Carriage of meticillin-resistant staphylococci between humans and animals on a small farm
Abstract
enBackground
Meticillin-resistant staphylococci (MRS) are pathogens of increasing importance to human and animal health worldwide. Transmission of meticillin-resistant Staphylococcus aureus (MRSA) between animals and humans has been well documented. By contrast, information about transmission of other Staphylococcus spp. is limited.
Hypothesis/Objectives
The aim of this study was to screen animals and humans on a small farm for nasal carriage of MRS and to assess interspecies exchange.
Methods
After detection of MRSA in a lung sample of a deceased cat, which lived on a small mixed farm, nasal swabs were taken within two weeks, four and 16 months from other animals of various species and humans living on the farm. Swabs were cultured for MRS which were then characterized molecularly.
Results
MRSA and meticillin-resistant coagulase negative staphylococci (MRCoNS), including Staphylococcus haemolyticus, S. epidermidis and S. fleurettii, were isolated from humans and different animal species. Typing of the MRS revealed isolates with the same characteristics in different human and animal hosts.
Conclusions and clinical importance
To the best of the authors’ knowledge, this is the first report of carriage of both MRSA and MRCoNS among humans and various animals within a shared environment. The detection of strains with indistinguishable molecular characteristics strongly suggested transmission of these MRS between the various animal species and humans.
Résumé
frContexte
Les MRS (staphylocoques résistants à la méticilline) sont des pathogènes d'importance croissante en médecine humaine et animale dans le monde entier. La transmission des Staphylococcus aureus résistants à la méticilline (MRSA) entre l'homme et l'animal a été bien documentée. En revanche, l'information sur la transmission des autres Staphylococcus spp. est limitée.
Hypohèses/Objectifs
La but de cette étude était de passer en revue les animaux et les hommes d'une petite ferme pour le portage nasal de MRS et d’évaluer les échanges inter-espèces.
Méthodes
Après la détection de MRSA dans un échantillon pulmonaire d'un chat décédé, qui vivait dans une petite ferme mixte, les écouvillons nasaux ont été prélevés après deux semaines, quatre et 16 mois sur tous les autres animaux et les hommes vivant dans la ferme. Les écouvillons ont été mis en culture pour MRS, caractérisés ensuite par biologie moléculaire.
Résultats
MRSA et MRCoNS (meticillin-resistant coagulase negative staphylococci), comprenant Staphylococcus haemolyticus, S. epidermidis et S. fleurettii, ont été isolés des différentes espèces animales et de l'homme. Le typage des MRS a montré des souches aux caractéristiques identiques pour les différents hôtes, hommes et animal.
Conclusions et importance clinique
A la connaissance des auteurs, ceci est la première description de portage à la fois de MRSA et de MRCoNS parmi des animaux variés et l'homme partageant un même environnement. La détection de souches avec des caractéristiques moléculaires indifférenciables suggère fortement la transmission de ces MRS entre les différentes espèces animales et l'homme.
Resumen
esIntroducción
los estafilococos resistentes a medicina (MRS) son patógenos que van cobrando mayor importancia para la salud animal y humana a nivel mundial. La transmisión del Staphylococcus aureus resistente a meticilina (MRSA) entre animales y humanos ha sido bien documentada. Por contra existe poca información acerca de la transmisión de otras especies de estafilococos.
Hipótesis/Objetivos
el propósito de este estudio fue investigar animales y humanos de una granja pequeña por la posible presencia de MRS nasal y evaluar el intercambio entre especies.
Métodos
tras la detección de MRSA de una muestra de pulmón en un gato fallecido que vivía en una pequeña granja mixta, se tomaron hisopos nasales en las dos semanas siguientes, a los 4 y a los 16 meses de otros animales de varias especies y de humanos que vivían en la granja. Los hisopos fueron cultivados para la presencia de MRS, que fue caracterizado a nivel molecular
Resultados
se aislaron de humanos y diferentes especies animales MRSA y estafilococ coagulasa negativo resistente a meticilina (MRCoNS), incluyendo Staphylococcus haemolyticus, Staphylococcus epidermidis, y S. fleurettii. La tipificación de MRS reveló aislados con las mismas características en diferentes humanos y animales.
Conclusión e importancia clínica
a nuestro entender este es el primer informe de portadores de MRSA y MRCoNS entre humanos y varios animales en un ambiente compartido. La detección de cepas con características moleculares distinguibles sugiere la transmisión de MRS entre diferentes humanos y animales.
Zusammenfassung
deHintergrund
Methicllin-resistente Staphylokokken (MRS) sind weltweit beim Menschen wie auch beim Tier Pathogene von zunehmender Bedeutung. Die Übertragung von Methicillin-resistenten Staphylococcus aureus (MRSA) zwischen Tieren und Menschen ist gut dokumentiert. Im Gegensatz dazu sind Informationen über die Übertragung von anderen Staphylococcus spp. limitiert.
Hypothese/Ziele
Das Ziel dieser Studie war es Tiere und Menschen auf einer kleinen Farm auf nasale Träger von MRS zu untersuchen und einen Austausch zwischen den Spezies zu erfassen.
Methoden
Nach dem Nachweis von MRSA in einer Lungenprobe einer verstorbenen Katze, die auf einer kleinen gemischten Farm gelebt hatte, wurden innerhalb von zwei Wochen, sowie nach vier und 16 Monaten Nasentupfer von den anderen Tieren verschiedener Spezies und von Menschen, die auf der Farm lebten, genommen. Die Tupfer wurden auf MRS angezüchtet, die dann mit molekularen Methoden charakterisiert wurden.
Ergebnisse
MRSA und Methicllin-resistente Koagulase negative Staphylokokken (MRCoNS), inklusive Staphylococcus haemolyticus, S. epidermidis und S. fleurettii, wurden von Menschen und verschiedenen Tierspezies isoliert. Die Typisierung der MRS zeigte bei verschiedenen menschlichen und tierischen Wirten Isolate mit denselben Merkmalen.
Schlussfolgerung und klinische Bedeutung
Nach bestem Wissen der Autoren handelt es sich hierbei um den ersten Bericht über Träger von sowohl MRSA als auch MRCoNS bei Menschen und verschiedenen Tieren in der gleichen Umgebung. Der Nachweis von Stämmen mit nicht unterscheidbaren molekularen Merkmalen ist ein starker Hinweis auf die Übertragung dieser MRS zwischen den verschiedenen Tierspezies und dem Menschen.
要約
ja背景
メチシリン耐性ブドウ球菌(MRS) は世界規模でヒトや動物の健康にとって重要性を増している病原菌である。動物とヒトの間でのメチシリン耐性黄色ブドウ球菌(MRSA)の伝達は十分に立証されている。その一方、その他のブドウ球菌種の伝達に関する情報は限られている。
仮説/目的
この研究の目的は小さな農場の動物およびヒトのMRSの鼻腔内保菌を検査すること、および動物種間の伝達を評価することである。
方法
小さな混合農場に住んでいたネコの死亡した肺の材料からMRSAを検出した後、農場に住む様々な種の他の動物およびヒトから2週間、4ヶ月および16ヶ月以内に鼻腔内スワブを採取した。スワブはMRSに合わせて培養され、分子学的に断定された。
結果
MRSAおよびStaphylococcus haemolyticus、S. epidermidisおよびS. fleurettiiを含むメチシリン耐性コアグラーゼ陰性ブドウ球菌(MRCoNS)がヒトおよび異なる動物種から分離された。MRSのタイピングにより、異なったヒトや動物宿主において同じ特徴を示す分離株の存在が明らかとなった。
結論および臨床的な重要性
筆者らが知るところによれば、同じ環境に住むヒトおよび様々な動物間でMRSAとMRCoNSの両方を保菌しているという最初の報告である。区別不可能な分子的特徴を示す菌株の検出は、様々な動物種とヒトの間でのこれらのMRSの伝達を強く示唆していた。
摘要
ch背景
全球范围内,甲氧西林耐药的葡萄球菌(MRS)对人和动物的健康威胁越来越严重。现已证明,甲氧西林耐药的金黄色葡萄球菌(MRSA)可在人与动物之间传播。相比之下,有关其他葡萄球菌的传播信息很有限。
假设/目的
这项研究的目的为筛查一个小农场中动物和人类鼻腔携带的MRS,并且进一步评估种间传播。
方法
在一个小型混合种植农场,一只已死亡猫的肺部样本中检测到MRSA,在第2周、4月和16月分别取该农场的其他动物和人的鼻腔拭子样本。经分子学特征鉴定,培养出的均为MRS。
结果
MRSA和甲氧西林耐药且凝固酶阴性的葡萄球菌(MRCoNS),包括溶血性葡萄球菌、表皮葡萄球菌和fleuretti葡萄球菌均能从人类和不同动物中分离出。分离自不同人和动物宿主的MRS类型具有相同的特征。
总结和临床意义
据作者所知,这是第一篇有关共同环境中,人类和不同动物携带MRSA和MRCoNS的报道。经检测,菌株分子学特征非常相似,强烈提示MRS可在人类和不同种动物间传播。
Introduction
Staphylococci are commonly found on the skin and mucous membranes of humans and animals where they represent an important part of the host microbiota. As opportunistic pathogens, they may cause a wide range of disease in humans and animals. Both coagulase positive species (in particular Staphylococcus aureus) and coagulase negative staphylococci (CoNS; in particular S. epidermidis) are among the most frequent causes of nosocomial infections in humans, the majority of which have been estimated to be resistant to meticillin.1 Numerous reports have also suggested nosocomial spread of meticillin-resistant S. aureus (MRSA) in veterinary settings. Phenotypic resistance to meticillin and other beta-lactam drugs expressed by the Staphylococcaceae is mediated mainly by acquisition of mecA or mecC genes. These genes are carried on mobile genetic elements termed Staphylococcal Cassette Chromosomes mec (SCCmec).2 The distinct increase in the presence of meticillin-resistant staphylococci (MRS) in humans and animals increases the likelihood of transmission of MRS in both directions, resulting in co-carriage or infections. In recent years, numerous reports have suggested animal to human transmission of MRSA, especially of strains belonging to clonal complex (CC) 398.3 Compared to MRSA, information regarding interspecies transmission of other MRS is limited.
The objective of this study was to investigate nasal carriage of MRS in animals and humans on a small farm, and to assess interspecies transfer.
Materials and methods
A 3-month-old male Maine coon cat which lived on a small farm in Lower Austria died suddenly and was submitted for postmortem examination. Histopathology of the lungs revealed severe suppurative bronchopneumonia. PCR for feline herpes and calici viruses were negative. Bacteriological examination yielded MRSA. Besides cats (n = 20), several other animal species resided on the farm, including three dogs, six horses, two pigs, five goats and two llamas. The cats and dogs were allowed to roam freely, whereas other animals were kept in paddocks separated by fences. To identify potential MRSA colonization in these animals and in the five people who had been in contact with the deceased cat (owner, family members and two farm workers), nasal swabs were collected by a veterinarian within two weeks, four and 16 months after identification of the index case and submitted to the laboratory within four hours of sampling. Written informed consent was provided by human participants and the farm owner.
All samples were incubated overnight in tryptic soy broth medium (BD; Heidelberg, Germany) with 6.5% NaCl and then streaked on BBL™ CHROMagar™ MRSA II (BD) and Mueller Hinton agar (MHoxa, Oxoid; Basingstoke, UK) with 2.5% NaCl, 2 mg/L oxacillin and 20 mg/L aztreonam. From the BBL™ CHROMagar™ MRSA II plates, colonies that showed typical morphology of MRSA were sub-cultivated on Columbia CNA Improved II Agar with 5% v/v sheep blood (BD). From the MHoxa plates, one colony representing each distinct morphotype with features of staphylococci was subcultured onto the CAN agar. All colony types were tested with the tube coagulase test. Cefoxitin resistance was confirmed by agar disk diffusion4 and mecA PCR.5
Isolates were subjected to SCCmec typing and detection of antibiotic resistance genes including ant(6)-Ia and str, as well as pyrogenic exotoxin genes.5, 6 MRSA isolates were genotyped by spa typing, Multi Locus Sequence Typing (MLST) and by two different multiple locus variable number of tandem repeat analyses (MLVA).5 To provide more evidence of their origin, all MRSA strains were tested by PCR for the presence or absence of genes including scn, chp, φ3 int, φ6 int, φ7 int, rep7, rep27 and cadDX, which are commonly located on mobile genetic elements (MGEs).7 Furthermore, using Fourier Transform Infrared (FTIR) spectroscopy, all isolates were phenotypically subtyped and biotyped for capsular polysaccharide (CP) expression.8
Meticillin-resistant coagulase negative staphylococci (MRCoNS) were genotyped using random amplification of polymorphic DNA (RAPD) and the Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR as described previously.9, 10 Isolates were considered to be indistinguishable if they shared identical RAPD and ERIC PCR profiles, and also had all other characteristics in common. The initial antimicrobial susceptibility testing was performed by agar disk diffusion with the antimicrobial agents described previously.5 In addition, MICs of different antimicrobial agents for selected MRSA isolates and representative MRCoNS isolates were determined by broth microdilution.4 In addition, the same isolates were analysed by a DNA microarray (S. aureus Genotyping Kit 2.0, Alere; Jena, Germany). Array procedures were performed according to the manufacturer's instructions. MRCoNS were identified to the species level by rpoB sequencing.11
All MRSA isolates were screened for fnbB with FNBB-1 (GTAACAGCTAATGGTCGAATTGATACT) and FNBB-2 (CAAGTTCGATAGGAGTACTATGTTC) primers.12
Results
The first sampling resulted in 10 MRSA positive samples, originating from one human, one horse and eight cats; there were 15 MRCoNS positive samples from one human, five horses, seven cats and two pigs (Table 1). During the second and third sampling, different numbers of MRSA and MRCoNS were detected (Table 1). All 20 MRSA isolates carried a SCCmec type IVa cassette, belonged to spa type t011 and sequence type (ST) 398, shared the same genotype after MLVA-14Orsay (7 7−1 5 2 2 5 0 5.5 4 5 3 3 2) and multiplex PCR MLVA (MLVAm), and could not be differentiated from each other by FTIR spectroscopy. All strains were resistant to ciprofloxacin, gentamicin, streptomycin and tetracycline, as determined by agar disk diffusion and confirmed by broth microdilution (Table 1). Besides mecA, the genes blaI/R/Z, aacA-aphD, str and tet(M) were detected in all isolates. DNA microarray analysis revealed that the MRSA isolates were positive for α and δ haemolysin, agr group I and capsule type 5 genes (Table 1). However, none of the MRSA isolates expressed the capsule (CP5) phenotypically, because they were nontypeable (NT) using the CP typing system based on FTIR spectroscopy. None of the MRSA isolates carried the MGE-located genes scn, chp, φ3 int, φ6 int, φ7 int, rep7, rep27 and cadDX. The MRSA isolates carried the same set of microbial surface components recognizing adhesive matrix molecules (MSCRAMM) genes, with the exception of fnbB (fibronectin binding protein B), which was absent in some isolates (Table 1). Genotyping and rpoB sequence analysis of MRCoNS revealed that four distinct strains were present, belonging to the three species: Staphylococcus fleurettii, Staphylococcus haemolyticus, and Staphylococcus epidermidis. Their pheno- and genotypic characteristics are summarized in Table 1.
| Bacterial species a | Host cat | Isolate (s) IDh | Isolation frequency b | SCCmec type | spa type | ST | MLVAm | MLVA-14Orsay | FTIR | ERIC/RAPD | Antimicrobial resistance | Virulence genes | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Phenotype c | Genotype d | ||||||||||||
| MRSAe | Cat | 4135 | Index case | IVa | t011 | 398 | a | A1 | NTg | NA f | BLA, GEN, TET, STR, FQ | mecA, blaZ/I/R, aacA-aphD, tet(M), str | lukF, lukS, hlgA, lukX, lukY, hl, hla, hlIII, aur, sspA, sspB, sspP, setC, set6-var1, set6-var4, ssl01/set6, ssl02/set7, ssl04/set9, ssl05/set3, ssl10/set4, setB1, isdA, cap 5, capH5, capJ5, capK5, icaA, icaC, icaD, MSCRAMM [bbp, clfA, clfB, cna, ebh, ebpS, eno, fnbA, fnbBi, map, sdrC, sdrD, vwB] |
| 7, 10, 11 | 1/✝ | ||||||||||||
| 30, 31i, 42i | 1/2/neg | ||||||||||||
| 8i, 9i | 1/2/3 | ||||||||||||
| Horse | 16 | 1/neg/neg | |||||||||||
| Human | Gia | –/2/– | |||||||||||
| Gib i | –/–/3 | ||||||||||||
| MaNaW i | 1/–/– | ||||||||||||
| MRSH | Cat | 80 | 1/neg/neg | V | NA | NA | NA | NA | NA | a/a | BLA, GEN, ERY, FQ | mecA, aacA-aphD, erm(C)i, msr(A) | isdA |
| Horse | 16b, 17b | neg/neg/3 | |||||||||||
| 21 | 1/2/neg | ||||||||||||
| Pig | 54b, 55b | neg/neg/3 | |||||||||||
| Human | 1w-11ors | 1/2/neg | |||||||||||
| MaNaNbW | neg/neg/3 | d/d | BLA, GEN, ERY, TET, SXT, FQ | mecA, blaZ/I/R, aacA-aphD, msr(A), mph(C), aphA3, sat, tet(K), tet(M) | isdA | ||||||||
| MRSF | Cat | 13, 14, 41, 45, 47, 48 | 1/neg/neg | irreg. | NA | NA | NA | NA | NA | b/b | BLA | mecA | isdA |
| Horse | 17, 18b | 1/neg/neg | |||||||||||
| 21b | 1/2/neg | ||||||||||||
| Human | 1w-11on | 1/2/neg | |||||||||||
| Pig | 54, 55 | 1/neg/neg | |||||||||||
| MRSE | Human | NaOHWA | neg/2/neg | IV | NA | n.t. f | NA | NA | NA | c/c | BLA, ERY, CLI | mecA, blaZ/I/R, erm(C)c, dfrA, aadD, qacA | hlgA, ACME, arcA-SCC, arcB-SCC, arcC-SCC, ssl10/set4,isdA |
- a MRSA, meticillin-resistant Staphylococcus aureus; MRSH, meticillin-resistant S. haemolyticus; MRSF, meticillin-resistant Staphylococcus fleurettii; MRSE, meticillin-resistant Staphylococcus epidermidis.
- b 1, tested positive during first examination; 2, tested positive during second examination; 3, tested positive during third examination; neg, tested negative during the respective examination; †, deceased; –, not available for the respective examination.
- c BLA, β-lactam antibiotics; CLI, clindamycin; ERY, erythromycin; FQ, fluoroquinolones; GEN, gentamicin; STR, streptomycin; SXT, sulfamethoxazole/trimethoprim; TET, tetracyclines.
- d erm(C)I , inducibly expressed erm(C); erm(C)c, constitutively expressed erm(C).
- e All MRSA isolates shared the same characteristics (i.e. SCCmec, spa and MLVA type, had the same resistance pheno- and genotype). In addition selective isolates shared the same set of other genes listed.
- f NA, not applicable; n.t., not tested.
- g NT, nontypeable.
- h Isolates in bold face were analysed by a DNA microarray.
- i fnbB positive isolates. All MRSA isolates were screened with FNBB-1 (GTAACAGCTAATGGTCGAATTGATACT) and FNBB-2 (CAAGTTCGATAGGAGTACTATGTTC) primers12 and selective MRSA were additionally analysed by a DNA microarray.
- ST, sequence type; MLVA, multiple locus variable number tandem repeat analyses; FTIR, Fourier Transform Infrared (FTIR) spectroscopy; ERIC, Enterobacterial Repetitive Intergenic Consensus; RAPD, random amplification of polymorphic DNA. Bold was used to stress which MRS isolates were selected for microarray analysis (selected (MRSA) or representative (other MRS) isolates).
Discussion
In this study, MRSA ST398 as well as MR S. haemolyticus (MRSH) and MR S. fleurettii (MRSF) were isolated from different host species and during different time periods. Further differentiation within each bacterial species (MRSF, MRSH) based on pheno- and genotypic characteristics was not detected. The only exception was one human MRSH. This observation points towards persistence and dissemination of the respective isolates between different hosts on the same farm. It is assumed that because fnbB is usually detected in CC398 isolates, its apparent loss or deletion occurred during transmission on the farm. Because the present study was retrospective, it was not possible to identify the directionality of transmission. For MRSA ST398, which has low host specificity, transmission has been documented between humans and animals, and between animals of different species.13, 14
Previous studies have revealed that loss of capsule expression is associated with chronic infections in human as well as animals and may constitute an important strategy for successful persistence in different hosts.8 While MRSA ST398, MRSH and MR S. epidermidis (MRSE) have been associated with infections in humans and companion animals, S. fleurettii has only rarely been implicated in human and animal infections.15, 16 Interestingly, none of the dogs or llamas on the farm tested positive for MRS at any point, whereas others (e.g. horses) were colonized temporarily and a third group of animals (e.g. two cats) was repeatedly positive in all three samplings. Even though close interspecies contact is a risk factor for becoming colonized with MRSA or other MRS, it does not necessarily lead to colonization or infection. In the present case, all feline MRSA carriers were kittens and none of the mother cats tested positive. The lack of φ3 int, scn, chp, cadDX and rep27 genes, which are considered to be human-associated genetic markers, and the presence of tet(M), which is a typical livestock associated genetic marker, suggest an animal origin of MRSA isolates.
The results of this screening study suggest that transmission of MRS – including CoNS species – may occur amongst various species of animals that live in close proximity to one another.
