Introduction

Nocardia spp., strictly aerobic actinomycetes, are Gram-positive, weakly acid-fast and dichotomous branching bacilli. They are essentially soil saprophytes worldwide and involve in the decomposition of plant material. They are not normal flora in humans or animals [18]. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections. Worldwide, about 150 million people are diagnosed with UTIs each year. The most common infection caused by opportunistic Enterobacteriaceae is a urinary tract infection (UTI). Enterobacteriaceae are Gram-negative, facultatively anaerobic rods belonging to the γ-class of proteobacteria. The improper and uncontrolled use of many antibiotics resulted in the occurrence of antimicrobial resistance, which becomes a major health problem worldwide. Multiple drug resistance has significantly increased in recent years [2, 14, 16]. Multiple antimicrobial resistances among Gram-negative organisms have been a long-term and well-recognized problem with urinary tract infections. GC-MS is used for the identification and quantification of bioactive compounds of N. brasiliensis [8, 12].

Experimental designs are excellent techniques for the optimization of culture conditions to achieve optimal production [1, 3]. One of the most promising sources of bioactive compounds is N. brasiliensis, which was reported as an antibiotic producer [6, 9, 11]. This study was designed with the aim of screening of antibacterial compounds production by N. brasiliensis PTCC 1422 against isolated Enterobacteriaceae from UTIs.

Materials and Methods

Samples (urines) were collected from UTI patients of North Hospital of Iran. Preliminary isolation and identification were based on the microscopic, cultural characteristics and other standard biochemical analysis [2, 16]. Bacterial strains isolates were cultured on nutrient agar and incubated at 30 °C. The strains were carried out for Gram staining and shape under light microscope. Various biochemical tests were performed for the identification of isolated bacteria according to Bergey’s Manual of Systematic Bacteriology.

The most prevalent organisms were chosen and subjected to antimicrobial sensitivity test. The AST for each isolate was carried out on Muller–Hinton agar by Kirby–Bauer disk diffusion technique. The microorganism suspensions used for inoculation were prepared at 108 CFU (colony-forming units)/ml by diluting fresh cultures at McFarland 0.5. The several antibiotics (Himedia Co.) were used for the antibiotic sensitivity test. Standardization of the technique controls variation in results, and interpretation is based on comparison of inhibition zones with published criteria for zone diameters [13, 14].

N. brasiliensis PTCC 1422 was grown in 50 ml of starch casein broth by submerged culture containing in 250-ml flasks, incubated at 28 °C in a shaker (150 rpm) for 7 days and centrifuged at 4000 rpm for 10 min, and the clear supernatant broth samples were tested for their antagonistic activity against the isolated pathogens by agar well diffusion method [15, 20]. Wells of 6 mm diameter were prepared in the nutrient agar plates. Isolated pathogenic bacteria were swabbed onto the nutrient agar surface [10]. The wells were filled with the 70 μl of culture supernatant, and the diameter of inhibition zones was measured after incubation for 24 h at 37 °C.

GC-MS Analysis

In this technique, the carrier gas was helium (99.999 %), linear velocity was 30 cm/s, and temperature of the split–splitless inlet was 220 °C (splitless time −1.0 min). Oven temperature program was as follows: The initial temperature of 60 °C was kept for 2 min and then raised 8 °C/min to 220 °C (kept for 4 min). The sample was ionized by electron ionization (EI) at an energy of 70 eV and mass spectrometry (quadrupole) ion range m/z = 35/500 atomic mass unit (amu) (quant. ions 95, 107, 108, 195, 197, 212, 112, 125 and 149, or ions for selected ion monitoring). The temperature of the interface was 200 °C [7].

Results

The microorganisms were confirmed as Escherichia coli (72.5 %), Klebsiella pneumoniae (3.2 %), Serratia marcescens (0.8 %) and Staphylococcus aureus (14.1 %) (Fig. 1). By standard confirmatory tests, all the isolates were found to be Gram-negative. According to Bergey’s Manual of Determinative Bacteriology and the Laboratory Manual for Identification of Bacteria, the isolates were identified. The four isolates obtained were subjected to study morphological and biochemical characteristics (Table 1).

Fig. 1
figure 1

Frequency of bacterial uropathogens isolated from UTI suspected patients

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Fig. 2
figure 2

Antimicrobial activity of N. brasiliensis against isolated bacteria from UTIs

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Table 1 Morphology and biochemical characteristics of isolated bacteria from UTIs
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Antibiotic resistance pattern revealed that these bacteria were highly resistance to ceftriaxone and showed the lowest percentage of resistance to cephalexin. High rate of multiple drug resistance was recorded among all isolates. The resistance rates of bacteria isolated from UTIs against different antibiotics are presented in Table 2.

Table 2 Antimicrobial resistance of isolated bacteria from UTIs to antibiotics
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N. brasiliensis PTCC 1422 was able to produce antibacterial supernatant against Enterobacteriaceae organisms. The formation of inhibition zone around the pathogenic strains was due to the production of secondary metabolites by N. brasiliensis PTCC 1422 (Fig. 3). Frequency of antimicrobial activity of N. brasiliensis against isolated bacteria from UTIs is shown in Fig. 2.

Fig. 3
figure 3

Antimicrobial activity of N. brasiliensis against isolated bacteria from UTIs in mm

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The highest concentrations of determined compounds in real-level samples of N. brasiliensis by GC-MS method are presented in Table 3. Phthalic acid as highest concentration of supernatant of N. brasiliensis was determined.

Table 3 Concentrations of determined compounds in N. brasiliensis by GC-MS method
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Discussion

The increasing rate of UTIs pathogens resistance to commonly used antibiotics, coherent prescription and use of antibiotics is advocated. With the increasing use of antibiotics, the serious problem of antibiotic resistance is gradually increasing [4, 17]. Thus, the result of the present investigation revealed that N. brasiliensis PTCC 1422 is the potent source of novel antibiotics and was found to be of potential antagonistic against test organisms which can control variety of pathogenic organisms. The GC-MS method for the determination of bioactive compounds in N. brasiliensis, which was optimized in this study, can be useful in the routine analysis in laboratories. The isolates showed their resistance to different antibiotics. This widespread resistance could be attributed to excessive or indiscriminate use of antibiotics. N. brasiliensis PTCC 1422 exhibited high antibacterial activity against Enterobacteriaceae including E. coli (48 %), K. pneumonia (36.3 %), P. mirabilis (24.1 %) and S. marcescens (33.3 %) in well diffusion method, and the inhibition zones were in the range of 7–19 mm. Similar study indicated the antagonistic activity of actinomycetes isolates such as Nocardia against human pathogen (S. aureus, Proteus vulgaris, P. aeroginosa, E. coli, B. subtilis, B. megaterium, K. pneumoniae, C. albicans, A. niger, S. cervisiae), and the inhibition zones were in the range of 7–20 mm [5, 15, 19]. Thus, in our present investigation, the result indicates that the substances present in the N. brasiliensis PTCC 1422 could be used to inhibit the growth of human pathogen. The supernatant of N. brasiliensis PTCC 1422 was analyzed by GC-MS, and phthalic acid was most fraction. The chemical analysis compounds can find places in the database for the development of antimicrobial substances. This study has demonstrated that N. brasiliensis PTCC 1422 has a high potential for the treatment of UTIs.

Conclusion

Antibacterial resistance among bacterial uropathogen is an evolving process. Therefore, in the field on the need of re-evaluation of empirical treatment of UTIs, our present investigation indicates that the substances present in the N. brasiliensis PTCC 1422 could be used to inhibit the growth of human pathogen. The study has demonstrated that N. brasiliensis PTCC 1422 has a high potential for the treatment of urinary tract infections (UTIs).