Isolation, characterization and prevention of various microbial strains in NIC unit and PIC unit

The health of the hospital associated persons, particularly those dealing directly with insertion of devices, are serious cause of concern for hospitals. In this study, the most prevalent organism on the surface of medical devices in PICU were CoNS (16.66%) and Staphylococcus aureus (16.66%), while in NICU the most prevalent organism was Klebsiella spp. (11.25%) among Entero-bacteriaceae group followed by Acinetobacter baumannii (10%), Escherichia coli (2.5%), CoNS (6.25%), S. aureus (6.25%) and Enterococcus faecalis (6.25%). The most common species identified from blood specimen of clinical samples shows the maximum presence of Candida sp. (60/135) followed by A. baumannii (21/135), Klebsiella Pneumoniae (20/135), Enterococci (12/135), Burkholderia cepacia complex (8/135), S. aureus (6/135), E. coli (5/135), Pseudomonas aeruginosa (3/135). Different antibiotics have been used against these micro-organisms; but Cotrimoxazole, Vancomycin have been found more effective against CoNS bacteria, Clindamycin, Tetracycline for S. aureus, Nitofurantoin for Acinetobacter, and for E. faecalis, A. baumanii, and Klebsiella, erythromycin, Colistin, and Ceftriaxone have been found more effective respectively.

. Few colonies of culture was inoculated for about 18-24 on the surface of the slant of bile esculin agar, then again incubated at 35°-38° in an ambient air for 48 h.
Nitrate reduction test 6 . Procedure. Nitrate broth was inoculated with 1-2 drops from the young broth culture. After completion of incubation, 1 ml of each reagent A and B was added to the test medium.
Oxidase test 8 . Procedure. Filter paper was moistened with the substrate or a commercially available paper disk that has been impregnated with the substrate is used. Using a platinum wire or wooden stick, a small portion of bacterial colony from the agar surface were removed and rubbed on the filter paper or commercial disk.
Than observed the inoculated area of paper or disk for a color changes to deep blue or purple color within 10 s.
Urease test 8 . Procedure. The surface of urea agar slant was streaked with a small portion of well isolated colony and the slant was inoculated with 1-2 drops from an overnight brain-heart infusion broth culture. The cap was loosed and the tubes were incubated at 35°-38° in ambient air for 48 h to 8 days.
L-Pyrrolidonyl Arylamidase (PYR) test 8 . Procedure. Before inoculation, the disk was moistened lightly with Analytical grade water. A wooden applicator stick was used to rub a small amount of several colonies for about 18-24 h with pure culture on small area of PYR disk then incubated at room temperature for 2 min. A small portion of detector reagent N, N-dimethylamino-cinnamaldehyde was added and observed for red color development within 1 min.
Fermentation media 8 . Procedure. A tube was inoculated with 1 drop of 18-24 h brain-heart infusion broth culture and was then incubated at 35-38 °C for about 8 days. Acid and gas production is examined.
Decarboxylase test 8 . Procedure. Tubes were inoculated with a drop of brain-heart infusion broth culture, and a thin layer of sterile mineral oil was added to each tube. The cultures were incubated for 4 days at 35°-38° in ambient air and were examined at 24, 48, 82, 96 h.
Triple sugar iron 8 . Procedure. By using a straight inoculation needle, the top of a well isolated colony was touched. TSI was first inoculated by stabbing through the medium to the middle of the tube and then streaking the surface of the agar slant. The cap was loosed and the tubes were incubated at 35°-38°c in excess air for 18-24 h.
Deoxyribonuclease test 6 . Procedure. 42.0 g of dehydrated media was suspended in 1000 ml in AR grade water and then heated with frequent agitation to mix the medium completely. The mixture after sterilization by autoclaving at 118°-121° for 15 min, was then made to cool to 45°, finally poured in sterile Petri plates. Then 100 mg toluidine blue was added before sterilizing the medium or the plates were flooded with 0.1% toluidine blue after incubation as desired.
Phenolphthalein phosphatase test 6 . Procedure. 28.01 g of dehydrated powder was mixed in 1000 ml of distilled water and then heated to boil for complete dissolution of medium. Do not autoclave. Dispense as desired.
Gelatin hydrolysis test 6 . Procedure. 34.00 g of dehydrated powder was dissolved in 1000 ml of distilled water with continuous heating to dissolve the medium completely, then autoclaved at 121 °C for 30 min in a dispensed plate.
Antibiotic susceptibility test 8 . Antibiotic susceptibility test was performed on the basis of organism isolated and antibiotic were selected according to CLSI guidelines. The antimicrobial susceptibility test was carried out Kirby Bauer disk diffusion method.
Muller Hinton agar 6 . Procedure. Nearly 38.0 g were dissolved in 1000 ml of distilled water, with continuous heating for the dissolution of medium completely, and then sterilization was done by autoclaving at 15lbs pressure at 121 °C for 15 min. Muller Hinton's agar is used for isolation of gram negative bacilli and gram positive cocci except Streptococcus species.
Kirby Bauer disk diffusion method 8 . Procedure. With a wire loop the 4 to 5 colonies of well isolated similar colonies was touched from the top of agar plate and transferred to the tube that contains 4-5 ml of suitable broth medium, followed by incubation at 35 °C until it matches the 0.5 Mc Farland usually 2-6 h.
A sterile nontoxic cotton swab was put into the inoculum after it adjusts its turbidity of that of standard and the swab was made to rotate many times with continuous pressure on the inside wall of the tube in order to remove excess fluid.
Then on the dried surface of Muller Hinton's agar plate, the swab was inoculated to bring the swab to room temperature by rotating the entire agar surface to near about 60 degrees each time and finally swab was rimmed www.nature.com/scientificreports/ of the agar. Finally the dish lid was replaced and allowed to dry on the surface of the agar before adding the antibiotic disk.
Observations. In the study period of about 6 months a total of 260 samples were collected from instruments, hand and nasal from neonatal and pediatrics ICUs. Total no of samples collected from instruments in PICU is 60 and 30 each from nasal and hand (Tables 1, 2 and 3). In NICU the samples collected from instruments were 80 and 30 from hand and nasal each (Tables 4, 5 and 6). The samples were collected with sterile cotton tipped swabs. The swabs were moistened with sterile normal saline and streaked across a 12 mm 2 areas and were immediately inoculated on Blood agar, MacConkey agar and the isolates were characteristics by colony identification, gram staining followed by standard biochemical reaction, and the antimicrobial activity was carried out by Kirby Bauer disk diffusion method.
Out of 30 nasal specimen, predominant isolate was CoNS with a good sensitivity for linezolid (13/15), vancomycin (12/15) and MRSA were produced by 3 isolates in contrast to S.aureus, which show good sensitivity Table 1. Spectrum of micro-organism isolated from medical devices associated infection in PICU. Different organisms isolated from different instruments of PICU. The organisms which were isolated are CoNS (10), S.aureus (10), Acinetobacter baumanii (6), candida spp. (2), Micrococcus (6), Bacillus (5).   www.nature.com/scientificreports/ for tetracycline (8/8) followed by linezolid (8/8) and Nitrofurantoin (6/8) and 3 isolates produce MRSA as mentioned in Fig. 5a,b. Surveillance of health care associates is a well-Constituted preventive measure for reduction of infections in HAIs, which are expected to be the carrier of a medium to long term infector's systematically 8 . Nosocomial infections (NIs) are common and are increasing treat for hospitalized patients, as they are the major causes of death and disability worldwide. As per the reports of World Health Organization, up to 15% of hospitalized patients get affected by infections associated with health care workers. Also the hospitals worldwide are facing the crisis of the upsurge and dissemination of resistant bacteria, particularly associated with nosocomial infections in ICU patients 9 . Regular checkup of device-associated infections in any healthcare center becomes the fundamental issue and an informative not only to health care professionals but also to administration in making various strategies for the prevention and control of such infections 10 . The result of this study is confirming our expectation that the ICU's are of more risk for the occurrence of nosocomial infection. In the current study as mention in Table 7 the most prevalent organism on the surface of medical devices in PICU were CoNS (16.66%) and S.aureus (16.66%) ( Table 1) while in NICU the most prevalent organism was Klebsiella spp. (11.25%) among Entero-bacteriaceae group followed by A. baumani (10%), E.coli(2.5%), CoNS (6.25%), S. aureus (6.25%) and E. faecalis (6.25%) ( Table 4). Similar to this result, another study on environment surface of hospital wards showed that CoNS and Klebsiella spp. as predominant isolates and was the most prevalent isolated bacteria 11,12 . A study from Northwest Ethiopia also shows the CoNS as the most predominant pathogen 13 Oxygen hood (n = 8) Bed with mattress (n = 8) Total 5 (6.25%) 5 (6.25%) 9 (11.25%) 8 (10%) 5 (6.25%) 2 (2.5%) 16 (20%) 30 (38.5%)          23 . Equal no of a CoNS and S.aureus isolated from NICU and both of them show a good sensitivity pattern for linezolid, co trimoxazole, vancomycin, levofloxacin and in each isolates 2 MRSA isolates were detected as mentioned in Fig. 6a,b. Among Entero-bacteriaceae, 9 Klebsiella pneumoniae and 2 E.coli both of them show sensitivity for colistin and imipenem Acinetobacter spp., were least resistance to colistin (2/8) imipenem (8/8), Enterococcus faecalis shows more sensitivity towards Tigecycline (5/5) followed by linezolid (3/5) as mentioned in Fig. 7a,b. CoNS as the predominant isolates shows a least resistant to tetracycline (1/13) and linezolid (2/13) with 3 MRSA isolated. For S.aureus, linezolid (8/8) susceptibility was observed with (2/8) MRSA detected as mentioned in Fig. 8a,b. Out of total CoNS isolated maximum sensitivity was shown for Linezolid (4/4) and Vancomycin (4/4) whereas for S aureus tetracycline (5/5) and Vancomycin (4/5) showing maximum sensitivity pattern Fig. 9a,b. A good sensitivity pattern was observed for both A. baumanii as well as Klebsiella spp. for colistin (5/5, 6/6) shown in Fig. 10a,b. Antibiogram of isolates from medical devices of ICU In this study, organisms grown from medical equipments of PICU, showed CoNS and S. aureus chief organism, and have been found susceptible to linezolid, tetracycline and vancomycin. Highest sensitivity of S.aureus for vancomycin was found in studies of A. D. khosravi et al. A. baumanii shows susceptibility to colistin and Imipenem 24 . Similar picture has been observed by priya dutta et al. 25 . The organisms isolated from medical equipments of NICU revealed similar picture as in PICU. CoNS and S.aureus although shows resistance to other commonly used drugs, yet linezolid, co-trimoxazole and vancomycin were found to be most effective while for E. faecalis; tigecycline and linezolid were sensitive (Table 4). From Entero-bacteriaceae family, colistin and imipenem has higher sensitivity pattern and same was observed for A. baumanii. Akash deep et al. in his study isolated 33.3% Klebsiella spp. and observed maximum sensitivity for amikacin 26 . Organism and the antibiogram of isolates from health care workers total of 30 health care workers each from PICU and NICU are included in the study, with a mean age of 20-30 years. Nasal and hand swabs were taken from each individual. Nasal swabs of HCWs from PICU shows a colonization of CoNS (15/30; Table 3), S.aureus (8/30; Table 3) with 6 MRSA strains detected by cefoxit in disk diffusion test. In this study the antibiotics sensitivity test revealed that CoNS and S.aureus remained sensitive to linozolid, vancomycin, tetracycline and nitro furan to in with low level of susceptibility to erythromycin, Gentamicin and Clindamycin. Ashish pathak et al. in his study isolated S.aureus from nasal carriage in healthy preschool children found highest sensitivity to Clindamycin 27 . At HRP Princess MCS Medical center, Thailand, S. aureus was found resistant to erythromycin, Clindamycin to tetracycline, chloramphenicol and fusidic acid were found resistant with the observation at www.nature.com/scientificreports/ the following rates: 63.2%, 63.2%, 34.2%, 2.6% and 2.6%, respectively 28 . Similar study is done for detection of microbes in hands of health care workers for which swabs from hands were taken and cultured. All participants were found to be colonized with CoNS (12/30; Table 2), S.aureus (6/30 Table 2), MRSA was found in 4 HCW ( Table 2) where antibiotic susceptibility pattern revealed their maximum sensitivity for vancomycin, tetracycline, linezolid and co trimoxazole and their resistance for Clindamycin, erythromycin and nitro furan. Similarly a sum of 30 nasal and hand swabs were also collected from health care associates of NICU, which shows the presence of CoNS (13/30 Table 5) and S.aureus (8/30; Table 5) from the anterior nares and CoNS (4/30    (Fig. 12). Prevention is more important than management of device associated infection, simple measures of asepsis and strict adherence to hand hygiene can decrease the incidence of nosocomial infection. The continuous surveillance of hospital environment for microbial infections with continuous inspection over infection control and prevention practices will prevent HAI.

Conclusion
A large number of factors are associated with health care associated infections in the hospitals. The Health care associated infections have significant impact on patients out comings including morbidity, mortality, hospital stay and cost of care. Therefore, a direct focus towards HCAI's may provide a way for the health quality of health care associates and their impact on the patients. Nursing care measures have direct impact on the prevention of various types of infections, central line infections, urinary system infections, sepsis and antibiotic resistant infections. Control over patient to patient transmission of infection by using hand hygiene's and control over general infection practices are the efficient ways for controlling the spread of resistant organisms. All persons who enter the unit should change into fresh gowns to prevent cross infection of susceptible infants. Environmental hygiene should be improved by removal of dust and dirt using vacuum cleaners with air exhaust and scrubbing of equipment's and inanimate objects will provide a really clean environment. HEPA filter can be used for air