Novel Chlamydia species isolated from snakes are temperature-sensitive and exhibit decreased susceptibility to azithromycin

Abstract

Chlamydia species have recently been recognized as emerging pathogens in snakes. However, isolation of novel snake chlamydiae is critical and their growth characteristics are largely unknown. In this study, two novel chlamydial species are described: Chlamydia serpentis and Chlamydia poikilothermis, isolated after attempts on 23 cloacal and choanal swabs from 18 PCR-positive captive snakes originating from different Swiss snake collections. Isolation success, growth curve and infectivity rates over a 48-hour time period were dependent on temperature (37 °C for C. serpentis, 28 °C for C. poikilothermis). C. serpentis and C. poikilothermis were sensitive to tetracycline and moxifloxacin during evaluation by in vitro antibiotic susceptibility assay but intermediate to resistant (2-4 μg/ml) to azithromycin. Whole genome sequencing of the isolates provided proof of the novel species status, and gives insights into the evolution of these branches of genus Chlamydia.

Figure 1

Immunofluorescence images of C. pneumoniae K6, H15-1957-3C and H15-1957-10C at 32 and 48 hours post infection (hpi). Shown are representative immunofluorescence images illustrating the morphology of C. pneumoniae K6 (a–h), H15-1957-3C (i–p), H15-1957-10C (q–x) at 32 hpi (columns 1 and 3) and 48 hpi (columns 2 and 4) in the presence (columns 1–2) or absence (columns 3–4) of cycloheximide. Strains were grown at 37 °C (top line per strain; lines 1, 3, 5) and 28 °C (bottom lane per strain; lines 2, 4, 6). The size bar indicates 5 µm. Chlamydial inclusions are shown in green, the LLC-MK2 nuclei (DAPI) are shown in blue.

Figure 2

Immunofluorescence images of S15-834C and S15-834K at 32 and 48 hours post infection (hpi). Shown are immunofluorescence images illustrating the morphology of S15-834C (a–h) and S15-834K (i–p) at 32 hpi (columns 1 and 3) and 48 hpi (columns 2 and 4) in the presence (columns 1–2) or absence (columns 3–4) of cycloheximide. Strains were grown at 37 °C (top line per strain; lines 1, 3,) and 28 °C (bottom lane per strain; lines 2, 4,). The size bar indicates 5 µm. Chlamydial inclusions are shown in green, the LLC-MK2 nuclei (DAPI) are shown in blue.

Figure 3

The average inclusion size of C. pneumoniae K6, H15-1957-3C and H15-1957-10C is significantly decreased following incubation at 28 °C compared to 37 °C. Shown is a boxplot comparing the inclusion size (µm²) distribution for strains a) C. pneumoniae K6, H15-1957-3C and H15-1957-10C, and b) S15-834C and S15-834K following 48 h of incubation at 28 °C (dark grey) or 37 °C (light grey). Filled circles represent outliers (>1.5x interquartile range), while asterisks represent extreme values (>3x interquartile range). The inclusion size was determined using a Leica DMLB fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and a UI-2250SEC-HQ camera (uEye, IDS Imaging Development Systems GmbH, Obersulm, Germany) and analysed with the BonTec measuring and archiving software (BonTec, Bonn, Germany). Boxplots were created by the SPSS Statistics software. An unpaired t test was used for statistical analysis, and a p-value of less than 0.05 was considered significant.

Figure 4

Titration by sub-passage confirmed that the infectivity of C. pneumoniae K6, H15-1957-3C and H15-1957-10C is significantly higher at 37 °C while the infectivity of strains S15-834C and S15-834K is increased at 28 °C. LLC-MK2 cells were infected with one chlamydial strain of a) either C. pneumoniae K6, H15-1957-3C, H15-1957-10C, or b) S15-834C, S15-834K and incubated for 48 hours at 28 °C or 37 °C in the presence (white bars) or absence (black bars) of cycloheximide. At 48 hours post infection, monolayers were scraped into the supernatant and used for titration by sub-passage. Inclusion forming units (IFU/ml) are shown in a logarithmic scale. Asterisks indicate statistical significance (p < 0.05) by unpaired t test.

Figure 5

The ultrastructure of C. pneumoniae K6, H15-1957-3C and H15-1957-10C is similar with fully developed inclusions at 37 °C as opposed to 28 °C, while strains S15-834C and S15-834K form mature inclusions at 28 °C and show indication for the presence of aberrant bodies (ABs) at 37 °C. Cultures were fixed in glutaraldehyde at 48 hours post infection and processed for transmission electron microscopy. Shown is the ultrastructure of C. pneumoniae K6, H15-1957-3C, H15-1957-10C, S15-834C and S15-834K (top to bottom) following incubation at 37 °C (left column) or 28 °C (right column). The size bar indicates 10 µm. N = nucleus, Nc = nucleolus, Chlc = chlamydial inclusion, Cy = cytoplasm, I = intercellular space.

Figure 6

Phylogeny of the genus Chlamydia including the new species. The phylogeny was reconstructed based on the concatenated alignment of 9 phylogenetically informative protein markers, and includes one representative of each species whenever genomic data is available. Bootstrap values are shown as percentages. The scale bar indicates the number of amino acid substitutions per site.

Figure 7

Circular representation of the chromosomes illustrating protein sequence conservation compared with other representatives of the Chlamydiaceae. The predicted CDSs are shown on the outer circle in forward or reverse frames. rRNA genes are shown in blue. The inner circles represent identities of the closest orthologue of each CDS identified using Orthofinder, compared against reference genomes. The genomes are ordered based on average sequence identity (see key) as below. Regions showing lower conservation are indicated. (a) C. serpentis H15-1957-10C: C. pneumoniae CWL029, C. poikilothermis. 834 K, C. psittaci 6BC, C. felis Fe/C-56, C. caviae GPIC, Ca. C. sanzinia 2742-308, C. abortus S26/3, C. pecorum E58, C. gallinacea 08–1274/3, Ca. C. ibidis 10–1398/6, C. muridarum Nigg, C. trachomatis D/UW-3/CX, C. avium 10DC88, C. suis MD56, Waddlia chondrophila WSU 86-1044. (b) C. poikilothermis S15-834K: C. psittaci 6BC, C. caviae GPIC, C. felis Fe/C-56, C. abortus S26/3, C. serpentis 10 C, C. pneumoniae CWL029, Ca. C. sanzinia 2742-308, C. gallinacea 08-1274/3, Ca. C. ibidis 10-1398/6, C. pecorum E58, C. muridarum Nigg, C. trachomatis D/UW-3/CX, C. avium 10DC88, C. suis MD56, Waddlia chondrophila WSU 86-1044. The two inner plots indicates the G + C content (blue for above average and red for below average) and G + C skew (blue for positive and green for negative).

Figure 8

Comparison of the genomes of (top to bottom) C. pneumoniae CWL029, C. serpentis H15-1957-10C, C. poikilothermis S15-834K and C. caviae GPIC. Each horizontal line represents the genome, with CDSs shown directionally as arrow heads. Identity (tblastx) between the genomes is shown according to the scale bar. Pink CDSs represent pmp genes, and brown disrupted pmp genes. The genomes of S15-834K and C. caviae GPIC have been reverse complemented to illustrate the synteny. The plasticity zone (PZ) locus is indicated.