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Gordonia species as a rare pathogen isolated from milk of dairy cows with mastitis

While Gordonia species have long been known to cause severe inflammation in humans, the pathogenic effects of Gordonia species in veterinary medicine have rarely been described. Between 2010 and 2019, we collected microorganisms of the genus Gordonia isolated from milk samples from dairy cows with mastitis. We describe the growth properties of these microorganisms and their prevalence, virulence factors and susceptibility to antimicrobial agents. From 31,534 quarter milk samples processed by standard culture methods, 27 isolates of Gordonia species (0.086% prevalence) were identified by a molecular phenotyping method. The isolates originated from 17 farms in 12 districts of the Czech Republic. Twenty-one isolates were tested for susceptibility to 7 antimicrobials by the disc diffusion method. Notably, 100% of these isolates were susceptible to streptomycin and neomycin, 85.7% to cefovecin and tetracycline, 76.2% to penicillin G, 47.6% to trimethoprim/sulfamethoxazole and 0% to clindamycin. The species was determined to be Gordonia paraffinivorans by whole genome sequencing for 9 isolates (from 8 farms in 7 districts). These isolates showed the highest similarity to two reference strains from the environment. In all these isolates, we identified genes encoding virulence factors that are very similar to genes encoding virulence factors expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis. However, genome analysis revealed 61 unique genes in all 9 sequenced isolates.


Results
Sample cultivation, confirmation and characterization of isolates. Out of 31,534 quarter mastitis milk samples, a total of 27 pure cultures of Gordonia sp. were isolated from 27 dairy cows over a period of ten years. These 27 samples constituted 0.086% of the samples, with detection of Gordonia sp. as the dominant pathogen. Figure 1 shows the numbers of annually examined milk samples and Gordonia sp. isolates. The annual prevalence was 0-0.3%. Table 1 provides anamnestic and geographic data for individual isolates. The isolates originated from 17 (9.7%) farms in 12 (20.3%) districts that provided milk samples, across five regions (with a total of 26 districts) of the Czech Republic. Gordonia sp. was isolated across all seasons.
Due to the primarily microbiological focus of the work, a detailed clinical history was retrospectively obtained for only 12 isolates, i.e., for 12 cows, 10 were diagnosed with parenchymatous mastitis, 8 of them with acute parenchymatous mastitis, i.e., a severe form of clinical mastitis (severity score: 3), and two with recurrent chronic mastitis.
After 24 h of incubation, the colonies of Gordonia sp. on Meat Peptone Blood Agar (MPBA) were 0.5-3.0 mm in diameter, they were non-haemolytic, catalase positive, oxidase negative, small, slightly convex or flat, rough and dull with uneven margins; they looked pale pink, orange and, later, reddish coloured (Fig. 2) and emitted various rotting odours (stronger after prolonged incubation). Semiquantitative assessments showed moderate (+++) or heavy (++++) growth. Using MALDI-TOF MS, all isolates were identified as Gordonia rubripertincta, with an identification score of 1.723-2.319 (probable genus identification, probable species identification; Supplementary file 1). For 9 selected isolates (from 8 farms in 7 districts; with an identification score of 1.781-2.162), species identification was performed by whole-genome sequencing using Illumina technology. These 9 isolates showed the closest relatedness to G. paraffinivorans NBRC 108238 (Fig. 3). Five genes encoding virulence factors similar to the virulence genes of Mycobacterium tuberculosis were found in the genome of all isolates sequenced in the present study, with the exception of 1 gene in 2 isolates in all cases. These virulence factor genes were also present in both reference strains of G. paraffinivorans isolated from the environment ( Table 2).  www.nature.com/scientificreports/ Genome fragments unique to Gordonia paraffinivorans isolates from cows with mastitis. Detailed comparison of our nine G. paraffinivorans isolate sequences to G. paraffinivorans reference NBRC 108238 and MTZ 041 genomes revealed a couple genome fragments common to all nine selected mastitis isolates but not present in the environmental strains. Fourteen of them were larger than 1000 bp (Supplementary file 2). The largest unique fragment consisted of 40,400 bp with 37 annotated coding sequences. In the genome fragment contig00009.v75000-79000, two copies of a gene encoding mammalian cell entry protein (locus_ tag = "c00009_00002" and locus_tag = "c00009_00003") were found to be similar to a gene encoding mammalian cell entry protein from Mycobacterium smegmatis str. MC2 155, but similar genes were also present in the NBRC 108238 and MTZ 041 genomes. Antimicrobial susceptibility. The results of susceptibility testing of 21 isolates to 7 antimicrobials are shown in Table 3. All tested isolates (100%) were resistant to clindamycin and susceptible to neomycin and streptomycin. There were both susceptible and resistant isolates to each of the other four antimicrobials.
Resistance profiles for Gordonia species mastitis isolates are shown in Table 4. Our frequent patterns of antimicrobial susceptibility were resistance only to clindamycin (n = 7) followed by resistance to clindamycin . Genetic relationship of Gordonia paraffinivorans mastitis isolates (Field Gordonia paraffinivorans mastitis isolates (n = 9) are marked as F9-F12 and G1-G5. Neighbor-joining tree was constructed on the matrix of presence-absence of genes. Numbers in nodes represents bootstrap values, branch length represent relative genetic distance. In the Roary matrix (middle section), the presence of gene common to at least two genomes is depicted by blue stripe. Number of common genes in genome is shown next to genome identification.) and Gordonia sp. reference genomes. Table 2. Virulence factor genes present in Gordonia paraffinivorans a according to virulence factor database. a Mastitis isolates (n = 9) and NBRC 108238 and MTZ 041 strains; † Only virulence genes with percentage of coverage greater than 40% and percentage of identity greater than 75% to virulence factor database genes are present; + gene present; nd not detected; ‡ Field G. paraffinivorans mastitis isolates are marked as F9-F12 and G1-G5. www.nature.com/scientificreports/ and trimethoprim/sulfamethoxazole (n = 6). Five isolates were considered multidrug resistant (resistance to ≥ 3 groups of antimicrobials). A number of multidrug resistance genes with high similarity to genes in a number of other Gordonia species (Stp: 98-100% coverage, 80-86% identity; EbrA: 93-98% coverage, 72-78% identity; MdtD: 92-97% coverage, 75-87% identity; EmrY: 89% coverage, 73% identity only to G. rubripertincta and Gordonia jinghuaiqii genes) were found in all nine sequenced isolates, as well as in the NBRC 108238 and MTZ 041 strains. The same result applies for tetracycline resistance protein class C and the daunorubicin/doxorubicin resistance genes DrrA, DrrB and DrrC (51-100% coverage, 71-95% identity to genes in number of other Gordonia species). One of the Stp gene variants found in all compared G. paraffinivorans genomes had 66% nucleotide similarity to the lmrC gene associated with lincomycin resistance in Streptomyces lincolnensis, a lincomycin producer.

Discussion
Hungarian and German studies 12,13 and our results show that clinical lesions attributable to the pathogenic effects of Gordonia sp. can also be encountered in numerous animal cases, contrary to earlier reports with only one case per study 19,20  The difference in prevalence in these Czech and Germany sets (0.086% versus 0.003%) of milk samples is relatively large, i.e., the value is almost thirty times lower in the whole German set (n = 708,330), which probably reflects a different sampling strategy consisting of predominantly preventive examinations/cultures. Taking into account only German samples from cows with udder health disorders (n = 223,008; G. paraffinivorans was isolated from these samples) 13 , the prevalence (0.0094%) of G. paraffinivorans infection was nine times lower. Our annual prevalence (0-0.3%) and portions of samples with Gordonia sp. delivered on a given day from a farm (2.4-100%; Table 1) were probably influenced by a broader change during this decade that many farms in the Czech Republic have introduced rapid on farm culture testing to diagnose mastitis pathogens and only sent atypical cases to the laboratory.
The detections of Gordonia spp. in human patients have been substantially more diverse. Nine Gordonia spp. have been isolated from pathological processes following cardiothoracic surgery as well as from lesions of the skin, soft tissues, bones and joints 2,17,21 , and these microorganisms have also been isolated from biological materials collected from septic patients 3 . Two Gordonia spp. have also been identified as causative agents of two cases of human mastitis 15,16 .
It follows that the diversity of pathogenic Gordonia spp. and their sources are much more varied in human medicine than in the veterinary sphere. This can be partly explained by the fact that modern diagnostic methods (MALDI-TOF MS, PCR) have arrived into wide or routine use in veterinary diagnostic laboratories later than in human medicine. Furthermore, the differences between human and animal immunity and their changes may also www.nature.com/scientificreports/ play a role. In a number of reported Gordonia infections 5,22,23 , immunocompromised status was the major risk factor, but infections were also reported in 15 immunocompetent patients who were mostly elderly persons with major primary comorbidities or who underwent exacting sternotomy-type surgery 18 . In contrast, farm animals in particular are not allowed to die of old age (especially if seriously ill), and they do not commonly undergo extensive surgery. In dairy cows, a significant risk of mammary gland infection is generally a consequence of high milk yields 24 and intensive dairy farming (carried out particularly in Europe and North America). Mastitis is the most economically important disease in dairy cows worldwide. Because Gordonia sp. isolated in our study was detected on 17 farms (also simultaneously in several cows and repeatedly on the same farms) in almost half of the districts of the large main catchment area in the eastern part of the Czech Republic, a wide area of occurrence of this pathogen can be assumed. The German study isolated G. paraffinivorans on nine dairy farms in the region of Saxony, i.e., the federal state next to the northwest border of the Czech Republic. The Hungarian study isolated G. paraffinivorans on five dairy farms in four regions of northwestern Hungary. Therefore, it is probably a pathogen associated with dairy cow mastitis not only in the Czech Republic but also in a substantial part of Central Europe, albeit in sporadic cases.
From a genetic point of view, the nine Gordonia sp. isolates sequenced in our study were most closely related to Gordonia paraffinivorans NBRC 108238 (Fig. 3), which was isolated from oil-contaminated water, and MTZ 041 (isolated from compost). However, five genes encoding virulence factors similar to virulence genes of Mycobacterium tuberculosis were found in the genome of the isolates from mastitis cases and both environmental reference strains 25 . This coincidence suggests that G. paraffinivorans has the potential to cause disease as an opportunistic pathogen. Furthermore, in contrast to the two copies of the mammalian cell entry protein gene in the reference genome of G. paraffinivorans NBRC 108238, mastitis isolates tested in our laboratory contained a unique genome fragment with another two mammalian cell entry protein-like genes. All isolates tested in the present study contained 61 unique genes that were not present in NBRC 108238 or MTZ 041. Almost half of these genes are hypothetical, and their function is unknown, but approximately one-third of them are involved in the cholesterol degradation pathway. This suggests a nonaccidental occurrence of these isolates in mastitis samples and a probable adaptation of these strains to the internal environment of the mammalian body as a minor environmental mastitis pathogen, which is in accordance with the other two studies 12,13 .
A potential explanation for the discrepancy in species identification between MALDI-TOF MS and whole genome sequence analysis is the incompleteness of the MALDI-TOF MS database, which currently contains a spectrum of 6 Gordonia spp. only (G. aichiensis, G. alkanivorans, G. bronchialis, G. rubripertincta, G. sputi and G. terrae), but G. paraffinivorans spectra are still missing there.
Differences in antimicrobial susceptibility were observed in veterinary isolates tested in our study as well as in human clinical isolates of Gordonia spp. Because of the various susceptibility testing methods used in different studies, the examination of different Gordonia spp. and the limited number of cases tested in the studies, it is difficult to adopt a clear position on this issue. Nevertheless, in accordance with the detected susceptibility to streptomycin and neomycin in all isolates tested in our study, human clinical isolates of Gordonia were also susceptible to aminoglycosides, whereas there was a discrepancy in the results obtained by susceptibility testing results for penicillin and trimethoprim/sulfamethoxazole reported in different papers 18,26,27 . Regarding clindamycin, there is a study in which Gordonia spp. (specifically G. terrae and G. sputi) were susceptible, i.e., had low minimum inhibitory concentrations 27 , and a study in which a clindamycin-resistant isolate of G. bronchialis was found 14 . The genomes of all G. paraffinivorans isolates from mastitis cases and the strains NBRC 108238 and MTZ 041 from the environment contain a variant of the multidrug resistance Stp gene similar to the lincomycin resistance gene of Streptomyces lincolnensis, a lincosamide antibiotic producer 28 . The resistance of all 21 isolates tested for clindamycin susceptibility in our study can also be associated with the fact that clindamycin is a lincosamide antibiotic, whose other representatives, especially lincomycin, were previously used in the Czech Republic mainly to treat severe mastitis, whether directly infused into the udder or given as an injection, or was used as a first-option antibiotic. In our intensive recent study of antimicrobial resistance of mastitis-causing gram-positive bacteria from the same intake area/catchment area, clindamycin was the antimicrobial with the third highest rate of resistant isolates of Streptococcus sp. 29 . A certain parallel can also be seen with trimethoprim/ sulfamethoxazole, which are representatives of the group of potentiated sulfonamides previously used (even in the intramammary form) for the treatment of mastitis and many other primary and secondary bacterial infections of dairy cows in the Czech Republic. Multidrug resistance was detected in 23.8% of our isolates of Gordonia sp., which is a lower frequent rate than that observed in the most prevalent gram-positive bacteria causing mastitis in the Czech Republic (S. uberis), with 38.7% of its isolates 29 . This is one of first studies reporting isolation of Gordonia species from numerous animal cases. Expected reports of Gordonia sp. from other veterinary laboratories may primarily reflect improved diagnostics. At the same time, mastitis caused by Gordonia sp. can probably be considered a small part of the global shift in the spectrum of mastitis agents in intensively reared dairy cows, from the original predominance of contagious agents to environmental agents 12 .

Conclusion
In the present study, we identified bacteria of the genus Gordonia as mastitis pathogens in dairy cattle in a large area of the Czech Republic over a decade, albeit in sporadic cases. Whole genome sequencing showed a very close relationship of the isolates tested in our study with reference strains of Gordonia paraffinivorans. However, a unique cluster of genes was found in all mastitis isolates. Antimicrobial susceptibility rates of the twenty-one tested isolates revealed high resistance to trimethoprim/sulfamethoxazole and low resistance to some beta-lactam antibiotics and tetracycline. All tested isolates were resistant to clindamycin and susceptible to neomycin and streptomycin. Five isolates were considered multidrug resistant. with one exception of two samples from one farm but eight months apart) from randomly selected cows were sequenced to obtain their whole genome sequences. The number of selected isolates was limited by the cost per sequencing. Genomic DNA of these Gordonia sp. isolates (further referred to as F9-F12 and G1-G5) was isolated from colonies grown on MPBA plates using a Qiagen DNeasy Blood and Tissue Kit (QIAGEN GmbH, Hilden, Germany). A sequencing library was prepared using a Nextera XT DNA Library Preparation Kit (Illumina, Inc., San Diego, California) and sequenced on a NextSeq 500 (Illumina, Inc., San Diego, California). Pairend 2 × 150 bp reads were processed by the Tormes pipeline 31 . This whole genome shotgun sequencing project data has been deposited at DDBJ/ENA/GenBank under the accessions listed in Table 5. To reveal phylogenetic relationship between mastitis isolates and Gordonia reference genomes 32 , a neighbor-joining tree (Fig. 3) was constructed on the base of presence-absence of genes. Such analysis was done by Roary 33 . Tree was drawn by Mega 34 . For detailed comparison of gene content, the mastitis isolates were compared with the environmental isolates G. paraffinivorans NBRC 108238 type strain (genome assembly number GCA_000344155.1, BioSample number SAMD00041809) 9 and MTZ 041 strain isolated from compost (genome assembly number GCA_003121315.1, BioSample number SAMN03785434) 10 . These genomes were annotated using prokka 35 , and annotation was compared by GenAPI 36 . Virulence factors were identified within the Tormes pipeline by screening the virulence factor database (VFDB) 37 .
Antimicrobial susceptibility testing. In all, 21 isolates of Gordonia sp. were tested for susceptibility to 7 antimicrobial agents. No more than one isolate collected from the same farm per six-month period was included in antimicrobial susceptibility testing. Disc-based antimicrobial susceptibility testing was performed using Mueller-Hinton agar with sheep blood (TRIOS, spol. s r.o., Prague, CZ) and discs (Oxoid Ltd., Basingstoke, Ethical approval. Ethical statement is not applicable to this study as the data were gathered through routinely laboratory analysis of samples non-invasively collected from animals on the farms without any animal experimentation. The owners of these animals gave their written permission to use the samples in ongoing studies after the original veterinary use.

Data availability
Most data generated and analysed during this study are included in this published article and its supplementary information files (Supplementary files). The sequences of Gordonia isolates are freely accessible as GenBank assemblies: https:// www. ncbi. nlm. nih. gov/ assem bly/. Some animal clinical data that support the findings of this study (e.g., names of the farms) are available from State Veterinary Institute Olomouc, Czech Republic, but restrictions apply to the availability of these data, which were used under licence for the current study and so are not publicly available. Data are, however, available from the authors upon reasonable request and with permission of State Veterinary Institute Olomouc, Czech Republic. Short basic description of Czech dairy operations is available in our recent study 40 .  www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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