一只猫爪部感染拟无枝酸菌属及相关组织病理学结果-Amycola...

王帆

Amycolatopsis spp. infection with correlative histopathological findings from the paw of a cat

一只猫爪部感染拟无枝酸菌属及相关组织病理学结果

翻译：罗雪

This report describes the clinical presentation and diagnosis of a deep cutaneous Amycolatopsis spp. infection in a cat. Diagnosis was based on a combination of methods including culture, 16s rRNA sequencing and histopathological evaluation. Histopathological findings demonstrated unique melanin production. This report highlights the potential for infection by Actinomycetales beyond Nocardia and Actinomyces.

本报告描述了一例猫深部皮肤拟无枝酸菌属的临床表现和诊断。诊断基于培养、16srRNA测序和组织病理学评估等综合方法。组织病理学表现出独特的黑色素产生。本报告强调了除诺卡氏菌和放线菌以外的放线菌目感染的可能性。

Deep cutaneous and subcutaneous infections of cats may be caused by atypical bacteria including Nocardia spp., Actinomyces spp. and Mycobacterium spp., and fungi. Lesions include chronic, nonhealing wounds and severely swollen tissues typical of cellulitis. Inoculation of atypical bacteria often is secondary to penetrating foreign bodies or bite wounds.Diagnostic tests include histopathological evaluation of the affected region and various laboratory cultures (i.e. aerobic, anaerobic, fungal and mycobacterial) or molecular diagnostic methods to identify a variety of potential pathogens.

猫的深层皮肤和皮下感染可能是由非典型细菌引起的，包括诺卡氏菌属，放线菌属，还有分枝杆菌属和真菌。病变包括慢性、不愈合的伤口和严重肿胀的典型蜂窝组织炎。非典型细菌的感染常继发于贯穿异物或咬伤。诊断检查包括对患病区域的组织病理学评估和各种实验室培养（如需氧、厌氧、真菌和分枝杆菌）或分子诊断方法，以识别各种潜在的病原体。

A 3-year-old, castrated male domestic short hair cat was presented for evaluation of swollen paws on the left forelimb and hind limbs (Figure 1a & b). The cat had a two year history of progressive paronychia, nail loss and dermatitis first involving the second and fifth forelimb digits and subsequently ascending into the paw. Within a year, similar lesions were noted in the left hind limb. The cat had tested negative for feline immunodeficiency virus and feline leukaemia virus (FIV/FELV SNAP ComboTest, Idexx Laboratories; Westbrook, MN, USA). Previous therapy had included cefovecin, tobramycin (topically) and itraconazole with no improvement. A six week course of oral doxycycline (10 mg/kg/day), completed two weeks before presentation, did not lead to clinical improvement; the owner perceived worsening of the swelling. Amputation was not feasible because of prior amputation of the right forelimb as a consequence of fracture.

一只3岁的雄性已去势家养短毛猫的左前肢和后肢爪部肿胀(图1a和b)。这只猫有两年的进行性甲沟炎、指甲脱落和皮炎病史，首先累及第二和第五前趾，随后累及爪部。在一年内，在左后肢也出现了类似的病变。这只猫的猫免疫缺陷病毒和猫白血病病毒检测呈阴性。以前的治疗包括头孢维星、妥布霉素（外用）和伊曲康唑，但无改善。在就诊前两周结束了6周口服多西环素(10mg/kg/天)，没有临床改善；主人认为肿胀加重了。由于骨折导致右前肢已经截肢，因此截肢不可行。

Punch biopsy samples were collected from the digit and processed as described in Appendix S1 (Supporting information). Histopathological evaluation revealed dermal and subcutaneous coalescing nodular aggregates of pyogranulomatous inflammation including neutrophils, epithelioid macrophages and multinucleated giant cells centred around large black-brown pigmented granular bodies (Figure 2 a,b). Gram stain highlighted colonies of Gram-positive filamentous rods (Figure 2c); Ziehl–Neelsen modified acid fast and Grocott methenamine silver stains were negative. A Fontana–Masson stain for melanin was performed for which the pigment stained positive (Figure 2d). Von Kossa and Prussian blue stains for calcium and iron, respectively, were negative.

从指部打孔活检采样，并按照附录S1（支持信息）所述进行处理。组织病理学显示，真皮和皮下合并脓性肉芽肿性炎症聚集物，包括中性粒细胞、上皮样巨噬细胞和多核巨细胞，以大的黑棕色颗粒体为中心(图2a，b)。革兰氏染色凸显出革兰氏阳性丝状杆菌(图2c)；齐尼抗酸染色和乌洛托品银染色均为阴性。对黑色素进行氨银液染色，其染色呈阳性(图2d)。硝酸银和普鲁士蓝分别给钙和铁染色为阴性。

Aerobic and anaerobic bacterial culture, fungal culture and mycobacterial culture were performed. A Grampositive filamentous rod-shaped bacterium consistent with the histopathological sections was the only organism isolated. PCR for bacterial 16s rRNA gene was performed and sequenced. Comparison by BLAST in the NCBI blastn database (accessed 26/8/2021) of an 893 bp sequence (GenBank accession no. MZ945439) showed a 99.62% similarity to Amycolatopsis spp. (GenBank accession no. KJ572284.1). See Appendix S1 for further details of culture and molecular methodologies. Amycolatopsis is a Gram-positive member of the Actinomycetales related to Nocardia spp., Actinomyces spp. and Streptomyces spp.

分别进行需氧和厌氧细菌培养、真菌培养和分枝杆菌培养。一种与组织病理学切片一致的革兰氏阳性丝状杆菌是唯一分离的微生物。对细菌16srRNA基因进行PCR检测并进行测序。在NCBI通过BLAST对比数据库（2021年8月26日访问）的893bp序列(基因库登录号MZ945439)与拟无枝酸菌属相似性为99.62%。(基因库登录号KJ572284.1)。关于培养和分子方法的详细信息见附录S1。拟无枝酸菌是放线菌目中与诺卡氏菌属、放线菌属和链霉菌属有关的革兰氏阳性菌。

To the best of the authors’ knowledge, this is the first report of an infection caused by Amycolatopsis spp. In small companion animals. However, a study of Streptomyces spp. infections described a histologically similar inflammatory and pigment pattern in cats. Because there has been rapid expansion of the genetic characterisation of organisms within this genus, it is possible that reevaluation of previously reported Streptomyces spp. infection might reveal some Amycolatopsis spp. infections. Extracellular production of melanin is documented in members of the Streptomyces genus via secreted tyrosinases.The genus Amycolatopsis was first proposed in 1986 to differentiate nocardioform actinomycetes organisms that lack mycolic acid in their cell wall.One important member of this genus is A. orientalis which was the original source of the antimicrobial vancomycin; this group has been studied as agents of nocardioform placentitis (along with Crosiella equi) in mares.

据作者所知，这是在伴侣小动物身上第一例由拟无枝酸菌属感染引起的报道。然而，一项关于链霉菌属感染的研究描述了组织学上相似的炎症和色素模式。由于该属内生物体的遗传特征已经迅速扩大，因此有可能对以前报道的链霉菌进行重新评估，可能是拟无枝酸菌属感染。在链霉菌属中通过分泌的酪氨酸酶产生细胞外黑色素。拟无枝酸菌属1986年首次被提出，用于区分细胞壁中缺乏分支菌酸的诺卡菌样放线菌生物。该菌属的一个重要成员是东方拟无枝酸菌，它是抗菌万古霉素的原始来源；该组已被研究作为母马诺卡菌样胎盘炎（以及克罗氏菌）的药物。

There is no validated method for veterinary antimicrobial susceptibility testing of Amycolatopsis. Based on one study of minimum inhibitory concentrations of mare isolates, drugs of choice may include doxycycline, trimethoprim-sulfamethoxazole (TMS) and linezolid. Given the suspected clinical progression while receiving  doxycycline, TMS was selected for subsequent therapy. It is the authors’ strong opinion that linezolid use should be restricted in animals owing to its clinical importance in humans. Potentiated-sulfonamide drugs also are a mainstay of treatment for infections caused by the related Nocardia spp.The owner reported completing two weeks of therapy of oral TMS at 15 mg/kg twice daily before the owner stopped therapy when the cat was diagnosed with congestive heart failure (CHF). The cat lived for approximately two more months before humane euthanasia was carried out as a consequence of perceived poor quality of life. No postmortem examination was performed and it is unknown whether the CHF and Amycolatopsis infection were linked. Infection with Amycolatopsis spp. and Streptomyces spp. may be rare in small animals. Notwithstanding, it is important for clinicians to be aware of actinomycetes beyond Nocardia and Actinomyces, that may lead to cutaneous infection.

目前尚无有效的对拟无枝酸菌的药敏试验方法。根据一项对母马分离株最低抑菌浓度的研究，选择的药物可能包括多西环素、甲氧苄啶-磺胺甲恶唑(TMS)和利奈唑胺。考虑到在接受多西环素治疗时怀疑有临床发展，我们选择TMS进行后续治疗。作者强烈认为，由于利奈唑胺在人类中的临床重要性，应限制其在动物中的使用。增强磺胺类药物也是治疗相关诺卡氏菌引起的感染的主要治疗方法。据主人报告，当猫被诊断患有充血性心力衰竭(CHF)时，在停止治疗之前，完成了为期两周的每日两次15 mg/kg的口服TMS治疗。这只猫又活了大约两个月，然后由于认为生活质量差而实施了人道安乐死。没有进行尸检，也不清楚CHF和拟无枝酸菌感染是否有关。拟无枝酸菌属和链霉菌属在小动物中可能很少见。尽管如此，重要的是，临床医生要了解诺卡氏菌属和放线菌属以外的放线菌，这可能导致皮肤感染。

Figure 1. a&b. Views of the severe swelling of the paw of a 3-year-old, castrated male domestic short hair cat following a two year course of paronychia and subsequent dermatitis, resulting from infection with Amycolatopsis spp.

图1.a&b。一只3岁雄性已去势家养短毛猫在感染了两年的甲沟炎和后来的皮炎，爪子严重肿胀。

Figure 2. Stained histopathological sections of a punch biopsy of haired skin from the dorsal left front foot of a 3-year old, castrated male domestic short hair cat with Amycolatopsis infection.

（a, b） Low and higher power views of the biopsy showing the surface is ulcerated and covered in a serocellular crust. The dermis and subcutis are effaced and expanded by coalescing nodular aggregates of pyogranulomatous inflammation surrounding pigmented bodies. Haematoxylin and eosin. c) Within these bodies are large colonies of Gram-positive, Ziehl–Nielson modified acid fast negative filamentous rods (Gram stain). d) The pigment is consistent with melanin and is present in peripheral epitheloid macrophages and multinucleated giant cells. (Fontana Masson stain).

图2；一只3岁，雄性已去势家养短毛猫拟无枝酸菌感染的左前足部带毛皮肤穿刺活检的染色组织病理学切片。（a, b）活检低倍镜和高倍镜显示表面溃疡伴有血清性结痂。真皮和皮下组织因色素小体周围的化脓性肉芽肿性炎症聚集而消失和扩张。苏木精和伊红染色。c)在这些小体内有大量的革兰染色阳性，齐尼抗酸染色阴性的丝状杆菌（革兰染色）。d)该色素与黑色素一致，存在于外周上皮样巨噬细胞和多核巨细胞中。（氨银液染色剂）。