Nasal carriage of coagulase positive staphylococci in patients of a Primary-Healthcare-Center: genetic lineages and resistance and virulence genes

Abstract

Introduction

Staphylococcus aureus and Staphylococcus pseudintermedius are highly important due to their capacity for producing diseases in humans and animals, respectively. The aim of the study was to investigate and characterize the coagulase positive Staphylococcus (CoPS) carriage in a Primary Healthcare Center population.

Methods

Nasal swabs were obtained from 281 non-infectious patients. The CoPS isolates recovered were typed, and their resistance phenotype and genotype, as well as their virulence profiles, were analyzed.

Results

CoPS isolates were recovered from 56/281 patients (19.9%). Fifty-five were S. aureus (19.6%), 54 were methicillin susceptible (MSSA) and one was methicillin resistant (MRSA). The remaining isolate was S. pseudintermedius (0.4%). A high diversity of spa-types (n = 40) was detected, with 6 of them being new ones. The multi-locus-sequence-typing of 13 MSSA and one MRSA selected isolates was performed and the STs detected were: ST8, ST15, ST30, ST34, ST121, ST146, ST398, ST554, ST942, ST2499, and ST2500 (the last two STs being new). One MSSA isolate was typed as t1197-ST398-(Clonal complex)CC398. The MRSA isolate was typed as t002-ST146-CC5-SCCmec-IVc, and exhibited a multiresistance phenotype. The detected resistances were: penicillin (76%), macrolides (7%), tetracycline (7%), trimethoprim-sulfamethoxazole (7%), quinolones (7%), and lincosamides (5%). Five isolates contained lukF/lukS-PV genes, 17 tst gene, one eta gene, and two etb gene. The S. pseudintermedius isolate presented a new spa-type (t57) (belonging to a new ST180) and the genes lukS/F-I, siet, se-int, and expB.

Conclusions

A high genetic diversity of S. aureus was detected. Mention must be made of the identification of MSSA CC398 and S. pseudintermedius isolates in two patients, one of them with animal contact. The detection of the genes lukF/lukS-PV and tst should be noted.

Resumen

Introducción

Staphylococcus aureus y Staphylococcus pseudintermedius son 2 especies de gran importancia que pueden producir enfermedades tanto en personas como en animales. El objetivo del trabajo fue estudiar el estado de portador nasal de aislados de Staphylococcus coagulasa positiva (SCoP) en pacientes de un centro de atención primaria.

Métodos

Se analizaron muestras nasales de 281 pacientes sin patología infecciosa. Se tiparon los aislados SCoP y se estudiaron sus fenotipos y genotipos de resistencia y sus perfiles de virulencia.

Resultados

Se aislaron SCoP en 56/281 pacientes (19,9%): 55 de los aislados fueron S. aureus (19,6%), 54 sensibles a la meticilina (SASM) y uno resistente a la meticilina (SARM). El aislado restante correspondió a S. pseudintermedius (0,4%). Se detectó una alta diversidad de tipos de spa (n = 40), identificándose 6 nuevos tipos. Se realizó el multi-locus-sequence-typing de 13 cepas SASM y una cepa SARM seleccionadas y se detectaron los siguientes STs: ST8, ST15, ST30, ST34, ST121, ST146, ST398, ST554, ST942, ST2499 y ST2500 (los 2 últimos nuevos). Una de las cepas SASM se tipó como t1197-ST398-(Clonal Complex)CC398. La cepa SARM se tipó como t002-ST146-CC5-SCCmec-IVc y mostró un fenotipo de multirresistencia. Se detectó resistencia a: penicilina (76%), macrólidos (7%), tetraciclina (7%), trimetoprim-sulfametoxazol (7%), quinolonas (7%) y lincosamidas (5%). Se identificaron los genes (número de cepas): lukF/lukS-PV (5), tst (17), eta (1) y etb (2). La cepa de S. pseudintermedius presentó un spa nuevo (t057), una secuencia tipo nueva (ST180), y contenía los genes lukS/F-I, siet, se-int y expB.

Conclusiones

Se detectó una alta diversidad genética entre los aislados de SASM. Destaca la identificación de una cepa SASM CC398 (en un veterinario) y otra de S. pseudintermedius, y la frecuente detección de los genes lukF/lukS-PV, tst, eta o etb entre las cepas SASM.

Introduction

Staphylococcus spp. are known to be colonizer agents of humans and animals. Some of the species included in this genus are also important pathogens, which are able to produce numerous virulence factors. Moreover, these microorganisms can acquire different resistance genes, highlighting the increase of methicillin resistance mediated by the gene mecA. There are two main coagulase positive Staphylococcus (CoPS) species, Staphylococcus aureus and Staphylococcus pseudintermedius, which are highly important due to their capacity of producing important diseases in humans and animals, respectively.

S. aureus is the most known and virulent species in humans. This pathogen can produce slight skin infections, food poisoning or life threatening diseases such as pneumonia, meningitis, or septicemia, among others. Up to one-third of the healthy human population is intermittently colonized and another third is S. aureus persistent nasal carrier.¹ It has been demonstrated that S. aureus nasal carriers have a higher risk of developing an infection with this microorganism.² There has been an important increase of the rate of methicillin resistant S. aureus (MRSA) in hospital environments (HA-MRSA) and in the community (CA-MRSA), with some clones being predominant in determined geographic areas.^3,4 Initially, sporadic cases caused by CA-MRSA clones were described in our country.^5,6 These community associated cases were detected in immigrant patients especially in those coming from South American countries.⁶ However, nowadays, the differences between HA-MRSA and CA-MRSA have diminished and infections apparently caused by CA-MRSA have been increasingly detected in our hospitals.^7,8 Moreover, in 2005, a new clonal lineage named MRSA ST398 was reported.^9,10 Since then, its relationship with farm animals, especially pigs, has been observed. The risk of colonization and infection of farmers has been identified,¹¹ this clone being also identified in healthy humans without animal contact.¹²

S. pseudintermedius is the main colonizing agent of healthy dogs and cats¹³ and also is a common pathogen of these animals, usually causing skin and soft tissue infections (SSTIs).¹⁴ The transmission of S. pseudintermedius isolates between pets and their owners has been suggested in some studies.^15,16 Moreover, cases of infections in humans caused by this microorganism have been described.¹⁷ Therefore, the presence of this microorganism as a colonizer or infecting agent in humans cannot be discarded.

There are few data about the real prevalence of S. aureus and S. pseudintermedius carriage in patients of the community in our country. The aim of this study was to investigate the CoPS carriage (CoPSC) in a population attending to a Primary Healthcare Center (for non-infectious diseases), located in a region of Spain characterized by a high percentage of immigration and high density of pig farms. In addition, it was intended to know the genetic lineages of recovered isolates and to study their antibiotic resistance mechanisms and virulence factors.

Materials and methods

Bacterial isolates

Nasal swabs of 281 non-infectious patients were obtained from October 2009 to March 2011 in a Health Primary Center of Lérida (Catalonia, Spain). Informed consent was obtained from all the patients. Individuals tested were from different geographic locations (number of patients): Lérida (Catalonia, Spain) (154), Center-Europe (18), Asia (15), Africa (69), South America (23) and unknown (2). Samples were seeded on blood agar (Oxoid^®), CNA agar (bioMérieux^®), and ORSA (Oxoid^®) plates for the recovery of CoPS, and plates were incubated at 35 °C for 36 h. Identification of S. aureus and S. (pseud)intermedius was performed by conventional methods, coagulase and API STAPH (bioMérieux®), and by specific PCR.¹⁵ PCR-RFLP of pta gene with MboI endonuclease was performed to differentiate between S. intermedius and S. pseudintermedius isolates.¹⁵

Molecular typing of isolates

Single-locus DNA sequencing of the gene spa encoding S. aureus protein A^7,15 was carried out in all S. aureus and S. pseudintermedius isolates and S. aureus sequences obtained were analyzed using Ridom Staph-Type software version 1.5.21 (Ridom GmbH). SCCmec-typing was performed by multiplex PCR strategy in MRSA.⁷ Multilocus Sequence Typing (MLST) was implemented in 14 selected S. aureus isolates (S. aureus isolates which presented new spa-types, or spa-types related to ST398, or isolates which contained the genes lukF/lukS-PV encoding the Panton-Valentine leucocidin, [PVL], or presented methicillin resistance) (www.saureus.mlst.net) and in one S. pseudintermedius isolate.¹⁵ In the 14 S. aureus isolates, their Clonal Complexes (CCs) were achieved using BURST analyses.

Susceptibility testing and detection of antimicrobial resistance genes

Susceptibility testing was carried out by VITEK 2 system (bioMérieux^®) and disk-diffusion agar method following the CLSI guidelines.¹⁸ Antibiotics tested were as follows: penicillin, oxacillin, cefoxitin, erythromycin, telithromycin, clindamycin, quinupristin-dalfopristin, gentamicin, streptomycin, kanamycin, tobramycin, tetracycline, ciprofloxacin, levofloxacin, chloramphenicol, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, mupirocin, fusidic acid, linezolid, fosfomycin, nitrofurantoin, and rifampin.

The presence of blaZ, mecA, erm(A), erm(B), erm(C), erm(F), erm(T), msr(A)/msr(B), tet(K), tet(L), tet(M), tet(O), dfrS1, dfrD, dfrG, dfrK, and vanA resistance genes was investigated by PCR.^7,15,19

Virulence factors

The presence of genes encoding PVL (lukF/lukS-PV), TSST-1 (tst), Exfoliative Toxin A (ETA) (eta), B (ETB) (etb), and D (ETD) (etd) was studied by PCR in all S. aureus isolates.⁷ In S. pseudintermedius the virulence genes tested by PCR were: lukS/F-I, siet, se-int, expB, sec_canine, and expA.^15,20,21

Results

Bacteria isolates detected

CoPS isolates were recovered from 56 of the 281 tested samples (19.9%) and one isolate per positive sample was further studied. Among the 56 CoPS isolates, 55 of them were S. aureus (19.6%): 54 methicillin susceptible S. aureus (MSSA) and one methicillin resistant S. aureus (MRSA). The remaining isolate was S. pseudintermedius (0.4%).

Characteristics of S. aureus isolates

Fifty-five S. aureus isolates were obtained from nasal samples of patients of different nationalities (% of recovery respect to the studied samples of each nationality): 36 isolates from patients of Lérida, Spain (23.4%), one from Center Europe (5.5%), 2 from Asia (13%), 10 from Africa (14%), 4 from South America (17%), and 2 of unknown nationality. A high diversity of spa-types was detected among these isolates obtained (Table 1). Thus, 40 different spa-types were identified, 6 of them being new ones. These new spa-types were registered in Ridom database with the accession numbers t6387, t6388, t6389, t6390, t6391, and t6392. Fourteen selected isolates were studied by MLST (one MRSA and 13 MSSA). The MRSA isolate was typed as t002-ST146-CC5-SCCmec-IVc. The seven MSSA isolates with new spa-types belonged to ST8-CC5, ST15-CC5, ST34-CC30, ST121-CC121, and ST554-CC5. One MSSA isolate presented the spa type t1197 and belonged to ST398-CC398. This isolate was obtained from a veterinarian who worked with cattle and pigs. Other 5 MSSA isolates were selected because they contained the genes lukF/lukS-PV and belonged to ST30-CC30, ST942-CC942, ST2499-CC22, and ST2500-CC942, the last two STs being new.

Table 1.

Characteristics of the 55 S. aureus isolates recovered from nasal samples of ambulatory patients.

Strain number	spa-type^a	MLST (CC)^b	Toxin genes	Antimicrobial resistance phenotype^c	Antimicrobial resistance genes	Nationality of patient (Continent)
C2823	t1197	ST398 (CC398)		PEN-TET-ERY-CLI-CIP-LEV-SXT	blaZ, tet(M), erm(C), dfrS1, dfrK	Spanish (Europe)
C2741	t002	ST146 (CC5)		PEN-OXA-FOX-ERY-CLI-TOB-CIP-LEV	mecA, erm(C), ant(4′)-Ia, aph(3′)-IIIa	Spanish (Europe)
C2758	t002		tst			unknown
C2743	t002					Spanish (Europe)
C2992	t008			PEN	blaZ	Romanian (Europe)
C2993	t008			PEN	blaZ	Nigerian (Africa)
C2989	t008			TET	tet(M)	Unknown
C2770	t010			ERY-CIP-LEV	msr(A)/msr(B)	Spanish (Europe)
C2754	t078			PEN	blaZ	Spanish (Europe)
C2789	t084			TET-SXT	tet(K), dfrS1	Malian (Africa)
C2790	t084			PEN	blaZ	Angolan (Africa)
C2778	t084		eta	PEN-SXT	blaZ, dfrS1	Spanish (Europe)
C2771	t094		tst	PEN-TET	blaZ, tet(K)	Spanish (Europe)
C2994	t189			PEN	blaZ	Mauritanian (Africa)
C2753	t346			PEN	blaZ	Nigerian (Africa)
C2751	t6387	ST15 (CC5)				Spanish (Europe)
C2755	t6387	ST15 (CC5)				Spanish (Europe)
C2739	t6391	ST8 (CC5)		PEN		Spanish (Europe)
C2769	t6392	ST554 (CC5)				Spanish (Europe)
C2824	t164			SXT	dfrS1	Indian (Asia)
C2888	t164			PEN	blaZ	Moroccan (Africa)
C2773	t005			PEN	blaZ	Spanish (Europe)
C2772	t005	ST2499 (CC22)	lukF/lukS-PV	PEN		Indian (Asia)
C2748	t012		tst	PEN	blaZ	Spanish (Europe)
C2756	t012		tst	PEN	blaZ	Spanish (Europe)
C2742	t012		tst	PEN	blaZ	Spanish (Europe)
C2759	t021		tst	PEN	blaZ	Colombian (South America)
C2780	t018		tst	PEN	blaZ	Spanish (Europe)
C2776	t253		tst	PEN	blaZ	Spanish (Europe)
C2988	t789	ST30 (CC30)	lukF/lukS-PV, tst	PEN	blaZ	Moroccan (Africa)
C2991	t789	ST30 (CC30)	lukF/lukS-PV, tst	PEN	blaZ	Moroccan (Africa)
C2750	t1076			PEN	blaZ	Spanish (Europe)
C2757	t1333			PEN	blaZ	Spanish (Europe)
C2777	t6388	ST34 (CC30)	tst	PEN	blaZ	Spanish (Europe)
C2760	t6390	ST34 (CC30)		PEN	blaZ	Spanish (Europe)
C2740	t015			PEN	blaZ	Spanish (Europe)
C2744	t031			PEN	blaZ	Spanish (Europe)
C2784	t065			PEN-ERY-CLI	erm(A), blaZ	Spanish (Europe)
C2990	t230			PEN	blaZ	Moroccan (Africa)
C2995	t230			PEN	blaZ	Moroccan (Africa)
C2788	t330					Spanish (Europe)
C2781	t3537			PEN	blaZ	Spanish (Europe)
C2821	t269			PEN	blaZ	Spanish (Europe)
C2786	t272		etb			Spanish (Europe)
C2782	t272		etb	PEN	blaZ	Spanish (Europe)
C2787	t6389	ST121 (CC121)	tst	PEN	blaZ	Spanish (Europe)
C2747	t1445	ST942 (CC942)	lukF/lukS-PV, tst	VAN^I		Colombian (South America)
C2822	t1445	ST2500 (CC942)	lukF/lukS-PV, tst	PEN		Spanish (Europe)
C2746	t360		tst	PEN	blaZ	Spanish (Europe)
C2779	t779			PEN	blaZ	Spanish (Europe)
C2745	t869			PEN	blaZ	Ecuadorian (South America)
C2775	t1326		tst	PEN	blaZ	Spanish (Europe)
C2783	t1900			PEN-CIP-LEV	blaZ	Spanish (Europe)
C2889	t2353					Spanish (Europe)
C2785	t2646		tst			Brazilian (South America)

a

The new spa-types are marked in bold.

b

The new STs are marked in bold.

c

PEN, penicillin; TET, tetracycline; ERY, erythromycin; CLI, clindamycin; TOB, tobramycin; CIP, ciprofloxacin; LEV, levofloxacin; SXT, trimethoprim-sulfamethoxazole

Forty-two S. aureus isolates showed penicillin resistance (76%) and all of them presented the blaZ gene. Four isolates were macrolide resistant (7%), three of them being lincosamide resistant (5%). In these isolates, the msr(A)/msr(B), erm(A) or erm(C) genes were identified. Other detected resistances were: methicillin resistance (2%) encoded by mecA gene, tetracycline resistance (7%) mediated by tet(K) and tet(M) genes, trimethoprim-sulfamethoxazole resistance (7%) encoded by dfrS1 and dfrK genes, and quinolone resistance (7%). One isolate showed diminished susceptibility to vancomycin (MIC = 3 mg/L) (Table 1).

Moreover, five isolates contained the lukF/lukS-PV genes (9%), 17 isolates the tst gene (31%), one isolate the eta gene (2%), and two isolates the etb gene (4%). Two of the lukF/lukS-PV-positive isolates also harbored the gene tst (Table 1). The PVL positive isolates were obtained from five patients of different nationalities: Europe (1 isolate), South America (1 isolate), India (1 isolate), and Africa (2 isolates) (Table 1).

Characteristics of the MRSA isolate

Only one sample contained a MRSA isolate (0.4%). This isolate (C2741) presented the spa type t002 and was typed as ST146-CC5, and its SCCmec was type IVc and presented resistance, in addition to beta-lactams, to macrolides, lincosamides, tobramycin, and quinolones. The patient was an 80-years-old Spanish man who presented multiple pathologies (liver cirrhosis, chronic arterial hypertension, atrial fibrillation, etc.), and had a high contact with the Primary Healthcare Center. However, he did not have hospital admissions in the previous year.

Characteristics of the S. pseudintermedius isolate

One methicillin susceptible S. pseudintermedius isolate was obtained (0.4%) from an Indian patient that referred no animal contact. This isolate (C2536) presented a new spa-type t57 which consisted of a new repeat combination (r01 r09 r21 r02 r02 r03 r03 r06 r05). Moreover, the isolate C2536 also belonged to a new ST named ST180 which presented two new alleles (cnp60_41 and pta_29). This isolate was susceptible to all tested antibiotics and presented the toxin genes lukS/F-I, siet, se-int, and expB.

Discussion

A rate of S. aureus of 19.6%, a low percentage of MRSA (0.4%) and a low percentage of S. pseudintermedius (0.4%) were found as colonizer agents in the studied patients. Some authors refer that the S. aureus nasal colonization rate in the general population is about 30%.²² Nevertheless, some recent studies have reported lower percentages (about 20%). In this sense, Bode et al.²³ reported a prevalence of 18.8% using real-time PCR. Den Heijer et al.²⁴ had similar results (21.6%) in a cross-sectional study which included 9 European countries. The differences detected among the different countries were from 12.7% (Hungary) to 29.4% (Sweden); the percentage detected in Spain, in that study,²⁴ was of 18.8%. In another study carried out in Spain¹² the percentage detected (19.1%) was very similar to the one found in our studied population. In our work, the highest rate of colonization was found in European patients (20%) and the lowest value in Asian patients (13%). However, it should be taken into account that the majority of samples studied were from patients of our country (European patients).

The prevalence of MRSA in our study was 0.4%. The percentages detected in other studies performed among healthy people^12,24 varied between 0.2 and 2%. However, in a very recent study in our country, a higher value was identified (6%).²⁵

Regarding S. pseudintermedius rate, this microorganism is more commonly found in dogs than in humans. Different studies have reported S. pseudintermedius colonization values on healthy dogs highly variable (23 and 92%).²⁶ However, these uneven results mainly depend on the body-site in which the samples are taken.²⁷ In humans, the presence of this microorganism is associated with individuals having regular contact with pets such as veterinarians and owners.^15,28 Nevertheless, our S. pseudintermedius isolate was obtained from an Indian patient that referred no contact with animals.

Interestingly, one MSSA isolate belonging to CC398 was obtained from a patient who worked as a veterinarian in a farm with pigs and cattle. In the case of methicillin resistant strains, this CC is related to livestock animals. It has been observed that people in contact with farm animals are more frequently carriers of MRSA CC398 than people who do not have that contact.²⁹ Recently, a second epidemiological event consisting in the emergence of MSSA CC398 human infections has been detected.³⁰ Indeed, it is thought that the origin of livestock-associated MRSA CC398 could be MSSA of humans.^31,32 So, MSSA CC398 strains have previously been detected as colonizer agents in healthy humans in Spain¹² and in other countries.³²

A high diversity of spa-types was identified which is in accordance with other studies about MSSA strains.^12,33 Remarkably, the strains C2739, C2989, C2992, and C2993 showed characteristics typical to community strains. These isolates were susceptible to all antimicrobial tested except penicillin or tetracycline, were obtained from non-infectious patients and three of them presented the spa-type t008. One of the most important community MRSA clones is known as USA300 which is typed as ST8-t008 and contains the PVL genes.^34,35 In our case, isolates were methicillin susceptible. It has been hypothesized that MSSA of CC8, sequence type 8, is the presumptive ancestor of the first MRSA USA500 and USA300 strains.³⁶

Interestingly, the PVL toxin genes were not found in these isolates but in strains belonging to CC30. The presence of this toxin in CC30 clones has frequently been identified³⁷ and the same applies with the tst gene.^12,38 The CC30 is one of the major S. aureus lineages and it is related to hospital-acquired and community-acquired infections worldwide.

Only one strain was methicillin resistant. This isolate was typed as ST146-CC5, spa-type t002 and SCCmec IVc and presented a multiresistance phenotype. Strains with similar characteristics are considered hospital acquired (HA) MRSA. Although strains with the spa-type t002 are very common in the hospitals of our country, the ST146 is found less frequently.^7,39 Moreover, in a very recent study the spa type t002 was the most commonly detected among nasal MRSA from European patients.²⁴

High percentage of penicillin resistance was detected among our S. aureus isolates (76%), in agreement with other studies.^7,24 The values of resistance to methicillin, macrolides and tetracycline were also in accordance with the ranges observed by den Heijer et al.,²⁴ highlighting the trimethoprim-sulfamethoxazole resistance rate which was very elevated (7%).

With respect to the S. pseudintermedius strain isolated in this study the detection of a new spa-type (t57) and a new ST (ST180) is remarkable. However, this result was expected as this isolate was methicillin susceptible (MS) and a high clonal diversity has been previously observed among MS S. pseudintermedius isolates.^15,40 Moreover, this isolate harbored the lukS/F-I, siet, se-int, an expB toxin genes. The expB gene was recently described,²¹ and it seems to be related to cases of superficial dermatitis in dogs.⁴⁰

Conclusions

In conclusion, a high genetic diversity of S. aureus was detected in nasal samples of ambulatory patients. The identification of MSSA CC398 and S. pseudintermedius isolates in two patients, one of them veterinarian, is highly remarkable. The detection of virulent strains in general population which contained the genes lukF/lukS-PV and tst should be noted and considered in the development of control measures.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

Part of this work has been financed by Project SAF2012-35474 of the Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF). C. Lozano has a contract associated with Project SAF2012-35474.