The Isolation Rate of Pseudomonas aeruginosa Opportunistic Pathogen and their Antimicrobial Responses in HIV-1 Positive and Negative Diarrhoea Patients at North-West Part of Ethiopia

HIV/AIDS pandemic is still extremely dynamic and growing worldwide. In the world, about 33.2 million people are estimated to be infected with HIV/AIDS [1]. From this more than 22.5 million are expected to live in Sub-Saharan Africa [1]. HIV is now the leading cause of death worldwide among the age group of 15-24. Among the most heavily HIV/AIDS affected countries, Ethiopia is the third next to India and South Africa in 2005 [2]. The prevalence of HIV infection in Ethiopia is currently over 3.5% [3]. HIV, in Ethiopia, is mostly associated with opportunistic infections such as candidiasis, tuberculosis, CNS mass lesion [4] and diarrhea [5].

Diagnosis of P. aeruginosa infection depends up on isolation and laboratory identification of the bacterium. It grows well on most laboratory media. It is identified on the basis of its Gram morphology, inability to ferment lactose, positive oxidase reaction, its fruity odor and ability to grow at 42°C [18]. Fluorescence under ultraviolet light is helpful for early identification of P. aeruginosa colonies [19,20].
Pseudomonas aeruginosa is notorious for its resistance to antibiotics and is therefore, a particularly dangerous and dreaded pathogen [21,22]. The bacterium is naturally resistant to many antibiotics due to the permeability barrier afforded by its outer membrane lipopolysaccharides (LPS). Also, its tendency to colonize surfaces in a biofilm form makes the cells impervious to therapeutic concentrations antibiotics [22]. Since its natural habitat is the soil, living in association with the bacilli, actinomycetes and molds, it has developed resistance to a variety of their naturally occurring antibiotics [23]. Moreover, P. aeruginosa maintains antibiotics resistance plasmids, both R-factors and RTFs, and it is able to transfer these genes by means of transduction and conjugation [24]. Generally, P. aeruginosa develops resistance to nearly any antimicrobial agent. Resistance is problematic at three levels: intrinsic resistance, acquired resistance and emergence resistance during therapy [25]. P. aeruginosa is frequently resistant to many commonly used antibiotics. Each of them must be considered when choosing an antibiotic regiment for patients infected with P. aeruginosa [26][27][28].
In Ethiopia, as far as my exploration is concerned, there are no studies conducted on the prevalence and sensitivity pattern of P. aeruginosa against the commonly used antibiotics in diarrheic patients with and without HIV infection. Therefore, the objective of this study is to determine the frequency of isolation and sensitivity pattern of P. aeruginosa against the commonly used antibiotics in HIV-1 positive and negative diarrheic patients. The outcome of this study may help as baseline information for empirical treatment of HIV 1 positive diarrheic patients affected by P. aeruginosa.

Study design and subjects
In this cross-sectional study, consecutive diarrheic patients diagnosed at the outpatient department of the University of Gondar Hospital, in Gondar, Ethiopia were included. Informed consent was obtained from all subjects and the study was approved by the Research Ethics Committee of the University.
Chronic diarrhea was defined as an average of three or looser watery bowl movement per day for at least two weeks before the study [29]. Acute diarrhea was defined as an episode that lasted less than 14 days [29]. All subjects underwent physical examination. Socio-demographic and clinical data were recorded by medical doctors on structured and pre-tested questionnaires.

The sample size
The sample size for the study was calculated by considering a 95% level of confidence, 90% proportion of the study done in Addis Ababa [30] and Jimma [31] respectively, 5% margin and 20% contingency. As the result, 312 patients with diarrhea were included in the study, out of which 199 were HIV seropositive and 113 HIV seronegative.

HIV serology
After obtaining informed consent and appropriate pre-test counseling, about 5 ml of venous blood was taken from each subject and sera were separated by centrifugation and stored at -20°C until used. The presence of HIV antibodies was determined by an enzyme linked immunosorbent assay following the manufacturer's instruction (Vironostica HIV Uni-Form II plus O, Organon Teknika, Boxtel, the Netherlands) [32].

Stool collection and isolation of pathogens
Diarrheic stool specimens were collected as fresh in sterile containers and processed immediately after collection following standard procedures [33]. The specimens were cultured on the MacConkey agar (BBL). All the inoculated plates of McConkey agar were incubated at 42°C for 48 hrs.
The P. aeruginosa isolates were identified based on colony characteristics, gram staining methods and by various biochemical tests as given below. In brief, they were identified on the basis of their Gram morphology, inability to ferment lactose, oxidase and hydrogen peroxide reaction, its fruity odor and its ability to grow at 42°C [34]. A single colony of non-lactose fermenting type from MacConkey plates was aseptically picked and inoculated in about 3 ml of nutrient broth and incubated at 35-37°C for 2-4 hours until growth was ascertained by turbidity. From this broth culture, identification of P. aeruginosa was performed with the help of biochemical tests which routinely included triple sugar iron (TSI) agar slant, lysine iron (LI) agar slant, Urea agar slant, Simmon's citrate agar slant, Shahidi Indole Motility (SIM) medium, Mannitol broth (1%) and glucose broth (1%). Biochemical results were interpreted by using Bergey's manual [35].

Antimicrobial sensitivity testing
Susceptibility testing of all strains was done on Muller-Hinton agar with commercial antibiotic discs using Kirby-Bauer method [36]. Each isolate was sub-cultured on a plate of MacConkey agar and incubated at 37°C for 18 to 24 hours. From a pure culture of each isolate 4 to 5 similar colonies were randomly selected and transferred to a test tube of Trypticase Soy broth by touching the top of each colony with a sterilized wire loop [34]. Trypticase Soy broth incubated at 37°C for 8 hours to obtain moderate turbidity. The suspension (a portion of it) was diluted with a sterile saline to obtain turbidity visually equal to that of the standard of McFarland 2 [37].
Finally, diameters of inhibition zones were measured in millimeters using a sliding caliper, which is held on the back of the inverted plate. The sizes of the zones of inhibition were interpreted by using zone diameter interpretive standards [38]. The intermediate readings were considered as sensitive for a purpose of assessment of the data. Multiple drug resistance was defined as a given bacterium which is resistant to more than one antibiotic.

Statistical analyses
Data were analyzed using SPSS version 16 statistical package.
Comparisons P. aeruginosa positive among HIV patients with diarrhoea were made using both Chi-square test at significance level P < 0.05 and compared means (student t test; independent samples test). P values less than 0.05 were considered statistically significant. Table 1 shows socio-demographic and clinical features of the patients by HIV serostatus. A total of 312 diarrheic patients were included in the study. Of this, one hundred eighty (57.7%) were males and 132 (42.3%) were females. Their mean age was 29 (range 15 to 67 years). A significant number of the patients were in the age group of 25 to 44 years. On the age group 15-24 HIV seropositive diarrheoa patients, the proportion of males and females was 3.5% to 4.5%, respectively. It was also observed that the proportion of males and females within the age groups greater than 34 years were 14.7% and 8.3%, respectively (P < 0.043). The prevalence of HIV in diarrhea patients with regard to marital status (P < 0.01) and occupation distribution (P < 0.01) has shown significant variation in the study subjects.  The prevalence of P. aeruginosa isolates in HIV positive and negative subjects was investigated ( Table 2). The most important finding of this study was that out of the total diarrhoea patients, 92 (29.5%) was P. aeruginosa positive in both HIV positive and negative study subjects. Out of the total P. aeruginosa isolates, 64 (20.5%) were isolated from HIV positive and 28 (9%) from HIV negative diarrhoea patients (P < 0.05). Of the total 199 (100%) H. zoster infected diarrheic patients, 161 (80.9%) and 38 (19.1%) were HIV positive and negative, respectively. From both HIV and P. aeruginosa positive study subjects (64), 47 (73.4%) were positive for H. zoster while the rest 17 (26.6%) negative for H. zoster (P < 0.05) ( Table 2).

Results
Clinical symptoms and diarrhea status in association with that of P. aeruginosa isolates in HIV seropositive and negative diarrhoea patients is presented in Table 2. In this study, nature of diarrhea and abdominal cramp of such diarrhoea were not statistically associated with P. aeruginosa isolates (P > 0.05). On the other hand, marked weight loss was observed in 32.2% and 24.8 of patients with HIV seropositive and HIV seronegative, respectively. The proportion of patients with weight loss and P. aeruginosa infection was significantly higher in those with HIV co-infection (P < 0.05). Based on the independent samples test analysis of HIV serostatus result, weight loss, and parasites as independent variables and P. aeruginosa as dependent variable, has been shown significant association (P < 0.05) between the parameters and prevalence of P. aeruginosa.
Resistance against one or more antibiotics is shown in Table 3. Out of the 112 strains, 109 (97.3%) showed multiple resistance. In all, 36 different patterns of resistance were noted. The frequency of Amp, Sxt, Nal, Nor, Gn, Kn, Ch, Ttc, Cip, and Amp, Sxt, Ch, Tet pattern of resistance are 19 (17%) and 28 (25%), respectively, which are the highest in comparison with other pattern of resistance. Three and five pattern of resistance were observed in 16 (14.3%) and 17 (15.3%) P. aeruginosa isolates, respectively. Pseudomonas aeruginosa isolates with greater than three pattern of antimicrobial resistance account for 89 (79.6%). In this finding, there is no any strain, which is sensitive, or resistance against to all antimicrobial agents.

Discussion
HIV infection has become the dominant health problem in many parts of sub-Saharan Africa, including Ethiopia [39]. The result of this study in terms of HIV infection agrees with that indicated on the 2005 UNAIDS report [3]. It was observed that the highest infection rates are concentrated in the 15-34 year age group. Within this age group, prevalence among females was found to be greater than that of males. Other than gender, marital status and occupation have also shown impact on the distribution of HIV. The association of H. zoster infection and HIV was also found to be significantly (p < 0.05) high 161/199 (80.9%). H. zoster infection was one of the most common HIV-related opportunistic infections and diseases in HIV seropositive patients [40,41]. According to this study, the infection rate of H. zoster in both HIV and P. aeruginosa positive was 73.4%. This is may be due to weak immune response of HIV positive study subjects. As well known, both H. zoster [40,41] and P. aeruginosa [15] infections were the most common opportunistic infections in HIV positive individuals.
One of the major manifestations of the HIV disease in the region is the diarrhea [42,43]. In this investigation, chronic diarrhoea was found to be more common than acute diarrhoea in HIV positive subjects. On the other hand, acute diarrhoea was more common than chronic diarrhoea in HIV negative subjects.
Intestinal parasitism occurs widely throughout Ethiopia [44]. It was reported that intestinal parasites were one of the causative agents of diarrhoea in HIV seropositive patients [5,45]. In line with these reports; we found a high prevalence (19.6%) of intestinal parasites in HIV seropositive diarrheic patients included in the present study. Earlier data suggest that intestinal parasite infections can affect the nutritional status of infected people through modification of food intake, digestion and absorption capacity [46]. Acquired immunodeficiency syndrome (AIDS) caused by HIV infection predisposes individuals to several diarrhoeagenic bacterial diseases of various species [47] in addition to several viral, protozoal and parasitic etiological agents [48]. This study was designed to investigate the prevalence and antimicrobial responses of P. aeruginosa isolated from HIV seropositive and negative diarrheic patents.
Pseudomonas aeruginosa is important opportunistic pathogens of bacteria in HIV-infected host [15]. This study showed a high prevalence of P. aeruginosa in diarrheic patients with or without HIV co-infection in Northwest Ethiopia (29.5%). In this investigation, the prevalence of   the isolation of P. aeruginosa in HIV seropositive subjects was 64/199 (20.5%), which was lower than 41/135 (30.7%) isolates from HIVinfected patients [49]. However, the result of this study was almost the same with the report of (19.2%) Osazuwa et al. [50], in Benin but higher than the isolated (8.7%) and (4.7%) strains reported by Olayinka et al. [51] and Hernandez et al. [52], respectively. Even the isolation rate is high in some studies [51,52], P. aeruginosa infection is not frequent bacterial disease in highly immunodeficient HIV-infected patients. It may be fatal in the advanced stages of HIV diseases, especially when patients have taken the common and cheap available antibiotics [53] and the society with poor hygiene conditions. The incidence of P. aeruginosa infections in HIV/AIDS patients appears to be on the rise [54].
The results of this study revealed that the prevalent rate of P. aeruginosa is high in HIV seropositive diarrheic patients. In this finding, HIV positive patients without P. aeruginosa were also affected by diarrhoea. This shows that diarrhoea may be associated with other diarrhoeagenic agents or mechanisms rather than P. aeruginosa [55]. Then, detail investigation has to be made in order to get comprehensive information for better treatment of diarrhoea in HIV/AIDS patients.
In this study, all the isolates were resistant to the cheap, commonly available antibiotics, such as Tet, Sxt and Chl in comparison with that of expensive and unavailable antibiotics such as Pol, Gen, Nor and Cip. Even Kan and Nal although shows some in vitro activities in this study, they are equally ineffective due to their phamacodynamic or pharmacokinetic characteristics [56]. However, the susceptibility patterns of P. aeruginosa vary geographically, and susceptibility tests should be done as an alternative means to select antimicrobial therapy [57]. According to this finding, Nor, Gen, Pol and Cip may be used as drug choice for empirical treatment. On the other hand, Amp, Tet, Sxt and Chl may not be used as the drugs of choice for the treatment of P. aeruginosa infection unless culture and sensitivity tests are done prior to treatment. The high prevalence of multidrug resistant indicates a serious need for broad-based, local antimicrobial resistance surveillance for continuous tracking of antibiotic resistance trends among all clinical relevant isolates and introduction of effective interventions to reduce multidrug resistance in such pathogens [58][59][60].
The occurrence of resistant strains in this study may be the spread use of these drugs. This may cause selective pressure of resistance problems on the enteric bacteria as a whole circulating in the community [61].
Culture and drug sensitivity results generally determine the choice of antibiotic for enteric bacterial infections. The choice of antimicrobial agents should be dictated by local resistance patterns and the patients' history antibiotic exposure [62,63]. Regardless of the choice of antibiotics, doses should be selected to maximize serum levels for enhanced activity. Combination therapy is also recommended to prevent the emergency of resistant strains. However, the effectiveness of combination therapy in preventing the emergency of antibiotic resistance in P. aeruginosa infections remains controversial. According to Carmeli et al. [64], combination therapy was not associated with a disease in the emergency of resistance. On the other hand, Jacobson et al. [65] found that combination therapy was associated with less frequent development of resistance than mono-therapy.
In this study, high prevalence of P. aeruginosa was observed in diarrheic patients with HIV infection suggesting that there is high human fecal contamination. This calls for efforts for strengthening control programs such as quality sanitation at community level to reduce risk factors of acquiring this diarrhoeagenic bacterium. In addition, routine examination of stool for P. aeruginosa with sensitivity test could significantly benefit the HIV infected and uninfected individuals in reducing morbidity and improving quality of life. This study was the first of its kind on the prevalence and antimicrobial responses of P. aeruginosa in HIV-1 positive and negative diarrhoea patients in Ethiopia. Therefore, the information obtained in this study is significant for empirical treatment of diarrheic patients' affected P. aeruginosa.
In this finding, Amp, Tet, Sxt and Chl may not be used for the treatment of P. aeruginosa infection, unless culture and sensitivity tests are done prior to treatment. In underdeveloped countries like Ethiopia, besides limiting therapeutic options and the high cost of alternative effective agents, resistant organisms may lead to longer hospitalization and an increased risk of death. The development of new antimicrobial agents may offer short-term solution to this problem but in the long run more effective measures such as health education and further research on the prevention of infections through quality sanitation, vaccination and immunization should be emphasized. Based on this study, I would recommend that public health measures should continue to emphasize the importance of environmental and personal hygiene.