INTRODUCTION:

Newborns admitted to intensive care units (ICUs) are at a high risk of developing nosocomial infections (NIs), because of the severity of their illness and exposure to invasive medical devices and the presence of multi resistant microorganisms^1-3. Nosocomial infections (NIs) occur in 15% to 25% of very low birth weight (VLBW) infants^4,5 NIs increase mortality⁶ morbidity⁷,length of stay^8,9), treatment costs¹⁰ and the risk of long-termdisabilities^11,12. Health care associated infections are a problem for neonatal intensive care units¹ and frequently arise as a result of formation of biofilms on the surfaces of indwelling medical devices¹³. These have become major tools in the clinical management of hospitalized patients, particularly those requiring life supporting devices¹⁴.

A biofilm is a functional multilayered community of microorganisms, adhering to a surface and organized within a selfproduced exopolymeric matrix¹³. The encasing slimy exopolysaccharide matrix, protects microcolonies from external insults by blocking entry of biocides, surfactants and predators and rendering them more resistant to antibiotics compared to free floating bacteria^15,16. In addition to acting as transport barriers to agents harmful to biofilms¹⁷ , exopolysaccharide matrix polymers also bind to and neutralize antibiotics prior to their interaction with bacteria¹⁸. Further more bacteria within biofilms are dormant and less responsive to antibiotics¹⁹.

Molecular typing like pulsed field gel electrophoresis is a useful epidemiologic tool in the elucidation of transmission routes of microbial pathogens and determining the interrelationship of bacterial isolates²⁰. DNA fingerprinting by pulsed-field gel electrophoresis (PFGE) is considered to be one of the most reliable, discriminatory, and reproducible typing procedures to allow the detection of high degree DNApolymorphism²¹.

The aim of the present study: is to determine the relationship between bacterial isolates from neonates in intensive care units with the surrounding environment and to assess the role of biofilm formation in the indewelling medical devices

MATERIALS AND METHODS:

Microorganisms:

Sources of tested isolates:

A total number of 470 samples were collected from neonates and their surrounding environment over the period from February 2010 to March 2011 from neonatal intensive care units of Zagazig University Hospitals.

Collection of samples:

The collected specimens compromised of :

A- Neonates 'specimens: swabs from ear, anterior nares, axilla and neck regions as well as blood sent for laboratory investigation.

B NICU environmental specimens, by swabs from incubators as well as from nursing facilities and equipment in NICU.

C- Specimens from hands of health care workers (HCW) were collected by using modified glove juice method²² “both hands of each worker were sequentially put into a sterile polyethylene bag containing 50 ml of sampling solution.

D- Specimens from used indwelling medical devices.

Processing of specimens:

Swabs were plated onto Nutrient agar, MacConkey agar, Blood agar. Blood samples were cultured in trypticase soya broth and incubated for 24 hrs at 35-37ºC. Followed by, subcultures on Nutrient agar, MacConkey, Blood agar and incubated at 35-37ºC for 24-48 h. For hand disifection solution and baby foods 1-2 ml were inoculated into 10 ml of nutrient broth and incubated for 24 h at 35-37ºC , then subcultures were made on Nutrient, MacConkey, Blood agar and incubated at 35-37ºC for 24-48 hrs. The most predominant isolates for each specimens was processed for identification

Identification of the isolated strains:

Isolates were identified by standard microbiologic techniques by Gram staining,colony characteristics, and biochemical properties including catalase, mannitol salt agar, and haemolysis on blood agar plates, for Gram positive isolates, and triple sugar iron (TSI), lysine iron agar (LIA), motility, indole, citrate, urease and oxidase for Gram negative bacilli^23,24 .

Antibiotic susceptibility testing:

The antibiotic susceptibility testing was performed by disc diffusion method according to Clinical and Laboratory Standards Institute guideline (CLSI, 2008)²⁵. Antimicrobial agents used were: Amikacin (AK), Amoxicillin clavulanic acid (AMC), Erythromycin (E), Sulbactam-Ampicillin (SAM), Cefoperazone (CEP), Cefotaxime (CTX), Ciprofloxacin (CIP), Vancomycin (VA), Gentamicin (CN), Imipenem (IPM) the results were interpreted according to CLSI(2008.

Detection of biofilm formation:

(i)Detection of biofilm formation by viable count:

The method was used after modification to Deighton²⁶. Briefly, a bacterial suspension of turbidity equal to 0.5 McFarland (~10⁸CFU/ml) was diluted 1:100 into Trypticate Soya Broth (TSB) and 100 µl of the diluted bacterial suspensions were pipetted into different wells in a 96-well flat-bottom microtiter plate and incubated for 18-24 h at 35°C without shaking. After overnight incubation, the bacterial suspensions were aspirated and the wells were rinsed twice with 100 μL of phosphate buffer saline (PBS) per well to remove non-adherent bacteria. The biofilm were disrupted by sonication then the colony were counted according to Villain-Guillot²⁷.

(ii) Detection of biofilm formation by Scanning electron microscopy (SEM):

The samples of catheters, feeding tube and cannulas used by neonates were subjected to (SEM). Samples obtained were prepared for (SEM) after air drying. Tubing was cutinto pieces and plated with gold according to Morck²⁸ . The scanning was studied bya Hitachi model 450 scanning electron microscope.

The samples from wells of microtiter plates were broken using pliers, rinsed once with 0.9% saline to disrupt loosely adherent planktonic cells and air dried for 120 h before mounting and examinaed as above .

Pulsed-field gel electrophoresis (PFGE):

Pulsed field gel electrophoresis for SmaI digested genomic DNAwas performed as described by Tassios²⁹ Brifely ,genomic DNA was prepared in agarose plugs that had been treated with lysozyme, lyzostaphin, and proteinase K, using the Gene Path Reagent kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. The DNA was digested with 20 U of SmaI . The DNA fragments were separated in a 1% agarose gel and were run in Tris-borate ethylene diamine tetra acetic acid (EDTA) buffer on a pulsed field apparatus (Gene Path System; Bio-Rad) at 6.0V/cm for 20 h, with pulse times ranging from 5.3 t Interpretation of the PFGE banding patterns followed established guidelines according to Tenover³⁰ isolates were considered identical if they shared every band, closely related if they differed by 1 to 3 bands, possibly related if they differed by 4 to 6 bands, and unrelated if a difference of more than 7 bands was observed .

RESULTS:

Out of 470 samples of different types taken from neonates and NICU 450 bacterial isolates were recovered The data in table (1) revealed that the most commonly isolated microorganisms were Staphylococcus. aureus (31.9%), Coagulase negative staphylococcus (31.9%), Klebseilla .pneumoia (21.3%), and Psudomonas. aerguinosa (10.6%).

Table (2)Antibiotic resistance of Staph. aureus and CoNS isoaltes.

Antimicrobial	*Staph.aureus*		CoNS
Antimicrobial	No	%	No	%
Amikacin	43	28.7	53	35.3
Amoxacillin- clavulanic acid	64	42.7	70	46.7
Cefoperazone	60	40	80	53.3
Cefotaxime	75	50	90	60
Ciprofloxacin	10	6.7	16	10.7
Erythromycin	50	33.3	48	32
Gentamycin	30	20	35	23.3
Imipenem	10	6.7	12	8
Sulbactam- Ampicillin	76	50.7	86	57.3
Vancomycin	0	0	0	0

Table (3)Antibiotic resistance of Ps. aerguinosa and K. pneumoniae isolates .

Antimicrobial	*Ps. aeruginosa*		*K. pneumoniae*
Antimicrobial	No	%	No	%
Amikacin	14	28	20	20
Amoxacillin- clavulanic acid	30	60	40	40
Cefoperazone	28	56	40	40
Cefotaxime	50	100	80	80
Ciprofloxacin	10	20	12	12
Erythromycin	28	56	28	28
Gentamycin	20	40	40	40
Imipenem	7	14	7	7
Sulbactam ampicillin	46	92	46	46

Detection of biofilm formation by viable count

The results of biofilm formation of the tested isolates were detected by viable count method and were presented in table (4) Viable counts of S. aureus, CoNS, P. aeruginosa and K. pneumoniae were ranged from 14.4- 30.0x10⁵ per well. The obtained results revealed that the bacterial isolates grew readily as planktonic organisms in liquid culture media. Also these organisms would form biofilms under cultural conditions. The duration of heavy biofilms formation on the wells (>10000cfu/well) was detected after 24 hours .

Table (4): Viable bacterial counts in biofilms formed in the 96 well microtiter plate after 24 h. growth

S. aureus (2)	14.4 x10⁵	6.16±0.1
S. aureus ( 9)	15.2 x10⁵	6.18±0.1
S. aureus (60)	14.6 x10⁵	6.16±0.2
S. aureus (123)	14.6 x10⁵	6.16±0.2
CoNS (30 )	16.2 x10⁵	6.2±0.1
CoNS ( 80)	16.8 x10⁵	6.23±0.1
CoNS ( 137)	16.8 x10⁵	6.23±0.1
Ps. aeruginosa (3)	26.6 x10⁵	6.4±0.2
P. aeruginosa (11)	26.6 x10⁵	6.4±0.2
P. aeruginosa (30)	28 x 10⁵	6.45±0.1
P. aeruginosa (50)	30 x10⁵	6.48±0.1
K. pneumonia (5)	26.6 x10⁵	6.4±0.2
K. pneumonia ( 14)	26.6 x10⁵	6.4±0.2
K. pneumonia ( 40)	26.6 x10⁵	6.4±0.2
K. pneumonia ( 95)	30 x10⁵	6.48±0.1

Scanning electron microscopy (SEM) was used for the examination and characterization of biofilms formed in a 96-well flat-bottom micro plate and inside the feeding tubes, cannulas and catheters taken from neonates. The SEM micrographes demonstrated the biofilm formation , as seen in figures (1-4) and the presence of individual microcolonies within the biofilms.

(A)

(B)

Fig. (1)(A):SEM of lumen of unused cannula (B): SEM of lumen of used cannula revealed that bacteria were bound to each other and form microcolonies (magnification 1500x) .

(C )

(D)

Fig. (2):(C)SEM of lumen unused feeding tube . (D): SEM of lumen of used feeding tube showing whole surface of feeding tube covered with abundant biofilm (magnification 1500x) .

(E)

(F)

Fig. (3):((E) SEM of unused catheter . (F): SEM of lumen of used catheter showing abundant slime and debrids of biofilm (magnification 1500x) .

(G)

(H)

(I)

Fig. (4): (G) SEM of cross section in unused well . (H) and (I): SEM of cross section in used well (magnification 1500x) showing biofilm formation microorganisms protected beneath a tough polysaccharides slime layer.

Figure 5. Pulsed-Field Gel Electrophoresis of Representative Strains of S. aureus isolated from neonates and the surrounding environment in the Neonatal Intensive Care Unit .Lane M shows the molecular-size ladder; lane 1, blood from neonate; lane 2,isolate from cannula; lane 3, isolate from catheter; lane 4, isolate from skin of neonate3; lane 5, from hand of nurse; lane 6, from hand of doctor ; lane 7, from incubator 1; lane 8,isolate from feeding tube ; lane 9, from gown ; lane 10, from hand disinfectant solution; lane 11, from table of medication ; lane 12, from wall of intensive care unit.

Lane M shows the molecular-size ladder; lane 1, blood from neonate; lane 2,isolate from cannula; lane 3, isolate from catheter; lane 4, isolate from skin of neonate3; lane 5, from hands of nurse; lane 6, from hand of doctor ; lane 7, from incubator; lane 8,isolate from feeding tube ; lane 9, from gown ; lane 10, from hand disinfectant solution; lane 11, from table of medication ; lane 12, from wall of intensive care unit.

Figure 7. Pulsed-Field Gel Electrophoresis of Representative Strains of P. aerguinosa isolated from neonates and the surrounding environment in the Neonatal Intensive Care Unit .

Lane M shows the molecular-size ladder; lane 1, blood from neonate; lane 2,isolate from cannula; lane 3, isolate from feeding tube; lane 4, isolate from skin of neonate3; lane 5, from hands of nurse; lane 6, from incubator; lane 7, from hand of doctor 1; lane 8,isolate from sink ;lane 9, from gown ; lane 10, from hand disinfectant solution; lane 11, from table of medication ; lane 12, from wall of intensive care unit.

Figure 8. Pulsed-Field Gel Electrophoresis of Representative Strains of K. pnumoniae isolated from neonates and the surrounding environment in the Neonatal Intensive Care Unit.

Lane M shows the molecular-size ladder; lane 1,blood from neonate; lane 2,isolate from catheter; lane 3, isolate from feeding tube; lane 4, isolate from gown; lane 5, from floor; lane 6, from incubator; lane 7, from skin of neonate ; lane 8,isolate from sink;lane 9, table of medication lane 10, from hand disinfectant solution; lane 11, from hand of nurse ; lane 12,hand of doctor.

The results of restriction fragment polymorphism using SmaI digested genome were represented in figures (5-8) revealed that Staph. aureus isolated from blood of neonates was identical to isolate from cannula while, there was a closely related relation between isolates from catheter, skin of neonate , incubator ,hand of nurse, hand of doctor, gown, feeding tube,hand disinfecting solution and isolate from blood of neonate .On the other hand isolates from wall and table of medication were considered to be possibly related to isolate from blood of neonate .

PFGE of isolates of CoNS showed that the isolate had a closely related relation with isolates from cannula , catheter , skin , hand of nurse , hand of doctor , incubator , feeding tube , hand disinfectant solution , gown , table of medication and wall of intensive care unit .

PFGE of isolates of Ps. aeruginosa revealed that isolates from blood , cannula and feeding tube of neonate were identical while, isolates from skin of neonate , incubator and hand of nurse were closely related to isolate from blood .On the other hand Isolates from hand of doctor, hand disinfectant solution , ` of medication , wall, sink and gown showed a possibly related relation with isolate from blood .

PFGE of isolates of K. pneumoniae showed that isolates from blood were closely related to isolates from catheter , feeding tube ,incubator , skin of neonate, hand of nurse , hand of doctor and hand disinfectant solution while , isolates from gown, floor, sink and table of medication had a possibly related relation with isolate from blood .

DISCUSSION:

The newborns represent one of the most vulnerable populations amongst the paediatric group, especially neonates hospitalised in a neonatal intensive care unit (NICU) where large-scale use medical devices, antimicrobial drugs and lack of maturation of a child's immune system increase the chances of acquiring nosocomial infection¹.

Neonates starting life in a NICU acquire their flora from their contacts with doctors and nurses and the environmental of the ward, possibly resulting in early colonization with antibiotic resistant bacteria³¹.

The present study was designed to investigate the most common organisms isolated from NICUs, to find a relation between different isolates and to detect presence of biofilm formation in indwelling medical devices. Four hundred and seventy isolates were collected from neonatal intensive care units of Zagazig University hospitals over the period from February 2010 to March 2011. The results of the study showed that the most commonly isolated microorganisms from blood of neonates were CoNS,K. pneumonia , staphylococcus aureus and Ps. aeruginosa with percentage (28.75%), (28.75%), (21.4%) and (14.28%)respectively . These results are in concordance with some of other studies . Aletyeb et al.³², Mireya et al.³³and West and Peterside³⁴. In this study the most commonly isolated organisms from incubators were CoNS (33.75%, Staph. aureus (31.25%), K. pneumoniae (16.25%) and Ps. Aerguinosa (8.75%). This results were in agreement with that reported by Abd El Haleim et al.³⁵ and Crivaro et al.³⁶

Isolates from hands of health care workers were CoNS (37.5%), Staph. aureus (31.25%), K.pneumoniae (21.25%), Ps. aerguinosa (3.75%) this in agreement with that reported by Aiello et al.³⁷ and Crivaro et al.³⁶

The environmental samples revealed that the percentage of isolates was Staph. aureus (33.75%) , CoNS(28.75%) , Ps. aerguinosa (18.75%) and K. pneumoniae (18.75%). Also ,sapmles from hand disinfectant solutions revealed the presence of Staph. aureus , CoNS , Ps. aerguinosa and K. pneumoniae .These results were in agremment with that reported by Crivaro et al.³⁶, Shrestha et al.³⁸Also , Krishna et al.³⁹ Reported the presence of K. pneumonia in hand washing soap and Reiss et al.⁴⁰informed that Klebseilla may spread rapidly from medical devices , soap and disinfectants and the hands of hospital staff.

Samples from nursing facilities revealed the presence of Staph. aureus , CoNS , Ps. aerguinosa and K. pneumoniae This in agreement with that reported by Büyükyavuz et al.⁴¹, Sánchez-Carrillo et al.⁴²and Hurrel et al.⁴³

In our study the most frequently isolated organisms were Staph. aureus, CoNS , Ps. aerguinosa and K. pneumonia ,this was in agreement with Auriti et al.⁴⁴The antibiotic susceptibility results revealed that Staph. aureus and CoNS showed high resistance to cefotaxime , ampicillin-calvulanic acid , cefoperazone and sulbactam ampicillin. While, all isolates showed high sensitivity to ciprofloxacin and imipenem. All isolates were 100% sensitive to vancomycin. These results are in agreement with that reported by Seif El-Din et al.⁴⁵and Abd El Haleim³⁵.

The antibiotic susceptibility data of Ps. aerguinosa and K.pneumoniae showed 100% and 80% resistance to cefotaxime respectively in agreement with Aletayeb et al³². Isolates of P. aerguinosa and K. pneumoniae showed a low rate of resistance to Amikin , ciprofloxacin and imipenem in concordance with Mutlu et al.⁴⁶

Bacterial biofilm has long been considered as a virulence factor contributing to infection associated with various medical devices and causing nosocomial infection Arciola et al.⁴⁷. Suggested mechanisms by which biofilm producing bacteria cause disease are detachment of cells from medical device biofilm causing blood stream or urinary infection, endotoxin production, resistance to immune system and generation of resistance through plasmid exchange Donlan and Costerton¹⁴. In the present study we detect biofilm formation from diffent isolates by viable count. . The viable cell counts is the most commonly used procedure for detection of biofilm by plate counting. This observation was also reported by Harrison et al.⁴⁸.All tested isolates could for biofilm. This in agreement with those reported by Olson et al.⁴⁹and Harrison et al⁴⁸.The SEM of well of micro plate, lumen of catheter , feeding tube and cannula revealed the presence of biofilm inside them and the biofilm structures appeared as thick cell layers with high density , clumps, microorganisms protected beneath a tough polysaccharides slime layer. These results are in concordance with those reported by Rodeny⁵⁰, 2001 that Biofilms on indwelling medical devices may be composed of gram-positive or gram-negative bacteria or yeasts. Bacteria commonly isolated from these devices include the gram positiveStaphylococcus aureus, Staphylococcus epidermidisand the gram-negative Klebsiella pneumonia and Pseudomonas aeruginosa⁵⁰. These organisms may originate from the skin of patients or health care workers, tap water to which entry ports are exposed, or other sources in the environment.

PFGE finding relation between isolates from blood and those isolated from cannula, catheter, feeding tube. The relation between isolates from health care workers, skin of neonates, incubators and disinfectant solution showed a closely related relationship with those isolated from blood of neonates. On the other hand there were a possibly realtion between environmental isolates and those isolated from blood. This finding rules out the possibility of cross infection. These Data are in agreement with those reported by Reese et al.⁵¹

The vast majority of clustered cases for any pathogen have been found to be genetically similar strains with transmission due to cross contamination from a small number of health care workers. Also the reservoirs for transmission are numerous: Soap bottles and sinks ⁵² , hand lotion⁵³, hands⁵⁴. Some studies proved that P. aeruginosa can be introduced to the NICU via a number of routes, including environmental contamination, transmission by HCWs, transfer of colonized patients and through the use of contaminated water to prepare milk or other nutrition⁵⁵.In another study of infections in neonates caused by gram-negative bacilli, the pathogen was transmitted by hand contact in 42%of cases⁵⁶.

CONCLUSION:

The study proved that there is a relation between different isolates isolated from different sites in neonatal intensive care units .The results of the study emphasis the detection of biofilm formation in indwelling medical devices commonly used in neonatal intensive care units .Understanding the relation between different isolates would serve in reducing spreading of infection among neonates. Understanding and including the relatedness between the bacterial isolates from neonates and their surrounding environment helps in tracing the possible reservoirs of infection and in advising infection control policies.

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Received on 28.04.2013 Modified on 22.05.2013

Research J. Pharm. and Tech 6(7): July 2013; Page 794-801

Types (number)		Number and (percentage) of isolates
Types (number)	No of negative samples	*No .of S.* *aureus* (%)	No. of Co NS (%)	*No .of* *P. aeruginosa* (%)**	No. of *K. pneumonia (*%)
Blood(70)	5	15(21.4)	20(28.75)	10(14.28)	20(28.75)
Skin of neonates(80)		30(37.5)	30(37.5)	5(6.25)	15(18.75)
Incubators(80)	8	25(31.25)	27(33.75)	7(8.75)	13(16.25)
Skin of hands’workers(80)	5	25(31.25)	30(37.5)	3(3.75)	17(21.25)
*Environmental Samples(80)		27(33.75)	23(28.75)	15(18.75)	15(18.75)
Disinfectants solutions (10)	2	3(30)	2(20)	2(20)	1(10)
Nursing facilities(20)		4(20	6(30)	4(20)	6(30)
Samples from gowns before and after using(10)		3(30)	3(3)	2(20)	2(20)
Indwelling medical devices (40)		18(45)	9(22.5)	2(5)	11(27.5)
Total samples(470)	20	150(31.9)	150(31.9)	50(10.6)	100(21.3)