ORIGINAL ARTICLE
Hippokratia 2012, 16, 4: 317-323
Katsiari M1, Roussou Z2, Tryfinopoulou K3, Vatopoulos AC4, Platsouka ED2, Maguina A1
1Intensive Care Unit, Konstantopouleio General Hospital, 2Department of Microbiology, Konstantopouleio General Hospital, 3Central Public Health Laboratory, Hellenic Centre of Disease Control and Prevention, 4Department of Microbiology, National School of Public Health, Athens, Greece
Abstract
Background: To investigate an outbreak of Burkholderia cenocepacia bacteremia. Observational study and chart review in a multidisciplinary adult Intensive Care Unit (ICU) at a tertiary care hospital.
Methods: Patients’ demographic variables, comorbid conditions, ICU admission diagnosis, disease severity and outcome were analyzed. In case-patients, time and possible sources of bacteremia, molecular assays, antimicrobial susceptibility and response to therapy were also recorded.
Results: During a 9-month period, 30 episodes of B. cenocepacia bacteremia were diagnosed in 21 patients. Median time for a positive blood culture was 9 days after admission. None of the case-patients had respiratory colonization prior to onset of bacteremia. Pathogen was susceptible to meropenem, piperacillin/tazobactam, ciprofloxacin and trimethoprim /sulphamethoxazole. Surveillance involved environmental and patient/personnel cultures. All samples were negative for B. cenocepacia. However, extensive assessment revealed lapses in infection control procedures. PFGE molecular typing showed that all isolates were indistinguishable. Prior surgery and septic shock on ICU admission were significantly more frequent among case-patients. These patients needed significantly prolonged mechanical ventilation, central venous catheterization and ICU hospitalization. All patients responded to antimicrobial therapy and the attributed mortality was zero. Complete elimination of the outbreak was achieved only after strict enforcement of infection control policies and ICU disinfection.
Conclusions: The outbreak influenced ICU morbidity but it did not affect mortality. Although extensive environmental investigations failed to identify the source of infection, B.cenocepacia disappeared after implementation of control measures. Effective outbreak elimination cannot be limited to offending reservoir removal but needs to extend to efficient infection control practices.
Keywords: Burkholderia cenocepacia, bacteremia, outbreak, molecural investigation, infection control
Correspoding author: Katsiari Maria, 3-5, Agias Olgas street, 142 33 Nea Ionia, Athens, Greece, tel: +302132057113, fax: +302132057126, e-mail:cosma@otenet.gr
Introduction
Burkholderia (previously Pseudomonas) cepacia is an aerobic Gram-negative bacillus commonly found in various aquatic environments and capable of surviving and growing in nutrient-poor water1. This ubiquitous bacterium can survive even in the presence of certain disinfectants2. B.cepacia is an organism of low virulence and often colonizes the lungs of patients with cystic fibrosis3. However, it can cause significant morbidity and mortality particularly in outbreak and ICU settings4.
B.cepacia rarely causes infection in healthy hosts but has emerged as an important opportunistic pathogen in hospitalized and immunocompromised patients5 . Small hospital outbreaks are frequent and are usually due to a single contaminated source such as water6, disinfectants7,8, heparin and intravenous solutions9, nebulizer solutions10, and medical devices, including respiratory therapy equipment11, temperature12 and sublingual probes13.
We hereby report a nosocomial outbreak of B.cenocepacia bacteremia that occurred during a 9-month period in the Intensive Care Unit (ICU) of our General Hospital. The epidemiology, demography, clinical features, laboratory results, treatment and outcome were analyzed. We also report the results of the outbreak’s environmental and infection control investigation and of the molecular identification of the Burkholderia sp. isolates.
Materials and methods
Study setting and case definition
The study was conducted in the nine-bed general ICU (medical and surgical) of Konstantopouleion General Hospital, Nea Ionia, Greece, a 279-bed tertiary care hospital. An observational study was conducted in the whole ICU population during the outbreak period to assess for possible differences between patients who developed B.cenocepacia bacteremia (Group A) and those who did not (Group B). The patients’ medical records were reviewed and the following data were collected: age, gender, underlying diseases, ICU admission diagnosis (medical or surgical), severity score on day of admission and final outcome. All patients were also assessed for Burkholderia cepacia respiratory colonization during ICU hospitalization. In case-patients, time and possible sources of bacteremia, antimicrobial susceptibility and response to therapy were also recorded.
A B.cenocepacia bacteremia case was defined as any ICU inpatient with at least one B.cenocepacia positive blood culture and with clinical signs of septicemia. All consecutive cases with B.cenocepacia positive blood culture from December 1, 2009 to August 31, 2010 were included in the analysis.
In the cases where B.cenocepacia was isolated from a specific body site at the time of the bacteremic episode, that body site was considered to be the source of bacteremia. In the cases where B.cenocepacia was not isolated from any laboratory culture other than blood, the source was considered to be unknown. Bacteremia was considered to be catheter-related (catheter-related bloodstream infection, CRBSI) in those cases where a) a semiquantitive culture of the tip of the catheter revealed B.cenocepacia during the same period of blood culture, or b) the differential time to positivity was ≥ 120 min in blood cultures drawn simultaneously from the catheter and a peripheral vein. A recurrent bacteremia was defined as a case where a blood culture collected more than a week after intravenous catheter removal and antimicrobial therapy was also positive.
Appropriate antimicrobial therapy was defined as administration of one or more antimicrobial agents including at least one agent with effective in vitro activity against the isolate with a dosage, route of administration, and treatment duration in line with up to date treatment guidelines.
The outcome data included overall mortality and mortality related to B.cenocepacia bloodstream infection. The cause of death was considered to be B.cenocepacia bacteremia in the cases where death occurred within 14 days after institution of treatment for bacteremia, unless clinical data clearly suggested a different cause of death.
Μicrobiological and Molecular Investigation
Blood cultures were obtained when septicemia was suspected. Blood samples were inoculated into both aerobic and anaerobic media for processing with the BacT/ALERT 3D (bioMérieux, Inc, Durham, NC) blood culture system. Identification of the microorganisms to the species level was performed with WIDER (Francisco Soria Melguizo, S.A., Madrid, Spain) semi-automated system according to the manufacturer’s instructions. Preliminary species identification was confirmed with the API 20 NE commercial system (bioMérieux, Marcy l’ Etoile, France). All isolates were stored at -70oC in trypticase soy broth with 20% glycerol for further analysis. The MICs of aminoglycosides, piperacillin/tazobactam, aztreonam, third-generation cephalosporins, carbapenems, ciprofloxacin, colistin and trimethoprim/sulphamethoxazole were determined by the WIDER microdilution system and E-test (AB Biodisk, Solna, Sweden) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines14. Catheter tips were cultured using a semi-quantitative technique15.
Further speciation of the clinical isolates was achieved by PCR amplification of B. cepacia complex recA gene with primers BCR1 and BCR2, as described by Mahenthiralingam et al and subsequent nucleotide sequencing of the 1.0 kb amplicons by BioGenomica using ABI 3730, Applied Biosystems16. Sequence identity was confirmed by analysis using BLAST at NCBI (Bethesda, Md).
Molecular typing of B.cepacia complexisolates was performed by Pulsed Field Gel Electrophoresis (PFGE). Chromosomal DNA blocks were digested with SpeI and electrophoresis was assessed using a Bio-Rad CHEF-DR III system, Biorad Laboratories, Milan at 6.0 V/cm with a switch time ramped from pulse 5.3 to 34.9s over 20h at 14°C, as described by Kidd TJ et al17. PFGE patterns were inspected visually and strains relatedness was determined by using criteria developed by Tenover et al18.
Environmental and personnel specimen cultures
Environmental investigations were conducted on several dates during the outbreak in order to identify the source and route of infection. Environmental samples that were potential reservoirs of B.cenocepacia included: chlorhexidine and povidone-iodine disinfectants, heparin solutions, ventilator equipment, bronchial secretions suction equipment, ventilator tubing condensate, water taps, sink drains, patient skin, personnel hands, blood gas analyser, intravenous lines and saline and dextrose solutions.
Infection control investigation
Hand hygiene compliance among staff in our ICU was assessed. Adequacy of general environmental cleaning and disinfection of reusable medical equipment was also evaluated. Procedures which were observed for identification of possible infection control lapses included: catheter insertion, line care/manipulation, medication administration, ventilator cleaning procedure, suctioning of bronchial secretions.
Statistical analysis
Results were expressed as mean ± standard deviation (SD) or median with lower quartile to upper quartile (continuous variables) or percentages of the group from which they were derived (categorical variables). Comparative analyses of continuous variables were executed using Student’s t-test or the Mann-Whitney U test for normally and non-normally distributed variables, respectively. Categorical variables were compared by using the χ2. A p-value less than 0.05 was considered significant.
Results
Description of the outbreak
A total of 30 cases of B.cenocepacia bacteremia were identified during the study period. Of the 21 patients involved, 15 (71.4%) were men and 6 (28.6%) were women, with a mean age of 66±15 years. Upon ICU admission, all patients were mechanically ventilated and had a nasogastric tube, a urinary catheter and at least one central venous catheter (CVC) was inserted. None of the case-patients had respiratory colonization prior to onset of bacteremia. Also control patients were never colonized by B.cenocepacia during their ICU hospitalization. More than 50% (17/30) of the episodes occurred in patients who had undergone a surgical procedure. Table I summarizes demographic characteristics, comorbidities and admission diagnosis of the study population. The two groups had similar characteristics with regard to age and gender, disease severity and underlying conditions. However, septic shock on ICU admission and prior surgery were significantly more frequent among B.cenocepacia bacteremic patients (p<0.001 and p=0.001, respectively).
During the study period, B.cenocepacia was not isolated from any laboratory culture other than blood culture and one case was reported where B.cenocepacia was isolated from a catheter tip. The portal of entry of B.cenocepacia was determined in 4 cases, where the route of infection was via a CVC (catheter-related infections). Recurrent bacteremia occurred in three patients.
The clinical manifestations of B.cenocepacia were similar to those found in other types of Gram-negative bacteremia. Septic shock was present in 22 (73.3%) episodes, whereas the remaining ones presented with systemic inflammatory response syndrome.
Patients with B.cenocepacia bacteremia (Group A) needed prolonged mechanical ventilation (p<0.001) and CVC catheterization (p=0.002) and were hospitalized in ICU for significantly longer time (p<0.001) when compared to Group B (Table II). Overall mortality did not statistically differ among the two groups (Group A: 28.6%, Group B: 20%). However, attributed mortality accounted for zero since for five out of six non-survivors death occurred after 14 days of receiving appropriate treatment. All patients died of septic shock and multiorgan failure due to other multi-resistant Gram negative pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, and Proteus mirabilis). Only one patient died within 5 days after identification of B.cenocepacia bacteremia and her death was related to severe infection by H1N1 influenza virus. All patients with catheter-associated bacteremia where CVC was immediately removed survived.
Microbiological investigation
All infections were considered hospital-acquired since median time for a positive blood culture was 9 days (range 5-14 days) after admission. Out of the 30 episodes of bacteremia, 27 were monomicrobial, and 3 were polymicrobial. The accompanying microorganisms in the polymicrobial cases were Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus epidermidis. B.cenocepacia grew in blood cultures after 3.3±1 days of incubation. Overall, the isolates were susceptible to piperacillin/tazobactam (100%), meropenem (100%), trimethoprim/sulphamethoxazole (76.7%), ciprofloxacin (76.7%) and less susceptible to ceftazidime (30%). In contrast, all isolates were resistant to other third-generation cephalosporins, aminoglycosides, imipenem/cilastin and aztreonam.
All isolates were initially identified as Burkholderia sp. with the API 20NE system (code number 0067577) and finally as B.cenocepacia according to molecular identification with recA gene sequence analysis exhibiting 99% similarity with EU079000 (GENEBANK).
All isolates shared major similarities in their in vitro susceptibilities but they were not identical. As far as the antimicrobial profile, a unique pattern among the isolates was not found. However, molecular typing of B.cenocepacia isolates revealed that all isolates were genetically indistinguishable and different from epidemiologically unrelated isolates (Figure 1).
Figure 1: Pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA of representative B.cenocepacia isolates recovered from patients. Lanes 1&15 lambda ladder size marker, lanes 2-12 isolates obtained from patients, lane 13 control A, lane 14 control B (control A & B are B.cenocepacia isolates from patients of Hospital A and Hospital B respectively).
Epidemiological investigation and infection control assessment
Due to the nature of the infecting organism, we suspected a fluid reservoir of B.cenocepacia. Cases occurred most frequently during the winter and spring months (Figure 2). During the first episodes, the cases clustered in a short period, pointing to direct access of the pathogen to the bloodstream and to the existence of a common and transient exogenous source. Because the cases were identified only in the ICU during this period, our hypothesis was that the organism was contaminating a fluid or equipment used only in our department. However, repeated culturing of environmental samples failed to reveal any source for the 9-month presence of B.cenocepacia. Since all cases were noted to involve bloodstream and CVCs, we concentrated our efforts initially on the observation of insertion and maintenance techniques. General environmental cleaning and disinfection of reusable medical equipment were properly executed. During CVC insertion, maximal sterile barrier precautions were used. However, CVC’s care was not always done with aseptic techniques. Also, catheter hubs and needleless connectors were not always disinfected before assessing the catheter for medication administration. Other faulty infection control procedures included utilization of the same multiple-dose bottle of heparin and disinfectants for 2-3 patients. However, the most significant finding after observation of health-care workers was the low hand hygiene compliance.
Figure 2: Number of B.cenocepacia bacteremias by month.
The identified lapses in infection control procedures revealed the need for institution of appropriate training and educational programs. We implemented an intensive educational program toward all aspects of infection control, particularly hand hygiene. Proper precautions against CVCs maintenance and accession were also taken. Although the tested multi-use bottles of heparin and disinfectants were negative for contamination, practices changed to single-patient use of these vials.
Regarding the geographic distribution of case-patients, results did not reveal any clustering in specific beds of the ICU. Additionally, after documentation of a bacteremic episode, isolation precautions were promptly executed.
Discussion
B.cepacia is being increasingly recognized as an important pathogen of humans in both immunocompromised and immunocompetent patients who are infected by contact with contaminated equipment during hospitalization1,2. Several predisposing factors have been suggested as the major determinants for developing B.cepacia bacteremia. These factors include ICU hospitalization, prior history of major surgery and invasive procedures, including urinary catheter, intravenous catheter and intubation19. Nasser et al reported a large nosocomial outbreak and identified prior receipt of antimicrobials, presence of a stopcock in the intravenous setup, dextrose in water solution, heparin infusions, urinary catheter and more than two intravenous catheters prior to B.cepacia bloodstream infection, as risk factors6. Our study population, being hospitalized in an ICU setting, shared more than one of the above mentioned risk factors. However, we observed that patients who had undergone surgical operation or presented with septic shock on ICU admission developed significantly more frequently B.cenocepacia bacteremia. Bressler et al conducted a case-control study in ICU setting and also concluded that recent abdominal surgery and presence of CVC before detection of B.cepacia bacteremiawere independently associated with B.cepacia bacteremia20.
Correct identification of B.cepacia is important because of the high rate of cross-infection and associated virulence. Over the last twenty years, B.cepacia has been transformed into a species complex which has currently been dissected into 17 validly named species (genomovars) and still there is a significant number of unnamed Bcc species21. In this outbreak, although there was variability in the antimicrobial-susceptibility profiles of these isolates, genotyping revealed that all patients were infected with the same strain, indicating that antibiogram appeared to be a poor marker for identification of the outbreak strain. Molecular identification with recA gene sequence analysis was consistent with Burkholderia cenocepacia (genomovar III). It has been observed that B.cenocepacia comprises the most virulent and transmissible bacterial clones22.
B.cepacia exhibits broad-range resistance to many antimicrobial agents in vitro. The high level of antibiotic resistance limits the therapeutic options, and this may be considered an important determinant of virulence. B.cepacia is intrinsically resistant to antimicrobial agents such as polymyxin, aminoglycosides, first- and second-generation cephalosporines and traditional antipseudomonal penicillins23. Some antibiotics such as ceftazidime, carbapenem, and ciprofloxacin display some in vitro activities against this bacterium, while various combinations have shown in vitro synergy24. In our study, patients responded clinically to treatment with meropenem plus ciprofloxacin, or meropenem alone, piperacillin/tazobactam, or ciprofloxacin.
Attributed mortality rates range between 2-18% in various reports6,25. In a study by Huang et al,mortality rate was positively associated with infections of unknown origin, respiratory failure, shock, an inappropriate antimicrobial therapy, and a period in the ICU25. In our study B.cenocepacia bacteremia may have affected patients’ ICU length of stay and the consequent duration of mechanical ventilation and CVC catheterization but did not affect patients’ mortality since death was clearly attributed to other causes. Prompt institution of appropriate antimicrobial treatment may have influenced the patients’ good clinical response. Interestingly, all patients in our cohort who suffered from CRBSI survived. In these patients removal of CVC may have affected their chance of survival.
Despite an extensive investigation, the source of B.cenocepacia strain associated with this outbreak remained unknown. No environmental or other point source for B.cenocepacia was identified, although horizontal spread was suspected. Since we were unable to identify a definite source, we could only speculate that an unidentified environmental niche and poor infection control techniques were responsible. Several findings suggested that the outbreak may have spread from a single source. Firstly, the case clustered over a period from December 2009 to April 2010. In addition, the outbreak strains of B.cenocepacia had identical patterns by PFGE. The occurrence of scattered cases after the aforementioned period suggested that following its introduction into hospital environment, B.cenocepacia acquisition may have been facilitated by multiple modes of transmission, including person-to-person transmission via the hands of health-care workers26. Consistent with the nature of nosocomial epidemics, breaks in infection control as well as the high transmissibility of the microorganism are mostly responsible for B.cepacia epidemics. In several B.cepacia outbreaks previously reported, no environmental or other point source had been identified11,20,26-28. In these studies, institution of appropriate infection control policies resulted in complete elimination of outbreaks. Similarly, in our ICU, comprehensive infection control assessment revealed several lapses during daily procedures. Poor hand hygiene compliance in combination with loosening of other infection control measures may have accounted for this outbreak. After implementation of an intensive educational program toward all aspects of infection control and strict enforcement of infection control policies, in combination with ICU disinfection, the organism was successfully eliminated.
In conclusion, B.cepacia infection may ensue a significant illness, particularly in the ICU setting, where patients are at greater risk of nosocomial infection due to their underlying illness, proximity and need for invasive devices and procedures. Our experience of this outbreak is not unique. The failure to detect a common source for genetically related isolates is likely underreported because of reporting bias against negative results. The first major finding of our study is that antibiogram appeared to be a poor marker for identification of the outbreak strain. The determination of genomovar, through molecular investigation, has the potential to enhance the investigation of apparent nosocomial outbreaks of B.cepacia. The second conclusion is that thorough environmental investigation is of paramount significance in eliminating outbreaks. However, effective outbreak elimination cannot be limited to the offending reservoir removal but needs to extend to efficient infection control practices. Implementation of educational programs and adherence to infection control policies are the cornerstone for complete elimination of an outbreak.
Disclosures: All authors report no conflict of interest relevant to this article.
The study has been reported in abstract form in the 21st European Congress of Clinical Microbiology and Infectious Diseases and 27th International Congress of Chemotherapy, Milan 7-10/5/2011.
Financial support: None.
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