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The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential
Accepted Manuscript Title: The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential Authors: Ciro C´esar Rossi, Ingrid da Silva Dias, Igor Mansur Muniz, Walter Lilenbaum, Marcia Giambiagi-deMarval PII: DOI: Reference:
S0378-1135(17)30104-9 http://dx.doi.org/doi:10.1016/j.vetmic.2017.01.029 VETMIC 7528
To appear in:
VETMIC
Received date: Revised date: Accepted date:
5-7-2016 23-1-2017 24-1-2017
Please cite this article as: Rossi, Ciro C´esar, da Silva Dias, Ingrid, Muniz, Igor Mansur, Lilenbaum, Walter, Giambiagi-deMarval, Marcia, The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential.Veterinary Microbiology http://dx.doi.org/10.1016/j.vetmic.2017.01.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential
Ciro César Rossia,1, Ingrid da Silva Diasa,1, Igor Mansur Munizb, Walter Lilenbaumc, Marcia Giambiagi-deMarvala,*
a
Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, RJ, Brazil
b
c
Departamento de Medicina Veterinária - Universidade Federal de Rondonia, RO, Brazil
Laboratório de Bacteriologia Veterinária, Universidade Federal Fluminense, Niterói, RJ, Brazil
*Corresponding author at: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Cidade Universitária, Rio de Janeiro, 21941902, Brazil. E-mail adress: [email protected]; Phone: +55 21 2560-8028; Fax: +55 21 25608344. 1
These authors contributed equally to this work
1
Highlights 1. A wide variety of Staphylococcus species (n=13) was isolated from the oral microbiota of domestic cats. 2. Many species isolated are potentially pathogenic for humans. 3. Atypical strains reveal the need for alternative strategies for species identification. 4. CRISPRs elements are rare, consistent with the strains role as gene reservoirs.
2
Abstract This study aimed to characterize the species, antimicrobial resistance and dispersion of CRISPR systems in staphylococci isolated from the oropharynx of domestic cats in Brazil. Staphylococcus strains (n=75) were identified by MALDI-TOF and sequencing of rpoB and tuf genes. Antimicrobial susceptibility was assessed by disk diffusion method and PCR to investigate the presence of antimicrobial-resistance genes usually present in mobile genetic elements (plasmids), in addition to plasmid extraction. CRISPR – genetic arrangements that give the bacteria the ability to resist the entry of exogenous DNA – were investigated by the presence of the essential protein Cas1 gene. A great diversity of Staphylococcus species (n=13) was identified. The presence of understudied species, like S. nepalensis and S. pettenkoferi reveals that more than one identification method may be necessary to achieve conclusive results. At least 56% of the strains contain plamids, being 99% resistant to at least one of the eight tested antimicrobials and 12% multidrug resistant. CRISPR were rare among the studied strains, consistent with their putative role as gene reservoirs. Moreover, herein we describe for the first time their existence in Staphylococcus lentus, to which the system must confer additional adaptive advantage. Prevalence of resistance among staphylococci against antimicrobials used in veterinary and human clinical practice and the zoonotic risk highlight the need of better antimicrobial management practices, as staphylococci may transfer resistance genes among themselves, including to virulent species, like S. aureus. Keywords: Domestic cats, feline, Staphylococcus, antimicrobial resistance, CRISPR.
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1. Introduction Cats are among the most common domestic animals in the world, totaling more than 500 million individuals living with humans, in a relationship dated from thousand years ago, as evidenced by fossil remains (Vigne et al., 2016). Pets can carry a variety of bacteria, worms, viruses and fungi. In addition, cats’ outdoor habits contribute for them to carry greater numbers and more diverse microbiota than dogs (Buma et al., 2006). Because of the intimate relationship that cats and humans may have, many of these microrganisms can be transmitted from pet to owner and vice-versa, including important pathogens like methicillin-resistant Staphylococcus aureus (MRSA), responsible for several hard-to-treat infections in humans (Muniz et al., 2013). Most of the almost 50 species of the Staphylococcus genus are harmless residents of the normal microbiota of skin and mucous membranes of mammals (Becker et al., 2014). However, species sharing the same environment may exchange genetic material among themselves, especially plasmids, providing genes that confer antimicrobial resistance to pathogenic species, such as S. aureus (Rossi et al., 2016). Thus, species historically and mistakenly referred to as inoffensive can threat human health for acting as reservoirs of genes for closely related pathogens (Otto, 2013). We hypothesize that this characteristic must be accompanied by the absence of CRISPR systems, which are arrays of repeated sequences and associated genes that give the bacterium the ability to develop adaptive immunity against exogenous genetic material, like plasmids and bacteriophages (Marraffini, 2015). Understanding the bacterial populations that inhabit companion animals is an important way of restraing their spreading and securing the health of both pet and human. Thus, the aims of this work were i) to identify a population of Staphylococcus spp. isolated from different cats in Rio de Janeiro (Brazil) and ii) to characterize their antimicrobial resistance and CRISPR content in order to evaluate the potential threat that these microorganisms may signify to the cats and their owners.
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2. Materials and methods 2.1. Microorganisms, culture conditions and DNA purification We analyzed 75 strains belonging to the Culture Collection of Bacteria of Veterinary Interest of the Laboratory of Veterinary Bacteriology of Universidade Federal Fluminense (www.labv.uff.br). Those were previously collected from the oropharynx of different clinically healthy adult cats from Rio de Janeiro and characterized by biochemical methods as belonging to the genus Staphylococcus (Muniz et al., 2013). All strains were cultivated at 37°C for 24h in brain-hearth infusion (BHI) before the tests. Genomic DNA was isolated with the WizardTM Genomic DNA Purification Kit (Promega, USA) following the manufacturer’s instructions.
2.2. Bacterial species identification The identification of the bacterial strains was performed by combining different molecular approaches. First, the differentiation of protein profiles of bacterial cultures was analyzed in triplicate by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF) as previously described (Tomazi et al., 2014). Mass spectral data were collected within the m/z range of 2,000 to 20,000, and the data were acquired using the FlexControl software 3.3 (Bruker Daltonics). MALDI-TOF results were complemented and confirmed by the partial sequencing of the rpoB and tuf genes. Briefly, these target genes were amplified from 25 ng of DNA by PCR with the primer pairs rpoBF/rpoBR (Drancourt and Raoult, 2002) and tufF/tufR (Heikens et al. 2005) and the GoTaq® G2 Green Master Mix (Promega, USA), following the manufacturer’s protocols. Amplification steps for the rpoB gene were performed as indicated by the primers authors specified above.
2.3. Antimicrobial susceptibility testing and plasmid extraction The susceptibility of the strains was tested by the disk diffusion method against eight antimicrobials of different classes: aminoglycosides (gentamicin), β-lactams (oxacillin and ampicillin), 5
amphenicols (chloramphenicol), cephalosporins (cefoxitin), tetracyclines (tetracycline), macrolides (erythromycin) and mupirocin, (Cefar, Brazil). All tests were performed according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2015). Because of the lack of breakpoints for mupirocin in CLSI, susceptibility and differentiation of levels of resistance to this antibiotic were interpreted following the criteria previously established by our group (Oliveira et al., 2007). In order to correlate the resistance profiles with the presence of mobile genetic DNA, all strains were submitted to plasmid extraction, following protocols established by our group (Giambiagi-Marval et al., 1990).
2.4. PCR for antimicrobial resistance genes The presence of resistance genes commonly encountered in mobile genetic elements was investigated by PCR. All reactions were performed with the GoTaq® G2 Green Master Mix (Promega, USA), 25 ng of purified DNA and primers specified below. The S. haemolyticus strain MD2 (Rossi et al., 2016) was used as positive control for all reactions. The aminoglycosideresistance
genes
aadD
and
aacA-aphD,
respectively coding
for
the
aminoglycoside
adenyltransferase AadD and the bifunctional aminoglycoside modifying enzyme AacA-AphD, were amplified with the primer pairs aadDF/aadDR and aac6-aph2aFw/aac6-aph2aRev (Schiwon et al. 2013), following the amplification steps suggested by the author. The methicillin-resistance gene mecA was amplified with the primer pair MRS1/MRS2, as recommended by the author (Del Vecchio et al., 1995). Finally, the mupirocin-resistance gene mupA was amplified with the primer pair M1/M2, following the amplification steps preconized by Nunes et al.(1999)
2.5. Investigation of CRISPRs The dispersion of CRISPR systems among the 75 strains was examined by PCR for the detection of the essential CRISPR-associated protein Cas1. A cas1F (5’-AGAAGCACAGGCTGCAAGAA-3’) and cas1R (5’-TCACACTATCAAGTAACCTCACCA-3’) primer pair was designed based on a 6
conserved region of the gene obtained from the alignment made with the sequences of the following CRISPR-containing strains (and Genbank ID): S. aureus 08BA02176 (CP003808), S. capitis CR01 (NZ_CBUB000000000), (NZ_CUFQ01000000),
S. S.
epidermidis lugdunensis
RP62A HKU09-01
(NC_002976), (NC_013893),
S.
haemolyticus S.
schleiferi
W_75 1360-13
(NZ_CP009470), S. simulans ACS-120-V-Sch1 (NZ_AGZX00000000) and S. warneri 738_SWAR (NZ_JUVB00000000). Because for now there are not representatives of all staphylococci species containing CRISPR systems available from the public sequence databases, type II and type III CRISPR system-contaning S. haemolyticus MD03 and S. epidermidis RP62A were used as positive controls of the reaction. Amplifications were performed with with the primer pairs cas1F/cas1R, 25 ng of purified DNA and the GoTaq® G2 Green Master Mix (Promega, USA). Amplification consisted in initial denaturation at 94°C for 3 mins, followed by 35 cycles (94°C for 30s, 51°C for 1 min and 72 °C for 30s) and a final extension step at 72°C for 3 mins. Amplicons were visualized in 1.0% agarose gels stained with ethidium bromide. To confirm the identity of the target amplified, the fragments obtained were purified and submitted to DNA sequencing.
3. Results 3.1. Bacterial species identification The species of 75 Staphylococcus spp. strains were first identified by the MALDI-TOF technique. With this method, 57 (76%) strains were identified with scores ≥ 2.000, indicating, according to the FlexControl software 3.3 manufacturer, a “secure genus identification and probable species identification”. The remaining 18 strains yielded unreliable identification scores (≤ 1.699). Because of these inconclusive results, the MALDI-TOF data were complemented with the partial sequencing of the housekeeping genes rpoB and tuf. The sequences obtained for every strain were deposited in NCBI’s Genbank database under access number intervals KU883152-KU883217 and KX198012KX198027 (for the tuf gene); and intervals KU867251-KU867306 and KX215896-KX215913 (for the rpoB gene). Table 1 shows the MALDI-TOF results and the definitive identification of the 7
strains, achieved when the results were coincident for at least two of the three methods employed. The average confidence of the MALDI-TOF method was 82.5%, as 47 out of 57 strains were identified in accordance to the definitive identification. A wide variety of Staphylococcus species (n=13) was found in our samples, particularly S. lentus (28.0% - 21/75) and S. nepalensis (20.0% - 15 / 75), followed by S. cohnii (8.0% - 6/75), S.sciuri (8.0% - 6/75), S. xylosus (5.3% - 4/75), S. equorum (5.3% - 4/75), S. epidermidis (4.0% 3/75), S. aureus (2.7% - 2/75), S. saprophyticus (1.3% - 1/75), S. felis (1.3% - 1/75), S. pettenkoferi (1.3% - 1/75), S. haemolyticus (1.3% - 1/75) and S. fleurettii (1.3% - 1/75). In addition, nine (12%) atypical strains could not be identified to the species level, despite being identified as belonging to the genus Staphylococcus.
3.2. Antimicrobial susceptibility and plasmids The susceptibility of the 75 strains against eight antimicrobials is presented in Fig. 1. All strains but one (S. equorum strain 1) were resistant to at least one drug tested. Most strains were resistant to oxacillin (95% - 71/75) and tetracycline (76% - 57/75), followed by erythromycin (33.3% - 25/75), ampicillin (28% - 21/75), chloramphenicol (24% - 18/75), mupirocin (12% - 9/75), gentamicin (4% - 3/75) and cefoxitin (2.7% - 2/75). Among the strains, 45% (34/75) were resistant to at least three different classes of antimicrobials, being thus considered multidrug-resistant. It is likely that many of these resistance determinants are located in mobile genetic elements, as at least 56% (42/75) of the strains presented plasmids (Fig. 1).
3.3. PCR for antimicrobial resistance genes The presence of genes coding for aminoglycosides (aacA-aphD and aadD), methicillin (mecA) and mupirocin (mupA) resistance was investigated by PCR. From the genes for aminoglycoside resistance, expected to be present in the gentamicin-resistant strains 43(2), 132(1) and 114 (Fig. 1), only the aadD gene was found, in one S. cohnii strain 132(1). The mecA gene was widespread, 8
being found in 63% (47/75) of the strains (Fig. 1). In relation to the mupA gene, it was found in only one, out of three, of the mupirocin-resistant strains, S. sciuri 199.
3.4. Investigation of CRISPRs The CRISPR molecular marker cas1 gene was investigated by PCR in all 75 strains studied in this work. Only 4% of them (3/75) were positive to the amplification: S. lentus 162 and S. spp. 134a and 145 (data not shown). The fragments obtained were submitted to DNA sequencing and, by homology search, they were all confirmed to be cas1 genes. These sequences were deposited in Genbank under the access numbers KU883215-KU883217.
4. Discussion In this study, staphylococci strains isolated from the oropharynx of healthy domestic cats, living in different residences located in Rio de Janeiro (Brazil), were characterized to the species level and phenotypic and genetic aspects of antimicrobial resistance. Since biochemical tests for the identification of species from the Staphylococcus genus can yield inconsistent results, inferior to those observed with molecular biology procedures (Hasan et al., 2014), the 75 strains used in this study were initially identified by MALDI-TOF. This method has emerged as a potential tool for the identification and diagnosis of microorganisms for being fast, sensitive, relatively cheap and easy to perform, as the identification of species can be made using intact cells or cell extracts (Singhal et al., 2015). However, one limitation of the technique is the fact that it depends on spectra databases containing fingerprints of peptides masses from the microorganism of interest, which may be limited when working with rare species or from an origin other than human (Singhal et al., 2015). The results we obtained by MALDI-TOF are consistent with this statement, since a significant portion of the isolated strains showed atypical peptide profiles, hampering their identification.
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Some Staphylococcus species isolated in the last two decades are still poorly studied, such as S. nepalensis, first identified in 2003 in the respiratory tract of goats in the Himalayan region (Spergser et al., 2003) and S. pettenkoferi, isolated in 2002 from human clinical specimens in Germany (Trulzsch et al., 2002). Because of the limitations we experienced with the MALDI-TOF, other molecular techniques were necessary to complement the identification results and to confirm the previously defined strains. The partial sequencing of the rpoB gene, encoding the beta subunit of RNA polymerase, is a central candidate for phylogenetic analysis and identification of bacteria, but the partial sequencing of the tuf gene, which encodes the elongation factor Tu, is considered the most accurate for the identification of staphylococci species (Carpaij et al., 2011). As expected, the sequence of both rpoB and tuf genes enabled us to identify the species of most strains previously not determined by MALDI-TOF. Many of these are still poorly studied in companion animals, such as S. lentus, S. nepalensis and S. pettenkoferi. In addition, some atypical strains yielded different results for the three methods employed, not being identified to the species level. The prevalent species were S. lentus and S. nepalensis. S. lentus is a commensal bacterium that colonizes the skin of several species of animals, commonly isolated from products of animal origin, which in some occasions was related to onset of localized infections, as the bacterium was isolated from urine, peritoneal fluid, blood, culture wounds and cerebrospinal fluid (Mazal and Sieger, 2010). S. nepalensis, as aformentioned, is a recently defined species, and although it has been isolated in episodes of urinary tract infections in humans, its clinical significance is not clear (Novakova et al., 2006). This is the first time, as the authors are aware, that S. nepalensis strains – highly representative in our sample – are described in feline microbiota. With the exception of S. aureus, S. intermedius and S. pseudintermedius, all other recovered strains found are coagulase-negative staphylococci (CoNS). The CoNS were long considered commensal and rarely pathogenic microorganisms. However, in recent decades, these bacteria have been recognized as etiologic agents of a number of infectious processes (Becker et al., 2014). 10
Among the CoNS found, S. haemolyticus and S. saprophyticus stand out as opportunistic pathogens in humans. S. haemolyticus is often isolated from blood cultures and can cause transient bacteremia when introduced into their hosts, generally linked to invasive medical devices (Czekaj et al., 2015). Members of S. saprophyticus are major causative agents of urinary tract infections (UTI) in humans (Eriksson et al., 2013). Other species are infrequently associated with human infections, such as S. cohnni isolated in cases of bacteremia (Soldera et al., 2013), S. sciuri identified in different infectious processes (Stepanovic et al., 2005) and S. xylosus associated with infective endocarditis (Sirmatel et al., 2015). Despite those reports in human beings, their role in animal health remains to be elucidated. The intimate relationship and contact between humans and their pets contribute to the transmission of pathogens between the two, including the transfer of multidrug resistant bacteria and genetic mobile elements carrying antimicrobial resistance genes (Costa et al., 2013; Muniz et al., 2013). Most strains of the studied Staphylococcus spp. are resistant to tetracycline and oxacillin, used in both veterinary and human medicine. Although the cefoxitin disk is generally preferable to be used in search of strains containing the mecA gene, as recommended by the CLSI (2014); for some species, like S. pseudointermedius, the oxacillin disk is recommended. Because we worked with many understudied Staphylococcus species, using both cefoxitin and oxacillin to search for mecA-positive strains enable us to retrieve richer information from our sample. Resistance genes can be located on the chromosome or on plasmids and can be horizontally transferred from one bacterium to another, contributing to the spread of resistance, originally caused by indiscriminate use of antimicrobials in human and veterinary medicine (Prescott, 2014). Mupirocin is particularly important in the prevention and treatment of MRSA, but bacterial resistance to this antimicrobial is increasing, related to either point mutations in the chromosomal ileS gene or associated with plasmids carrying the mupA gene (Deeny et al., 2015). The aadD gene, which encodes an enzyme for aminoglycoside resistance was described being located on a plasmid also integrated in the SCCmec (Chambers, 1997). The mecA gene encodes the penicillin binding protein PBP2 and is 11
located in the SCCmec chromosome cassette, a mobile genetic element which is widespread among staphylococci, usually related to multidrug resistance (Hanssen and Sollid, 2006). The abundance of methicillin-resistance among companion animals’ microorganisms has been previously described (Muniz et al., 2013). Even though the mupA and aadD genes were not as widespread as mecA, their presence still poses a threat to human health, once CoNS can act as reservoirs of antimicrobial resistance genes. After horizontal gene transfer, these genes may increase the potential of more pathogenic species, such as S. aureus, to colonize the host, increase virulence and resist treatment with drugs (Otto, 2013). This is consistent with the fact that more than half of the bacteria analyzed harbor plasmids, which probably carry genetic determinants to one or more of the resistance genes observed. In addition, the presence of bacteria in cats with high resistance to commonly used drugs in human medicine may indicate the exchange of microorganisms between the pet and the owner, consisting on an important zoonotic concern. Also consistent with the possible role of the gene reservoirs of CoNS, the great majority of the strains here studied apparently do not have CRISPR systems, generally widespread among bacteria and archaea, as investigated by the marker gene cas1, present in all active systems (Makarova et al., 2015). Because the CRISPR can confer to the bacteria the ability to develop resistance against exogenous genetic material (Marraffini, 2015), their presence in the bacteria analyzed could act as preventing or reducing their role as gene reservoirs. 5. Conclusions This study demonstrates that domestic cats may harbor many bacteria of the Staphylococcus genus, including atypical and understudied species, without threatening the animal health. Many of these bacteria are multidrug resistant and may act as gene reservoirs for pathogenic species, which is indicated by the abundance of plasmids and the lack of restrictive CRISPR systems. Conflict of interest None.
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Acknowledgments This work was supported by FAPERJ [grant E-26/112.649/2012], CNPq [grant 476119/2012-0] and CAPES-Proex [grant 23038.001255/2011-29]. C.C.R. was recipient of a scholarship from FAPERJ [grant E-26/202.799/2016].
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Singhal, N., Kumar, M., Kanaujia, P.K., Virdi, J.S., 2015. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front. Microbiol. 6, 791. Sirmatel, F., Yetkin, G., Sirmatel, P., Guzel, N., 2015. Infective endocarditis in native value by Staphylococcus xylosus: case report and review. Case Study Case Rep. 5, 142-148. Soldera, J., Nedel, W.L., Cardoso, P.R.C., d'Azevedo, P.A., 2013. Bacteremia due to Staphylococcus cohnii ssp. urealyticus caused by infected pressure ulcer: case report and review of the literature. São Paulo Med. J. 131, 59-61. Spergser, J., Wieser, M., Taubel, M., Rossello-Mora, R.A., Rosengarten, R., Busse, H.J., 2003. Staphylococcus nepalensis sp. nov., isolated from goats of the Himalayan region. Int. J. Syst. Evol. Microbiol. 53, 2007-2011. Stepanovic, S., Jezek, P., Dakic, I., Vukovic, D., Seifert, L., 2005. Staphylococcus sciuri: an unusual cause of pelvic inflammatory disease. Int. J. STD AIDS 16, 452-453. Tomazi, T., Goncalves, J.L., Barreiro, J.R., de Campos Braga, P.A., Prada e Silva, L.F., Eberlin, M.N., dos Santos, M.V., 2014. Identification of coagulase-negative staphylococci from bovine intramammary infection by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 52, 1658-1663. Trulzsch, K., Rinder, H., Trcek, J., Bader, L., Wilhelm, U., Heesemann, J., 2002. "Staphylococcus pettenkoferi," a novel staphylococcal species isolated from clinical specimens. Diagn. Microbiol. Infect. Dis. 43, 175-182. Vigne, J.D., Evin, A., Cucchi, T., Dai, L., Yu, C., Hu, S., Soulages, N., Wang, W., Sun, Z., Gao, J., Dobney, K., Yuan, J., 2016. Earliest "Domestic" Cats in China Identified as Leopard Cat (Prionailurus bengalensis). Plos One 11, e0147295.
Figure captions Fig. 1. Antimicrobial resistance, antimicrobial-resistance genes and detection of plasmidial forms in Staphylococcus strains used in this study. The presence of squares indicate the resistance to a given 16
antimicrobial, the presence of resistance genes or plasmidial forms. Lighter shades of a color in the same column indicate intermediate resistance, according to CLSI guidelines (2015).
17
Table 1 Species identification of Staphylococcus strains used in this work by MALDI-TOF complemented with sequencing of the tuf and rpoB genes. MALDI-TOF (number of strains)
Definitive identification (tuf and/or rpoB genes)1 (number of strains)
Confidence of MALDI-TOF results
S. cohnni (5)
S. cohnni (5)
100%
S. haemolyticus (1)
S. haemolyticus (1)
100%
S. pettenkoferi (1)
S. pettenkoferi (1)
100%
S. saprophyticus (1)
S. saprophyticus (1)
100%
S. sciuri (5)
S. sciuri (5)
100%
S. xylosus (2)
S. xylosus (2)
100%
S. felis (1)
S. felis (1)
100%
S. lentus (18)
S. lentus (16), S. nepalensis (1), S. spp. (1)
88,9%
S. nepalensis (6)
S. nepalensis (5), S. spp. (1)
83,3%
S. epidermidis (4)
S. epidermidis (3), S. spp. (1)
75%
S. aureus (3)
S. aureus (2), S. lentus (1)
66,7%
S. equorum (6)
S. equorum (4), S. nepalensis (1), S. lentus (1)
66,7%
S. fleurettii (2)
S. fleurettii (1), S. spp. (1)
50%
S. lugdunensis (1)
S. lentus (1)
0%
S. arlettae (1)
S. nepalensis (1)
0%
2
Not determined (18)
S. nepalensis (7), S. lentus (3), S. xylosus (2), S. cohnni (1), S. sciuri (1), S. sp. (4)
1
not applicable
Species were defined when at least two of the three identification techniques were concordant. Strains with inconclusive results to the species level were designated as S. spp. 2 Results yielded from MALDI-TOF were unreliable (scores ≤1.699).
18
19
S0378-1135(17)30104-9 http://dx.doi.org/doi:10.1016/j.vetmic.2017.01.029 VETMIC 7528
To appear in:
VETMIC
Received date: Revised date: Accepted date:
5-7-2016 23-1-2017 24-1-2017
Please cite this article as: Rossi, Ciro C´esar, da Silva Dias, Ingrid, Muniz, Igor Mansur, Lilenbaum, Walter, Giambiagi-deMarval, Marcia, The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential.Veterinary Microbiology http://dx.doi.org/10.1016/j.vetmic.2017.01.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential
Ciro César Rossia,1, Ingrid da Silva Diasa,1, Igor Mansur Munizb, Walter Lilenbaumc, Marcia Giambiagi-deMarvala,*
a
Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, RJ, Brazil
b
c
Departamento de Medicina Veterinária - Universidade Federal de Rondonia, RO, Brazil
Laboratório de Bacteriologia Veterinária, Universidade Federal Fluminense, Niterói, RJ, Brazil
*Corresponding author at: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Cidade Universitária, Rio de Janeiro, 21941902, Brazil. E-mail adress: [email protected]; Phone: +55 21 2560-8028; Fax: +55 21 25608344. 1
These authors contributed equally to this work
1
Highlights 1. A wide variety of Staphylococcus species (n=13) was isolated from the oral microbiota of domestic cats. 2. Many species isolated are potentially pathogenic for humans. 3. Atypical strains reveal the need for alternative strategies for species identification. 4. CRISPRs elements are rare, consistent with the strains role as gene reservoirs.
2
Abstract This study aimed to characterize the species, antimicrobial resistance and dispersion of CRISPR systems in staphylococci isolated from the oropharynx of domestic cats in Brazil. Staphylococcus strains (n=75) were identified by MALDI-TOF and sequencing of rpoB and tuf genes. Antimicrobial susceptibility was assessed by disk diffusion method and PCR to investigate the presence of antimicrobial-resistance genes usually present in mobile genetic elements (plasmids), in addition to plasmid extraction. CRISPR – genetic arrangements that give the bacteria the ability to resist the entry of exogenous DNA – were investigated by the presence of the essential protein Cas1 gene. A great diversity of Staphylococcus species (n=13) was identified. The presence of understudied species, like S. nepalensis and S. pettenkoferi reveals that more than one identification method may be necessary to achieve conclusive results. At least 56% of the strains contain plamids, being 99% resistant to at least one of the eight tested antimicrobials and 12% multidrug resistant. CRISPR were rare among the studied strains, consistent with their putative role as gene reservoirs. Moreover, herein we describe for the first time their existence in Staphylococcus lentus, to which the system must confer additional adaptive advantage. Prevalence of resistance among staphylococci against antimicrobials used in veterinary and human clinical practice and the zoonotic risk highlight the need of better antimicrobial management practices, as staphylococci may transfer resistance genes among themselves, including to virulent species, like S. aureus. Keywords: Domestic cats, feline, Staphylococcus, antimicrobial resistance, CRISPR.
3
1. Introduction Cats are among the most common domestic animals in the world, totaling more than 500 million individuals living with humans, in a relationship dated from thousand years ago, as evidenced by fossil remains (Vigne et al., 2016). Pets can carry a variety of bacteria, worms, viruses and fungi. In addition, cats’ outdoor habits contribute for them to carry greater numbers and more diverse microbiota than dogs (Buma et al., 2006). Because of the intimate relationship that cats and humans may have, many of these microrganisms can be transmitted from pet to owner and vice-versa, including important pathogens like methicillin-resistant Staphylococcus aureus (MRSA), responsible for several hard-to-treat infections in humans (Muniz et al., 2013). Most of the almost 50 species of the Staphylococcus genus are harmless residents of the normal microbiota of skin and mucous membranes of mammals (Becker et al., 2014). However, species sharing the same environment may exchange genetic material among themselves, especially plasmids, providing genes that confer antimicrobial resistance to pathogenic species, such as S. aureus (Rossi et al., 2016). Thus, species historically and mistakenly referred to as inoffensive can threat human health for acting as reservoirs of genes for closely related pathogens (Otto, 2013). We hypothesize that this characteristic must be accompanied by the absence of CRISPR systems, which are arrays of repeated sequences and associated genes that give the bacterium the ability to develop adaptive immunity against exogenous genetic material, like plasmids and bacteriophages (Marraffini, 2015). Understanding the bacterial populations that inhabit companion animals is an important way of restraing their spreading and securing the health of both pet and human. Thus, the aims of this work were i) to identify a population of Staphylococcus spp. isolated from different cats in Rio de Janeiro (Brazil) and ii) to characterize their antimicrobial resistance and CRISPR content in order to evaluate the potential threat that these microorganisms may signify to the cats and their owners.
4
2. Materials and methods 2.1. Microorganisms, culture conditions and DNA purification We analyzed 75 strains belonging to the Culture Collection of Bacteria of Veterinary Interest of the Laboratory of Veterinary Bacteriology of Universidade Federal Fluminense (www.labv.uff.br). Those were previously collected from the oropharynx of different clinically healthy adult cats from Rio de Janeiro and characterized by biochemical methods as belonging to the genus Staphylococcus (Muniz et al., 2013). All strains were cultivated at 37°C for 24h in brain-hearth infusion (BHI) before the tests. Genomic DNA was isolated with the WizardTM Genomic DNA Purification Kit (Promega, USA) following the manufacturer’s instructions.
2.2. Bacterial species identification The identification of the bacterial strains was performed by combining different molecular approaches. First, the differentiation of protein profiles of bacterial cultures was analyzed in triplicate by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF) as previously described (Tomazi et al., 2014). Mass spectral data were collected within the m/z range of 2,000 to 20,000, and the data were acquired using the FlexControl software 3.3 (Bruker Daltonics). MALDI-TOF results were complemented and confirmed by the partial sequencing of the rpoB and tuf genes. Briefly, these target genes were amplified from 25 ng of DNA by PCR with the primer pairs rpoBF/rpoBR (Drancourt and Raoult, 2002) and tufF/tufR (Heikens et al. 2005) and the GoTaq® G2 Green Master Mix (Promega, USA), following the manufacturer’s protocols. Amplification steps for the rpoB gene were performed as indicated by the primers authors specified above.
2.3. Antimicrobial susceptibility testing and plasmid extraction The susceptibility of the strains was tested by the disk diffusion method against eight antimicrobials of different classes: aminoglycosides (gentamicin), β-lactams (oxacillin and ampicillin), 5
amphenicols (chloramphenicol), cephalosporins (cefoxitin), tetracyclines (tetracycline), macrolides (erythromycin) and mupirocin, (Cefar, Brazil). All tests were performed according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2015). Because of the lack of breakpoints for mupirocin in CLSI, susceptibility and differentiation of levels of resistance to this antibiotic were interpreted following the criteria previously established by our group (Oliveira et al., 2007). In order to correlate the resistance profiles with the presence of mobile genetic DNA, all strains were submitted to plasmid extraction, following protocols established by our group (Giambiagi-Marval et al., 1990).
2.4. PCR for antimicrobial resistance genes The presence of resistance genes commonly encountered in mobile genetic elements was investigated by PCR. All reactions were performed with the GoTaq® G2 Green Master Mix (Promega, USA), 25 ng of purified DNA and primers specified below. The S. haemolyticus strain MD2 (Rossi et al., 2016) was used as positive control for all reactions. The aminoglycosideresistance
genes
aadD
and
aacA-aphD,
respectively coding
for
the
aminoglycoside
adenyltransferase AadD and the bifunctional aminoglycoside modifying enzyme AacA-AphD, were amplified with the primer pairs aadDF/aadDR and aac6-aph2aFw/aac6-aph2aRev (Schiwon et al. 2013), following the amplification steps suggested by the author. The methicillin-resistance gene mecA was amplified with the primer pair MRS1/MRS2, as recommended by the author (Del Vecchio et al., 1995). Finally, the mupirocin-resistance gene mupA was amplified with the primer pair M1/M2, following the amplification steps preconized by Nunes et al.(1999)
2.5. Investigation of CRISPRs The dispersion of CRISPR systems among the 75 strains was examined by PCR for the detection of the essential CRISPR-associated protein Cas1. A cas1F (5’-AGAAGCACAGGCTGCAAGAA-3’) and cas1R (5’-TCACACTATCAAGTAACCTCACCA-3’) primer pair was designed based on a 6
conserved region of the gene obtained from the alignment made with the sequences of the following CRISPR-containing strains (and Genbank ID): S. aureus 08BA02176 (CP003808), S. capitis CR01 (NZ_CBUB000000000), (NZ_CUFQ01000000),
S. S.
epidermidis lugdunensis
RP62A HKU09-01
(NC_002976), (NC_013893),
S.
haemolyticus S.
schleiferi
W_75 1360-13
(NZ_CP009470), S. simulans ACS-120-V-Sch1 (NZ_AGZX00000000) and S. warneri 738_SWAR (NZ_JUVB00000000). Because for now there are not representatives of all staphylococci species containing CRISPR systems available from the public sequence databases, type II and type III CRISPR system-contaning S. haemolyticus MD03 and S. epidermidis RP62A were used as positive controls of the reaction. Amplifications were performed with with the primer pairs cas1F/cas1R, 25 ng of purified DNA and the GoTaq® G2 Green Master Mix (Promega, USA). Amplification consisted in initial denaturation at 94°C for 3 mins, followed by 35 cycles (94°C for 30s, 51°C for 1 min and 72 °C for 30s) and a final extension step at 72°C for 3 mins. Amplicons were visualized in 1.0% agarose gels stained with ethidium bromide. To confirm the identity of the target amplified, the fragments obtained were purified and submitted to DNA sequencing.
3. Results 3.1. Bacterial species identification The species of 75 Staphylococcus spp. strains were first identified by the MALDI-TOF technique. With this method, 57 (76%) strains were identified with scores ≥ 2.000, indicating, according to the FlexControl software 3.3 manufacturer, a “secure genus identification and probable species identification”. The remaining 18 strains yielded unreliable identification scores (≤ 1.699). Because of these inconclusive results, the MALDI-TOF data were complemented with the partial sequencing of the housekeeping genes rpoB and tuf. The sequences obtained for every strain were deposited in NCBI’s Genbank database under access number intervals KU883152-KU883217 and KX198012KX198027 (for the tuf gene); and intervals KU867251-KU867306 and KX215896-KX215913 (for the rpoB gene). Table 1 shows the MALDI-TOF results and the definitive identification of the 7
strains, achieved when the results were coincident for at least two of the three methods employed. The average confidence of the MALDI-TOF method was 82.5%, as 47 out of 57 strains were identified in accordance to the definitive identification. A wide variety of Staphylococcus species (n=13) was found in our samples, particularly S. lentus (28.0% - 21/75) and S. nepalensis (20.0% - 15 / 75), followed by S. cohnii (8.0% - 6/75), S.sciuri (8.0% - 6/75), S. xylosus (5.3% - 4/75), S. equorum (5.3% - 4/75), S. epidermidis (4.0% 3/75), S. aureus (2.7% - 2/75), S. saprophyticus (1.3% - 1/75), S. felis (1.3% - 1/75), S. pettenkoferi (1.3% - 1/75), S. haemolyticus (1.3% - 1/75) and S. fleurettii (1.3% - 1/75). In addition, nine (12%) atypical strains could not be identified to the species level, despite being identified as belonging to the genus Staphylococcus.
3.2. Antimicrobial susceptibility and plasmids The susceptibility of the 75 strains against eight antimicrobials is presented in Fig. 1. All strains but one (S. equorum strain 1) were resistant to at least one drug tested. Most strains were resistant to oxacillin (95% - 71/75) and tetracycline (76% - 57/75), followed by erythromycin (33.3% - 25/75), ampicillin (28% - 21/75), chloramphenicol (24% - 18/75), mupirocin (12% - 9/75), gentamicin (4% - 3/75) and cefoxitin (2.7% - 2/75). Among the strains, 45% (34/75) were resistant to at least three different classes of antimicrobials, being thus considered multidrug-resistant. It is likely that many of these resistance determinants are located in mobile genetic elements, as at least 56% (42/75) of the strains presented plasmids (Fig. 1).
3.3. PCR for antimicrobial resistance genes The presence of genes coding for aminoglycosides (aacA-aphD and aadD), methicillin (mecA) and mupirocin (mupA) resistance was investigated by PCR. From the genes for aminoglycoside resistance, expected to be present in the gentamicin-resistant strains 43(2), 132(1) and 114 (Fig. 1), only the aadD gene was found, in one S. cohnii strain 132(1). The mecA gene was widespread, 8
being found in 63% (47/75) of the strains (Fig. 1). In relation to the mupA gene, it was found in only one, out of three, of the mupirocin-resistant strains, S. sciuri 199.
3.4. Investigation of CRISPRs The CRISPR molecular marker cas1 gene was investigated by PCR in all 75 strains studied in this work. Only 4% of them (3/75) were positive to the amplification: S. lentus 162 and S. spp. 134a and 145 (data not shown). The fragments obtained were submitted to DNA sequencing and, by homology search, they were all confirmed to be cas1 genes. These sequences were deposited in Genbank under the access numbers KU883215-KU883217.
4. Discussion In this study, staphylococci strains isolated from the oropharynx of healthy domestic cats, living in different residences located in Rio de Janeiro (Brazil), were characterized to the species level and phenotypic and genetic aspects of antimicrobial resistance. Since biochemical tests for the identification of species from the Staphylococcus genus can yield inconsistent results, inferior to those observed with molecular biology procedures (Hasan et al., 2014), the 75 strains used in this study were initially identified by MALDI-TOF. This method has emerged as a potential tool for the identification and diagnosis of microorganisms for being fast, sensitive, relatively cheap and easy to perform, as the identification of species can be made using intact cells or cell extracts (Singhal et al., 2015). However, one limitation of the technique is the fact that it depends on spectra databases containing fingerprints of peptides masses from the microorganism of interest, which may be limited when working with rare species or from an origin other than human (Singhal et al., 2015). The results we obtained by MALDI-TOF are consistent with this statement, since a significant portion of the isolated strains showed atypical peptide profiles, hampering their identification.
9
Some Staphylococcus species isolated in the last two decades are still poorly studied, such as S. nepalensis, first identified in 2003 in the respiratory tract of goats in the Himalayan region (Spergser et al., 2003) and S. pettenkoferi, isolated in 2002 from human clinical specimens in Germany (Trulzsch et al., 2002). Because of the limitations we experienced with the MALDI-TOF, other molecular techniques were necessary to complement the identification results and to confirm the previously defined strains. The partial sequencing of the rpoB gene, encoding the beta subunit of RNA polymerase, is a central candidate for phylogenetic analysis and identification of bacteria, but the partial sequencing of the tuf gene, which encodes the elongation factor Tu, is considered the most accurate for the identification of staphylococci species (Carpaij et al., 2011). As expected, the sequence of both rpoB and tuf genes enabled us to identify the species of most strains previously not determined by MALDI-TOF. Many of these are still poorly studied in companion animals, such as S. lentus, S. nepalensis and S. pettenkoferi. In addition, some atypical strains yielded different results for the three methods employed, not being identified to the species level. The prevalent species were S. lentus and S. nepalensis. S. lentus is a commensal bacterium that colonizes the skin of several species of animals, commonly isolated from products of animal origin, which in some occasions was related to onset of localized infections, as the bacterium was isolated from urine, peritoneal fluid, blood, culture wounds and cerebrospinal fluid (Mazal and Sieger, 2010). S. nepalensis, as aformentioned, is a recently defined species, and although it has been isolated in episodes of urinary tract infections in humans, its clinical significance is not clear (Novakova et al., 2006). This is the first time, as the authors are aware, that S. nepalensis strains – highly representative in our sample – are described in feline microbiota. With the exception of S. aureus, S. intermedius and S. pseudintermedius, all other recovered strains found are coagulase-negative staphylococci (CoNS). The CoNS were long considered commensal and rarely pathogenic microorganisms. However, in recent decades, these bacteria have been recognized as etiologic agents of a number of infectious processes (Becker et al., 2014). 10
Among the CoNS found, S. haemolyticus and S. saprophyticus stand out as opportunistic pathogens in humans. S. haemolyticus is often isolated from blood cultures and can cause transient bacteremia when introduced into their hosts, generally linked to invasive medical devices (Czekaj et al., 2015). Members of S. saprophyticus are major causative agents of urinary tract infections (UTI) in humans (Eriksson et al., 2013). Other species are infrequently associated with human infections, such as S. cohnni isolated in cases of bacteremia (Soldera et al., 2013), S. sciuri identified in different infectious processes (Stepanovic et al., 2005) and S. xylosus associated with infective endocarditis (Sirmatel et al., 2015). Despite those reports in human beings, their role in animal health remains to be elucidated. The intimate relationship and contact between humans and their pets contribute to the transmission of pathogens between the two, including the transfer of multidrug resistant bacteria and genetic mobile elements carrying antimicrobial resistance genes (Costa et al., 2013; Muniz et al., 2013). Most strains of the studied Staphylococcus spp. are resistant to tetracycline and oxacillin, used in both veterinary and human medicine. Although the cefoxitin disk is generally preferable to be used in search of strains containing the mecA gene, as recommended by the CLSI (2014); for some species, like S. pseudointermedius, the oxacillin disk is recommended. Because we worked with many understudied Staphylococcus species, using both cefoxitin and oxacillin to search for mecA-positive strains enable us to retrieve richer information from our sample. Resistance genes can be located on the chromosome or on plasmids and can be horizontally transferred from one bacterium to another, contributing to the spread of resistance, originally caused by indiscriminate use of antimicrobials in human and veterinary medicine (Prescott, 2014). Mupirocin is particularly important in the prevention and treatment of MRSA, but bacterial resistance to this antimicrobial is increasing, related to either point mutations in the chromosomal ileS gene or associated with plasmids carrying the mupA gene (Deeny et al., 2015). The aadD gene, which encodes an enzyme for aminoglycoside resistance was described being located on a plasmid also integrated in the SCCmec (Chambers, 1997). The mecA gene encodes the penicillin binding protein PBP2 and is 11
located in the SCCmec chromosome cassette, a mobile genetic element which is widespread among staphylococci, usually related to multidrug resistance (Hanssen and Sollid, 2006). The abundance of methicillin-resistance among companion animals’ microorganisms has been previously described (Muniz et al., 2013). Even though the mupA and aadD genes were not as widespread as mecA, their presence still poses a threat to human health, once CoNS can act as reservoirs of antimicrobial resistance genes. After horizontal gene transfer, these genes may increase the potential of more pathogenic species, such as S. aureus, to colonize the host, increase virulence and resist treatment with drugs (Otto, 2013). This is consistent with the fact that more than half of the bacteria analyzed harbor plasmids, which probably carry genetic determinants to one or more of the resistance genes observed. In addition, the presence of bacteria in cats with high resistance to commonly used drugs in human medicine may indicate the exchange of microorganisms between the pet and the owner, consisting on an important zoonotic concern. Also consistent with the possible role of the gene reservoirs of CoNS, the great majority of the strains here studied apparently do not have CRISPR systems, generally widespread among bacteria and archaea, as investigated by the marker gene cas1, present in all active systems (Makarova et al., 2015). Because the CRISPR can confer to the bacteria the ability to develop resistance against exogenous genetic material (Marraffini, 2015), their presence in the bacteria analyzed could act as preventing or reducing their role as gene reservoirs. 5. Conclusions This study demonstrates that domestic cats may harbor many bacteria of the Staphylococcus genus, including atypical and understudied species, without threatening the animal health. Many of these bacteria are multidrug resistant and may act as gene reservoirs for pathogenic species, which is indicated by the abundance of plasmids and the lack of restrictive CRISPR systems. Conflict of interest None.
12
Acknowledgments This work was supported by FAPERJ [grant E-26/112.649/2012], CNPq [grant 476119/2012-0] and CAPES-Proex [grant 23038.001255/2011-29]. C.C.R. was recipient of a scholarship from FAPERJ [grant E-26/202.799/2016].
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Figure captions Fig. 1. Antimicrobial resistance, antimicrobial-resistance genes and detection of plasmidial forms in Staphylococcus strains used in this study. The presence of squares indicate the resistance to a given 16
antimicrobial, the presence of resistance genes or plasmidial forms. Lighter shades of a color in the same column indicate intermediate resistance, according to CLSI guidelines (2015).
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Table 1 Species identification of Staphylococcus strains used in this work by MALDI-TOF complemented with sequencing of the tuf and rpoB genes. MALDI-TOF (number of strains)
Definitive identification (tuf and/or rpoB genes)1 (number of strains)
Confidence of MALDI-TOF results
S. cohnni (5)
S. cohnni (5)
100%
S. haemolyticus (1)
S. haemolyticus (1)
100%
S. pettenkoferi (1)
S. pettenkoferi (1)
100%
S. saprophyticus (1)
S. saprophyticus (1)
100%
S. sciuri (5)
S. sciuri (5)
100%
S. xylosus (2)
S. xylosus (2)
100%
S. felis (1)
S. felis (1)
100%
S. lentus (18)
S. lentus (16), S. nepalensis (1), S. spp. (1)
88,9%
S. nepalensis (6)
S. nepalensis (5), S. spp. (1)
83,3%
S. epidermidis (4)
S. epidermidis (3), S. spp. (1)
75%
S. aureus (3)
S. aureus (2), S. lentus (1)
66,7%
S. equorum (6)
S. equorum (4), S. nepalensis (1), S. lentus (1)
66,7%
S. fleurettii (2)
S. fleurettii (1), S. spp. (1)
50%
S. lugdunensis (1)
S. lentus (1)
0%
S. arlettae (1)
S. nepalensis (1)
0%
2
Not determined (18)
S. nepalensis (7), S. lentus (3), S. xylosus (2), S. cohnni (1), S. sciuri (1), S. sp. (4)
1
not applicable
Species were defined when at least two of the three identification techniques were concordant. Strains with inconclusive results to the species level were designated as S. spp. 2 Results yielded from MALDI-TOF were unreliable (scores ≤1.699).
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