Recruitment of Patients, Urine Collection, and Bacterial Culture of Urine

Urine samples were collected during January 2008–January 2010 at the Military Medical University, Hospital 103, in Ha Dong, Hanoi. Patients with clinical symptoms of UTI (i.e., >1 of the following symptoms: frequent urination; painful urination; hematuria; cloudy urine; or pain in pelvic area, flank, or low back) were referred from nearby pharmacies and informed about the project. A midstream urine sample was collected at the hospital under supervision of a nurse. Only patients with uncomplicated UTIs were included; patients reporting underlying diseases, such as hematologic disorders, respiratory infections, diarrhea, diabetes, cancer, HIV/AIDS, liver cirrhosis, alcoholism, anatomic malformations of urinary tract, nephrolithiasis, or urolithiasis were excluded, as were patients with hospital-acquired UTIs. The urine was cultured immediately after collection. Thirty-one UTI patients met the study criteria of having E. faecalis CFU >10³/mL isolated from a urine sample in pure culture and were raising poultry in their households.

The urine samples were cultured on Flexicult agar plates (Statens Serum Institut, Copenhagen, Denmark), where E. faecalis grows as small green/blue-green colonies and E. faecium as small green colonies (10). Three colonies were isolated from each UTI patient. All 31 participants were interviewed when urine samples were collected. Personal information recorded included age, sex, and underlying diseases. The following clinical symptoms were recorded: frequent urination, painful urination, cloudy urine, blood in urine, pain in pelvic area, flank pain, pain in low back, and fever. In addition, information about duration of symptoms; previous UTIs; and medical treatment before arrival at the hospital, including type of antimicrobial drug used, was recorded.

Species identification of all 31 presumptive E. faecalis isolates from urine and 83 isolates from poultry were confirmed by species-specific PCR as described by Dutka-Malen et al. (11). Only isolates identified as E. faecalis by PCR were further characterized.

All study participants were informed orally and in writing about the study and provided written consent. The ethics committee at Army Hospital 103 approved the study protocols.

Collection of Cloacal Swabs from Poultry

When a urine sample was positive for E. faecalis, the patient’s household was visited within 1 week, and cloacal swabs were taken from 2–4 chickens in the household. Fecal samples were taken with a sterile cotton swab and immediately placed in Cary-Blair media (Oxoid, Basingstoke, Hampshire, UK) for transportation to the laboratory. Samples were then streaked on Slanetz and Bartley agar medium (Merck, Darmstadt, Germany) the same day and incubated for 24–48 h at 37°C. Subsequently, 2 individual colonies were randomly selected and subcultured on nonselective LB-agar, Lennox plates (Difco, Becton Dickinson, Sparks, MD, USA), which were incubated overnight at 37°C to obtain pure cultures. Colonies were then grown in brain–heart infusion broth (Oxoid) overnight at 37°C and stored for further characterization at –80°C in cryotubes containing 30% glycerol.

MLST and PFGE

To investigate whether isolates of E. faecalis from urine and poultry belonged to identical STs, we characterized isolates from urine and poultry by MLST. Urine isolates were characterized by sequencing of all 7 housekeeping genes used in the MLST scheme: gdh, gyd, pstS, gki, aroE, xpt, and yqil. To confirm that the UTIs were caused by a single strain, 1 additional colony from 9 (29%) of 31 urine samples was characterized by sequencing the gki and yqil genes. Two isolates from each chicken were characterized by sequencing the gki and yqil genes. When sequences of both genes in 2 isolates corresponded to the sequence of the same genes in the urine isolate, which occurred in 11 cases, 1 of the 2 isolates from poultry was randomly selected and further characterized. When gene sequences in only 1 isolate from poultry were identical to the isolate from urine, the isolate was further characterized. Primers and PCR conditions are described on the E. faecalis MLST website (http://efaecalis.mlst.net/). Amplicons were sequenced in both directions by Macrogen (Seoul, South Korea). DNA sequences obtained were assembled using CLC Main Workbench 5.2 software (CLC bio, Aarhus, Denmark) and compared with published alleles, and an ST was assigned to each strain (http://efaecalis.mlst.net/). PFGE was performed as described (12) by using the restriction enzyme smaI (New England BioLabs, Ipswich, MA, USA).

Virulence Genes

The presence and sequence of the following 6 virulence genes were used to further characterize the isolates from urine and poultry: asa1, CylA, efaA, Esp, gelE, and EF0591 (13). After detecting the virulence genes by PCR (13), we sequenced the genes in both directions using Macrogen. DNA sequences were compared, and possible nucleotide differences were calculated by using Smith-Waterman local alignment (EMBOSS) available online from the European Bioinformatics Institute: (www.ebi.ac.uk/).

Antimicrobial Drug Susceptibility Testing

MICs were determined for 16 antimicrobial drugs for comparison analyses by using the Sensititer system (Trek Diagnostics Systems, East Grindstead, UK) according to the manufacturer’s guidelines. These drugs were ampicillin (2–32 μg/mL), avilamycin (4–32 μg/mL), chloramphenicol (2–64 μg/mL), daptomycin (0.25–16 μg/mL), erythromycin (0.5–32 μg/mL), gentamicin (16–1,024 μg/mL), kanamicin (128–2,048 μg/mL), linezolid (0.5–8 μg/mL), moxifloxacin (0.25–8 μg/mL), penicillin (2–32 μg/mL), salinomycin (2–16 μg/mL), streptomycin (64–2,048 μg/mL), quinupristin-dalfopristin (0.25–16 μg/mL), tetracycline (1–32 μg/mL), tigecycline (0.015–2 μg/mL), and vancomycin (1–32 μg/mL).

MLST

Sequencing the 7 housekeeping genes in the 31 E. faecalis strains showed the following 14 STs: 4, 16, 17, 93, 116, 136, 141, 314, 410, 411, 412, 413, 415, and 417, with ST16 shown by 16 (51.6%) isolates. Three isolates belonged to ST4, and each of the remaining STs was represented by only 1 isolate. In 7 of 31 households, the same ST was obtained from poultry and urine (Table 1). In 3 households, ST16 was isolated from urine and poultry. In the remaining 4 households, STs 93, 141, 413, and 415 were identified (Table 1). Because each pair of isolates from all selected patients (28%) showed identical gki and yqil gene sequences, we concluded that the UTI cases were associated with 1 E. faecalis strain.

PFGE

We detected 6 PFGE patterns (A1–A6). Of these, 4 pairs from urine and poultry from the same households showed indistinguishable patterns (Table 1; Figure).

Antimicrobial Drug Susceptibility Testing

When we compared isolates from urine and poultry from individual households, we detected similar MICs of each tested antimicrobial drug, showing a 1-dilution factor deviation (Table 2). For several isolates, an MIC could not be established because the MIC fell outside the test intervals. We detected different MICs for 7 antimicrobial drugs when we compared strains 204U and 204P. All isolates were fully susceptible (lowest or second lowest MIC tested) to ampicillin, avilamycin, linezolid, penicillin, salinomycin, tigecycline, and vancomycin (results not shown in Table 2).

Virulence Genes

PCR for the 6 virulence genes showed that the isolates from urine and poultry from an individual household contained identical virulence genes that varied from 1 to 5 genes, except for 1 household in which the isolate from urine (90U) did not contain the asa1 gene (Table 1). When we compared the DNA sequences from the epidemiologically related urine and poultry strains, we found that all 23 sequenced gene pairs showed 100% similarity.