Phenotype and Genotype Characteristics of Staphylococcus aureus Resistant to Methicillin/Oxacillin Carrying Gene mecC in the Czech Republic from 2002 to 2017

Abstract

The aim of this study was to detect and characterize isolates of methicillin-/oxacillin-resistant Staphylococcus aureus (MRSA) carrying gene mecC (MRSA/mecC) and occurring in the Czech Republic within the period from 2002 to 2017. Altogether, 18 from 3,472 isolates of MRSA were mecC positive (0.52%). The first detection of MRSA/mecC in the Czech Republic is dated to 2004. MRSA/mecC isolates were susceptible to almost all tested antibiotics with few exceptions. Resistances to erythromycin (n = 2), clindamycin (n = 1), trimethoprim-sulfamethoxazole (n = 1), and rifampicin (n = 1) were found in the collection. Multilocus sequence typing and spa typing revealed a genetic heterogeneity of MRSA/mecC strains: three CCs (130, 425, and 2361), five STs (1245, 130, 2361, 425, and a new ST5480), and eight spa types (t843, t978, t1048, t1535, t1736, t6104, t8842, and t17153), which were detected in the study, with the highest prevalence of CC130/t843 lineage (n = 8, 44%). Except for two strains, none from 18 examined isolates harbored genes encoding any of S. aureus toxins: enterotoxins a–u, exfoliative toxins A, B, and D, toxic shock syndrome toxin-1, and the Panton-Valentine leukocidin.

Introduction

Staphylococcus aureus is the most important human Staphylococcus species associated with nosocomial and community-acquired infections.¹ Since the first detection of diseases caused by methicillin-/oxacillin-resistant S. aureus (MRSA) (1961, UK), this pathogen has expanded into the whole world.² MRSA isolates can exhibit resistance to a broad spectrum of antibiotics. Decreased susceptibility to methicillin/oxacillin as well as to other antibiotics is caused by acquired transpeptidase penicillin binding protein 2a (PBP2a) characterized by low affinity to β-lactams, with the exception of ceftaroline and ceftobiprole, so called antiMRSA cephalosporins. PBP2a is coded by mecA occurring in the staphylococcal chromosome cassette mec (SCCmec). SCCmec consists of variable and conserved genetic elements, including mec operon with mecA and regulatory (mecI and mecR1) genes, as well as recombinase genes (ccrA, ccrB, and ccrC) responsible for excision/integration of this genetic element within chromosome.³ Till now, 13 types of SCCmec (I–XIII) have been identified.⁴

In 2002 (Scotland) was detected a new variant of mecA gene in humans; mecA_LGA251 (later called mecC) producing an altered transpeptidase (PBP2c), also with a low affinity to β-lactam antibiotics.⁵ MecA and mecC homologs share similarity at the DNA level around 70%,³ and only 63% at amino acid level.⁵ Due to a nucleic acid divergence between mecA and mecC genes, all methods commonly used for mecA detection failed in mecC uncovering. One of the successful and simple methods used in the detection of MRSA isolates carrying gene mecC (MRSA/mecC) is PCR in combination with primers specific for gene mecC.^5,6

Despite the fact that the presence of MRSA isolates carrying gene mecC was confirmed almost from all over Europe in humans,^5,7 there is missing information about mecC detection in the Czech Republic. Therefore, the objective of this work was to examine the occurrence, an antibiotic susceptibility profile, virulence potential, as well as the genetic diversity of MRSA isolates carrying gene mecC in the long-term period, from 2002 to 2017 in the Czech Republic.

Materials and Methods

Bacterial isolates

Screening of MRSA/mecA isolates is provided as routinely in the National Reference Laboratory for Antibiotics (National Institute of Public Health, Prague, Czech Republic) as a part of European Antimicrobial Resistance Surveillance Network (EARS-Net). MRSA strains with an absence of mecA gene were further examined for the presence of mecC gene. Overall, 3,472 isolates of MRSA were isolated in the National Reference Laboratory for Antibiotics from 2002 to 2017. The majority of the clinical specimen, altogether 78.6% (n = 2,728), were of invasive origin (blood and cerebrospinal fluid), and 21.4% (n = 744) of MRSA strains were isolated from noninvasive materials (surgical wound, urine, sputum, and swab: nose, nasopharynx, and throat). All staphylococci were cultivated on nutrient agar (Oxoid) overnight at 36°C aerobically. Identification of S. aureus strains was performed by Matrix-assisted laser desorption ionization–time of flight mass spectrometry (Maldi-TOF; Microflex Brucker, Bremen, Germany).

Susceptibility testing

Screening for MRSA was performed by disk diffusion method using cefoxitin disc (30 μg); zone diameter <22 mm was interpreted as oxacillin resistance according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations.⁸ Minimal inhibition concentration of oxacillin (OXA), erythromycin (ERY), clindamycin (CLI), trimethoprim-sulfamethoxazole (SXT), rifampicin (RIF), ciprofloxacin (CIP), gentamicin (GEN), vancomycin (VAN), fusidic acid (FUS), chloramphenicol (CMP), linezolid (LNZ), and tigecycline (TGC) was determined by broth microdilution method according to ISO 20776-1. Susceptibility to tetracycline (TET) and tobramycin (TOB) was determined by disc diffusion method.⁹ Interpretation of susceptibility testing results was performed as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (later the Clinical & Laboratory Standards Institute: CLSI Guidelines)¹⁰ and EUCAST (version according to a corresponding year).¹¹ Minimal inhibitory concentration of oxacillin was interpreted according to the NCCLS (later CLSI)¹⁰ and the EUCAST-ECOFF value (version according to a corresponding year).¹²

Molecular typing

PCR

DNA was isolated according to the procedure of isolation kit (GenElute™ Bacterial Genomic DNA Kits, Sigma Aldrich). The presence of mecA and mecC genes in MRSA isolates was identified by PCR. Gene mecA was amplified using primers F 5′ -GTA GAA ATG ACT GAA CGT CCG ATA A- 3′ and R 5′ -CCA ATT CCA CAT TGT TTC GGT CTA A- 3′. Conditions for PCR were 2 minutes at 94°C followed by 30 cycles of 30 seconds at 94°C, 1 minute at 55°C, 1 minute at 72°C, and finished by 10 minutes at 72°C (Bio-rad, DNA Engine Dyad^® Dual–Bay Thermal Cycler). Gene mecC was detected according to the protocol of National Food Institute.⁶ PCR products were resolved in 1% agarose (Agarose SFR, VWR Life Science) in electrophoresis 5 V/cm for 50 minutes. S. aureus, NCTC13552 (Salisbury, UK), was used as positive control for mecC gene.

Spa and MLST typing

Spa typing: MRSA/mecC isolates were further analyzed using genotypic methods; repetitive sequences of protein A were amplified using the following primers: 1113f (5′- TAA AGA CGA TCC TTC GGT GAG C -3′) and 1514r (5′-CAG CAG TAG TGC CGT TTG CTT -3′), according to the procedure¹³ by analyzer (Applied Biosystems 3130xL). Data were analyzed using Bionumerics 7.6.2 (Applied Maths, Ghent, East Flanders, Belgium). The minimum spanning tree (MST) algorithm (gap creation cost 250%, gap extension cost 50%, duplicate creation cost 25%, and duplicate extension 25%, maximum duplication of three repetitions) used for cluster analysis of spa isolates was carried out also by Bionumerics 7.6.2.

Multilocus sequence typing (MLST) of mecC strains: seven housekeeping genes arcC, aroE, glpF, gmk, pta, tpi, and yqi were amplified according to the standard procedure described earlier.¹⁴ All strains were sequenced by analyzer (Applied Biosystems 3130xL). Data were analyzed by Bionumerics 7.6.2 and eBurst analysis was performed (database is no longer active).

Toxin detection

Isolates of S. aureus carrying mecC gene were also subjected to PCR analysis for confirmation or refutation of presence of genes encoding staphylococcus toxins: enterotoxins a-u (sea–seu), exfoliative toxins A, B, and D (eta, etb, and etd), staphylococci superantigens: toxic shock syndrome toxin-1 (tst), and the Panton–Valentine leukocidin (lukS/F-PVL; consisting of the LukS and LukF fragments). The list of primers and PCR conditions used for toxins amplification is in the Supplementary Table S1. S6 (sea, seb, seq, and sek), FRI137 (sec, seh, sel, sem, sen, seo, and seu), 01HMPL280 (sed, seg, sei, sej, sep, and ser), and FRI326 (see) were reference strains used for the detection of enterotoxins (all acquired from the French Food Safety Agency, Paris, France). Strains CCM7056 (eta and etb), CCM2331 (etd) (both acquired from Brno, Czech Republic), CNCTC5931 (Prague, Czech Republic, positive for tst,), and ATCC49775 (Virginia, positive for lukS/F-PVL,) were used for the detection of exfoliative toxins, tst, and the lukS/F-PVL.

Results

Antibiotic susceptibility of MRSA/mecC isolates

Altogether, 3,472 isolates of S. aureus resistant to CXT were tested for the presence of mecA/mecC gene in the period from 2002 to 2017. Eighteen of them harbored the mecC gene (0.52%). Resistance to methicillin/oxacillin in the rest of the MRSA samples (99.48%) was caused by the presence of mecA gene (data not shown). The magnitude of spreading differed from year to year, but the first detection of mecC in the Czech Republic was dated to the year 2004 (Table 1, isolate number 2132). MRSA/mecC isolates were detected in Ceske Budejovice, Pribram, Prostejov, Kyjov, Uherske Hradiste, Benesov, Prague, and Plzen. Positive strains were isolated especially from noninvasive material: swab (nose, nasopharynx, and throat) (n = 8), chirurgical wound (n = 5), and sputum (n = 1). Only four strains were acquired from blood.

Table 1.

The List of Methicillin-/Oxacillin-Resistant Staphylococcus aureus/mecC Isolates Involved in the Study

Isolate	Age	Sex	Source	Year of isolation	Location	mm			mg/L
						CXT	TET	TOB	OXA	ERY	CLI	SXT	RIF	CIP	GEN	VAN	FUS	CMP	LNZ	TGC	spa	ST	CC	En	tst	Exf	lukS/F-PVL
2132	59	M	Blood	2004	Prague	12	24	21	>8	1	0.25	≤0.125	≤0.03	0.5	0.5	1	0.25	8	2	0.5	t1736	5480	130	−	−	−	−
4747	67	M	Sputum	2004	Ceske Budejovice	14	28	22	>8	1	0.25	≤0.125	≤0.03	0.25	0.5	1	0.125	8	2	0.5	t843	130	130	−	−	−	−
4259	79	M	Swab	2010	Ceske Budejovice	12	27	22	8	0.5	0.125	≤0.125	≤0.03	0.5	0.25	0.5	0.125	8	2	0.125	t843	1245	130	−	−	−	−
7468	79	M	Blood	2010	Ceske Budejovice	14	25	22	>32	0.5	0.25	≤0.125	≤0.03	0.5	0.25	0.5	0.06	4	1	0.06	t843	130	130	−	−	−	−
8223	79	M	Blood	2011	Ceske Budejovice	13	28	23	8	0.25	0.125	≤0.125	≤0.03	0.5	0.5	1	0.06	4	2	0.125	t843	130	130	−	−	−	−
18063	73	M	Chirurgical wound	2013	Benesov	17	26	21	2	>8	0.125	≤0.125	≤0.03	0.5	0.5	2	0.125	3	2	0.06	t978	2361	2361	#	+	−	−
25255	65	M	Swab	2014	Pribram	18	25	22	1	0.5	0.125	≤0.125	≤0.03	0.5	≤0.125	1	0.125	8	2	0.06	t843	1245	130	−	−	−	−
25994	6	M	Swab	2014	Kyjov	11	27	22	16	0.5	0.125	≤0.125	≤0.03	0.5	0.25	1	0.125	8	2	0.06	t6104	425	425	−	−	−	−
26510	68	F	Blood	2014	Plzen	16	27	21	8	0.25	0.06	≤0.125	≤0.03	0.5	≤0.125	1	0.125	8	2	0.5	t8842	1245	130	−	−	−	−
29732	62	M	Chirurgical wound	2015	Prerov	14	23	20	4	0.25	0.25	≤0.125	≤0.03	0.5	0.25	1	≤0.03	8	2	0.5	t1048	130	130	−	−	−	−
30299	65	M	Chirurgical wound	2015	Prerov	17	27	21	4	0.25	0.125	>4	>2	0.5	0.25	1	0.125	4	2	0.06	t1048	130	130	−	−	−	−
30720	37	F	Swab	2015	Pribram	16	25	21	8	0.25	0.125	≤0.125	≤0.03	0.5	0.5	1	0.06	8	2	0.06	t843	1245	130	−	−	−	−
30721	5	F	Swab	2015	Pribram	16	28	23	8	0.5	0.25	≤0.125	≤0.03	0.5	0.25	1	0.125	8	2	0.125	t843	1245	130	−	−	−	−
30722	5	F	Swab	2015	Pribram	15	27	21	4	0.5	0.25	≤0.125	≤0.03	0.5	0.5	1	0.125	8	2	0.125	t843	1245	130	−	−	−	−
30730	84	M	Chirurgical wound	2015	Uherske Hradiste	16	27	22	4	>8	>4	≤0.125	≤0.03	0.5	0.5	2	0.06	8	2	0.125	t1048	130	130	−	−	−	−
30792	57	M	Swab	2015	Pribram	15	26	22	8	0.5	0.25	≤0.125	≤0.03	0.5	0.5	1	0.06	8	2	0.125	t1535	1245	130	−	−	−	−
35220	71	F	Swab	2016	Prerov	13	28	23	8	0.5	0.125	≤0.125	≤0.03	1	0.5	1	0.06	4	2	0.125	t17153	130	130	−	+	−	−
37553	53	M	Chirurgical wound	2017	Brno	11	26	21	8	1	0.25	0.5	≤0.03	0.5	≤0.125	1	0.125	8	2	0.125	t1048	130	130	−	−	−	−

CIP, ciprofloxacin; CLI, clindamycin; CMP, chloramphenicol; CXT, cefoxitin; ERY, erythromycin; FUS, fusidic acid; GEN, gentamicin; LNZ, linezolid; OXA, oxacillin; RIF, rifampicin; SXT, trimethoprim-sulfamethoxazole; TET, tetracycline; TGC, tigecycline; TOB, tobramycin; VAN, vancomycin.

#, genes for enterotoxins (sec, seg, sei, sel, and sem) detected in MRSA/mecC isolates; +, positive; −, negative; CC, clonal complex; En, enterotoxins; Exf, exfoliative toxins; F, female; lukS/F-PVL, Panton–Valentine leukocidin; M, man; spa, Staphylococcus protein A; ST, sequence type; tst, toxic shock syndrome toxin-1.

Antimicrobial susceptibility testing revealed that the majority of isolates was susceptible to most of tested antibiotics. Altogether, two isolates of MRSA/mecC (18063 and 25255) were resistant to CXT, but sensitive to OXA. One isolate showed resistance to ERY. The isolate 30730 was resistant to both ERY and CLI. The strain 30299 was resistant to SXT and RIF (Table 1).

Molecular typing

Among 18 isolates, eight different spa types were found in the collection. Spa type t843 (n = 8), followed by t1048 (n = 4) were the most frequent spa types detected in the study. The remaining spa types of MRSA/mecC isolates were as follows: t978, t1535, t1736, t6104, t8842, and t17153 (Table 1). The core of the group/the closest genetic relationship (one difference in repeat sequence) was observed between four spa types: t843, t1048, t1535, and t8842. The rest of isolates (t978, t1736, t6104, and t17153) differed from the core of the group by more than just one repeat sequence in the spa gene. The cluster analysis (MST) based on similarity of spa profiles is shown in Fig. 1.

FIG. 1.

MST analysis of MRSA/mecC isolates (n = 18) illustrating the relationship within cluster. The nodes/one-colored circles consist of isolates with the same genotype, the size of node/circle correlates with the number of genetically identical isolates, and the distance between nodes represents the genetic relationship between nodes. MST, minimum spanning tree; MRSA, methicillin-/oxacillin-resistant Staphylococcus aureus. Color images are available online.

Altogether, there were five sequence types (STs) detected in MRSA/mecC isolates: ST130 (n = 8), ST425 (n = 1), ST1245 (n = 7), and ST2361 (n = 1). A new ST5480 (a new allele tpi 611) was found in the first MRSA-/mecC-positive isolate (number 2132) detected in the Czech Republic. MLST analysis revealed the prevalence of clonal complex (CC) 130 (89%, n = 16), including STs 1245, 130, and 5480. Other STs detected in the study, ST2361 and ST425, belonged to different CCs: CC2361 (ST2361) and CC425 (ST425) (Table 1).

Toxins

Strains were negative for virulence toxin genes encoding exfoliative toxins, and lukS/F-PVL (Table 1), with the exception of genes for enterotoxins sec, seg, sei, sel, sem (found in 18063), and tst that were detected in two isolates (18063 and 35220) (Table 1).

Discussion

Identification of MRSA isolates is key for controlling dissemination of this pathogen in human population and it is important for suitable management of colonized and infected patients. Except for mecA, there is also a new homologue of this gene, mecC, which has been already sporadically reported in MRSA strains all over Europe.^15,16 Except for S. aureus, mecC has been already detected in other Staphylococcus species, namely Staphylococcus stepanovicii,¹⁷ Staphylococcus xylosus,¹⁸ and Staphylococcus sciuri.¹⁹

The most reliable tools used in the MRSA/mecC detection are PCR with specific primers for mecC gene and disc diffusion test with CXT. Two (18063 and 25255) from 18 strains were susceptible to OXA and resistant to CXT, along with a positive detection of mecC gene by PCR. The majority of mecC-positive MRSA was susceptible to the most of examined antibiotics, which was in line with results of other authors.^7,20,21

Molecular typing is an important tool for control and prevention of infections. One of such methods used especially for analysis of genetic variability of S. aureus is spa typing. Spa analysis revealed the presence of eight spa types associated with mecC strains in the trial. The most prevalent spa type t843 (44%),²² altogether with t978, t1535, t8842,²³ t1736,²⁴ and t1048,^7,25 has been already confirmed in mecC-positive strains in epidemiological studies all over Europe. Except for a susceptible S. aureus from France (Ridom SpaServer, Ridom GmbH, Würzburg, Germany), spa type t6104 was detected in this study too. MLST is also an epidemiology tool useful for understanding the molecular evolution of bacteria. Isolates of MRSA were assigned to three CCs: 130, 425, and 2361. CC130 has been previously reported in MRSA isolates of animal and human origin and it is a CC commonly associated with the occurrence of MRSA isolates carrying gene mecC in Europe.^6,25,26 CC2361 and 425 were previously observed in MRSA/mecC isolates in Denmark and Spain.^27,28

A virulence potential of mecC-positive MRSA strains has been also studied. A whole genome sequencing analysis has been pointed at an absence of genes coding exfoliative toxins as well as staphylococcal tst, and enterotoxins (sea–see and seg–sej).^7,16 The absence of lukS/F-PVL in MRSA/mecC isolates was confirmed.²⁵ On the other hand, it seems that ST profile could be associated with toxin production; an absence of toxin genes in the most samples of CC130 has been already described before.^7,16 The only two toxins-positive isolates (enterotoxins sec, seg, sei, sel, sem, and tst) belonged to the CC130/t17153 and CC2361/t978, indicating that this toxin producing CCs/spa types might represent a risk to humans.

In line with our results, the spreading of MRSA/mecC isolates becomes more frequent in people older than 50 years.^7,16,27 This part of human population is considered high risk due to the weakening of immune system and frequent consumption of antibiotics.²⁷ Also, a hospitalization or contact with animals (zoonotic transmission) as well as travel activities belong to the risk factors for MRSA spreading.¹⁶

The limitation of this study lies in the kind of analyzed clinical specimen. The most of MRSA isolates was acquired from invasive material, preferentially sent to the National Reference Laboratory for Antibiotics. However, the majority of mecC-positive MRSA isolates, as showed this study, has been found in noninvasive clinical specimen. Most of isolates were acquired from swab (nose/throat).

The aim of this study was to detect and describe MRSA/mecC isolates occurring in the Czech Republic within a 15-year long period. And to our knowledge, this is the first report concerning the detection of these strains in the Czech Republic. The first detection of MRSA/mecC isolate was dated to 2004 (59 years, man, Prague), a new ST5480, a spa type t1763. The most of MRSA-positive mecC strains belonged to the susceptible lineage CC130/t843. A rare occurrence of MRSA/mecC isolates confirmed in this study (0.52%) was comparable with findings in Europe (2%, in Denmark).²⁵

Due to a clinical relevance of MRSA strains and their spreading possibilities, there should be attention on a right detection as well as monitoring of abovementioned strains associated, especially with hospital-acquired infections.

Disclosure Statement

No competing financial interests exist.

Funding Information

This project was supported by Ministry of Health, Czech Republic—conceptual development of research organization (The National Institute of Public Health – NIPH, 75010330).

Supplementary Material

Supplementary Table S1