Molecular epidemiology of Acinetobacter baumannii in Central Intensive Care Unit in Kosova teaching hospital.
Acinetobacter spp. cause a wide range of nosocomial infections, such as ventilator-associated pneumonia, bloodstream infections, urinary tract infections, surgical site infections and meningitis, especially in immunocompromised patients staying in ICU . Other risk factors for colonization and infection are recent surgery, central vascular catheterization, tracheostomy, mechanical ventilation, enteral feeding and treatment with antibiotics (third-generation cephalosporins, fluoroquinolones or carbapenems)[6,7]. Extensive use of antimicrobials within hospitals has contributed to the emergence and increase of antimicrobial resistance among Acinetobacter strains .
Numerous reports implicates A. baumannii as a major pathogen involved in nosocomial infections causing epidemic outbreaks or endemic occurrence with a documented high mortality rates [9-12]. An increase in the number of A. baumannii isolates from clinical samples has been observed in microbiology laboratory over the past few years in ICU of university hospital in Kosova. But, this was not accompanied by detailed epidemiological and clinical investigation.
Knowledge regarding species, strains and clones of Acinetobacter circulating in Kosova hospitals is lacking. Published data concerning the clinical implications of Acinetobacter spp. infections in Kosova are scarce. A study regarding clinical samples of Acinetobacter spp. isolates and their susceptibility pattern undertaken during 2001-2004, showed a total of 242 Acinetobacter spp., of which A. baumannii predominate with 81.2% . The majority of samples were revealed from patients staying in ICUs (62%). Based on laboratory report between March 2005 and August 2006, A. baumannii was responsible for 100 of the 719 infections, which occurred in the CICU (13.9%). Other most common isolated pathogens were P. aeruginosa (22.1%), S. aureus (15.39%), and Klebsiella pneumoniae (12.9%).
The present study was undertaken to elucidate the molecular epidemiology of Acinetobacter baumannii using pulse field gel electrophoresis (PFGE). Therefore, the objectives of the present study were (i) to assess the genetic relatedness of A. baumannii isolates in the ICU of our university hospital; and (ii) to study the clinical features of patients from whom A. baumannii had been isolated.
Material and Methods
Hospital Setting and Patients
The study was conducted at the University Clinical Centre of Kosova (UCCK), in Prishtina, the capital city of Kosova. The center has 2,100 beds with approximately 60,000 admissions per year and serves as the only referral tertiary care center for a population of around 2.1 million. The Central Intensive Care Unit is a mixed ICU with 12 beds. The bacterial isolates selected for the present study included 30 A. baumannii isolates from 30 patients from the ICU of UCCK, during the period from March 2006 to July 2006. Laboratory diagnosis of microbiological samples and susceptibility testing was done in the Department of Microbiology within the National Institute for Public Health of Kosova. The genotyping was performed in the Clinical Hospital Centre Zagreb, Department of Clinical and Molecular Microbiology in Zagreb, Croatia. Clinical specimens included cerebrospinal liquid, endotracheal aspirate, thoracal drain and tracheostoma. The following data were recorded from the medical charts of patients with A. baumannii infection or colonisation: age, gender, number of patient-days in hospital, underlying diseases or conditions, susceptibility pattern and clinical outcome. Nosocomial infections were classified according to standard CDC definitions, whilst A. baumannii was considered to be a colonising organism when it was isolated from clinical specimens, but the criteria for infection were not met . Only one sample of A.baumanni per patient was enrolled in the study.
A. baumannii strains were collected from clinical specimens by using standard methods, isolated in pure cultures on MacConkey agar plates. Organisms were identified by using the API system for the identification (bioMerieux, Marcy l'Etoile, France). From a fresh 18 hours plate culture of A. baumanii, a heavy, cloudy suspension of the organism was made in the CRYOBANK [TM] medium in the tube (COPAN Diagnostics Inc., CA, USA). Tube was mixed by shaking and inverting to allow the bacteria in the suspension to coat the beads. Using a sterile pipet the CRYOBANK [TM] medium was removed from the tube. Than, the tube was placed in a -70[degrees]C freezer to store the culture. Afterwards the samples were transported to Croatia where the bacteria were recovered removing the cap of the CRYOBANK [TM] tube. Using forceps one bead was rolled over the culture mediums (brain-heart infusion) and Kaufman-Muller broth. Isolates were verified in Croatia as A.baumannii using the Vitek 2 automatic system (bioMerieux, Marcy l'Etoile, France).
Antimicrobial resistance was determined by the disk diffusion method according to the Clinical and Laboratory Standards Institute criteria, former NCCLS . The following antimicrobial drugs were tested: Ampicillin 10 [mu]g, Ceftriaxon 30 [mu]g, Gentamicin 10 [mu]g, Amikacin 30 [mu]g, Imipenem 10 [mu]g, Pipercilin + tazobactam 100 [mu]g, Cefoxitin 30 [mu]g, Ceftazidime 30 [mu]g, Tobramycin 10 [mu]g, Cotrimoxasole 1.25+23.75 [mu]g and Ciprofloxacin 5 [mu]g.
Molecular Typing by Pulsed-Field Gel Electrophoresis (PFGE) and Dendrogram Analysis
The preparation of genomic DNA of A. baumannii isolates was performed as described by Schwartz and Cantor with minor modifications. Macrorestriction analysis of chromosomal DNA with XbaI was carried out by PFGE following published procedures . PFGE was run in a CHEF-DRIII apparatus (Bio-Rad Laboratories, CA, USA), with pulses ranging from 5 to 50 seconds at a voltage of 6 V/cm at 10-12[degrees]C for 20 h. Products were detected after staining with ethidium bromide (50 mg/mL) and photographed with Polaroid type 667 film. A ladder ofbacteriophage lambda concatemers (New England Biolabs) was used as molecular weight markers.
Clusters of possibly related isolates were identified by using the Dice coefficient of similarity and unweighted group method with arithmetic averages at 80%, which indicates fourto six fragment differences in gels. The relationships between all isolates were analysed using the GelComparII software package and presented as a dendrogram (Applied Maths NV, Belgium). DNA fingerprints were interpreted as recommended by Tenover et al. .
From March 16th to July 27th 2006, a total of 30 Acinetobacter baumannii isolates were obtained from 30 patients (24 males, 6 females) admitted to the CICU. Their age range was from 2 to 82 years (mean age 47.5, median age 52.5 years). Based on evaluation of clinical charts, 22 patients were classified as infected and had nosocomial infections and eight of them were considered colonized with A. baumannii.
Isolates were most frequently recovered from endotracheal aspirate (n = 26); the other isolates were recovered from tracheostoma (n=2), thoracal drain (n=1) and cerebrospinal fluid (n = 1). Twenty patients developed nosocomial pneumoniae; one patient had a diagnosis of meningitis, and two had coinfection with bloodstream infection and surgical site infection. The most common diagnoses upon admission to the ICU were politrauma and cerebral hemorrhage. Other pathogens were co-isolated from nine patients: Staphylococcus aureus from two patients, P. aeruginosa from 4 patients, and Klebsiella pneumonaie from three patients.
The clinical characteristics of patients from whom A. baumannii was isolated are shown in Table 1.
The length of stay in ICU ranged from 1-59 days with median time of 17 days. The median time that had elapsed between admission and isolation of A. baumannii was 8 days. During the ICU stay, 16 patients died (crude mortality 53.3%) and the A. baumannii-attributable mortality was 62.5% (10 / 16). The time-frame of admission, discharge and isolation of A. baumanni from ICU patients is presented in Figure 1.
The length of ICU stay for non-survivors and survivors was 14.5 and 18.5 days, respectively. The length of stay was significant in comparison between infected and colonised patients (19.5 vs. 9 days).
The length of ICU stay for epidemic strains and nonepidemic strains was 15.7 and 24.5 days, respectively.
Four patients yielded A. baumannii upon hospital admission and were transferred from other hospitals to ICU, while 22 patients yielded A. baumannii only following hospitalization. Twelve patients were transferred to ICU from other departments within the UCCK.
PFGE profiles of A. baumannii strains isolated from CICU is shown in the Figure 2.
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Genotypic analysis of A. baumannii isolates from ICU patients identified nine major PFGE patterns, which we named from A to I, that differed in migration of at least four DNA fragments and showed a similarity of < 80% at dendrogram analysis. Of these, PFGE pattern E predominate with isolates from nine patients. Eight isolates were resistant to carbapenems.
Although only 5-10% of all hospitalized patients are treated in ICUs, they account for approximately 25% of all nosocomial infections . The incidence of nosocomial infections in ICUs is 5-10 times higher than that observed in general hospital wards [19,20]. In developing countries the occurrence of nosocomial infections is 12-20 fold higher .
A. baumannii outbreaks have been reported previously, particularly in ICU wards [22-26]. Severe underlying diseases, invasive diagnostic and therapeutic procedures used in ICUs have been demonstrated to predispose patients to severe infections with A. baumannii [27-29]. Our results show that nosocomial infections and colonizations by A. baumannii in the ICU were prolonged for several months. The impact of A. baumannii on ICU-acquired infections and colonization was substantial from clinical samples received in our laboratory from CICU. From March 2006 to August 2007 Acinetobacter spp. were the second most prevalent identified microorganism with 13.9% (100/719). Other most frequent isolates were P aeruginosa (22.1%), S. aureus (15.3%) and Klebsiella spp.(12.9%). Acinetobacter strains (n=100) showed globally high resistance pattern to cephalosporins (76.9%). Imipenem and amikacine were the most effective drugs against A. baumannii with sensitivity rate of 92.4% and 85.7% respectively (unpublished data).
Previous prevalence studies in Kosova showed hight rates of health care associated infections in UCCK (17.4%) and in CICU with 68.7% of patients having nosocomial infections, with a predominance of ventilator associated pneumonia (72.7% of infections) [30,31].
There are many causes for high rate of nosocomial infections in ICU and A. baumannii outbreaks. Main factor remains the lack of support and implementation of prevention and control policies. The proportion of health care workers working in CICU to patients staying in ICU is only 5 HCW per 12 patients per shift. CICU is referent center for intensive care for all 6 regional hospitals and other depratments within the UCCK.
Single use devices were reused due to limited budget. Suction catheters for aspiration of respiratory tract were amongst most used equipment in this group. Audit in the ward during the study period proved that these catheters were placed in a containers containing diluted chlorhexidine. The same catheters after "disinfection" were used for more than one patient carrying a significant risk for cross-infection. Some equipment used in ICU were outdated and their maintenance services were not regular.
A study of compliance with hand hygiene in CICU showed the alert rate of only 19% . During the outbreak period alcoholic hand rubs were not in used in ICU. There are three washing sinks in the ward. Low number of wash sinks contributed to high rate of infection in ICU. Gloves were not changed after each contact with patients but they were used and maintained for successive patients intervention.
For many years in Kosova, the cephalosporins are the drugs of choice in empiric treatment in ICU and they have been used without any restrictions not only in ICUs but also in other hospital wards and ambulantory care. This could explain the high resistance rates of Acinetobacter baumannii to antimicrobials. For a decade in Kosova, all antimicrobials have been available in pharmacies without a physician's prescription.
CICU is reference center for patients from other hospital departments of CICU, from regional hospitals and also from the private hospitals. Delay of referral to this unit contributed to infections, severity of illness and poor outcome prognosis for the patients.
Delay is related to patients who are previously treated at the regional hospitals and they are not transferred on time to the CICU, which is the only ICU reference center for six regional hospitals and for 13 clinics within UCCK.
Genotypic analysis of A. baumannii isolates from ICU patients identified nine major PFGE patterns. The most predominant clones of A. baumannii (E and F) were related with more than one outbreak during the study period occurring sequentially. Case-control study was not performed in epidemiological investigation. The data were recorded from the medical charts of patients with A. baumannii infection or colonization. Some genetically indistinguishable A. baumannii isolates (931, 933 and 934) were isolated on the same day (May 2, 2006) and had similar antimicrobial susceptibility pattern, suggesting common source of infection. The median time from admission to isolation of this bacteria revealed that it's shorter than in other publications [23-27].
In cases where the genetically same strains were not related in timely manner, the only explanation would be poor hand hygiene of health care workers (HCW). Another argument is high endemic rate of MRSA, which is 61.3% of all S. aureus isolates . These facts suggests the horizontal transmission of the epidemic strains from one patient to another through the hospital staff.
As in other publications endotracheal aspirates were predominant clinical samples received from ICU [33,34].
The length of stay was significant in comparison between infected and colonised patients. This finding is consistent with reports of other outbreaks [6,29,33,34]. But there was no significant diference between non survivors and survivors. This can be explained with a fact that some patients spent some hospital days in regional hospitals before referral to CICU and also six patients were sent for treatment in other ICUs in neighbouring countries. As in previous studies, the respiratory tract was the most frequent site of isolation of A. baumannii in ICU patients [35,36]. Colonization with A. baumannii in not performed routinely at admission to ICU.
In conclusion, we show here that A. baumannii strains cause large and sustained hospital outbreak due to insufficient preventive measures. These results emphasize the need for preventive interventions in ICU.
Received on 6 May 2009; revised 20 November 2009.
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Lul Raka (1,2), Smilja Kalenc (3), Zrinka Bosnjak (3), Ana Budimir (3), Stjepan Katic (3), Dubravko Sijak (3), Gjyle Mulliqi-Osmani (1,2), Dick Zoutman (4) and Arberesha Jaka (1)
(1) Medical School, Prishtina University, Prishtina, Kosova; (2) National Institute for Public Health of Kosova, Prishtina, Kosova (3) Department of Clinical and Molecular Microbiology, Clinical Hospital Centre Zagreb, Zagreb, Croatia; (4) Queen's University and Kingston General Hospital, Kingston, Ontario, Canada
Address for correspondence: Dr. Lul Raka. "Emin Duraku", N=166, 71000 Kacanik, Kosova; Phone: +3813829080666 and +37744368289. Email: firstname.lastname@example.org.
Table 1. Clinical and PFGE data of the patients with Acinetobacter baumannii isolates. Nr Gender/ Day of Length of Diagnosis age isolation ICU stay 1 M/ 66 17 18 Tumor cerebri 2 M/ 33 4 28 Tumor cerebri 3 M/ 62 3 8 Tumor cerebri 4 M/ 17 1 14 Diabetes mellitus 5 M/ 11 12 37 Politrauma 6 M/ 62 4 6 Politrauma 7 M/41 4 28 Politrauma 8 F/33 10 14 Politrauma 9 M/ 51 14 59 Cerebral infarct 10 M/ 2 1 10 Politrauma 11 M/ 21 8 10 Politrauma 12 M/ 72 3 5 Cerebral infarct 13 M/ 43 1 13 cardiac arrest 14 M/ 55 4 8 Politrauma 15 M/ 74 18 23 Politrauma 16 M/ 71 9 34 Politrauma 17 M/ 57 2 8 Politrauma 18 M/ 46 14 20 Tumor cerebri 19 F/ 20 2 15 Pulmonal infiltrat 20 M/ 22 1 1 Politrauma 21 M/ 50 5 11 Tumor cerebri 22 F/ 24 15 30 Myocardiopathia 23 M/ 82 8 32 Peritonitis 24 F/ 54 26 44 Peritonitis 25 F/ 72 12 34 cardiac arrest 26 M/ 60 10 14 Cerebral infarct 27 M/ 74 8 17 Politrauma 28 F/ 71 10 19 Tumor cerebri 29 M/ 61 16 19 Cerebral infarct 30 M/ 20 8 11 Politrauma Nr Sensitive Outcome Sample 1 AMI Died EA 2 TOB, AMI, DM Died CLS 3 AMI, IMI, TOB, GEN Died EA 4 AMI, CIP Recovered EA 5 TOB, AMI, IMI Transferred EA 6 AMI, IMI Died EA 7 IMI Recovered EA 8 AMI, IMI Transferred EA 9 TOB, AMI, CAZ Died TRA 10 IMI Died EA 11 IMI, AMI Died EA 12 AMI, IMI Died EA 13 GEN, AMI, CAZ Recovered EA 14 AMI, IMI Recovered EA 15 TOB, AMI Recovered EA 16 AMI, CIP, IMI Recovered EA 17 GEN, TOB, AMI, IMI Transfer EA 18 AMI, IMI Recovered EA 19 AMI, IMI Died TC 20 IMI, CAZ, AMI Transferred EA 21 IMI Died EA 22 AMI Died EA 23 AMI, IMI Died EA 24 TOB, AMI, Transferred TRA 25 AMI, IMI Died EA 26 AMI Died EA 27 IMI,CAZ.AMI Recovered EA 28 AMI,IMI Transferred EA 29 AMI Died EA 30 AMI, IMI Died EA Nr Isolate PFGE type 1 1277 F4 2 1592 G1 3 1007 A3 4 930 E5 5 1570 C1 6 838 E2 7 972 B2 8 868 E3 9 1622 A2 10 1461 C2 11 1517 F6 12 768 E3 13 1191 B1 14 869 E1 15 933 D1 16 907 E5 17 1574 F5 18 943 D1 19 929 E5 20 1003 A1 21 575 E4 22 1438 F3 23 931 D1 24 1605 H1 25 1188 I1 26 815 F1 27 1009 A1 28 933 D1 29 907 E5 30 948 F2 EA=endotracheal aspirate; AMI=amikacin, IMI=imipenem, TOB=tobramycin, CIP=ciprofloxacine, CAZ=cephtazidime, GEN=gentamycine; TRA=tracheostoma; TC=Thoracal drain.