Microbial evaluation and public health implications of urine as alternative therapy in clinical pediatric cases: health implication of urine therapy.

Background Cultural means of pediatric treatment during ill health is a mainstay in Africa, and though urine has been known to contain enteric pathogens, urine therapy is still culturally applicable in some health conditions and also advocated as alternative therapy. The study therefore, is to evaluate the microbial contents and safety of urine. Methods Urinary bacteria from cows and healthy children aged 5-11 years were identified by conventional phenotypic methods and antimicrobial susceptibility testing was performed using modified agar disc and well-diffusion methods. Results A total of 116 bacterial isolates (n = 77 children; n = 39 cows) were identified as Bacillus (10.4%; 5.1%)), Staphylococcus (2.6%; 2.6%), Citrobacter (3.9%; 12.8%), Escherichia coli (36.4%; 23.1%), Klebsiella (7.8%; 12.8%), Proteus (18.2%; 23.1%), Pseudomonas (9.1%; 2.6%), Salmonella (3.9%; 5.1%) and Shigella (7.8%; 12.8%) spp. Antibiotic resistance rates of the Gram-positive bacteria were high (50.0100%), except in Bacillus strains against chloramphenicol, gentamicin and tetracycline (14.3%), while higher resistance rates were recorded among the Gram-negative bacteria except in Citrobacter (0.0%) and Proteus (8.5%) spp. against gentamicin and tetracycline respectively. The Gram-negative bacteria from ito malu (cow urine) were more resistant bacteria except in Citrobacter (20.0%) and Shigella spp. (0.0%) against tetracycline and Proteus spp. (11.1%), (22.2%) against amoxicillin and tetracycline respectively. Multiple antibiotic resistance (MAR) rates recorded in children urinal bacterial species were 37.5-100% (Gram-positive) and 12.5-100% (Gram-negative), while MAR among the cow urinal bacteria was 12.5-75.0% (Gram-positive) and 25.0-100% (Gram-negative). Similar higher resistance rates were also recorded among the Gram-negative bacterial species from urine specimens against the pediatric antibiotic suspensions. Conclusion The study reported presence of multiple antibiotic-resistant indicator bacteria in human urine and ito malu used as alternative remedy in pediatric health conditions like febrile convulsion.

midstream urine specimens of their children. The cow urine specimens were personally collected from Bodija and Moniya abattoirs, after obtaining the consents of the personnel in charge. All the urine specimens were cultured within 4 hours on same days of collection.

Isolation and characterization of bacterial species from the urine specimen
Urine culture were determined on plate count agar (PCA; LAB M), eosin methylene blue agar (EMB), MacConkey (MCC; LAB M) agar and cystein lactose electrolyte deficient (CLED; LAB M) agar at 350 degree Celsius for 24-48 hours. Different colonies on culture plates having ≥1.0 x 104 cfu ml-1 were randomly selected and colonies sub cultured on CLED and MacConkey agars. Pure bacterial isolates were identified to species level by the standard taxonomic protocol of the laboratory, based on the phenotypic profiles, including their cultural and microscopic morphologies as well as basic biochemical procedures [17].
In this study, the antibiotic resistance patterns of the bacterial isolates from the urine specimens of children (antibiotic discs) indicated that the antibiotic resistance of the Gram-positive bacterial species (Bacillus and Staphylococcus) were high (50.0-100%), except in Bacillus strains against chloramphenicol, gentamicin and tetracycline (14.3%). Higher resistance rates were also recorded among the Gram-negative bacteria against the test antibiotics except in Citrobacter (0.0%) and Proteus (21.4%) towards gentamicin. Multiple antibiotic resistance recorded among the Grampositive bacterial species were 37.5-87.5%, while 12.5-100% MAR were recorded among the Gram-negative bacterial species from the urine specimens of children. At least, thirteen of the bacterial strains had total resistance (100%), while only maximum of five strains had total (100%) susceptibility towards all the test antibiotics ( Table 1).
The Bacillus strains from the urine specimens of cows were more resistant to the test antibiotics (50.0-231 100%), except in chloramphenicol, in which no resistance was recorded. Similarly, the resistance patterns of the Gram-negative bacterial species from urine of cows were more resistant towards the test antibiotics (40.0-100%), except in Citrobacter, Klebsiella and Proteus against gentamicin (20.0%; 20.0%, 11.1%) and Citrobacter and Proteus against tetracycline (20.0%; 22.2%) respectively. No resistance was recorded among the Shigella strains against tetracycline. The MAR recorded among the bacterial species from the urine specimens of cows in this study was between 12.5 and 100%. Six of the bacterial strains from urine specimens of cows had total (100%) susceptibility, while only two strains had total (100%) resistance towards all the test antibiotics ( Table 2). Table 3, all the Bacillus and Staphylococcus strains from children urine were moderately or highly resistant to the pediatric antibiotic suspensions except in ampicillin/ cloxacillin (Emzoclox) and flucloxacillin (Floxapen) (12.5%); azithromycin (Zithromax) cotrimoxazole (Ranotrim), erythromycin (Erythrokid) and sulfamethoxazole/trimethoprim (Bactrim) (25.0%) to which the Bacillus strains were more susceptible. Total susceptibility was recorded in Bacillus strains towards ampicillin/cloxacillin (Jawaclox), in Staphylococcus strains towards amoxicillin/ clavulanic acid (Fleming), ampicillin/cloxacillin (Jawaclox) and azithromycin (Zithromax).

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Relatively higher resistance rates were recorded by the bacterial species from urine of cows towards the test paediatric antibiotics although total  Table 4).
Multiple antibiotic resistance patterns were also exhibited by the Gram positive bacterial strains from the urine of children (Bacillus; 28.6 -61.9%) and (Staphylococcus; 38.1 -81.0%) against the pediatric antibiotic suspensions (Table 3), while as high as 81.0% MAR was also exhibited by the bacterial species from cows towards the pediatric antibiotic suspensions (Table 4).

Discussion
Febrile seizures, which carry a good prognosis around the world are associated with a relatively high mortality and morbidity in Africa and other courtiers of the world [22,23]. Convulsion among children between six months and five years is a major contributor to childhood mortality in lessdeveloped societies, especially in sub-Saharan Africa [24]. In Nigeria, this has been attributed to the administration of some indigenous concoctions before the children are brought to hospital [25][26][27]. The traditional concoctions used in the treatment of convulsion vary with cultural practices [26], and based on the parental fears in cases of seizures, as well as accumulating epidemiological evidence indicating that febrile seizures are the most common recognized antecedent for epilepsy in childhood [28][29][30], various home remedies including human and cow urine, kerosene, fuel and crude oil are used in cases of infantile cases of febrile convulsion [2,25,26].
In this study, apart from the viable and culturable Gram-positive bacterial species (Bacillus and Staphylococcus), the most prevalent Gram-negative bacterial species obtained from the children urine were E. coli (36.4%) and Proteus (18.2%), although other Gram-negative bacterial uropathogens, Citrobacter, Salmonella, Klebsiella, Pseudomonas and Shigella were also isolated, in agreement with previous studies on the aetiology of pediatric uropathogens [9,13,14,16,[31][32][33]. Citrobacter, Klebsiella, Proteus, E. coli and Shigella were also the most recovered bacterial species from urine of cows in this study. These groups of pathogens are also similar to those obtained from some earlier studies [12,34,35].
Recovery of these group of indicator bacteria from urine that are consumed for the cure of clinical conditions in children is therefore, of great concern, especially because the isolated bacterial pathogens have been implicated in infantile and children gastroenteritis in some earlier studies [36][37][38][39][40][41][42]; and it is a well known fact that diarrhoeal diseases are a principal cause of childhood morbidity and mortality in the developing countries like Nigeria, being responsible for death of more than 4 million children each year [43][44][45].
It has also been well reported that antibiotic resistance demonstrates considerable geographic variability [31, 45,47], while studies on pediatric uropathogens in most countries also indicated a rise in resistance to common antibiotics and continuing evolution of resistance to antimicrobial agents, as well as large inter-regional variability [9,13,[31][32][33][48][49][50]. Moderate to high resistance (26-63%) to ampicillin, amoxicillin-clavulanic acid, cephalothin, cefuroxime and trimethoprim-sulfamethoxazole (SMZ/TMP) was noted among some of the bacteria in this study, which is similar to some earlier studies [13,33,51,52]. In the study of Pape et al. [11] as well, resistance rates to cotrimoxazole and 1st generation cefalosporines increased by about 20% compared with the previous analyses undertaken between 1990 and 1995, and therefore, concluded that the policies for treatment of UTI in children should be re-evaluated every 5 years according to local resistance rates. As an example, SMZ/TMP is a popular antibiotic in the treatment of pediatric UTIs and other clinical cases but the Infectious Disease Society of America had stated that with an SMZ/TMP resistance of 10-20% in adults, alternative first-line antimicrobial agents should be used [53]. This is based on adult uropathogen data but it is difficult to determine whether this same cut-off can be used in paediatric populations. Higher resistance (17.9-100%) to SMZ/TMP in this study therefore, raises some concerns.
Most of the bacteria isolated from urine of cows in this study also exhibited high rates of MAR towards 4 or more number of antibiotics. Globally, an estimated 50% of all antimicrobials serve veterinary purposes and literature of the last few years provides ample evidence that antibiotic resistance traits have entered the microflora of farm animals and the food produced from them [54]. Antimicrobial resistance has also emerged in zoonotic enteropathogens, commensal bacteria and bacterial pathogens of animals, although the prevalence of resistance varies [46,47,54]. In this present study, the recorded resistance rates as high as 87.5-100% observed among the bacterial species from urine of children, in spite of the fact that the bacterial species were non-UTI confirmed pathogens signifies that the introduction of strains that are drug resistant into a community plays a greater role in changing the prevalence of drug-resistant UTI [55]. In the five-year retrospective study by Ladhani and Gransden [55], bacterial isolates from children with underlying renal problems were generally more resistant to commonly used antibiotics in comparism with the children in the community, therefore, if such high antibiotic resistance (87.5-100%) as reported in this study were recorded among non-UTI confirmed bacterial strains, it then means that usage of urine of children and cows as alternative therapy in pediatric ill-health conditions like convulsion is quite hazardous.

6
Children are the most vulnerable members of any society since their immunity is not fully developed [45] but though the premium placed on children in African societies is so high, yet, even in the 21st century, the childhood mortality rates in the majority of African countries remain disturbingly high, with some 12 million children under-5 dying every year [56]. In 2006, there were 41 countries in which at least 10% of children under five died and all but three of the countries were in Africa. Ten of the 41 countries had higher rates of child mortality than 1990 and four were exactly the same. Among the worst 20, Nigeria ranked 12th with 181 deaths per 1000 [57]. Though the expected benefits of medical intervention should outweigh the possible harm, parents and guardians occasionally adopt some interventions which are futile, harmful and with no apparent curative or pathophysiologic rationale [25,58,59]. In Nigeria, as in most other developing countries, children are subjected to unorthodox treatment as first aid therapy in emergency conditions at home. In a study by Iyun and Tomson [60] in Nigeria, the reported dominating practice of mothers in cases of acute respiratory infections of children was either the use of irritants to get rid of the cause of the disease ('coldness') through vomiting, by forcing the child to swallow bitter remedies such as cow urine, or to use a remedy with warming and soothing properties.

Conclusion
Urine therapy is being advocated worldwide as an alternative therapy in many clinical cases. This study however, confirms that there could be introduction of multiple antibiotic resistant bacterial pathogens through consumption of cow and human urine in pediatric cases, more especially, since urine has not been reported to be of any medical benefit in cases of convulsion or other pediatric health condition. Although the magnitude of antibiotic resistance vary among regions but the rates are alarmingly higher in the developing countries. This study has been able to highlight the public health implications of urine as alternative therapy in paediatric convulsion in a developing country like Nigeria, which is of great concern.
Only the non-fastidious, viable and culturable bacterial species were isolated in this study, indicating that more pathogenic strains can be isolated with more sophisticated culture media and kits. It is strongly suggested that alternative therapies should be non-hazardous, and therefore, inappropriate administration of remedies, such as urine therapy in pediatric health conditions should be discouraged, considering the fact that no documented scientific / clinical evidence of the beneficial effect of urine therapy in clinical had been reported, while multiple antibiotic resistant bacterial species had also been recovered from such urine.

Competing interests
The authors declare no competing interest in the study.  Table 1: Antibiotic resistance patterns of isolated bacterial flora from urine samples of children using antibiotic discs Table 2: Antibiotic resistance of isolated bacterial flora from urine samples of cows using antibiotic discs Table 3: Antibiotic resistance pattern of isolated bacterial flora from urine samples of children using pediatric oral suspensions (mg/ml) Table 4: Antibiotic resistance pattern of isolated bacterial flora from urine samples of cows using pediatric oral suspensions (mg/ml)     B. cer [2] Staph [1] Citro [5] E. coli [9] Kleb [5] Prot [9] Pseud [1] Salm [2] Shig [5] Amoxicillin (