Abstract:
This study was conducted to determine the prevalence of bacteria recovered from bloodstream samples by Bactec 9240 at our hospital wards and to evaluate their antibacterial susceptibility patterns. During January 2001 through December 2004, 9407 referred blood samples in Bactec bottles from admitted patients at three main wards, neonates, pediatrics and adults at Nemazee Hospital, affiliated to Shiraz University of Medical Sciences in Shiraz were processed. Positive cultures were purified and identified according to standard methods. Sensitivity of bacteria to different antibiotics was determined by Kirby-Bauer disk diffusion method. Staphylococcus aureus 132(25%), Escherichia coli 64(12.1%) and Pseudomonas aeruginosa 52(9.8%) were the most pathogenic bacteria which were recovered from the blood samples. Pathogenic microorganisms were isolated from blood samples of 305 (57.8%) at pediatrics, from 181 (34.2%) at adults and from 42 (8%) at neonates wards. The highest antibiotics activities against gram positive isolates observed for vancomycin (98.4%), chloramphenicol (86.4%) and ciprofloxacin (77.4%), while in gram negative bacteria imipenem (96.1%), ciprofloxacin (83%) and amikacin (77.9%), were effective antibiotics. Frequency of isolated bacteria at pediatrics compared to adults and neonates wards were approximately two and seven folds high, respectively which indicates special attention should be paid to pediatrics patients both in prevention and treatment aspects. Vancomycin and imipenim are the effective antibiotics and could cover majority of gram positive and negative bacteria. Therefore, combined administrations of these antibiotics seems mandatory for empirical therapy.
INTRODUCTION
Rapid and reliable detection of microorganisms from blood is one of the most critical functions of a diagnostic microbiology laboratory.
The BACTEC 9240 (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) system was established in our center in 1999. This instrument is a continuous monitoring blood culture system that uses internal, fluorescent-CO2 sensors. It accommodates up to 240 blood culture bottles and serves as an incubator, agitator and detection system. Each bottle contains a fluorescent-CO2 sensor and the sensors are monitored on a continuous basis (every 10 min). This system is noninvasive and automated blood culture systems with continuous monitoring have introduced technology that reduces the time needed to detect positive blood cultures as well as decreases specimen handling (Nolte et al., 1993;Rohner et al., 1997). On such a system, computer algorithms analyze the rate and amount of CO2 increase, which corresponds to an increase in fluorescence, thereby enabling the instrument to recognize a positive culture. When inoculated blood culture bottles are incubated and tested in the BACTEC instrument, microorganisms are detected as they enter the logarithmic phase of growth. Different media have been developed to enhance recovery and detection of positive cultures with this instrument. BACTEC plus aerobic/F and plus anaerobic/F media, which contain mixed resins, have been shown to be superior in their ability to support the growth of positive blood cultures (Rohner et al., 1997; Wilson et al., 2001). It has been suggested that enhanced recovery in resin-containing BACTEC media is due to lysis of leukocytes and subsequent release of viable, phagocytized microorganisms (Rohner et al., 1997). The manufacturer of plus media states that to maximize the number of positive blood cultures detected, it is recommended that blind Gram stains and/or subcultures be performed.
The purpose of this study was to report our four year experiences with BACTEC 9240 system regarding the epidemiology of recovered bacteria and to report antibacterial susceptibility patterns of the isolated bacteria. The data generated from this study could help the clinicians to treat patients more appropriately.
MATERIALS AND METHODS
Place of study: This study was conducted during January 2001 to December 2004 at Nemazee Hospital, affiliated to Shiraz University of Medical Sciences, Shiraz, Southern Iran. This hospital is tertiary care facility with 1000 beds and located in Fars Province. Normal or complicated patients from Fars, Hormozgan, Boushehr, Khouzestan and Kohkiloyeh-Boyerahamd provinces are referred daily or admitted in this hospital.
Patients and sampling: Suspicious patients to blood infections which were admitted at three main hospital wards including: neonates, pediatrics and adults were studied. Ten and three milliliter blood samples from adult and pediatric/neonate patients under supervision of specialized physician were taken and inoculated to BACTEC bottles peds plus/F or adult plus aerobic/F aseptically. An indication for patient blood infection was confirmed by the specialized physician in each ward.
Processing of specimens: The bottles were incubated in BACTEC system as recommended by manufacturer for 7 consecutive days. At the end of 7 days, the negative bottles were removed from the instrument and subcultured as mentioned below for positive bottles. During seven day incubation, when system alerts for positive results, 3 to 5 drops of blood culture samples with 1 mL sterile syringe were inoculated on blood and chocolate agars containing 5% whole sheep blood and incubated aerobically overnight. The pure culture were then stained by Gram`s method. For data analysis, the following criteria were established. An isolate was defined as an organism recovered from a blood culture bottle. A culture that was detected by BACTEC 9240 and confirmed to be positive by both Gram stain and subculture was considered to be a true positive. A culture that was instrument negative and negative upon terminal subculture was considered to be a true negative. Any culture that was instrument negative but positive upon terminal subculture was considered to be a false negative. Further identification of bacteria in positive cultures was carried out according to standard biochemical tests and subculturing to appropriate media.
Antibacterial susceptibility testing: Sensitivity of identified bacteria to different antibiotics was determined according to standard disk diffusion (Kirby-Bauer) method using Mast Co (Mast Co, Merseyside, UK) or Difco (BBL, USA) disks and was interpreted as recommended by National Committee for Clinical Laboratory Standard (Approved standard M7-A5. Wayne, PA: NCCLS, 2003). E. coli (ATCC 25922) and S. aureus (ATCC 25923) were used as controls for antibiotic susceptibility determination.
RESULTS
Totally 9407 blood samples in BACTEC bottles have been received and processed. Numbers of received specimen, frequency of culture positive specimens during a four year period screening is shown in Table 1.
Frequency and percent of bacteria isolated from blood samples based on wards are shown in Table 2. Coagulase Negative Staphylococci (CNS), Bacillus sp., Diphthroid and Micrococcus sp. were common contaminants of patients` samples. Therefore, results are reported for all isolated bacteria except for potential contaminants (Table 2). S. aureus, E. coli and P. aeruginosa were the most pathogenic bacteria isolated from the blood samples. The pediatrics with 305 (57.8%), adults with 181 (34.2%) and neonates wards with 42 (8%) were sequential for incidence of the pathogenic microorganisms. Antibacterial susceptibility patterns of Gram negative and Gram positive bacteria are shown in Table 3 and 4. Vancomycin and ciprofloxacin were the most active antibiotics against gram positive and negative bacteria in vitro. Brucellosis as an endemic disease has been observed in some reigns of the country which needs specific antibiotic therapy; therefore, sensitivity of these bacteria to antibiotics is shown separately (Table 5). Brucella sp. were highly sensitive to imipenem, rifampin and chloramphenicol.
| Table 1: | No. of specimens
and positive culture isolated from BACTEC 9240 during year 2001-2004 |
| Table 2: | Frequency of true pathogenic bacteria isolated from patients with BACTEC 9240 |
| *: It is fungus isolated by BACTEC system | |
| Table 3: | Antibacterial susceptibility patterns of Gram negative bacteria isolated from patients with BACTEC 9240 |
| ND: Not Determined; Values in parenthesis show percentage | |
| Table 4: | Antibacterial susceptibility patterns of Gram positive bacteria isolated from patients with BACTEC 9240 |
| ND: Not Determined | |
| Table 5: | Antibiotic susceptibility
of Brucella sp. recovered from BACTEC 9240 |
C: Highly effective antibiotics, respectively: 1-Imipenem, 2-Rifampin, 3-Chloramphenicol. ®: Less effective antibiotics, respectively: 1-Cephalexin, 2-Ceftazidime, 3- Cefuroxime |
|
DISCUSSION
Due to constant evolving antimicrobial resistant patterns which results in present global public health problem, there is the necessity for constant antimicrobial sensitivity surveillance. This will help clinicians provide safe and effective empirical therapies, develop rational prescription programs and make policy decisions and finally assess the effectiveness of all.
During the study, the annual numbers of specimens and positive cultures have been increased rapidly (Table 1). This could happen due to the introduction of this equipped system to the physicians in hospitals, health centers and private sectors in our regain. It is well known that isolation of microorganisms is a golden standard for accurate detection of etiological agents of infectious diseases (Liu et al., 2005; Jervoe-Storm et al., 2005). Furthermore, early detection of bloodstream infections could prevent implantation of microorganisms into vital organs such as brain, heart or kidneys (Pirker et al., 2006; Alp et al., 2006; Safdar et al., 2005).
The number of positive cultures in the pediatrics ward is high as compared with adults and neonates wards. This is partly due to number of beds in pediatrics ward which admit a wide range of patients with ages ranging from 6 months to 16 years old. Moreover, children at pre or primary school ages may have not received an appropriate care regarding their hygiene as compared with adults. Besides, neonates` hygiene are continuously supported and monitored by their mothers.
Ampicillin, co-trimoxazole gentamicin and cefuroxime were less active against our isolates. This is in agreement with the reports of other studies (Mamishi et al., 2005; El Kholy et al., 2003). They showed that due to low activity of these antibiotics against bloodstream infections, it should not be administrated for treatment, prophylactic and empirical purposes of the bloodstream infections. It is documented that due to emerging resistance, the efficacy of antibiotics is decreasing gradually particularly those administrated for bloodstream infections (Cordero et al., 1999; Tantracheewathorn et al., 2007). These data support the hypothesis that determination of antibiotic sensitivity patterns in periodic intervals is mandatory in each region for choosing appropriate antibiotic therapy (Turnidge, 2003). Overall, present results indicate that imipenem and vancomycine are highly active against Gram negative and positive bacteria. These results are in concordance with other reports (Raja, 2007; Zapantis et al., 2005). However, it should not be expected that this activity continues for a longtime, as we observed during the last two years in our hospital (unpublished data) or in other centers (Swoboda et al., 2007), Vancomycin Resistant Entrococci (VRE) were emerged in bacteria isolated from the patients with urinary tract infections. These findings suggest that survey of antibiotics sensitivity would be preferable if performed in two years intervals.
One of the advantages of BACTEC system is to recover intracellular fastidious bacteria such as Brucella sp. This could help to treat brucella infected patients with appropriate antibiotics such as rifampin or chloramphenicol and cease of administration of inappropriate antibiotics such as cephalosporins. Detection of brucella infection in our regions is epidemiologically valuable especially in rural areas where consumptions of unpasteurized milk and dairy products is still noticeable (Roushan et al., 2005; Refai, 2002). Accurate detection and complete treatment of the human brucellosis could reduce frequency of morbidity or chronic infection cases which have clinical and economic benefits (Fallatah et al., 2005; Ruiz-Mesa et al., 2005; Lim et al., 2005). Besides, it is evident that BACTEC 9240 can successfully recover Brucella melitentsis in synovial fluid within 5-7 day incubation period (Yagupsky et al., 2001).
CONCLUSION
Present findings reveal the etiology of infectious diseases of the blood. The bacteria with high frequency recovered from blood samples at pediatrics ward indicate that special attention should be paid to our pediatrics patients both in prevention and treatment aspects. Vancomycin and imipenem were the most active antibiotics which could cover the majority of gram positive and negative bacteria. Therefore, an administration of the combination of these antibiotics seems mandatory for empirical therapy when an infection with fastidious intracellular bacteria such as Brucella sp. is ruled out.
ACKNOWLEDGMENT
This study was financially supported by Professor Alborzi Clinical Microbiology Research Center (grant 80-15).