2.1. Sample collection Four major wells that supply population with water for domestic use were visited during the dry (DS) and wet (WS) season of 2010 in Guinea-Bissau. Dry season spans from November to May, and wet season from June to October, according to annual rainfall pattern. The wells were chosen from a larger set according to the number of people they served and the microbial contamination previously found (Bordalo and Savva-Bordalo, 2007; Bordalo and Savva-Bordalo, 2012). Two wells where located within the city of Bissau (#1, #18), the country capital, and the other two in Bolama (#16, #50), a major island off the coast of Guinea-Bissau (11 1N), and the former colonial capital. The exact position of each well was obtained by means of GPS (Magellan 600). Water samples were collected using 500 ml plastic sterile flasks. All samples were kept in the dark, refrigerated in ice chests with shredded ice available locally, and processed no later than 3.5 h after collection, in a mobile laboratory similar to the one described in Bordalo and Savva-Bordalo (2007).
2.2. Physical and chemical parameters Temperature, conductivity, turbidity, dissolved oxygen, oxygen saturation, and pH were measured in situ, using a Hanna Instruments 9828 m. Samples for color, nitrate, nitrite, ammonium, aluminum, copper, iron, chromium and cyanide were assayed in a 12 V multiparameter Hanna HI83200 photometer (www.hannacom. pt), following the manufacturer instructions.
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2.4. Bacterial isolation and preliminary characterization Spread plate method was used with Yeast extract agar (Oxoid CM19) for examination of heterotrophic colony forming bacteria. This is a non-selective media suitable for the isolation of fresh-water aerobic heterotrophic viable bacteria. An appropriate volume or decimal dilutions in sterile saline were used for inoculation. Triplicate plates were incubated for 24 h at 37 1C. After visual examination, representatives of all colony types were randomly selected for further culture isolation and analyzes. Pure cultures were preserved at 80 1C in Luria–Bertani broth supplemented with glycerol (final concentration 25 percent). Colony morphology, Gram staining and lactose fermenting test were performed according to Smibert and Krieg (1994), for preliminary characterization of the bacterial isolates. 2.5. Antibiogram assay The antibiotic susceptibility of the isolates was assessed by the disk diffusion method on Mueller-Hinton agar according to the standard test method for antimicrobial disc susceptibility tests (CLSI, 2007). The inoculum density was adjusted to 0.5 McFarland turbidity standard and incubated at 37 1C for 24 h. The bacterial isolates were screened against the five most prescribed antibiotics in Guinea-Bissau according to records retrieved from Bolama regional Hospital logbook: ampicillin (10 mg), chloramphenicol (30 mg), doxycycline (30 mg), amoxicillin þclavulanic acid (20 þ 10 mg), and gentamicin (10 mg). The strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as quality controls. 2.6. Bacterial identification Total DNA from each bacterial isolate was extracted as described in Ausubel et al. (1990). The 16S rRNA sequences for bacterial identification were generated following the PCR conditions and primer set (27F/1492R), outlined in Lane (1991). The 1400 bp amplicons were excised from bands using Illustra GFX PCR DNA and Gel band purification (Amersham Biosciences, Buckinghamshire, UK), and sequenced by STABVIDA (Lisbon, Portugal). All sequences generated were used to query the Genbank database using the Basic Local Alignment Search Tool BLAST (Altschul et al., 1990), in order to identify the closest type strain. The 16S rRNA sequences determined in this study have been deposited in the GenBank database under accession numbers KF819659–KF819735.
3. Results 3.1. Water quality Since there are no guidelines in Guinea-Bissau (drinking water), the key water quality parameters were compared to parametric values from the WHO (2006), EU (1998) and UK (Tebutt, 1998), guidelines to ascertain if the quality of the water was fitted for human consumption. The water quality was analyzed seasonally at each study site (Table 1), revealing that all wells were grossly polluted with fecal materials with an increase in the wet season. The pH was very acidic (average 5.14), being all wells out of the suitable range for drinking water according to EU standards (6.5–9.0). The water temperature averaged 29.1 1C, above the 25 1C recommended temperature for drinking water according UK standards. The water conductivity was moderate, in the range 226–712 mS/cm (i.e. no salinity intrusion was detected), with rather low oxygen contents averaging 3.29 mg l 1 (41.3% dissolved oxygen saturation) confirming the presence of organic contamination. Extremely high ammonium concentrations, color and turbidity values were found at well #1, especially during the dry season. However, nitrate, nitrite and metal concentrations were under the parametric values for drinking water.
2.3. Bacterial analysis
3.2. Antibiotic susceptibility
Samples for presumptive fecal coliforms (FC), and presumptive fecal enterococci (FE) enumerations were filtered onto sterile cellulose nitrate membranes (0.45 mm pore size, 47 mm diameter, Whatman, UK), by means of a hand-pump, placed in mFC-agar (Difco) and Slanetz–Bartley agar (Oxoid) plates, respectively, and incubated at 44.5 1C for 24 h (FC) or 48 h (FE). Typical colonies were counted and results expressed as colony forming units (CFU) 100 ml 1.
Antibiotic resistance was found in all wells and seasons, and resistance phenotypes were observed to all antibiotic tested (Fig. 1). A total of ninety-two strains were isolated from the well water examined. Overall, 65 percent of the strains evidenced resistance to at least one antibiotic, with furthermore 12 percent
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Table 1 Key water quality parameters analyzed during the dry and wet seasons surveys in Guinea-Bissau (FC – Presumptive fecal coliform; FE – Presumptive fecal enterococci; Temp. – Temperature; Cond. – Conductivity; TUR – turbidity; D.O. – dissolved oxygen). Samples FC (CFU 100 ml−1)
FE (CFU 100 ml−1)
pH
Temp. (1C)
Cond. Turv (lS cm 1) (NTU)
Color (mg l−1 PtCo)
D.O. NO3 NH4þ Fe Cu Cr Al (mg l 1) (mg l 1) (mg l 1) (mg l 1) (lg l 1) (lg l 1) (mg l−1)
Cn (lg l 1)
Dry season
# # # #
1 16 18 50
5440 17550 1088 5400
4480 10800 36 240
5.99 4.91 5.08 4.74
27.0 28.9 32.0 29.1
598 200 582 273
4.79 2.40 2.07 0.67
30 16 n.d. n.d.
2.63 3.19 4.47 3.13
29.5 24.40 16.10 43.50
1.57 0.23 0.14 0.07
45 29 73 32
n.a. n.d. n.a. n.d.
n.a. n.d. n.a. n.d.
n.d. 110 n.d. 22
n.a. n.d. n.a. n.d.
Wet season
# # # #
1 16 18 50
9760 24300 0 14850
320 5600 30 4725
5.47 4.78 5.42 4.76
28.1 29.0 29.6 29.2
712 325 226 411
9.64 0.67 3.27 0.45
83 n.d. 17 n.d.
2.16 3.01 4.28 3.26
30.10 15.50 15.60 20.60
2.53 0.07 0.04 n.d.
68 49 62 42
151 185 56 1
20 n.d. 3 n.d.
n.d. 150 n.d. 20
1 10 1 n.d.
n.a. – non-assayed; n.d. – non-detected.
Burkholderia, Citrobacter, Hylemonella, Pseudomonas, Rhodococcus, and Salmonella one percent. The wet season showed a higher diversity with twelve of the sixteen genera found whereas in the dry season only eight different genera were present. Antibiotic resistance at least one antibiotic tested was observed for all but two genera (Rhodococcus, Arthrobacter). Isolates affiliated to the same genera found in the same well and season exhibited distinct antibiotic profiles (Table 2).
4. Discussion
Fig. 1. Percentage of isolates showing resistance to each antibiotic tested in the dry and wet season (n¼ 60).
showing some degree of intermediate resistance (Table 2). The number of isolates with some degree of resistance (resistance to 1 or more antibiotic) in the dry season (72 percent) was higher than in the wet season (59 percent). However, the wet season showed a higher number of isolates resistant to three or more antibiotics (22 percent of the isolates retrieved in the wet season compared with only nine percent in the dry season; Fig. 2). Ampicilin (55 percent), amoxicillin þclavulanic acid (33 percent) and gentamicin (32 percent) were the antibiotics with higher percentages of resistance (Fig. 1). Moreover, 60 percent of the environmental isolates showing resistance were simultaneously resistant to ampicilin and amoxicillin þclavulanic acid, the two most prescribed antibiotics among the five (Table 2). Resistance to ampicilin and amoxicillin þclavulanic acid was preferably evidenced by the resistant bacteria in the dry season, whereas resistance to gentamicin was present in a higher number of isolates from the wet season (Fig. 1). 3.3. Bacterial identification The large majority (85 percent) of the ninety-two isolates recovered from the well water samples were gram negative bacilli, and 43 percent were lactose fermenting bacteria. From the total number of ninety-two strains characterized based on 16S rRNA gene sequence, only 77 showed enough quality for further analysis. The sequences recovered shared 16–100 percent identities among each other, and 99 percent or more identity to known, culturable bacteria. The phylogenetic analysis revealed the existence of sixteen different genera (Table 2) as follows: Aquitalea nineteen percent; Acinetobacter eighteen percent; Ralstonia seventeen percent; Acidovorax ten percent; Chromobacterium ten percent; Chryseobacterium six percent; Enterobacter four percent; Klebsiella three percent; Xenophilus three percent; Arthrobacter,
In Guinea-Bissau, one of the poorest countries in the world according to the Human Development Index (rank 175 out of 177 countries in 2008), the sole source of water for daily needs, including drinking water, for the majority of the population are open/unprotected, shallow hand-dug wells (o15 m). This study was based on the hypothesis that well water may represent an important source of transmission of bacteria to humans, not only potential pathogenic bacteria but also antibiotic resistant microbes that can have an aggravated potential to cause disease. Moreover, to date there have been few studies investigating the role of antibiotics and bacterial resistances outside the clinical environment (Walsh, 2013). In respect to the water quality assessment, our results followed the trend of previous studies developed in the same area (Bordalo and Savva-Bordalo, 2007, 2012). All studied wells were fecal contaminated throughout the year but the situation deteriorated further in the wet season. The proximity of latrines (o 30 m) whose influence has been previously described (e.g. Abdelrahman and Eltahir, 2011, Escamilla et al., 2013), but also the presence of freely wandering domestic animals and the contact of the well bucket and rope with contaminated soil, contributed to the situation. However, the rain water infiltration and percolation, facilitated by the sandy nature of soil were probably the driving forces for the mobilization and consequent increment of presumptive fecal coliforms counts found in the wet season (e.g. Bordalo and Savva-Bordalo, 2007; Pritchard et al., 2007; Bennett et al., 2010, Abdelrahman and Eltahir, 2011). Within the physical and chemical parameters, the pH stand out with all the samples in the acid to very acid range as a result of the soil characteristics in West Africa (Jalloh et al.,2012). This problem can have a major impact in the population health, particularly in dental erosion (O'Sullivan and Milosevic, 2008), since the country health system is fragile and scarce, but also in the potential mobilization of metals (Calmano et al., 1993). The high temperature observed throughout the year is known to foster microbial growth (Shiah and Ducklow, 1997) exacerbating furthermore the poor water quality. The absence of rain and consequent extremely low water level in the
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Table 2 Antibiotic resistance pattern of isolated bacteria from well water samples (S/white: sensitive; I/light gray: intermediate resistance; R/gray: resistant). Number of isolates Dry season
Wet season
Aquitalea
7
8
Rhodococcus Arthrobacter Acinetobacter
1 1 9
0 0 5
Chromobacterium
3
5
12
1
Acidovorax
8
0
Xenophilus
2
0
Chryseobacterium
0
5
Pseudomonas Enterobacter
0 0
1 3
Klebsiella
0
2
Ralstonia
Isolate
D16_1 D16_5 D16_10 D18_9 D50_3 D50_4 D50_7 W1_10 W1_12 W14_4 W14_8 W14_11 W18_5 W50_2 W50_5 D16_2 D16_3 D1_1 D1_2 D1_3 D1_4 D1_5 D1_7 D1_9 D1_11 D1_13 W1_4 W14_12 W18_3 W50_4 W50_7 D50_1 D50_5 D50_6 W1_9 W1_13 W14_5 W50_3 W50_10 D16_4 D18_1 D18_2 D18_3 D18_4 D18_5 D18_6 D18_7 D18_8 D18_10 D50_2 D50_8 W14_2 D1_6 D1_8 D1_10 D1_12 D16_6 D16_8 D50_9 D50_10 D16_7 D16_9 W1_1 W1_6 W14_1 W14_7 W50_8 W1_8 W1_11 W18_6 W18_7 W14_10 W18_11
Resistance pattern Chloramphenicol
Doxycycline
Ampicillin
Amoxicillin þClavulanic acid
Gentamicin
S S S S S S S S S S S S S S I S S I I I I R R R I R R I R S R S S S S I S S S I S I I I I S I I I R R I S S S S S S S S R S R R I I I R S I S I S
S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S R S S R S S S R I I S I S
S S S S S I I S I R R R I S R S S R R R R R R R R I R I S S R R R R R R R R R S R R R S I S R R S S R S R R R R R S R R R R R R R R R R R R S R R
S S S S S S S S S R R R S S R S S I R R R R I R S I I S S S S R R R R R R R R S R R R S I S R R R S R S S S S S S R S S R R R R R R R R R R S R S
S S S S S S S S S R S S S S S S S S S S S S S S S R S S S S S S S S R R S R R R S S S S S S S S S R R R S S S S S S S S S S R R R I R S S I R I S
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Table 2 (continued ) Number of isolates Dry season Hylemonella Burkholderia Citrobacter Salmonella
0 0 0 0
Isolate
Wet season 1 1 1 1
W18_2 W14_3 W14_6 W14_9
Resistance pattern Chloramphenicol
Doxycycline
Ampicillin
Amoxicillin þClavulanic acid
Gentamicin
S S I I
S S I S
I R R I
S R R S
S R I S
Table 3 Ecological niches and major roles of the bacterial isolates from well water in Guinea-Bissau. Genera
Ecological niche
Major roles
Aquitalea Rhodococcus
Humic lakes Soil, water, eukaryotic cells Soil Soil
Denitrification Biodegradation, phyto and animal pathogen, nosocomial Bioremediation Nosocomial infections: skin/ wound infections, bacteremia, meningitis Inflammation of tissue and glands, septicemia Nosocomial infections: bacteremia, septicemia; phytopathogen Nosocomial infections; phytopathogen Nosocomial infections: peritonitis; Biodegradation Nosocomial infections: bacteremia Opportunistic human, plant and animal pathogen Nosocomial infections; biological control of plant disease Human opportunistic pathogens: pneumonia, urinary tract infections, septicemia N2O production Human, phyto pathogen; nosocomial, biodegradation Nosocomial infections: respiratory and urinary tract, blood, meningitis, septicemia; reduction of nitrate to nitrite; bioremediation Gastroenteritis, thyphoid fever, infection of liver spleen and bone marrow
Arthrobacter Acinetobacter
Chromobacterium Soil, water Fig. 2. Percentage of isolates showing sensitivity or resistance to one (R1), two (R2) and 3 or more (R3þ ) antibiotics in the dry and wet season surveyed (n¼ 92).
dry season leads to sediment resuspension due to higher water residence time and increased contact with earth walls and bottom, possibly explaining the higher values of ammonium, color and turbidity found. Moreover, sediments seem to play a major role in what well water quality is concerned in both seasons; not only are known reservoirs for potential pathogenic bacteria (Johnson et al., 2012; Bö er et al., 2013), but also can hamper disinfection (Holf, 1978). Indeed, the use of chlorine to disinfect wells during a cholera epidemic in Guinea-Bissau failed due to the rapid degradation of the disinfection agent (Rowe, 1998). Antibiograms of the bacterial isolates from well water reveal resistance to the most prescribed antibiotics in Guinea-Bissau. The majority of the isolates were resistant to at least one antibiotic and some were even resistant to all antibiotics at the tested concentrations. Although this study was not designed to discriminate between natural and acquired resistance, the presence of isolates from the same well and season, associated to the same genus but with distinct resistance profiles, suggested that the resistance phenotypes were not stable and may be acquired, being an ubiquitous process occurring in acidic well water. Consistently with other studies (Okonkwo et al., 2008; Odeyemi et al., 2012), our results revealed the presence of 16 bacterial genera. The increased number of bacterial genera complies with the higher number of fecal indicators founded in the wet season, can be explained by the infiltration and percolation from the surface that transport suspension particles with associated bacteria and fecal contamination of the nearby environment (Bö er et al., 2013; Escamilla et al., 2013). Members of the genera found in this study (Table 3) are commonly detected in natural environments, clinical samples and even related with beneficial functions (e.g. Bagley, 1985; Camargo et al., 2004; Ryan et al., 2006). Additionally to the presence of the microorganisms and the existence of antibiotic resistance genes in the natural or wild bacteria, the concern nowadays is the transference of this resistance to other bacteria, potentially pathogenic (D'Costa et al., 2011; Falcone-Dias et al., 2012). Indeed, potentially pathogens members of Salmonella, Klebsiella, Enterobacter, Pseudomonas, Acinetobacter, Burkholderia or Chromobacterium genera were isolated from the well water samples investigated in this study. The detection of members of the genera Salmonella in the water used for domestic
Ralstonia
Soil
Acidovorax
Soil, water
Xenophilus
Soil, Air
Chryseobacterium Soil, water, plants, clinical Pseudomonas Enterobacter
Klebsiella
Hylemonella Burkholderia
Soil, water, plants, animal tissue Soil, water, sewage, human and animal skin and intestinal tract Ubiquitous, skin, pharynx, or gastrointestinal tract in humans Water Soil, groundwater
Citrobacter
Ubiquitous, soil, water, sewage
Salmonella
Contaminated water, raw eggs, poultry and red meat
use, including drinking is of particular concern, since they have been associated with gastrointestinal infections like dysentery and diarrhea, as well as typhoid and paratyphoid fever (EPA, 2003). These diseases still remain a heavy burden in Africa and have been particularly complicated by the recent emergence of resistance to frontline antibiotics in the treatment of typhoid and non-typhoid Salmonella infections (e.g. chloramphenicol; Khandeparkar 2010; Okoro et al., 2012), and also by the African HIV epidemic (Okoro et al., 2012; Feasey and Gordon, 2013). Pseudomonas members are human opportunistic pathogens responsible for infections in the pulmonary and urinary tract and wounds, being particularly relevant in immunocompromised individuals (Iglewski, 1996). The low susceptibility to antibiotics and the facility in developing antibiotic resistance either by mutation or horizontal gene transfer (Poole, 2004), add importance and alarm to the presence of members of this genus in drinking water. The genera Klebsiella, Enterobacter, and Citrobacter detected are ubiquitous in nature and
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can be found as members of the normal intestinal flora of humans and animals, and in a variety of environmental sources (Guentzel, 1996). However, they are also opportunistic pathogens responsible for a wide range of infections and consequent major health problems worldwide (Guentzel, 1996). The presence of members of genera commonly associated with nosocomial infections such as Acinetobacter, Ralstonia, Chyseobacterium and Burkolderia can prove to be an additional major cause of concern since the multi-antibiotic resistance favor the thrive of these bacteria, and can make the antibiotic therapy problematic (Berger et al., 2006). Moreover, it seems that these potentially pathogens members were found preferentially in the wet season reinforcing the hypothesis of contamination of the well water by infiltration and percolation from the above surface (Table 3). This fact is of great health significance, even more when the isolates hold antibiotic resistance. Not only the water quality was compromised, being people exposed to those potential pathogenic bacteria and prone to disease, but also the chance of a successful antibiotic therapy decreases. The situation is further aggravated by the fact that, often, the prescribed antibiotic is the one currently available from a very limited choice, and second-line antibiotics are virtually non-existent in the country. Our results were in accordance with Farkas et al. (2013), reveling similarities in the route of introduction and spread of bacterial species carrying antibiotic resistance genes into the environment, mainly connected with animal and anthropogenic disturbance in two restricted areas, and very different drinking water sources investigated in Europe and Africa. In a country, like Guinea-Bissau, where the population is vulnerable (HIV, malaria, cholera epidemic, malnutrition), where the health system is fragile and not universally available, and the population has no access to other source of drinking water apart from contaminated well water, the exposure to antibiotic resistant bacteria can further exarcebate the health risk, particularly to the very young, the elderly, the immune compromised, or with chronic conditions (Falcone-Dias et al., 2012). Available studies showed that chlorination, a recommended disinfection method can prove to be ineffective as in the case of Guinea-Bissau conditions (Rowe, 1998), and additionally can favor the selection of antibiotic resistant bacteria (Armstrong et al., 1981; Murray et al., 1984; Shrivastava et al., 2004; Xi et al., 2009). Although overall it still remains an effective and inexpensive method to reduce the microbial contamination in water, this reality adds new challenges to the internationally accepted approach to improve water quality in developing countries and should not be overlooked.
5. Conclusions This study showed that the well water used for drinking and other domestic proposes was unfit for human consumption, being very acidic and heavily contaminated with fecal material. Additionally, well water was confirmed as a potential source of antibiotic resistant bacteria. Potentially pathogenic bacteria which showed resistance to the most prescribed antibiotics in GuineaBissau have been isolated from well water and pose a serious health risk. These bacteria can serve as reservoirs for antibiotic resistance genes that can enter, adapt and thrive in the ecosystem, and reduce the chances of a successful antibiotic therapy in the already vulnerable and more susceptible to disease population.
Acknowledgments We thank AMI (International Medical Assistance) and AIDA (Ayuda, Intercambio y Desarrollo), for logistic support, and Mr. Alfa
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for fieldwork assistance. This study was partially funded through a Ph.D. fellowship to A. Machado (SFRH/BD/46146/2008) cofinanced by POPH/FSE, and a grant to A. Bordalo (PTDC/AAC-CLI/ 103539/2008). This research was also partially supported by the European Regional Development Fund (ERDF) through the COMPETE – Operational Competitiveness Program and Portuguese national funds through FCT – Foundation for Science and Technology, Portugal under the Project “PEst-C/MAR/LA0015/2013”.
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Prevalence of antibiotic resistance in bacteria isolated from drinking well water available in Guinea-Bissau (West Africa) A. Machado a,b,n, A.A. Bordalo a,b a Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences (ICBAS-UP), University of Porto, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal b Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR ), University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal
art ic l e i nf o
a b s t r a c t
Article history: Received 4 November 2013 Received in revised form 21 April 2014 Accepted 23 April 2014 Available online 20 May 2014
The dissemination of antibiotic-resistant bacteria and the spread of antibiotic resistance genes are a major public health concern worldwide, being even proposed as emerging contaminants. The aquatic environment is a recognized reservoir of antibiotic resistant bacteria, and antibiotic resistance genes have been recently detected in drinking water. In this study, the water quality and the prevalence of antibiotic resistance of heterotrophic culturable bacteria were characterized seasonally in wells that serve the population of Guinea-Bissau (West Africa) as the sole source of water for drinking and other domestic proposes. The results revealed that well water was unfit for human consumption independently of the season, owing to high acidity and heavy fecal contamination. Moreover, potentially pathogenic bacteria, which showed resistance to the most prescribed antibiotics in Guinea-Bissau, were isolated from well water, posing an additional health risk. Our results suggest that well water not only fosters the transmission of potential pathogenic bacteria, but also represents an important reservoir for the proliferation of antibiotic resistant bacteria, that can aggravate the potential to cause disease in a very vulnerable population that has no other alternative but to consume such water. & 2014 Elsevier Inc. All rights reserved.
Keywords: Hand-dug well Water quality Antibiotic resistance Drinking water Africa
1. Introduction Water is indispensable for the maintenance of living organisms and ecosystem health, but 783 million people worldwide continued to use unimproved sources to meet their drinking water needs (WHO and UNICEF, 2010). Because of its importance to human existence and public health, much of the concern about drinking water has focused on microbial water quality, and the occurrence of pathogens (Szewzyk et al., 2000; Berry et al., 2006). The spread of antibiotic resistance bacteria and antibiotic resistance genes in the environment has been the subject of growing concern. Antibiotic resistance is naturally present in the environment, since most antibiotics used for treating infections have an environmental origin, being produced by microorganisms (Martínez, 2008). Indeed, natural environments like the aquatic one represents a reservoir of antibiotic resistance contributing for the spread and evolution of antibiotic resistance genes (e.g. Baquero et al., 2008; Martínez, 2008; Falcone-Dias et al., 2012). As the human population grow, anthropogenic pressures increase, including the use of antibiotics in medicine, agriculture, and n Corresponding author at: Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal. E-mail address: [email protected] (A. Machado).
http://dx.doi.org/10.1016/j.ecoenv.2014.04.037 0147-6513/& 2014 Elsevier Inc. All rights reserved.
veterinary. In this way, the increased prevalence of antibiotic resistance genes observed in the aquatic environment potentially favor further recombination and horizontal gene transfer (e.g. Baquero et al., 2008; Martínez, 2008; Zhang and Zhang, 2009; Lupo et al., 2012; Farkas et al., 2013). Presently, the presence of antibiotic resistance genes have been confirmed in different environments being recognized as a new emerging contaminant in the environment (Wells et al., 2007; Zou et al., 2011). Recent studies emphasized the presence of antibiotic resistance bacteria and antibiotic resistant genes in drinking water (e.g. Schwartz et al., 2003; Faria et al., 2009; Xi et al., 2009; VazMoreira et al., 2011; Falcone-Dias et al., 2012; Farkas et al., 2013; Shi et al., 2013), a potential risk to human and animals. Due to the potential direct transmission of antibiotic resistant pathogens to humans that may decrease the efficiency of the antibiotic therapy, and/or promote the transmission of antibiotic resistance genes to commensal or opportunist bacteria, drinking water may represent an important source of antibiotic resistance (Schwartz et al., 2003). This issue takes on special relevance in developing countries where the access to potable water is limited, and drinking water may be retrieved directly from contaminated environmental sources, and used without any treatment. The WHO and UNICEF (2010) reported that 37 percent of the people without access to improved sources of drinking water live in sub-Saharan Africa. In Guinea-Bissau, a West Africa country of
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1.6 million inhabitants living under extreme poverty, water supply and sanitation cover less than half of the population (INEC, 2004), and only 13.6 percent of the population has access to potentially potable water and no wastewater treatment is available (Bordalo and Savva-Bordalo, 2012). The population retrieves water for daily use, including drinking from open/unprotected shallow hand-dug wells, 80 percent of which are contaminated with fecal materials and have an acid pH (Bordalo and Savva-Bordalo, 2007). Traditionally, the population defecates on the wild, and the distance of the few existing latrines is often insufficient to avoid contamination of the water source with human-pathogenic microorganisms increasing the rise of contamination. In addition, animals such as cows, goats, chickens and pigs wander freely enabling the rise of contamination. Cholera periodically ravages the country, and during the 2008 epidemic 14228 cases of cholera were diagnosed, and 225 people died (Luquero et al., 2011). A 2012 outbreak yielded 3280 cases and 22 deaths (A.A. Bordalo, unpublished). Although some recent studies concerning water quality in Africa are available (e.g. Abdelrahman and Eltahir, 2011; Chigor et al., 2012), few were undertaken on shallow well water (Bordalo and Savva-Bordalo, 2007; Pritchard et al., 2007), and even fewer dealt with antibiotic resistance (e.g. Inyang, 2009; Emmanuel et al., 2011; Odeyemi et al., 2012). This study was designed to help respond to the need of knowledge concerning the water quality and antimicrobial resistance patterns in different environments towards the understanding of the associated human health risk and to develop global strategies to fight against infectious diseases. In this way, we investigated the water quality and antibiotic resistance susceptibility pattern of culturable bacteria from wells, during the dry and wet season, that serve the population as the sole source of water for drinking and other domestic uses.
2. Material and methods 2.1. Sample collection Four major wells that supply population with water for domestic use were visited during the dry (DS) and wet (WS) season of 2010 in Guinea-Bissau. Dry season spans from November to May, and wet season from June to October, according to annual rainfall pattern. The wells were chosen from a larger set according to the number of people they served and the microbial contamination previously found (Bordalo and Savva-Bordalo, 2007; Bordalo and Savva-Bordalo, 2012). Two wells where located within the city of Bissau (#1, #18), the country capital, and the other two in Bolama (#16, #50), a major island off the coast of Guinea-Bissau (11 1N), and the former colonial capital. The exact position of each well was obtained by means of GPS (Magellan 600). Water samples were collected using 500 ml plastic sterile flasks. All samples were kept in the dark, refrigerated in ice chests with shredded ice available locally, and processed no later than 3.5 h after collection, in a mobile laboratory similar to the one described in Bordalo and Savva-Bordalo (2007).
2.2. Physical and chemical parameters Temperature, conductivity, turbidity, dissolved oxygen, oxygen saturation, and pH were measured in situ, using a Hanna Instruments 9828 m. Samples for color, nitrate, nitrite, ammonium, aluminum, copper, iron, chromium and cyanide were assayed in a 12 V multiparameter Hanna HI83200 photometer (www.hannacom. pt), following the manufacturer instructions.
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2.4. Bacterial isolation and preliminary characterization Spread plate method was used with Yeast extract agar (Oxoid CM19) for examination of heterotrophic colony forming bacteria. This is a non-selective media suitable for the isolation of fresh-water aerobic heterotrophic viable bacteria. An appropriate volume or decimal dilutions in sterile saline were used for inoculation. Triplicate plates were incubated for 24 h at 37 1C. After visual examination, representatives of all colony types were randomly selected for further culture isolation and analyzes. Pure cultures were preserved at 80 1C in Luria–Bertani broth supplemented with glycerol (final concentration 25 percent). Colony morphology, Gram staining and lactose fermenting test were performed according to Smibert and Krieg (1994), for preliminary characterization of the bacterial isolates. 2.5. Antibiogram assay The antibiotic susceptibility of the isolates was assessed by the disk diffusion method on Mueller-Hinton agar according to the standard test method for antimicrobial disc susceptibility tests (CLSI, 2007). The inoculum density was adjusted to 0.5 McFarland turbidity standard and incubated at 37 1C for 24 h. The bacterial isolates were screened against the five most prescribed antibiotics in Guinea-Bissau according to records retrieved from Bolama regional Hospital logbook: ampicillin (10 mg), chloramphenicol (30 mg), doxycycline (30 mg), amoxicillin þclavulanic acid (20 þ 10 mg), and gentamicin (10 mg). The strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as quality controls. 2.6. Bacterial identification Total DNA from each bacterial isolate was extracted as described in Ausubel et al. (1990). The 16S rRNA sequences for bacterial identification were generated following the PCR conditions and primer set (27F/1492R), outlined in Lane (1991). The 1400 bp amplicons were excised from bands using Illustra GFX PCR DNA and Gel band purification (Amersham Biosciences, Buckinghamshire, UK), and sequenced by STABVIDA (Lisbon, Portugal). All sequences generated were used to query the Genbank database using the Basic Local Alignment Search Tool BLAST (Altschul et al., 1990), in order to identify the closest type strain. The 16S rRNA sequences determined in this study have been deposited in the GenBank database under accession numbers KF819659–KF819735.
3. Results 3.1. Water quality Since there are no guidelines in Guinea-Bissau (drinking water), the key water quality parameters were compared to parametric values from the WHO (2006), EU (1998) and UK (Tebutt, 1998), guidelines to ascertain if the quality of the water was fitted for human consumption. The water quality was analyzed seasonally at each study site (Table 1), revealing that all wells were grossly polluted with fecal materials with an increase in the wet season. The pH was very acidic (average 5.14), being all wells out of the suitable range for drinking water according to EU standards (6.5–9.0). The water temperature averaged 29.1 1C, above the 25 1C recommended temperature for drinking water according UK standards. The water conductivity was moderate, in the range 226–712 mS/cm (i.e. no salinity intrusion was detected), with rather low oxygen contents averaging 3.29 mg l 1 (41.3% dissolved oxygen saturation) confirming the presence of organic contamination. Extremely high ammonium concentrations, color and turbidity values were found at well #1, especially during the dry season. However, nitrate, nitrite and metal concentrations were under the parametric values for drinking water.
2.3. Bacterial analysis
3.2. Antibiotic susceptibility
Samples for presumptive fecal coliforms (FC), and presumptive fecal enterococci (FE) enumerations were filtered onto sterile cellulose nitrate membranes (0.45 mm pore size, 47 mm diameter, Whatman, UK), by means of a hand-pump, placed in mFC-agar (Difco) and Slanetz–Bartley agar (Oxoid) plates, respectively, and incubated at 44.5 1C for 24 h (FC) or 48 h (FE). Typical colonies were counted and results expressed as colony forming units (CFU) 100 ml 1.
Antibiotic resistance was found in all wells and seasons, and resistance phenotypes were observed to all antibiotic tested (Fig. 1). A total of ninety-two strains were isolated from the well water examined. Overall, 65 percent of the strains evidenced resistance to at least one antibiotic, with furthermore 12 percent
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Table 1 Key water quality parameters analyzed during the dry and wet seasons surveys in Guinea-Bissau (FC – Presumptive fecal coliform; FE – Presumptive fecal enterococci; Temp. – Temperature; Cond. – Conductivity; TUR – turbidity; D.O. – dissolved oxygen). Samples FC (CFU 100 ml−1)
FE (CFU 100 ml−1)
pH
Temp. (1C)
Cond. Turv (lS cm 1) (NTU)
Color (mg l−1 PtCo)
D.O. NO3 NH4þ Fe Cu Cr Al (mg l 1) (mg l 1) (mg l 1) (mg l 1) (lg l 1) (lg l 1) (mg l−1)
Cn (lg l 1)
Dry season
# # # #
1 16 18 50
5440 17550 1088 5400
4480 10800 36 240
5.99 4.91 5.08 4.74
27.0 28.9 32.0 29.1
598 200 582 273
4.79 2.40 2.07 0.67
30 16 n.d. n.d.
2.63 3.19 4.47 3.13
29.5 24.40 16.10 43.50
1.57 0.23 0.14 0.07
45 29 73 32
n.a. n.d. n.a. n.d.
n.a. n.d. n.a. n.d.
n.d. 110 n.d. 22
n.a. n.d. n.a. n.d.
Wet season
# # # #
1 16 18 50
9760 24300 0 14850
320 5600 30 4725
5.47 4.78 5.42 4.76
28.1 29.0 29.6 29.2
712 325 226 411
9.64 0.67 3.27 0.45
83 n.d. 17 n.d.
2.16 3.01 4.28 3.26
30.10 15.50 15.60 20.60
2.53 0.07 0.04 n.d.
68 49 62 42
151 185 56 1
20 n.d. 3 n.d.
n.d. 150 n.d. 20
1 10 1 n.d.
n.a. – non-assayed; n.d. – non-detected.
Burkholderia, Citrobacter, Hylemonella, Pseudomonas, Rhodococcus, and Salmonella one percent. The wet season showed a higher diversity with twelve of the sixteen genera found whereas in the dry season only eight different genera were present. Antibiotic resistance at least one antibiotic tested was observed for all but two genera (Rhodococcus, Arthrobacter). Isolates affiliated to the same genera found in the same well and season exhibited distinct antibiotic profiles (Table 2).
4. Discussion
Fig. 1. Percentage of isolates showing resistance to each antibiotic tested in the dry and wet season (n¼ 60).
showing some degree of intermediate resistance (Table 2). The number of isolates with some degree of resistance (resistance to 1 or more antibiotic) in the dry season (72 percent) was higher than in the wet season (59 percent). However, the wet season showed a higher number of isolates resistant to three or more antibiotics (22 percent of the isolates retrieved in the wet season compared with only nine percent in the dry season; Fig. 2). Ampicilin (55 percent), amoxicillin þclavulanic acid (33 percent) and gentamicin (32 percent) were the antibiotics with higher percentages of resistance (Fig. 1). Moreover, 60 percent of the environmental isolates showing resistance were simultaneously resistant to ampicilin and amoxicillin þclavulanic acid, the two most prescribed antibiotics among the five (Table 2). Resistance to ampicilin and amoxicillin þclavulanic acid was preferably evidenced by the resistant bacteria in the dry season, whereas resistance to gentamicin was present in a higher number of isolates from the wet season (Fig. 1). 3.3. Bacterial identification The large majority (85 percent) of the ninety-two isolates recovered from the well water samples were gram negative bacilli, and 43 percent were lactose fermenting bacteria. From the total number of ninety-two strains characterized based on 16S rRNA gene sequence, only 77 showed enough quality for further analysis. The sequences recovered shared 16–100 percent identities among each other, and 99 percent or more identity to known, culturable bacteria. The phylogenetic analysis revealed the existence of sixteen different genera (Table 2) as follows: Aquitalea nineteen percent; Acinetobacter eighteen percent; Ralstonia seventeen percent; Acidovorax ten percent; Chromobacterium ten percent; Chryseobacterium six percent; Enterobacter four percent; Klebsiella three percent; Xenophilus three percent; Arthrobacter,
In Guinea-Bissau, one of the poorest countries in the world according to the Human Development Index (rank 175 out of 177 countries in 2008), the sole source of water for daily needs, including drinking water, for the majority of the population are open/unprotected, shallow hand-dug wells (o15 m). This study was based on the hypothesis that well water may represent an important source of transmission of bacteria to humans, not only potential pathogenic bacteria but also antibiotic resistant microbes that can have an aggravated potential to cause disease. Moreover, to date there have been few studies investigating the role of antibiotics and bacterial resistances outside the clinical environment (Walsh, 2013). In respect to the water quality assessment, our results followed the trend of previous studies developed in the same area (Bordalo and Savva-Bordalo, 2007, 2012). All studied wells were fecal contaminated throughout the year but the situation deteriorated further in the wet season. The proximity of latrines (o 30 m) whose influence has been previously described (e.g. Abdelrahman and Eltahir, 2011, Escamilla et al., 2013), but also the presence of freely wandering domestic animals and the contact of the well bucket and rope with contaminated soil, contributed to the situation. However, the rain water infiltration and percolation, facilitated by the sandy nature of soil were probably the driving forces for the mobilization and consequent increment of presumptive fecal coliforms counts found in the wet season (e.g. Bordalo and Savva-Bordalo, 2007; Pritchard et al., 2007; Bennett et al., 2010, Abdelrahman and Eltahir, 2011). Within the physical and chemical parameters, the pH stand out with all the samples in the acid to very acid range as a result of the soil characteristics in West Africa (Jalloh et al.,2012). This problem can have a major impact in the population health, particularly in dental erosion (O'Sullivan and Milosevic, 2008), since the country health system is fragile and scarce, but also in the potential mobilization of metals (Calmano et al., 1993). The high temperature observed throughout the year is known to foster microbial growth (Shiah and Ducklow, 1997) exacerbating furthermore the poor water quality. The absence of rain and consequent extremely low water level in the
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Table 2 Antibiotic resistance pattern of isolated bacteria from well water samples (S/white: sensitive; I/light gray: intermediate resistance; R/gray: resistant). Number of isolates Dry season
Wet season
Aquitalea
7
8
Rhodococcus Arthrobacter Acinetobacter
1 1 9
0 0 5
Chromobacterium
3
5
12
1
Acidovorax
8
0
Xenophilus
2
0
Chryseobacterium
0
5
Pseudomonas Enterobacter
0 0
1 3
Klebsiella
0
2
Ralstonia
Isolate
D16_1 D16_5 D16_10 D18_9 D50_3 D50_4 D50_7 W1_10 W1_12 W14_4 W14_8 W14_11 W18_5 W50_2 W50_5 D16_2 D16_3 D1_1 D1_2 D1_3 D1_4 D1_5 D1_7 D1_9 D1_11 D1_13 W1_4 W14_12 W18_3 W50_4 W50_7 D50_1 D50_5 D50_6 W1_9 W1_13 W14_5 W50_3 W50_10 D16_4 D18_1 D18_2 D18_3 D18_4 D18_5 D18_6 D18_7 D18_8 D18_10 D50_2 D50_8 W14_2 D1_6 D1_8 D1_10 D1_12 D16_6 D16_8 D50_9 D50_10 D16_7 D16_9 W1_1 W1_6 W14_1 W14_7 W50_8 W1_8 W1_11 W18_6 W18_7 W14_10 W18_11
Resistance pattern Chloramphenicol
Doxycycline
Ampicillin
Amoxicillin þClavulanic acid
Gentamicin
S S S S S S S S S S S S S S I S S I I I I R R R I R R I R S R S S S S I S S S I S I I I I S I I I R R I S S S S S S S S R S R R I I I R S I S I S
S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S R S S R S S S R I I S I S
S S S S S I I S I R R R I S R S S R R R R R R R R I R I S S R R R R R R R R R S R R R S I S R R S S R S R R R R R S R R R R R R R R R R R R S R R
S S S S S S S S S R R R S S R S S I R R R R I R S I I S S S S R R R R R R R R S R R R S I S R R R S R S S S S S S R S S R R R R R R R R R R S R S
S S S S S S S S S R S S S S S S S S S S S S S S S R S S S S S S S S R R S R R R S S S S S S S S S R R R S S S S S S S S S S R R R I R S S I R I S
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Table 2 (continued ) Number of isolates Dry season Hylemonella Burkholderia Citrobacter Salmonella
0 0 0 0
Isolate
Wet season 1 1 1 1
W18_2 W14_3 W14_6 W14_9
Resistance pattern Chloramphenicol
Doxycycline
Ampicillin
Amoxicillin þClavulanic acid
Gentamicin
S S I I
S S I S
I R R I
S R R S
S R I S
Table 3 Ecological niches and major roles of the bacterial isolates from well water in Guinea-Bissau. Genera
Ecological niche
Major roles
Aquitalea Rhodococcus
Humic lakes Soil, water, eukaryotic cells Soil Soil
Denitrification Biodegradation, phyto and animal pathogen, nosocomial Bioremediation Nosocomial infections: skin/ wound infections, bacteremia, meningitis Inflammation of tissue and glands, septicemia Nosocomial infections: bacteremia, septicemia; phytopathogen Nosocomial infections; phytopathogen Nosocomial infections: peritonitis; Biodegradation Nosocomial infections: bacteremia Opportunistic human, plant and animal pathogen Nosocomial infections; biological control of plant disease Human opportunistic pathogens: pneumonia, urinary tract infections, septicemia N2O production Human, phyto pathogen; nosocomial, biodegradation Nosocomial infections: respiratory and urinary tract, blood, meningitis, septicemia; reduction of nitrate to nitrite; bioremediation Gastroenteritis, thyphoid fever, infection of liver spleen and bone marrow
Arthrobacter Acinetobacter
Chromobacterium Soil, water Fig. 2. Percentage of isolates showing sensitivity or resistance to one (R1), two (R2) and 3 or more (R3þ ) antibiotics in the dry and wet season surveyed (n¼ 92).
dry season leads to sediment resuspension due to higher water residence time and increased contact with earth walls and bottom, possibly explaining the higher values of ammonium, color and turbidity found. Moreover, sediments seem to play a major role in what well water quality is concerned in both seasons; not only are known reservoirs for potential pathogenic bacteria (Johnson et al., 2012; Bö er et al., 2013), but also can hamper disinfection (Holf, 1978). Indeed, the use of chlorine to disinfect wells during a cholera epidemic in Guinea-Bissau failed due to the rapid degradation of the disinfection agent (Rowe, 1998). Antibiograms of the bacterial isolates from well water reveal resistance to the most prescribed antibiotics in Guinea-Bissau. The majority of the isolates were resistant to at least one antibiotic and some were even resistant to all antibiotics at the tested concentrations. Although this study was not designed to discriminate between natural and acquired resistance, the presence of isolates from the same well and season, associated to the same genus but with distinct resistance profiles, suggested that the resistance phenotypes were not stable and may be acquired, being an ubiquitous process occurring in acidic well water. Consistently with other studies (Okonkwo et al., 2008; Odeyemi et al., 2012), our results revealed the presence of 16 bacterial genera. The increased number of bacterial genera complies with the higher number of fecal indicators founded in the wet season, can be explained by the infiltration and percolation from the surface that transport suspension particles with associated bacteria and fecal contamination of the nearby environment (Bö er et al., 2013; Escamilla et al., 2013). Members of the genera found in this study (Table 3) are commonly detected in natural environments, clinical samples and even related with beneficial functions (e.g. Bagley, 1985; Camargo et al., 2004; Ryan et al., 2006). Additionally to the presence of the microorganisms and the existence of antibiotic resistance genes in the natural or wild bacteria, the concern nowadays is the transference of this resistance to other bacteria, potentially pathogenic (D'Costa et al., 2011; Falcone-Dias et al., 2012). Indeed, potentially pathogens members of Salmonella, Klebsiella, Enterobacter, Pseudomonas, Acinetobacter, Burkholderia or Chromobacterium genera were isolated from the well water samples investigated in this study. The detection of members of the genera Salmonella in the water used for domestic
Ralstonia
Soil
Acidovorax
Soil, water
Xenophilus
Soil, Air
Chryseobacterium Soil, water, plants, clinical Pseudomonas Enterobacter
Klebsiella
Hylemonella Burkholderia
Soil, water, plants, animal tissue Soil, water, sewage, human and animal skin and intestinal tract Ubiquitous, skin, pharynx, or gastrointestinal tract in humans Water Soil, groundwater
Citrobacter
Ubiquitous, soil, water, sewage
Salmonella
Contaminated water, raw eggs, poultry and red meat
use, including drinking is of particular concern, since they have been associated with gastrointestinal infections like dysentery and diarrhea, as well as typhoid and paratyphoid fever (EPA, 2003). These diseases still remain a heavy burden in Africa and have been particularly complicated by the recent emergence of resistance to frontline antibiotics in the treatment of typhoid and non-typhoid Salmonella infections (e.g. chloramphenicol; Khandeparkar 2010; Okoro et al., 2012), and also by the African HIV epidemic (Okoro et al., 2012; Feasey and Gordon, 2013). Pseudomonas members are human opportunistic pathogens responsible for infections in the pulmonary and urinary tract and wounds, being particularly relevant in immunocompromised individuals (Iglewski, 1996). The low susceptibility to antibiotics and the facility in developing antibiotic resistance either by mutation or horizontal gene transfer (Poole, 2004), add importance and alarm to the presence of members of this genus in drinking water. The genera Klebsiella, Enterobacter, and Citrobacter detected are ubiquitous in nature and
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can be found as members of the normal intestinal flora of humans and animals, and in a variety of environmental sources (Guentzel, 1996). However, they are also opportunistic pathogens responsible for a wide range of infections and consequent major health problems worldwide (Guentzel, 1996). The presence of members of genera commonly associated with nosocomial infections such as Acinetobacter, Ralstonia, Chyseobacterium and Burkolderia can prove to be an additional major cause of concern since the multi-antibiotic resistance favor the thrive of these bacteria, and can make the antibiotic therapy problematic (Berger et al., 2006). Moreover, it seems that these potentially pathogens members were found preferentially in the wet season reinforcing the hypothesis of contamination of the well water by infiltration and percolation from the above surface (Table 3). This fact is of great health significance, even more when the isolates hold antibiotic resistance. Not only the water quality was compromised, being people exposed to those potential pathogenic bacteria and prone to disease, but also the chance of a successful antibiotic therapy decreases. The situation is further aggravated by the fact that, often, the prescribed antibiotic is the one currently available from a very limited choice, and second-line antibiotics are virtually non-existent in the country. Our results were in accordance with Farkas et al. (2013), reveling similarities in the route of introduction and spread of bacterial species carrying antibiotic resistance genes into the environment, mainly connected with animal and anthropogenic disturbance in two restricted areas, and very different drinking water sources investigated in Europe and Africa. In a country, like Guinea-Bissau, where the population is vulnerable (HIV, malaria, cholera epidemic, malnutrition), where the health system is fragile and not universally available, and the population has no access to other source of drinking water apart from contaminated well water, the exposure to antibiotic resistant bacteria can further exarcebate the health risk, particularly to the very young, the elderly, the immune compromised, or with chronic conditions (Falcone-Dias et al., 2012). Available studies showed that chlorination, a recommended disinfection method can prove to be ineffective as in the case of Guinea-Bissau conditions (Rowe, 1998), and additionally can favor the selection of antibiotic resistant bacteria (Armstrong et al., 1981; Murray et al., 1984; Shrivastava et al., 2004; Xi et al., 2009). Although overall it still remains an effective and inexpensive method to reduce the microbial contamination in water, this reality adds new challenges to the internationally accepted approach to improve water quality in developing countries and should not be overlooked.
5. Conclusions This study showed that the well water used for drinking and other domestic proposes was unfit for human consumption, being very acidic and heavily contaminated with fecal material. Additionally, well water was confirmed as a potential source of antibiotic resistant bacteria. Potentially pathogenic bacteria which showed resistance to the most prescribed antibiotics in GuineaBissau have been isolated from well water and pose a serious health risk. These bacteria can serve as reservoirs for antibiotic resistance genes that can enter, adapt and thrive in the ecosystem, and reduce the chances of a successful antibiotic therapy in the already vulnerable and more susceptible to disease population.
Acknowledgments We thank AMI (International Medical Assistance) and AIDA (Ayuda, Intercambio y Desarrollo), for logistic support, and Mr. Alfa
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for fieldwork assistance. This study was partially funded through a Ph.D. fellowship to A. Machado (SFRH/BD/46146/2008) cofinanced by POPH/FSE, and a grant to A. Bordalo (PTDC/AAC-CLI/ 103539/2008). This research was also partially supported by the European Regional Development Fund (ERDF) through the COMPETE – Operational Competitiveness Program and Portuguese national funds through FCT – Foundation for Science and Technology, Portugal under the Project “PEst-C/MAR/LA0015/2013”.
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