ABSTRACT
Patients undergoing hemodialysis are at an increased risk for bloodstream infections (BSIs). Infection usually occurs because of contamination of water supply, water treatment, distribution systems, or reprocessing dialyzers. Here, we report an outbreak of BSIs caused by Stenotrophomonas maltophilia (n = 21) and Burkholderia cepacia (n = 22) among dialyzed patients at a large hemodialysis center in Brazil. Overall, three patients died (7%), two of which had bacteremia caused by S. maltophilia and the other had a B. cepacia infection. We collected water samples from different points of the hemodialysis system for culture and typing. Genetic patterns were identified through polymerase chain reaction-random amplified polymorphic DNA (PCR-RAPD) and pulsed-field gel electrophoresis. The same genotypes of S. maltophilia and B. cepacia recovered from blood cultures were found in dialysis water. Also, multiple genetic profiles were identified among water isolates, suggesting heavy contamination. Bacteremia cases persisted even after implementing standard control measures, which led us to believe that the piping system was contaminated with microbial biofilms. Soon after we changed the entire plumbing system, reported cases dropped back to the number typically expected, and the outbreak came to an end.
INTRODUCTION
Chronic kidney disease (CKD) is a major public health issue. Current evidence estimates that more than two million people worldwide undergo long-term dialysis treatment or receive a renal replacement to stay alive. Infectious diseases, especially bacterial ones, are among the leading causes of mortality and morbidity in CKD patients.1–3
Dialysis is an invasive procedure that frequently requires the usage of catheters and needle insertion. If not appropriately executed, these steps can eventually introduce contaminants into the bloodstream. Moreover, infections can originate from the dialysis system itself. When water quality management protocols are not followed correctly, microbial colonization of pipelines and filters may occur. Microorganisms can attach themselves to surfaces and aggregate in a self-produced biopolymer matrix, giving rise to biofilms. Biofilms often release chemical, bacterial, and endotoxin contaminants that can cross the dialysis membrane and trigger infection. Once formed, this microbial matrix becomes challenging to remove and can bring about dialysis-related outbreaks.4,5
Stenotrophomonas maltophilia and Burkholderia cepacia are Gram-negative bacteria widely found in water and soil. These organisms are commonly found in aqueous habitats, including plant rhizospheres, animals, food, and water. Stenotrophomonas maltophilia is intrinsically resistant to several antibiotics, and it is considered an opportunistic pathogen because it rarely causes infections in healthy individuals. In clinical settings, this bacterium can be detected on surfaces of devices, equipment, and supplies. It usually affects immunocompromised patients with underlying diseases or the ones submitted to invasive procedures. Those abilities have made this pathogen an important agent connected to healthcare-associated infections.5–12
In December 2015, the infection control service of a tertiary public hospital in Salvador, Brazil, became aware of clusters of bloodstream infections (BSIs) caused by S. maltophilia and B. cepacia among patients undergoing hemodialysis at the dialysis center. Soon after, we started to investigate the water quality management protocols and infection control practices, aiming to identify and eradicate the source of infection. We closely observed the dialysis center’s routine to recognize lapses that could have led to the outbreak. Environmental sampling was performed to help identify possible sources of contamination.
METHODS
Description of outbreak.
The cases occurred at a 650-bed tertiary public hospital in Salvador, Bahia, Brazil. The hemodialysis service is a high-turnover unit that assists an average of 200 patients/month. It is located on the fourth floor of the hospital, and it uses dialyzers that are manually reprocessed and subsequently reused on the same patients. The dialyzers are first washed with dialyzed water and then filled with 2% peracetic acid, which is left on the machine for 48 hours.
The municipal water supply passes through carbon filters and a reverse osmosis unit before being distributed to the hemodialysis room and an intensive care unit (ICU) on the ground floor of the hospital. The quality control protocols advise that the reverse osmosis unit should be disinfected every month with 15 L of 3.5% peracetic acid. The acid is used to clean all the dialyzed water circuits for 4 hours, including the hemodialysis room and the ICU, which are connected by a 300-m looping pipe. Monthly, water samples from different post- and pre-osmosis points are collected. When more than 50 colony-forming units (CFU)/mL are detected, measures are taken to control microbial contamination.
On December 14, 2015, the first two cases of S. maltophilia BSI were detected. From December 2015 through January 2016, another six cases of S. maltophilia BSI were diagnosed in dialyzed patients. All of them were from the hemodialysis room, except for one from the ICU. To identify case patients and determine the background of BSI, microbiology records from July 2014 to November 2015 were reviewed. No other cases of S. maltophilia BSI were found; however, 10 cases of B. cepacia BSI were identified from November 2014 to January 2016. These cases of B. cepacia BSI occurred in dialyzed patients from the same locations where the S. maltophilia BSI outbreak was previously reported.
Ethics statement.
This protocol was approved by the Institutional Review Boards of Hospital Geral Roberto Santos (CAAE no 12448919.3.0000.5028 and approval no 3.340.490).
Outbreak investigation, study design, and population.
The outbreak investigation was conducted from November 2014 to December 2016. Patients were included when at least one blood culture was positive for S. maltophilia or B. cepacia associated with clinical signs or symptoms of infection. The medical records of these patients were examined for demographic and clinical characteristics (gender, age, type of catheter, presence of a fistula, and outcomes).
In 2014 and 2015, some of the microbiological controls of the dialysis water detected heterotrophic bacteria growth above 50 CFU/mL and fecal coliforms. The disinfection of the water system was intensified from one to two times a month with peracetic acid (PAA), and an ionization system was installed. Technical procedures for central venous catheter (CVC) insertion, maintenance, and manipulation were also assessed. On August 18, 2016, eight samples from different sites of the dialysis water were cultured. Two samples from the peracetic acid were also cultivated.
Microbiological and molecular analysis.
Clinical microbial identification and antimicrobial susceptibility testing.
Blood cultures were collected from all suspected patients according to the criteria that the hospital had previously established. Each blood specimen was directly analyzed using the automated BACTEC®9050 (Becton Dickinson, Franklin Lakes, NJ) system. The microbial identification and antimicrobial susceptibility testing were performed by the VITEK 2® (BioMérieux, Marcy-l’Étoile, France) automated apparatus. The interpretation was performed following the Clinical and Laboratory Standards Institute recommendations.13 The S. maltophilia and B. cepacia isolates were later sent to the Laboratory of Pathology and Molecular Biology at the Gonçalo Moniz Institute/Oswaldo Cruz Foundation (IGM/FIOCRUZ) for molecular analysis.
Microbial isolation from hemodialysis center samples.
The dialysis water was collected from different points of the hemodialysis room, ICU, and the looping pipe for microbiological analysis. Duplicate 50-mL aliquots were stored in sterile tubes, under aseptic conditions, from the following sites: the reverse post-osmosis points, reuse rooms, looping pipe, and five machines (numbered 2, 11, 22, 24, and 27). All the samples were also sent to the IGM/FIOCRUZ for microbial isolation and identification.
First, the aliquots were centrifuged and cultured on blood agar plates, MacConkey agar, and trypticase soy broth (Becton, Dickinson, Cockeysville, MD). We used loops of 1 µL and 10 µL for inoculation and incubated the agar plates at 35°C in a 5% CO2 atmosphere for 18–24 hours. The broths were inoculated and incubated under the same conditions.
After 24 hours of incubation at 35°C, all colonies were manually counted (CFU/mL). The identification was performed using standard microbiological methods, including Gram stain, colony morphology, and the triple sugar iron test (Difco Laboratories, Detroit, MI) to differentiate microorganisms according to their ability to ferment sugars. At least three different suspected colonies of non-fermentative gram-negative bacilli (NFGNB) were submitted to species identification using the MALDI-TOF MS system (BioMérieux). The microbial isolation process is illustrated in Figure 1.
Figure 1.
Flowchart of microbiological procedures. 1Dialyzed patients with clinical signs or symptoms of infection. 2Two aliquots (50 mL) of each site were centrifuged. 3The isolation was performed using standard microbiological methods, including Gram stain, colony morphology, and the triple sugar iron test. Suspected colonies for NFGNB were submitted to species identification using the MALDI-TOF/MS system.
Molecular characterization.
To investigate the genetic relationship between BSI and dialysis water isolates, the S. maltophilia and B. cepacia strains were submitted to a PCR-RAPD (fingerprinting technique–random amplified polymorphic DNA) for NFGNB using the previously described 272 primer (sequence 5′ to 3′ – AGCGGGCCAA).14
Pulsed-field gel electrophoresis (PFGE) was performed to determine electrophoretic patterns from chromosomal DNA using SpeI restriction enzyme.14 Pulsed-field gel electrophoresis patterns were clustered by the unweighted-pair group method using average linkages. A dendrogram was generated from a similarity matrix calculated using the Dice similarity coefficient with an optimization of 1.0% and a tolerance of 1.5%. Pulsed-field gel electrophoresis patterns were defined as isolates with a similarity of 80% or higher in the dendrogram.
RESULTS
Case characteristics.
A total of 21 cases of S. maltophilia were identified from December 2015 to August 2016 and 22 cases of B. cepacia from November 2014 to September 2016. Overall, 26 patients were included in the investigation because some of them had both pathogens causing BSIs (Figure 2). Clinical characteristics were obtained from all cases of S. maltophilia, but only from five of B. cepacia owing to lack of patient authorization. We experienced a loss of information because of the late identification of this outbreak and the transference of patients to other dialysis units.
Figure 2.
Epidemic curve of bloodstream infections (BSIs) caused by select organisms at the hemodialysis center, from July 2014 to December 2016. The arrow indicates the period when the hemodialysis tubes were replaced.
Most patients (n = 15; 57.7%) were men with a median age of 57 (29–91) years. Four patients infected by S. maltophilia (19%) died; however, just two deaths were directly related to the infection. Two patients infected with B. cepacia died, but the infection was the cause in only one. The majority of patients (n = 17; 65.4%) were dialyzed by short-term CVC, seven (26.9%) by a long-term CVC, and two (7.6%) by arteriovenous fistula. Stenotrophomonas maltophilia BSI recurrence was detected in six patients, and one died. Burkholderia cepacia BSI reoccurred in two patients, and one died.
Microbiological findings.
The microbiological result of the samples from post-osmosis points, reuse rooms, looping pipe, and five dialysis machines evidenced more than 104 CFU/mL of S. maltophilia, Cupriavidus pauculus, B. cepacia, and Ralstonia pickettii (Table 1). Peracetic acid cultures did not show any bacterial growth (Table 1). Although C. pauculus were recovered from dialysis water, none of the patients presented infection caused by this pathogen. Two patients had R. pickettii, but the strains were not stored to perform a molecular study.
Table 1.
Quantification and identification of Gram-negative bacteria from water samples collected from the main points of the purification system
| Sampling point | (CFU/mL)* | Microbial identification |
|---|---|---|
| Post-reverse osmosis | > 10.000 | C. pauculus |
| Looping of distribution | > 10.000 | B. cepacia/R. pickettii |
| Machine 2 | > 10.000 | R. pickettii/C. pauculus |
| Machine 11 | > 10.000 | B. cepacia |
| Machine 22 | > 10.000 | B. cepacia |
| Machine 24 | > 10.000 | B. cepacia/R. pickettii |
| Machine 27 | > 10.000 | R. pickettii |
| Reuse (room 1) | > 10.000 | Stenotrophomonas maltophilia |
| Reuse (room 3) | > 10.000 | R. pickettii |
| PAA (2%) (room 1) | NA | No growth after 72 hours |
| PAA (2%) (room 3) | NA | No growth after 72 hours |
B. cepacia = Burkholderia cepacia; C. pauculus = Cupriavidus pauculus; R. pickettii = Ralstonia pickettii; NA = not applied .
Colony-forming units/mL.
Overall, 10 environmental samples and 13 patient isolates (11 S. maltophilia and two B. cepacia) were submitted for RAPD and PFGE typing. Stenotrophomonas maltophilia isolates from three patients had a pattern indistinguishable from that isolated from water by RAPD (Supplemental Figure S1) and PFGE. The other eight isolates had different patterns by both methodologies (Figure 3). Of the two isolates of B. cepacia analyzed, only one had an identical pattern from those isolated from water by PFGE (Figure 3) and RAPD (Supplemental Figure S2).
Figure 3.
Dendrogram and pulsed-field gel electrophoresis (PFGE) profiles of SpeI digested chromosomal DNA of (A) Stenotrophomonas maltophilia and (B) Burkholderia cepacia.
Control measures.
In February 2016, the disinfection of the water system with PAA was intensified to every 15 days, instead of monthly. During the outbreak, the dialyzers from patients with S. maltophilia and B. cepacia BSI were replaced, and sulfamethoxazole/trimethoprim was added to the empiric antibiotic treatment. All strains in the study were sensitive to sulfamethoxazole/trimethoprim, except for two B. cepacia isolates. Therefore, the infection control team advised that all patients with signs of bacteremia should be treated with this antibiotic, in association with the empirical treatment for other bacteria (e.g., cefazolin). The sulfamethoxazole/trimethoprim regimen was performed for 14 days. The sulfamethoxazole/trimethoprim combination was not prophylactically used in all patients because of the risk of hyperkalemia in chronic renal patients.
Patients with S. maltophilia and B. cepacia BSI had their vascular catheters removed. Until the source of the outbreak was identified, the practice of reusing dialyzers was interrupted. Between April and July, the membrane filters were replaced, and an ionization purification system of the dialysis water was installed.
Because of the persistence of S. maltophilia and B. cepacia BSI cases between July and September 2016, it was suspected that biofilms might have been formed on the pipes. Although the pipes were not microbiologically evaluated to confirm this hypothesis, we decided to change the entire plumbing system of the hemodialysis unit. After the exchange of the pipes, no other case of S. maltophilia and B. cepacia was detected.
From October 2016 to March 2017, we performed a post-outbreak observation. During this period, the microbiological control of water was within the safety standards. After the interventions, the rates of BSI by all microorganisms in the hemodialysis sector decreased from 38% to 12%.
DISCUSSION
In this study, we described a prolonged outbreak of B. cepacia and S. maltophilia BSI, with a total of 43 cases occurring over 22 months. The dialysis water was the most likely source of infection.
The process of hemodialysis contains several steps that are susceptible to contamination by Gram-negative bacteria.15 Chemical, bacterial, and endotoxin contaminants are health threats to dialysis patients. Bacteria are often detected in the water of dialysis systems, and health risks are present when the concentrations are high enough.4 Microbial colonization of ultrafilters may occur if bacteria attach to surfaces and aggregate in a biopolymer matrix to form a biofilm. Because of inadequate disinfection protocols, the membrane is exposed to persistent bacterial contamination, and the biofilm is allowed to form and to grow.5 In our study, lack of proper disinfection of the water system, an error in the dilution of peracetic acid during the monthly disinfection of the system, and the maintenance of the membrane filters beyond the expiration date may have triggered biofilm formation in the pipes.
In 2015, a quality program was implemented in the hospital with changes in several sectors, such as the hospital infection control service. The new hospital infection control team detected some nonconformities during the outbreak investigation, including membrane filters beyond the expiration date. Even after the microbial count reached significantly high levels (> 10,000 CFU), the dialysis unit operations continued. This event occurred because 200 patients were undergoing hemodialysis and could not be transferred to other units owing to a severe lack of vacancy in the public health system.
The clonal diversity of the isolates recovered from water even after the measures implemented suggested the hypothesis of biofilms in the pipes. Preventing the initial growth of a biofilm is of utmost importance. One of the tactics to inhibit biofilm formation includes choosing appropriate materials for the dialysis water distribution system, along with a compatible disinfection method.16 The hydraulic chain from tap water, downstream to the dialysate, is an ideal place for biofilm formation. Microbial contamination, presence of organic nutrients, dead ends, low fluxes, and periods without flow represent the basis for biofilm growth.5 Acetic acid, used for the disinfection of hemodialysis water, prevents bicarbonate in the hydraulic system. However, biofilms are highly resistant to disinfectants owing to the mixed bacterial community’s structure and the formation of exopolysaccharides.5,16
Klebsiella pneumoniae, Pseudomonas aeruginosa, and B. cepacia complex are the primary Gram-negative biofilm producers.17 Stenotrophomonas maltophilia can also form biofilms that have already been identified in surfaces, both hydrophilic and hydrophobic, such as polystyrene microplates, borosilicate, or polypropylene tubes. These biofilms may exhibit greater resistance to antimicrobial drugs.18
Contaminated reprocessed dialyzers resulting from incomplete disinfection procedures are associated with BSI outbreaks affecting patients in multiple hemodialysis clinics. The practice of reusing and reprocessing dialyzers increases the risk of infection. However, this was not the reason for our outbreak. The contamination was detected in the water, and the dialyzers were dismissed throughout the period after use.19
The RAPD technique was used in our study to perform molecular characterization. Although this approach has less discriminatory power than PFGE, it has been widely used for typing bacterial isolates in outbreaks.20,21 Herein, the RAPD presented similar findings to PFGE. The genetic relatedness was confirmed through PFGE, which is considered a method with excellent discriminatory power and high epidemiological concordancebecause it addresses a large portion of the investigated genome (> 90%).22
Both RAPD and PFGE methods detected the same pattern of S. maltophilia and B. cepacia in the blood cultures and dialysis water, suggesting that the dialysis system was the source of the outbreak. The RAPD and PFGE also detected multiple clones that did not have similar electrophoretic patterns, indicating heavy bacterial contamination of water.
Our study has some limitations. First of all, most strains of B. cepacia isolated from blood cultures were not subjected to molecular investigation because they were not stored. Another drawback was that we could not perform a microbiological analysis of the pipes removed from the hemodialysis facility. Consequently, we were not able to confirm the presence of biofilms. Furthermore, we could not set up a case–control study. All patients in the hemodialysis center had undergone dialysis three times a week, and all of them were exposed to contaminated water. In this scenario, a case–control study was not feasible.
The persistence of S. maltophilia and B. cepacia BSI, even after the implementation of the standard measures to control the outbreak, suggested that the piping system was contaminated with microbial biofilm. Shortly after we changed the entire plumbing system, reported cases decreased sharply, and the outbreak was controlled.
Supplemental figures
ACKNOWLEDGMENTS
We would like to thank the teams at the Laboratory of Microbiology and the Infection Control Committee from Hospital Roberto Santos for all their logistical support in the storage and collection of the bacterial isolates.
Note: Supplemental figures appear at www.ajtmh.org.
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