Bacteriophages in clinical samples can interfere with microbiological diagnostic tools

Abstract

Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample.

Figure 1. Phages on antibiogram plates.

Two examples of antibiogram agar plates showing areas of confluent bacterial growth, with spots consistent with phage lysis plaques.

Figure 2. Occurrence of phages in clinical samples. Lysis plaques obtained from the suspension of antibiogram plates of urine cultures.

2.1: Spot tests of suspensions of samples A-D in E. coli WG5 and E in P. aeruginosa. PBS control. 2.2: Lysis plaques observed by the double agar layer method in E. coli WG5 in 10⁻⁷ and 10⁻⁸ dilutions of the suspension of plate A. 2.3: Electron micrographs of phages from plates (A–E). Bar 100 nm.

Figure 3. Morphological types of phages observed in samples.

(A) Podoviridae from an ascitic fluid sample after propagation in WG5. (B) Siphoviridae and C: Myoviridae from a urine sample. (B,C) correspond to the same urine sample, which when processed directly showed only B and when enriched showed both. Bar 100 nm.

Figure 4. Interference of phages in antibiogram plates.

(A) ChromID™ CPS^® Elite plate of a urine sample; (B) antibiogram agar plate inoculated directly from a colony close to the inoculation zone of this urine sample (arrow 1 in Fig. 4A); (C) antibiogram from an isolated colony far from the inoculation zone of this urine sample (arrow 2 in Fig. 4A). (A,B) show areas of confluent bacterial growth with spots consistent with phage lysis plaques but no spots were observed in (C). Bacteriophage of Podoviridae morphology isolated from (A,B). Bar 100 nm.

Figure 5. Interference of phages in bacterial isolation.

(A) ChromID™ CPS^® Elite plate of a urine sample; (B) Gram staining of the urine sediment; (C) antibiogram agar plate inoculated directly from a colony (arrow in B); (D) bacteriophage of Siphoviridae morphology isolated from A. Bar 100 nm.