Study Design and Subjects
This study was approved by the ethical committee of our institute and written informed consent was obtained from all patients for publication of this manuscript.
Patients
The study was conducted over a period of 12 months from January 2006 to December 2006 to all patients admitted at different adult ICUs at Assiut University Hospitals including: Coronary care unit (CCU), Chest ICU, Tropical ICU, Neurological ICU, Internal Medicine ICU, General ICU, Trauma ICU and Neurosurgical ICU. All patients admitted at different adult intensive care units (ICUs) were monitored daily by attending physicians for subsequent development of nosocomial BSI, which must meet at least one of the following criteria:
Criterion 1: Patient has a recognized pathogen cultured from one or more blood cultures and the organism cultured from blood is not related to an infection at another site.
Criterion 2: Patient has at least one of the following signs or symptoms: fever (> 38°C), chills, or hypotension and at least one of the following:
Common skin contaminant (e.g., diphtheroids, Bacillus sp., coagulase-negative staphylococci, or micrococci) is cultured from two or more blood cultures drawn on separate occasions. The Signs and symptoms of infection appear 48 hours to four days after admission, and there are no signs or symptoms of infection at the time of admission, proven by history and clinical examination [7].
Controls: included 159 Patients admitted to different ICUs during the same period but without any signs or symptoms of infection. Each case was matched with one control for age, sex and ICU specialty.
Environmental assessment: 475 environmental samples were collected from walls, floors, beds, bedside tables, and trays. These are the sites that we usually sample from based on the protocol followed in our institute.
Contact precautions were taken into consideration for the medical staff, hospital workers and visitors.
Sample collection
Blood samples: 5 to 10 ml of venous blood was collected from patients using sterile syringes. Blood samples were inoculated immediately under complete aseptic conditions into bottles containing 50 ml of brain heart infusion broth [8].
Environmental samples: sterile cotton swabs were moisten by a sterile physiological saline solution and used to collect samples from walls, floors, beds, bedside tables, and trays.
Processing of samples
Blood samples: the blood culture bottles were incubated aerobically at 37°C for 7 days. The bottles were examined daily for evidence of bacterial growth as haemolysis, gas production or turbidity above the red cell line. Subcultures using sterile syringes were done on blood agar, chocolate agar, MacConkey's agar and Bile Esculin Azide agar daily for 7 days before reporting blood cultures as negative [9]. Isolation of anaerobes is not considered.
Environmental samples: they were inoculated aerobically at 37°C for 24 hours on brain heart infusion broth for enrichment and then sub-cultured on blood agar and incubated for 24 hours at 37°C.
Identification of bacterial isolates
Identification of gram negative bacilli: gram negative bacilli were identified by API 20E system (Biomeriux SA, Montalien Vercica and France).
Identification of enterococcal isolates: suspected enterococcal isolates on Bile Esculin Azide agar were identified by the Colony morphology, Gram staining, the catalase test, the PYR test (production of pyrrolidonyl arylamidase), and the possession of Lancefield antigen D by using commercially available latex agglutination test. Omega Avipath Strep (Omega Diagnostica LTD., Scotland, UK).
Identification of staphylococcal isolates: according to Louie et al. [10], staphylococci were identified by standard methods including the gram stain, catalase test and tube coagulase test. Samples were cultured on Mannitol Salt Agar (MSA) (Oxoid, UK), where S. aureus produces yellow colonies (1 mm in diameter) surrounded by a yellow medium, and CNS forms small orange colonies surrounded by a red or purple medium [11].
Isolates which showed positive growth on Mannitol Salt Agar plates were subcultured on an Oxacillin Resistant Screening Agar Base (ORSAB) medium (Oxoid, UK) for Detection of oxacillin resistance.
Antimicrobial Susceptibility testing by modified Kirby-Bauer disc diffusion method: [10]
According to the protocol of the infection control lab, the following antibiotics were used: Antibiotics for Gram positive bacteria: B-Lactams (Ampicillin, Penicillin G, Cefazoline, Amoxicillin/clavulanic acid, Ceftriaxone, Cefotaxime and Oxacillin), Macrolides (Erythromycin), Aminoglycosides (Amikacin, Gentamicin), Tetracyclines (Tetracycline), Quinolones (Ciprofloxacin, Norfloxacin), Others (Chloramphenicol, Clindamycin, Vancomycin, Teicoplanin, Trimethoprim/sulfamethoxazole)
Antibiotics for Gram negative bacteria: B-Lactams (Ampicillin, Penicillin G, Cefazoline, Amoxicillin/clavulanic acid, Ceftriaxone, Ceftazidime, Piperacillin, Cefoperazone, Cefpodoxime, Cefuroxime, Aztreonam, Imipenem) Aminoglycosides (Amikacin, Gentamicin, Tobramycin), Tetracyclines (Tetracycline), Quinolones (Ciprofloxacin, Norfloxacin), Others (Chloramphenicol, Trimethoprim/sulfamethoxazole).
Detection of β-lactamase production
β-lactamase enzyme is detected among the gram negative bacilli by the chromogenic method (Nitrocefin test).
Detection of Extended Spectrum B-Lactamases
Selective testing for ESBL production is considered for all gram negative bacilli.
Screening for ESBL-Production: [12].
Isolates that exhibited reduced susceptibility to one or more of cefpodoxime, ceftazidime, aztreonam, cefotaxime or ceftriaxone were considered as potential producers of ESBL.
Confirmatory Tests:
The combined disk method or Inhibitor potentiated disc diffusion test [12]: ceftazidime (30 μg) versus ceftazidime/clavulanic acid (30 μg/10 μg), (Oxoid, UK), were used as a phenotypic confirmatory test where a greater than or equal to 5 mm increase in the zone diameter for the antimicrobial agent tested in combination with B-lactamase inhibitor versus its zone when tested alone indicates ESBL production.
Double-Disk Synergy "DDS" Test [13]: where any enhancement in the zone of inhibition between a beta-lactam disk and one containing the beta lactamase inhibitor was indicative of the presence of an ESBL.
ESBL – E – Test [14]: According to the manufacturer, a ceftazidime MIC/ceftazidime clavulanic acid MIC ratio which is equal to or greater than 8 indicates the presence of ESBLs (positive test).
Determination of the type of β-lactamase by polymerase chain reaction
In the present study, 18 Klebsiella pneumoniae strains which have caused primary bloodstream infection were investigated to determine the probable type of β-lactamase enzyme which was responsible for resistance. The isolates were picked up and tested for TEM, SHV, CTX-M-1, TOHO-1 genes by the PCR method [15].
Molecular typing by Random Amplified Polymorphic DNA (RAPD)
18 Klebsiella pneumoniae strains isolated from nosocomial BSI cases and 36 Klebsiella pneumoniae strains isolated from the environmental samples were typed by the RPAD method.
RAPD is a method for detecting strain differences. Its ability to type a wide variety of bacterial strains in a short time suggests that it will be a useful molecular epidemiological tool [16].
Statistical analysis
Data entry and statistical analysis was done using the SPSS ver.15 which included descriptive analysis, logistic regression for calculation of risk factors and dendrogram analysis. Figure 1 was done by using the MS excel 2003 program.
Consent
Written informed consent was obtained from the patients for publication of this manuscript.