Introduction

Urinary tract infections (UTIs) represent one of the most common diseases encountered in medical practice today and encompass a broad range of clinical entities that are associated with a common finding of a positive urine culture (Chomarat, 2000).UTIs can be caused by exogenous microorganisms such as P. aerugionsaor by endogenous faecal or uretheral microorganisms (Abou-Dobaraet al., 2010). The microorganisms responsible for UTIs are usually the microbial flora found in the gut and are always present as a potential source of reinfection (Hillier et al., 2007). The endogenous microorganisms causing UTIs include E. coli, Staphylococcus epidermidis, S. saprophyticus, Proteus spp. and Klebsiellaspp. (Godfrey and Evans, 2000; Papeet al., 2004 and Jacobsenet al., 2008).

Bacteria can cause UTIs when they invade the mucosa (Pinkerman, 1994), attach mucosal surfaces, grow in the host’s tissue, or interfere with host defense causing damage to the host (Smith, 1995).

Bacterial factors play an important role in the pathogenesis of UTI. Certain bacteria possess properties that enable them to attach or adhere to uroepithelial cells or to the surface of catheter materials (Watts et al., 2010). Mannose – specific ligands on the fimbria or pili of E. coli bind to mannose receptors on urethral and bladder epithelial cells. Once attachment has occurred, the ability to infect the urinary tract starts, causing local cystitis or renal parenchymal infection. Bacterial attachment depends on the strain of bacteria, the presence of urinary proteins and salts, and the pH of the urine (Stamm, 1991). The accumulation of bacteria, glycocalyx, protein, crystalline salts and amorphous cellular debris eventually causes encrustations that can obstruct the flow of urine and serve as a persistentnidus for infection. This study will focus on the prevalence of bacteria isolated from urine cultures received from hospitalized patients. Analysis of glycocalyx as partial pathogenic factor as well as the effect of urine chemical elements on urinary pathogens will be investigated.

Materials and Methods

Urine samples

The present study was conducted at Urology Dept., El-Hussein University Hospital during the period from September to November 2003. Urine samples were collected from patients (males and females) immediately after their admission to hospital.

Every patient got a sterile dry, wide opening, leak proof container. Female patients were instructed to cleanse the area around the uretheral opening with soap of Dakin solution (diluted solution of sodium hypochloride) and then rinsed with water, dried the area and collected the mid stream urine (labia) with the label held apart. Two specimens were collected from each patient, one for bacterial culture and sensitivity test and the other for fresh film examination.

Bacteriological media

Three different types of enrichment media were used for isolating proper aerobic and/or facultative anaerobic bacteria. All media were readily prepared (Oxoid, England).

a- Nutrient agar medium

It is a basic culture medium used without additives. It is suitable for cultivation of a wide range of saprophytic and potentially pathogenic bacteria especially enteriobacteriaceae.

b- Blood agar medium

Blood agar base is a non-selective medium widely employed for the growth of pathogenic and non-pathogenic bacteria. When blood is added, the medium become suitable for the determination of typical haemolytic reactions produced by certain species of bacteria.

c- MacConkey’s agar medium

It is a differentiated medium used for the isolation of coliform and intestinal pathogens.

Assessment and purification of bacterial isolates

Plates containing the three types of media were incubated after inoculation at 37°C for 24 and 48 hrs, respectively. The colonies grown were selected, picked up and transferred to agar slants containing the same medium. They were purified by several consecutive streaking on agar plates. Purity was checked by microscopic examination of the isolates using gram stain. Purified isolates were subjected to a scheme of experimental identification.

Bacterial identification

Many biochemical reactions were performed for identification of bacteria such as sensitivity to potassium cyanide (KCN), catalase, oxidase, coagulase, acid production from carbohydrates, IMViC, H₂S production and growth in triple sugar iron agar medium. The identification process was carried out according to the methods described in Cowan and Steel (1974), Manual of Methods for General Bacteriology (1981) and Medical Laboratory Manual for Tropical Countries, Vol. II: Microbiology (Cheesbrough, 1984).

Chemical environmental factors affecting the growth of the isolated bacteria

The natural selection, fitness, pathogenicity and communities of isolated bacteria could be affected by some ecological factors. The environmental changes were mainly different concentrations of each chemical constituent of urine especially glucose, uric acid, sodium chloride, Calcium carbonate, Calcium oxalate, albumin, creatinine and magnesium chloride.

Each substrate was prepared in different concentrations (as indicated in the results) with normal urine, which was used as natural medium. The urine and substrate were sterilized by filtration throughout bacteriological filter, 0.2 mm. Each bacterial strain was inoculated in different concentrations of each chemical constituent and incubated at 37°C for 48 hrs. The results were expressed as optical density(O.D) at 660 nm using UV/Vis spectrophotometer (Unicam, England).

Effect of different antibiotics on the bacterial growth

Nutrient agar medium seeded with the bacterial isolates were used for such a purpose. However, all bacterial isolates were screened concerning their sensitivity to different antibiotics. The disc diffusion method was applied using commercial paper discs (Oxoid) impregnated with antibiotics. The following antibiotics were utilized with the given concentration (mcg): Amikacin (30), Streptomycin (10), Ciprofloxacin (5), Tetracycline (30), Amoxycillin (30), Chloramphenicol (30), Colistin sulfate (10), Unasyn (20), Noracin (5), Ampicillin (10), nitrofurantoin (300) and Ofloxacin (5). The results were expressed as percentage of resistant strains.

Separation and analysis of bacterial capsule

A- Separation of the bacterial capsule

The method used for separation of the capsule from bacteria was preceded according to Humpheryet al. (1974). A set of flasks 250 mls capacity containing 100 mls nutrient broth amended with sucrose 10% (w/v) were inoculated each with bacterial isolates. The flasks were incubated on a shaker (New Bruinsweek, New Jersey) in 160 rpm at 37°C for 10 days. After the incubation period, the cultures were diluted with an equal volume of distilled water. The cells were centrifuged off at 12,000 g for 30 minutes using Sigma centrifuge (Germany). The supernatant was mixed with 3 volumes ethanol (abs.) and centrifuged again. The supernatant was drained and the precipitated dialysedusingdistilled water for 3 days. The samples were then ready for sugar and amino acids analysis.

The separation of capsule components was proceeded using TLC plates (cellulose F254, Merck, Germany). A set of plates were used for separation of sugars and another set were used for amino acids separation. After complete separation process whether sugars or amino acids, the appeared spots on plates were identified using authentic maps.

B- Capsule analysis

B-l- Determination of amino acids

Preparation of authentics

Solution of amino acids was prepared in aqueous isopropanol (10%, v/v). Amino acids (1.2 mg) were dissolved in one ml isopropanol as solvent.

Locating reagents:

Ninhydrin reagent (200 mg/100 ml acetone) was used to visualize the amino acids.

B-2- Determination of sugar:

Preparation of the standard solution:

The different sugars (400 mg for each one dissolved in 10 ml aqueous solution of isopropanol (10 %, v/v) were prepared.

Solvents:

Butan-1-ol: Glacial acetic acid: water; 90: 10: 29 (v/v) and phenol saturated water: ammonia; 200:1 (v/v).

Locating reagent:

It consists of aniline (1 volume); diphenylamine, 1% in acetone (100 volume) and phosphoric acid 85% (10 volume). After complete separation of sugars, the air dried TLC were sprayed with this reagent. The plates were heated at 95-100°C for few minutes, to yield green, blue, or brown colour. The reagent was effective in locating different sugars since marked variation in colour was obtained with different sugars.

Results and Discussion

Surveillance results regarding UTI

The total numbers of infected urine samples collected from the surveyed patients enrolled in this study were 300 samples. The fresh film examination was considered as a base line to differentiate between infected and noninfected patients.

Since age is considered to be one of the risk factors for UTI (Little et al., 2009), classification of patients in the current study were surveyed according to age group. Table (1 & 2) shows the incidence of UTI for patientssurveyed by sex distribution. It is obvious that the most infected age group (21/70 %) was found between 36-46 years old women, followed by age group 47-57, which give rise to (18/60 %) for the same sex. Meanwhile the younger males and females exhibited low UTI rate (2/ 6.6 %) and (3/10 %) respectively. On these bases; it is clearly established that older people were more susceptible to UTI than youngerones.Thereby indicating that senility plays an important role in UTI. Indeed; these results are in agreement with those obtained by Herruzo-Cabrera et al., (2001) and Ahmed (2003).

Table (1): Age group of males and number of infected samples.

Table 2: Age group of females and number of infected samples.

Identification and determination of bacteria as an etiological agent of UTI

In this study seventy-eight bacterial isolates were collected from infected samples. These isolates were tentatively identified to species level using international reference keys of Krieg (1984), Sneath (1986) and Holt et al. (1994). The isolates were found to belong to 10 genera: Staphylococcus (aureus, saprophyticus), Enterococcus faecalis, Streptococcus sp. (B) group, E. coli, Klebsiellapneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobactersp., Enterobactersp. and Citrobacterfreundii.

Incidence of etiological agents, sometimes, reflects the sensitivity of UTI. The number and percentage of these etiological agents were recorded in table (3). E. coli was the most predominant organism causing UTI in 21 isolates (26.92 %), followed by Klebsiellapneumonia in 9 isolates (11.54 %), and Streptococcus sp. (B) group in 8 isolates (10.26 %).

Table 3: Number and percentage of etiological agents of UTI.

Many investigations had been reported that E. coli and Klebsiellaspp. were the most frequent organisms isolated from UTIs (Goldman, 2001 and Shao et al., 2003). Others reported that, two additional organisms inlcudingEnterobacterand Pseudomonas spp. may cause UTI (Tanejaet al., 2004; Soodet al., 2008and Becerra et al. 2010). The results of the present study emphasized that different incidence of positively reported positive culture may be caused “partly’ bydifferences in procedures and microbiological methods rather than true differences in incidence.

It is generally acceptable that each microbial species has its unique biochemical and morphological properties which permit the development of such an organism in some environments but not in others. The distribution patterns of bacteria in relation to disease and/or patient, or the microenvironment in which they reside are determined by these traits. The prevalence of certain bacterial species and the non detection of others may be attributed to the specific culture media used in the isolation process.

AlthoughStaphylococcus aureuswere isolated from most cultures in 7.69 %,none of the previous report (Das et al., 2006)has mentioned it asan etiological agent for UTI.

It was reported that urine specimens with more than one bacterial spp. were considered to be contaminated with skin, vaginal or periurethral flora. However,Leblebicioglu and Esen (2003), andÇetinet al. (2005) stated that polymicrobial infection is seen in more than 15% of urine samples; thereby indicating thatpolymicrobialbacteriuria is the rule, rather than the exception. In this context; two to three different bacterial species are often recovered from aseptically collected urine specimens even when no symptoms of UTI are present (Garibaldi et al., 1982).

Similarly Kohler – Ockmore and Feneley’s (1996) found that 177 urine samples contained mixed cultures while 51 samples were pure culture. In this work 218 urine samples were found to contain mixed cultures especially E. coli with Pseudomonas aeruginosaor E. coli with Streptococcus sp. (B) group while 82 samples were found to be pure cultures, i.e. one organism only isolated from each sample.

Chemical constituents of urine and their relation to bacterial growth

To live in an environment, bacteria must be able to endure all the abiotic stress characteristics of that environment. These stress conditions include different chemical constituents of urine. If an organism in a culture is unable to survive and occasionally grows when exposed to these stresses, even under artificial conditions, it is unlikely to be an inhabitant of an environment in which those stresses occur. These are the stresses are easy to establish and to show the important factors in determining the absence of an organisms (Alexander, 1997). In this regards 10 bacterial genera were tested in order to investigate the effect of different chemical constituents of urine on their growth in order to figure out their adaptability powers.

Tables 4-7 shows the effect of urine chemical constituents and their different concentrations (g/l) such as CaC₂O₄(0.05, 0.1 & 0.15); CaCO₃ (0.5, 1.0 & 1.5); MgCl₂ (0.5, 1.0 & 1.5); NaCl (20, 25 & 30); Citric acid (1.0, 1.5 & 2.0); glucose (1.0, 2.0 & 3.0); albumin (1.0, 1.5 & 2.0) and creatinine (2.5; 3.0 & 3.5).

Gram-negative bacteria  showed a decrease in the OD of its growth as increasing the concentration of Ca-C₂O₄ (except Citrobacterspp.) or CaCO₃ (except (Citrobacterspp&Enterobacter spp.). Contrariwise Gram positive bacteria vizStaphylococcus spp., Enterococcus spp. and Streptococcus spp. showed an increase in the O.D of its growth as increasing the concentration of both CaC₂O₄ and CaCO₃.

On the other hand the O.D of growth of Gram-positive bacteria wasinfluenced by increasing concentration of citric acid, for example Streptococcus spp. recorded zero O.D at 2 g/l while Staphylococcus saprophyticusand Enterococcus recorded 0.077 & 0.073 O.D, respectively, at the same concentration.

As a matter of fact, glucose andalbumin were the most favorable chemical constituents of urine for the growth of 10 tested genera. The growth is increased as the concentration increased. Similarly Moore et al (2002) reported that the causative agents of UTI were propagated in the presence of hyperglycemia.

Interestingly,Acinetobacterspp. was sensitive to the presence of all chemical constituents of urine (except glucose and albumin); sincetheir increase in concentration causes the growth to markedly decrease. This indicates that Acinetobacterspp plays a minimal role in UTI.

Enterococcus spp., Streptococcus spp. and Citrobacterspp. were affected by increasing the concentration of NaCl and thereby recording a low growth rate.

Table 4: Growth of etiological agents of UTI in different concentrations of Ca C₂O₄and CaCO₃ (Results expressed as O.D. at 660 nm).

Table 5: Growth of etiological agents of UTI in different concentrations of MgCl₂ and NaCl. (Results expressed as O.D. at 660 nm).

Table 6: Growth of etiological agents of UTI in different concentrations of uric acid and glucose (Results expressed as O.D. at 660 nm).

Table 7: Growth of etiological agents of UTI in different concentrations of albumin and creatinine (Results expressed as O.D. at 660 nm).

Bacterial resistance to antibiotics

It is widely known that bacteria are able to develop resistance to antibiotics. The development of antibiotic resistant is essentially an adaptive process since it reflects the ability of organisms to survive by adjusting themselves to adverse environmental conditions.The etiology of UTIs and the antibiotic susceptibility of urinary pathogens have been changing over the past years and recently resistance to antibiotics has become a major problem worldwide (Chomarat, 2000).

The prevalence of Enterobacteras a hospital-acquired pathogen has greatly increased since the introduction of extended-spectrum cephalosporins into clinical practice (Kaminskaet al., 2002). Broad spectrum antimicrobial therapy promotes the acquisition of resistance to extended–spectrum cephalosporines, aminoglycosides and fluoroquinolines (Grandsen, 1997 and Domin, 1998).Enterobacter“in this study” showed resistance to aminoglycoside (71.4 %) and ofloxacin (28.6 %) as they contain quinoline constituents, while it was sensitive to other quinolines (ciprofloxacin and noracin) with a 100 % rate (table 8).

Akbar (2001) found ampicillin resistance strains of E. coli and Pseudomonas sensitive to gentamycin and ciprofloxacin. Also Lark et al. (2000) mentioned that E. colicould account for approximately 50 % of hospital – acquired bacteriuria.It is often resistant to both sulfonamides and ampicillin. Conversely a study undertaken by Sotto et al. (2001) described that E. coli hasa resistance rate of 20.3 % for ampicillin and it is sensitive to gentamycin and ciprofloxacin.

However; in the present study E. coli showed an absolute resistance (100%) to ampicillin and an absolute sensitivity to noracin (100%). Similarly Proteus showed the same ratio of resistance and sensitivity to both antibiotics, respectively.This was consistent with Chomarat (2000) results.

To the best of our knowledge we conclude that antibiotic resistance problem could arise from many reasons, including antibiotic use in animal feeds, inappropriate prescription of antibiotics, arbitrary administration of antibiotics, and poor infection control strategies.

NonethelessAcinetobacter was very sensitive to all tested antibiotics, except ampicillin in which Acinetobacter shows 50% resistance rate. Therefore; this finding is consistent with the previous conclusion that Acinetobacterhas no important role in UTI.

Table 8: Resistance (%) of etiological agents of UTI to some antibiotics.

Cip = Ciprofloxacin, S = Streptomycin, TE = Tetracycline, Amx = Amoxycillin, C = Chloramphenicol, CS = Colistinsulphate, Un = Uncisyn, Nor = Noracin, Am = Ampicillin, AN = Amkacin, NF = Nitrofurantoin, Ofx = Ofloxacin.

Analysis of extrapolymeric substance

A preliminary test of extrapolymeric substance on sucrose agar medium reviled that some bacterial strains possesses capsules or mucous. These strains were Staphylococcus saprophyticus, Streptococcus sp. (B) group, Klebsiellapneumoniae, Proteus mirabilis and Pseudomonas aeruginosa(tables 9 & 10).The presence of a capsule can be a major factor in determining the pathogenicity of these bacteria especially K. pneumoniae, Proteus mirabilis and P. aeruginosa. In some cases those bacteria have two variants; one that forms a capsule, representing the virulent pathogen, and a noncapsulated one whichis subjected to phagocytosis by blood cells involved in the immune response of the infected host organism. In this regard, Williams and Gibbons (1972) emphasized that the main function of SIgA antibody is to prevent bacterial adherence to the mucosal surface by coating bacterial cell wall.

On the other hand, phagocytizing blood cells involved in the immune response are properly unable or less able to engulf and digest those capsulated bacteria. Moreover, Van Demark and Batzing (1984) mentioned that because the organism can be sequestered within the capsular bag, adequate drug levels may not be present for sufficient time to kill the organism even after injection of antibiotics into the bag.As bacteria use their capsules as a mean of attachment and pathogenicity for UTI, the isolated bacterial capsules were subjected to structural analysis using this layer chromatography (TLC) technique. On the bases of the results shown in tables (9 & 10) bacteria was classified as capsule carbohydrate – containing, or capsule amino acid associated with carbohydrate – containing bacteria. In this context; Proteus mirabilis was found to be capsule carbohydrate containing bacterium while Pseudomonas aeruginosa was found to be capsule amino acid containing. Meanwhile others strains were possesses capsule carbohydrate and amino acid containing. It is merit to mention that L-rhamnose, glucuranolactone, D-mannose and D-fructose were detected only in capsule of Pseudomonas aeruginosa. However; this result was in concomitant with Stanieret al (1970) study.

Table 9: Analysis of sugar in extrapolymeric substance of etiological agents of UTI.

Table 10: Analysis of amino acid in extrapolymeric substance of etiological agents of UTI.

Conclusion

Coli and Klebsiellaspp. were the most frequent organisms isolated from UTIs.However, glucose and albumin were the most favorable chemical constituents of urine for bacterialgrowth since their concentration increased the growth of bacteria increased. In addition increasing creatineneconcetrantion increases the growth of Enterococcusfaecalis, Streptococcus sp. (b) group,Proteus mirabilis, Pseudomonas aeruginosa, and Citrobacterfruendii. Nevertheless, NaCl, and CaC₂O₄ effect on bacterial growth were influenced by their concentration and the type of bacteria tested.

Noracin has an absolute inhibition effect on all tested bacteria. However, Acinetobacter shows 50% resistance rate to Ampicillin while all other bacteria showed a resistance rate between 80.7 % to 100 % to the same antibiotic

Acknowledgment

The Authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project RGP-VPP-062.

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