Molecular analyses of Fusarium isolates recovered from a cluster of invasive mold infections in a Brazilian hospital

This study was conducted in the University Hospital, Federal University of Rio de Janeiro, Brazil. The University Hospital is a 13-floor tertiary care teaching hospital with ~480 beds. The BMTU is an isolated 18-bed ward located on the eighth floor of the building. Rooms in the BMTU are under positive pressure air flow, and 8 beds have high-efficiency particulate air (HEPA) filters. Sporadic cases of IF had been diagnosed in the BMTU at a rate of 1.47 cases per 1000 admissions between 2000 and 2006. In 2007, there was a sharp increase in the rate of fusariosis, reaching 16.78 cases per 1000 BMTU admissions. During this period, there was a concurrent increase of superficial fusariosis in outpatients seen at the dermatology clinic on the hospital’s second floor.

Fifty-five Fusarium isolates were recovered from 18 hematology patients (38 isolates from sterile site samples and 17 from non-sterile sites) in the BMTU as a part of standard medical care between December 2005 and September 2009. Forty-three Fusarium isolates were collected from skin and nail cultures of superficial lesions (onychomycosis and intertrigus) of 40 dermatology outpatients between June 2007 and October 2009 as part of standard medical care. After collection, all patient isolates were plated on Sabouraud dextrose agar (SDA), Mycosel, brain/heart infusion (BHI), and yeast extract agars and incubated at 25°C.

Hospital environmental sampling was performed periodically in the BMTU between June 2005 and Aug 2006 as part of an environmental surveillance program focused on air, water, and water-related surfaces. In September 2006 - October 2007, environmental sampling was performed in rooms of infected patients, with an emphasis on bathroom surfaces (shower heads, drains, and tile grout and sink surfaces and drains). Common areas of the BMTU were also swabbed as potential point sources of fungal contamination. Thereafter, routine environmental surveillance of the BMTU continued through July 2009. Environmental sampling of the outpatient dermatology clinic was not performed.

Surfaces were sampled using Culturette swabs (Becton Dickinson, Franklin Lakes, NJ, USA), inoculated onto SDA plates containing 50 mg/L chloramphenicol (SDA+C), and incubated at 25°C. Water (100 ml) was collected in sterile glass bottles and was filtered through 0.45 μm membranes (Millipore, Billerica, MA) which were placed on SAB plates and incubated at 25°C. Air was sampled from patient rooms in November 2006, August 2007, and July 2009 using a six-stage Anderson bioaerosol sampler (Anderson Instruments, Atlanta GA) with air impacting on an SDA plate for 30 min, for a total volume of 849 L per sample. Control air samples were also collected from other areas of the hospital, including the intensive care unit (2005, 2007 and 2009), pediatrics unit (2007) and the nephrology dialysis unit (2007).

Fusarium were isolated from 68 samples, ranging in time of collection from June 2005 to October 2009. Provisional identification of hematology patient Fusarium isolates were obtained in order to decide if more dramatic measures should be implemented in the BMTU. This identification was based on the micromorphology that is typical of this genus [6].

All available Fusarium isolates collected from patients and the environment were sent to the Centers for Diseases Control and Prevention, Atlanta, GA, USA for molecular identification. At the CDC, isolates were sub-cultured on SDA+C with 50 mg/ml gentamycin agar, and incubated at 30°C for approximately 72 hours. Genomic DNA was extracted using the Qiagen DNeasy tissue kit (Qiagen, Valencia, CA) with slight modifications to the manufacturer’s instructions. Briefly, small sections of sub-cultured Fusarium (1 cm²) were cut from mycelial mats and placed in 5 ml polypropylene tubes containing 900 μl Qiagen ATL buffer and 60 U proteinase K. The mycelia in each tube were homogenized using the Omni TH Mixer (Omni Intl., Kennesaw, GA) at slow speed for 30 s and then high speed for 30 s, using a clean probe between each isolate. Homogenates were capped and incubated at 55°C for 1 h with frequent vortexing and then cooled to RT. RNase A (Sigma-Aldrich Corp., St. Louis, MO) was then added to a final concentration of 1 mg/ml and incubated for 5 min at RT, followed by the addition of 900 μl Qiagen buffer AL and vortexing. Homogenates were incubated at 70°C for 10 min, then transferred to 1.7 ml microcentrifuge tubes and centrifuged at 15,000 X g for 10 min. Clear supernatants (1 ml each) were transferred to clean microcentrifuge tubes and 500 μl ethanol (Sigma-Aldrich Corp.) was added. The suspensions were vortexed and transferred to Qiagen DNeasy columns, and manufacturer’s instructions were followed throughout the remainder of the procedure.

A region of the translation elongation factor-1α (EF-1α) gene was used to resolve all Fusarium isolates to the species complex level [7] [Genbank: HM852032-HM852059]. Multi-locus sequence typing (MLST) was performed on selected isolates. The EF-1α and two additional genetic loci, the internal transcribed spacer (ITS) including D1/D2 and large nuclear subunit (LSU) rRNA regions (rRNA) and a portion of the RNA polymerase II subunit (RPB2) gene, were amplified using primers described by O’Donnell et al. to determine species type (ST) [7] (Table 1). Additional primers were developed for this study to sequence the 5’ (F2F and F1R) and 3’ (40-2f and 40-r) of RPB2 (Table 1). PCR was performed in 25 μl reactions containing 0.2 μM each forward and reverse primer, 2.5 mM MgCl_2, 0.2 mM each dNTP, and 0.625 U Taq DNA Polymerase (Roche Carolina Inc., Florence, SC) in a buffer of 10 mM Tris pH 8.3/50 mM KCl. Thermal cycling was performed in MicroAmp 96-well optical reaction plates using the GeneAmp PCR System 9700 (Applied Biosystems, Inc., Foster City, CA) for forty cycles as follows: 94°C for 5 min, 10 cycles of 94°C for 30 s, 60°C for 30 s (decreasing 1°C each cycle), 72°C for 30 s; then 30 cycles as above, but with the annealing temperature held at, 50°C for 30 s, and a final step of 72°C for 5 min. Amplicons were sized on 1.75% agarose and visualized with ethidium bromide under ultraviolet light. Amplicons were purified using Exo-SAP-IT (USB Corp., Cleveland, OH) according to manufacturer’s instructions, followed by sequencing with locus-specific primers using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc.) in a 3730 DNA Analyzer (Applied Biosystems, Inc.). Consensus sequences were generated from raw data using Sequencher 4.9 software (Gene Codes Corp., Ann Arbor, MI).

Fusarium species complexes and STs were identified using the FUSARIUM ID database BLAST search feature [8] [Genbank: JX156437-JX15644, Genbank: JX282603-JX282609]. Consensus sequences were aligned using Clustal W and cleaned within the BioEdit Sequence Alignment Editor [9, 10].

Neighbor-joining (NJ) trees were generated using Kimura’s two-parameter model and bootstrapped using 1000 random replicates with the Phylip 3.69 software package [11]. Resultant trees were then analyzed to generate a consensus NJ tree that was annotated using FigTree v.1.3.1 software [12]. Maximum Likelihood (MJ) and Maximum Parsimony (MP) algorithms were used to generate alternate trees for comparison to the NJ tree to ensure its validity.

A total of 166 Fusarium isolates were available for analysis. These included 98 clinical isolates (55 from 18 hematological patients, 43 from 40 dermatology patients) and 68 isolates from environmental samples, including 14 air samples, 44 swab samples, and 10 water samples. Comparative EF-1α sequence analysis using the FUSARIUM ID database BLAST feature allowed for identification of most study isolates to the species level, with sequence identities ranging from 94.1% to 100% (mean = 99.9, median = 100%) when compared with database isolates. Frequencies of different species complexes/species isolated from patients and the environment are shown in Figure 1. Isolates from BMTU patients and dermatology patients showed a similar distribution. The majority of isolates (68%, 113/166) were identified as Fusarium solani species complex (FSSC). Among clinical isolates, FSSC species 2 (FSSC 2) predominated, and comprised 69% (38/55) of those collected from hematology patients, and 74% (32/43) of those collected from dermatology patients (Figure 1). Fusarium oxysporum species complex (FOSC) isolates were found in much greater numbers in the hospital environment (50%, 34/68) than in those collected from hematology (5%, 3/55) and dermatology (7%, 3/43) patients. Gibberella fujikuroi (GFSC) were isolated exclusively from hematology patients and Fusarium incarnatum-equiseti (FIESC) exclusively from environmental sources. FSSC 1 and FSSC 2 clinical and environmental isolates shared between 99.7 -100% EF-1α identity with FUSARIUM ID isolates, and were chosen for MLST due to their predominance in patients.

Species type identifications of 104 FSSC isolates (FSSC 1 = 30, FSSC 2 = 74) by MLST and EF-1α species complex/ species identification (42 isolates) are shown in Table 2. The cladogram in Figure 2 represents all FSSC 2 isolates described in this study. No cladogram was prepared for FSSC 1-a and 1-b isolates, since these STs differ by only one base pair.

FSSC 2-d and FSSC 2-f were the most common STs isolated from patients (Figure 2). FSSC 2-d was collected from seven patients in the BTMU (20 isolates) and 12 dermatology outpatients, while FSSC 2-f was isolated from five BTMU patients (8 isolates) and five dermatology outpatients (6 isolates). FSSC 2 isolates were rarely found in the environment (n = 4); two FSSC 2-d isolates were collected from the same water source (BR07) on the same day, one FSSC 2-d was isolated from an air sample (BR03), and FSSC 2-f was collected from a floor swab of BR10. None of the environmental FSSC 2 isolates were collected within the same month as those collected from patients in the BMTU (Table 2).

FSSC 2-k (1 patient, 8 isolates) and FSSC 2-t (1 patient, 2 isolates) were the other STs identified by MLST in the BMTU, but none were collected in the environment (Figure 2). Other Fusarium isolated in hematology patients were provisionally identified by EF-1α sequence comparison and each was found in one BMTU patient. These were FSSC 3+4 (HP16, corneal swab), FSSC 7 (HP15, blood), FOSC 33 (HP13, blood and catheter tip), GFSC (HP12, blood), and unspeciated FSSC (HP01, blood) and FOSC (HP15, skin) (Table 2).

Among environmental isolates, FOSC 33 comprised almost half of all samples collected, and was isolated 32 times between September 2006 and July 2009, with the majority (24) sampled between August and October 2007. This species was found in water-related surface swabs of all 12 BMTU bathrooms, a nephrology wall swab (control), and water of two bathrooms (BR02, BR07). FOSC 33 was also isolated from blood and a catheter tip of a patient (HP13) in October 2008, but none were collected from the environment that month (Table 2).

Forty-three bathroom swabs were performed between September 2006 and July 2009, and 6 different Fusarium species/STs were recovered; FOSC (unknown species) and FOSC 33, FSSC 1-a, 1-b, 2-d, and 2-f. Water samples positive for Fusarium (n = 10) were collected in four bathrooms (BR01, 02, 03, 07) and yielded four STs; FOSC 183, FOSC 33, FSSC 1-b and FSSC 2-d. Only BR01 and BR07 were positive for FSSC (1-b, and 2-d, respectively), and water in these rooms also yielded FOSC STs. FIESC was collected from air in three bathrooms (BR03, BR04, BR06) and from 8 control samples. Three other STs were found in air, FSSC 1-a, 1-b, and 2-d, and all were collected from BR03.