Characterization of Staphylococcus aureus Strains Isolated from Czech Cystic Fibrosis Patients: High Rate of Ribosomal Mutation Conferring Resistance to MLS B Antibiotics as a Result of Long-Term and Low-Dose Azithromycin Treatment

Abstract

Staphylococcus aureus is one of the most frequent pathogens infecting the respiratory tract of patients with cystic fibrosis (CF). This study was the first to examine S. aureus isolates from CF patients in the Czech Republic. Among 100 S. aureus isolates from 92 of 107 observed patients, we found a high prevalence of resistance to macrolide–lincosamide–streptogramin B (MLS_B) antibiotics (56%). More than half of the resistant strains (29 of 56) carried a mutation in the MLS_B target site. The emergence of MLS_B resistance and mutations conferring resistance to MLS_B antibiotics was associated with azithromycin treatment (p=0.000000184 and p=0.000681, respectively). Methicillin resistance was only detected in 3% of isolates and the rate of resistance to other antibiotics did not exceed 12%. The prevalence of small-colony variant (SCV) strains was relatively low (9%) and eight of nine isolates with the SCV phenotype were thymidine dependent. The study population of S. aureus was heterogeneous in structure and both the most prevalent community-associated and hospital-acquired clonal lineages were represented. Of the virulence genes, enterotoxin genes seg (n=52), sei (n=49), and sec (n=16) were the most frequently detected among the isolates. The PVL genes (lukS-PV and lukF-PV) have not been revealed in any of the isolates.

Introduction

Cystic fibrosis (CF) is the most common life-shortening genetic disorder in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a chloride channel resulting in abnormal ion transport. CF targets many organs, including the pancreas, liver, and intestine, but the most commonly affected are the lungs. Production of thick mucus on the respiratory epithelial surface is the cause of decreased mucociliary clearance which, in turn, leads to chronic respiratory infection. This ongoing infection together with an inflammatory response leads to irreversible progressive lung damage, and pulmonary insufficiency is the primary cause of death in the majority of patients with CF.³¹ Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus aureus, Stenotrophomonas maltophilia, Burkholderia cepacia complex (Bcc), and Achromobacter xylosoxidans are the most common pathogens infecting the lungs of these patients.³⁶

S. aureus, historically the first bacterial pathogen to be associated with respiratory infections in patients with CF,¹ is still one of the most common isolates recovered from the sputum of these patients, and children in particular, with a prevalence ranging between 50% and 70% across Europe.^3,20,43 Recently, increasing prevalence of S. aureus, including methicillin-resistant S. aureus (MRSA), at some US CF care centers is an emerging challenge in CF.^10,36

S. aureus small-colony variants (SCVs) are often recovered from chronic and persistent infections, including CF lung infection.¹⁹ SCVs represent a persistent subpopulation of metabolically deficient/auxotrophic bacteria that usually grow slowly in nonpigmented and nonhemolytic colonies with pinpoint or fried egg morphology. A pinpoint colony isolate is approximately 10 times smaller in size than the parent strain.⁸ SCV formation is associated with prolonged antibiotic treatment with trimethoprim–sulfamethoxazole (TMP-SXT) or aminoglycosides.^9,20 The underlying mechanisms for SCV formation appear to be disruption of the electron transport chain or thymidylate synthase pathway for menadione/hemin- and thymidine-dependent SCVs, respectively.^22,33 These metabolic defects are responsible for SCV resistance or tolerance to TMP-SXT and aminoglycosides and because of slow growth of the affected cells also to the antibiotics targeting exponentially growing bacteria. The significance of SCVs in the context of chronic infections is also supported by their increased ability to persist intracellularly in different cell lines. Moreover, SCVs in CF were found to be associated with hypermutability which, in turn, results in a high rate of Macrolide–Lincosamide–Streptogramin B (MLS_B) resistance in these strains.^4,35 The high rate of MLS_B resistance could be a result of the long-term low-dose azithromycin therapy. Azithromycin is frequently used in CF patients for its beneficial effects on inflammation and lung physiology and also the potential to reduce P. aeruginosa biofilm formation.⁷

The impact of S. aureus on the pathogenesis of respiratory tract infections in CF patients is not fully understood, with the exception of MRSA and SCV strains, which are associated with poor clinical outcome.^5,42 Although the presence of pathogenic bacteria such as S. aureus in the respiratory tract is not normal, different studies disagree on the impact of S. aureus on the progression of CF.^13,25,39

The goal of our study was to analyze the prevalence of SCVs, virulence factors, and antibiotic resistance (together with the mechanisms underlying the resistance) among S. aureus isolates from Czech CF patients.

Methods

Patients and isolates

Between July 2011 and March 2013, 100 S. aureus isolates were recovered from 92 of 107 selected children and adult patients followed up at the Centre for CF of the Motol University Hospital, when presenting for periodical checkups or with exacerbation. The study group consists of patients with documented staphylococcal infection in their medical records followed up at the Prague CF Centre and selected in an attempt to maximize the number of S. aureus isolates to analyze. Written informed consent was obtained from all study participants or their parents.

The S. aureus-positive group included 44 males and 48 females and the median age when sampled was 16 years (age range from below one year to 37 years). Single unique S. aureus isolates were recovered from 84 patients and two phenotypically different (in colony size, shape, or pigmentation) isolates from the same sputum sample were available from eight patients. Most patients (n=85) had chronic staphylococcal infection as defined according to the modified Leeds criteria: >50% of the sputum samples positive during the last 12 months. At least four sputum samples were collected during that period.⁴⁴

Species identification was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics). Nonhemolytic and nonpigmented isolates with a small colony size or fried egg morphology were considered to be S. aureus SCVs. These isolates were also tested by the Pastorex Staph Plus^© latex agglutination test (Bio-Rad Laboratories) and biochemical STAPHYtest 16 (Erba Lachema).

When sampled or within three months, coinfection with S. aureus and the following bacteria was detected: P. aeruginosa (n=21), Bcc (n=5), S. maltophilia (n=8), P. aeruginosa and Bcc (n=1), and P. aeruginosa and S. maltophilia (n=3). Within one year before the sampling date, the patients were treated with long-term azithromycin (n=22), trimethoprim–sulfamethoxazole (TMP-SXT, n=55, for 25 days on an average, for a median of 20 days, long-term TMP-SXT in one patient), and other MLS_B antibiotics (clindamycin, clarithromycin, and spiramycin) (n=23, for 21 days on an average, for a median of 14 days). The use of other antibiotics was not reported.

Susceptibility testing

Antimicrobial susceptibility of the strains was determined by the disk diffusion method (DDM) according to the EUCAST guidelines for oxacillin, cefoxitin, erythromycin, clindamycin, gentamicin, tetracycline, amoxicillin/clavulanic acid, rifampicin, ofloxacin, vancomycin, teicoplanin, TMP-SXT, linezolid, azithromycin, and quinupristin/dalfopristin.¹² In addition, the minimum inhibitory concentrations (MICs) for oxacillin, vancomycin, gentamicin, erythromycin, or clindamycin were measured for strains with a borderline DDM value and for MRSA strains (vancomycin) using M.I.C. Evaluator strips (Oxoid). Inducible MLS_B resistance was tested by the D-test. S. aureus and SCV isolates were cultured on Mueller Hinton (MH) agar and Columbia agar supplemented with sheep blood (Oxoid), respectively. Plates were incubated at 37°C for 24 and 48 hours. S. aureus strain ATCC 25923 was used as a quality control.

Polymerase chain reaction detection of MLS_B, aminoglycoside, and methicillin resistance genes

Bacterial genomic DNA was extracted using the Amplicor^® Respiratory Specimen Preparation Kit (Roche) and used as a template for polymerase chain reaction (PCR).

Detection of the methicillin resistance gene mecA, MLS_B resistance genes ermA, ermB, ermC, ermT, and msrA, and aminoglycoside resistance genes aadC, aphA3, and aac-aphD was performed as described previously.^17,23,30,41

PCR amplicons were resolved in 2% (w/v) agarose gels in Tris-boric acid-EDTA (TBE) buffer at constant voltage (6 V/cm) and stained with ethidium bromide (0.5 mg/ml). Images were photographed under UV light using a gel documentation system.

Detection of mutations conferring resistance to MLS_B

Amplification and sequencing were conducted using the respective primers specific for the domain V of the 23S rRNA (rrl) and the genes for ribosomal proteins L4 (rplD) and L22 (rplV), as described previously.³⁵ PCR products were purified using MagneSil Paramagnetic Particles (Biomek 3000; Beckman Coulter) and sequenced by ABI 3130xl Genetic Analyzer (Biotechnology Center).

The sequence of each gene was compared with sequences of genes from susceptible strain Staphylococcus aureus subsp. aureus NCTC 8325 (GenBank accession number NC_007795) for the detection of mutations.

spa, MLST, and SCCmec typing

Single-locus DNA sequencing of the repeat region of the Staphylococcus protein A gene (spa) was performed on all 100 isolates according to the protocol described on the European Network of Laboratories for sequence-based typing of microbial pathogens website (www.seqnet.org). spa types were assigned by the Ridom StaphType software (Ridom). The BURP (Based Upon Repeat Pattern) algorithm was used to define spa clonal complexes (spa-CC). Multilocus sequence types (ST) and clonal complexes (CC) were inferred from the spa type in the spa server database (http://spaserver. ridom.de/). SCCmec and MLST typing of the MRSA isolates was performed as described previously.^11,26

Virulence gene detection

The exfoliatin genes eta, etb, and etd, staphylococcal enterotoxin genes sea, sej, see, sec, seh, seb, seg, sed, and sei, toxic shock syndrome gene tst, and Panton-Valentine leukocidin (PVL) genes lukS-PV and lukF-PV were detected using PCR and multiplex PCR, as described previously.^24,28,38

SCV auxotrophy tests

SCV strains were screened for auxotrophy using standard 5-μg hemin disks (ITEST plus) and sterile blank disks impregnated with 15 μl of thymidine or menadione solution (Sigma-Aldrich) at a concentration of 100 μg/ml, as described previously.³ Isolates were plated in duplicate on MH agar (Oxoid), with disks with auxotrophy supplements placed on one plate and the other plate serving as a negative control. The isolates growing only in the presence of an auxotrophic supplement were classified as auxotrophic.

Statistical analyses

Two-tailed Fisher's exact test was used to analyze binary variables, whereas continuous scaled variables were evaluated with the nonparametric Kruskal–Wallis test. A p-value of ≤0.05 was considered to be statistically significant for all analyses. Statistical analyses were performed using STATISTICA software, version 12 (StatSoft).

Results

Patients

During the study period, the number of CF patients in the Czech Republic varied from 542 in 2011 to 586 in 2013 and more than half of these patients (325) were followed up at the Centre for CF of the Motol University Hospital (data derived from the Czech Cystic Fibrosis Patient Registry www.cfregistr.cz/). The prevalence of S. aureus in our CF patients could not be determined due to incomplete data, but S. aureus was recovered at least once from at least 124 (38.8%) of about 325 patients. In our study, we screened samples from 107 selected patients for the presence of S. aureus. From July 2011 to March 2013, we collected 100 S. aureus isolates from 92 of the 107 patients. SCV phenotype prevalence among S. aureus isolates was about 9% (n=9).

Susceptibility to antibiotics

The prevalence of resistance to oxacillin/cefoxitin (MRSA) was 3.26% among patients (n=3) and 3% among S. aureus isolates (n=3). The respective rates of resistance to most other antibiotics tested ranged from 1% to 12% (Table 1), with the exception of resistance to MLS_B antibiotics found in 55% of isolates. Of the MLS_B-resistant isolates, 48% showed constitutive resistance to macrolides and lincosamides (cMLS_B), 32% possessed inducible resistance (iMLS_B), and 20% were resistant to macrolides only (ERY).

Table 1.

Prevalence of Antibiotic Resistance and Small-Colony Variant Isolates

Resistance	MRSA	TMP-SXT	GEN	TET	OFX	RIF	Total MLS_B	iMLS_B	cMLS_B	ERY	SCV
No. of isolates	3	12	11	5	4	1	56	18	27	11	9
No. of pts (%)	3 (3.26)	11 (11.96)	11 (11.96)	4 (4.35)	4 (4.35)	1 (1.09)	51 (55.43)	17 (18.48)	24 (26.09)	10 (10.87)	9 (9.78)

cMLS_B, constitutive MLS_B resistance; ERY, erythromycin; GEN, gentamicin; iMLS_B, inducible MLS_B resistance; MLS_B, macrolide-lincosamide-streptogramin B; MRSA, methicillin-resistant Staphylococcus aureus; OFX, ofloxacin; RIF, rifampin; SCV, small-colony variant; TET, tetracycline; TMP-SXT, trimethoprim–sulfamethoxazole.

MLS_B-resistant S. aureus was recovered from all patients treated with long-term azithromycin and the association between MLS_B resistance and azithromycin treatment reached unambiguous statistical significance (p=0.000000184). All of the 22 azithromycin-treated patients harbored MLS_B-resistant strains.

Resistance determinants

All oxacillin- and cefoxitin-resistant strains (n=3) harbored the mecA gene. The presence of the aphA3, aac-aphD, and aadC genes either alone or in combination was identified as the cause of resistance to aminoglycosides in 10 isolates. One aminoglycoside-resistant isolate with the SCV phenotype did not possess any aminoglycoside resistance determinant. A possible explanation is decreased aminoglycoside susceptibility in SCV strains. Disruption of the respiratory chain in SCV strains is resulting in changes in the membrane potential and the consequent decreased transport of the aminoglycosides into the bacterial cell that eventually leads to the resistance to the aminoglycosides.³³

The ermA, ermC, ermT, and msrA genes as well as antibiotic target-site mutations—domain V of the 23S rRNA (rrl) and ribosomal protein L4 gene (rplD) mutations—were identified as MLS_B resistance determinants. In one isolate, the cause of resistance was not determined (Table 2). The mutations conferring MLS_B resistance were associated with azithromycin treatment (13 of 26 patients with such mutations were treated with azithromycin, p=0.000681). The erm and msrA genes were not associated with azithromycin treatment (p=0.1078).

Table 2.

Mechanism of MLS_B Resistance

Resistance mechanism	Profile	No. of isolates (%)	spa-types
Gene		26 (46.43%)
ermA		9 (16.07%)	t003, t209, t786, t5098, t8826, t13812
	iMLS_B	5
	cMLS_B	4
ermC		10 (17.86%)	t002, t084, t091, t095, t177, t548, t579, t13495
	iMLS_B	9
	cMLS_B	1
ermA+ermC	cMLS_B	1 (1.79%)	t2249
ermT	iMLS_B	2 (3.57%)	t571, t1451
msrA	ERY	4 (7.14%)	t024, t091, t398, t550
Target-site alteration		29 (51.79%)	t002, t012, t015, t024, t056, t065, t091, t095, t153, t164, t311, t386, t643, t831, t1365, t1510, t1671, t2275, t2449, t6906, t10146, t12062, t13497, t13498, t13503
23S RNA	cMLS_B	23 (41.07%)
L4	ERY	6 (10.71%)
Unknown	ERY	1 (1.79%)	t10146
Total of isolates		56

Small-colony variant

Eight of nine S. aureus isolates with the SCV phenotype appeared to be thymidine auxotrophic and one readily reverted to wild type after subculture. All isolates exhibited resistance to TMP-SXT. The SCV-positive patients at a median age of 17 years (range from four to 31 years) were older than the remaining patients, but the difference did not reach statistical significance (p=0.5361). No association was observed between SCV positivity and TMP-SXT treatment (p=0.079), sex (p=0.302), or coinfection with P. aeruginosa (p=0.4419), S. maltophilia (p=0.0723), or Bcc (p=0.470).

Typing

The spa typing revealed 65 spa types among 100 S. aureus isolates from CF patients (n=92). The most common spa types were t065 and t127, each detected in five isolates. The spa types were further divided using the BURP algorithm of the Ridom Staph Type software (Fig. 1) into 12 clusters (spa-CC) and 16 singletons (spa types not assigned to any of the clusters). Five isolates were excluded from the cluster analysis due to an insufficient number of repeats. For the spa-CC, where both spa types and ST were assigned, the respective MLST CC are also indicated. The largest cluster CC-t065 (MLST CC45) included 19 isolates from 18 patients, followed by CC-t002 (MLST CC5) comprising 10 isolates from 10 patients (Table 3).

FIG. 1.

BURP clustering of spa types. The spa typing data from S. aureus isolates were analyzed by the BURP algorithm (http://spaserver.ridom.de/, StaphType software v. 1.5, Ridom). Each spa type identified is depicted with a circle. The size of the circle is proportional to the frequency of the spa type in the population. Related spa types are connected with a black line. (F) or (SF) founder or subfounder of each clonal complex.

Table 3.

spa and MLST Clonal Complexes and Their Frequency

spa-CC	MLST CC (STs)	Origin	spa-types	Frequency% (no.)
CC-t065	CC45 (ST45,ST46)	HA	t015, t040, t065, t371, t505, t630, t831, t1365, t1510, t2275, t8826, t12062, t13498	19 (19)
CC-t002	CC5 (ST5, ST231)	HA	t002, t179, t311, t548, t579, t2249	10 (10)
CC-t084	CC15 (ST1, ST15, ST18, ST772)	CA	t084, t085, t346	5 (5)
CC-t024	CC8 (ST8, ST247, ST250, ST254)	CA	t008, t024, t9340	5 (5)
CC-t127	CC15 (ST1, ST3)	CA	t127, t2000	7 (7)
CC-t349	(ST101)	ND	t056, t349, t1671	5 (5)
CC-t122	CC30 (ST30, ST34, ST1472)	CA	t012, t019, t122	5 (5)
CC-t148	CC8 (ST8, ST72)	CA	t148, t3682, t13495	4 (4)
CC-t091/13503	CC7 (ST7, ST1867)	CA	t091, t13503	5 (5)
CC-t095/550	CC45 (ST45)	HA	t095, t550	4 (4)
CC-t571/1451	CC398 (ST398)	LA	t571, t1451	2 (2)
CC-t160/156	(ST12, ST13)	ND	t156, t160	2 (2)
Singletons	CC5 (ST225)	HA	t003	3	22 (22)
	(ST20)	ND	t164	2
	(ST97)	ND	t224	1
	(ST109)	ND	t209	2
	CC45(ST45)	HA	t10146	14
	ND	ND	t786, t2449, t5098, t6155, t6906, t153, t13497, t13500, t13502, t13505, t13812
Excluded	CC15 (ST1)	CA	t386	2	5 (5)
	ND	ND	t398, t586, t643	3

spa type of the MRSA strains is underlined.

CA, community-associated strains of S. aureus; HA, healthcare-associated strains of S. aureus; LA–livestock-associated strains of S. aureus, ND, not determined.

MLST and SCCmec typing was performed on all MRSA isolates (n=3). All of them showed identical ST225 and identical SCCmec type II, respectively. Because of their shared genetic background (spa type t003, SCCmec type II and MLST type ST225), all MRSA strains were assigned to the widespread clonal complex CC5. Detected strain ST225-MRSA-II is a single-locus variant of ST5-MRSA-II known also as UK-EMRSA-3, New York-Japan Clone, and Rhine-Hesse Epidemic Strain.²⁹

Virulence genes

The most prevalent virulence genes detected in the study isolates were the staphylococcal enterotoxin genes seg (n=52), sei (n=49), and sec (n=16). The staphylococcal enterotoxin genes sea (n=8), seb (n=1), sed (n=6), seh (n=10), and sej (n=5), exfoliatin genes eta (n=2) and etd (n=3), and toxic shock syndrome toxin gene tst (n=1) were found sporadically in one to 10 isolates. The PVL genes (lukS-PV and lukF-PV), exfoliatin B gene (etb), or staphylococcal enterotoxin E gene (see) were not detected in any of the isolates.

Discussion

This is the first detailed study of S. aureus isolates from CF patients to be conducted in the Czech Republic. The study objective was to analyze the population structure of S. aureus isolates from CF patients and to investigate in detail antibiotic resistance and virulence of these isolates, with emphasis on the study of the MLS_B resistance mechanisms and occurrence of SCVs.

S. aureus is among the most commonly isolated bacterial pathogens in CF patients, with a prevalence rate ranging between 30 and 70% across Europe.^3,20,21,43 S. aureus isolates from CF patients included MRSA and SCV strains known to be associated with worse lung disease in CF patients.^5,42 Increasing prevalence of MRSA in some US CF care centers is an emerging challenge in CF.¹⁸ In comparison with the US data, the MRSA prevalence of 3.26% (three patients) in our study cohort was relatively low. All MRSA-positive patients were infected with the ST225-MRSA-II strain (t003/ST225/SCCmecII) assigned to the globally widespread hospital-acquired MRSA (HA-MRSA) CC5.⁴⁰

The prevalence of SCV strains in S. aureus-positive CF patients has been reported to range between 17% and 52%.^3,20,43 In our study cohort, the respective rate was 9.78% only (nine patients). However, the study design with the prospective collection of single-patient isolates only can be responsible for a slight underestimation of the overall prevalence of SCV strains. In six additional patients, SCV strains were detected in samples collected outside the time frame of this study. When taking into account these six patients, the SCV prevalence reaches 16.3%, which does not differ significantly from the data reported by other countries. Most SCV isolates (eight of nine) were recovered along with the normal colony variant (NCV) strain, isogenic in six cases, from the same sample. Prophylactic TMP-SXT, older age, and P. aeruginosa coinfection have been identified previously as the factors associated with SCV isolation.^3,27 In total, 55 patients were treated with TMP-SXT (from 10 days to continuous treatment, average duration of 26 days) and only eight of them harbored SCV strains; thus, correlation between TMP-SXT and SCV formation did not reach statistical significance (p=0.079). However, our findings are not in conflict with previous studies that confirmed an association between long-term prophylactic TMP-SXT treatment and the presence of SCV.^3,20 Eight of our nine patients with SCVs were treated with TMP-SXT, but none of them prophylactically. Since the SCV formation is caused by mutation²² and the mutations are stochastical processes, it is not surprising that without strong selective pressure of continuous prophylactic TMP-SXT treatment SCV appears only in a fraction of patients occasionally treated with TMP-SXT. P. aeruginosa coinfection was only detected in one patient. The mean age was higher in SCV patients than in non-SCV patients, but the difference did not reach statistical significance. These discrepancies from the data reported by others can be explained by the small number of SCV-positive patients as well as by long-term persistence of S. aureus along with SCV strains as a possible persistence mechanism.¹⁶ The factors associated with the emergence of the SCV phenotype may have played a role outside the time frame delimited for this study.

From the perspective of the population structure, the study collection was heterogeneous and consisted of six major clones of methicillin-sensitive S. aureus (MSSA) with hospital-acquired (HA)-MSSA (33 strains) represented by CC5 and CC45 and community-associated (CA)-MSSA (35 strains) represented by CC7, CC8, CC15, and CC30.^6,14,37 The proportion of the CA and HA strains is likely to reflect the probability of S. aureus transmission to CF patients from the community and hospital environment.

Increased prevalence of resistance to MLS_B antibiotics in S. aureus isolates from CF patients treated with azithromycin has been reported by several studies.^15,32 Nevertheless, the MLS_B resistance mechanisms in CF isolates have been studied insufficiently. The most frequent of the MLS_B resistance mechanisms are MLS_B target-site alteration due to erm methylase action or msrA efflux pump.²³ Prunier et al.^34,35 have also reported MLS_B target-site mutations in the ribosomal genes rrl (23S rRNA), rplD (L4 protein), and rplV (L22 protein) as the cause of resistance in CF isolates. Isolates in their study were from CF patients treated with azithromycin.³⁵ A limitation of both studies is a relatively small sample of CF patients (five and nine patients, respectively) from whom the isolates were collected and, moreover, neither of them has reported the prevalence of these mutations among the S. aureus isolates from CF patients. In our study, we also found a high prevalence of MLS_B resistance (56%), with nearly half of the isolates carrying a resistance gene from the erm methylase group or msrA efflux pump. A greater half of MLS_B-resistant isolates (52%) had a mutation in the gene encoding 23S rRNA or ribosomal protein L4. A relevant finding is the association between azithromycin treatment and MLS_B resistance and mutations conferring this resistance.

PVL-positive S. aureus has recently been reported as a possible severe pathogen in CF patients.² In our study, the PVL genes have not been detected in any CF isolate, but a large proportion of isolates carried enterotoxin genes, a smaller proportion of isolates possessed exfoliatin genes, and one isolate harbored gene for toxic shock syndrome toxin (tst). However, no study of the impact of these virulence factors on the severity of CF has been conducted to date.

In conclusion, this is the first report to provide information about S. aureus isolates from Czech patients with CF, that is, about their clonal structure, distribution of resistance mechanisms, virulence genes, and the presence of SCV. High prevalence of MLS_B resistance emerged as a major finding. Most significantly, the connection between long-term prophylactic use of azithromycin and increase in MLS_B resistance in S. aureus was described, in agreement with previous studies. Nevertheless, we were the first to observe mutation of the MLS_B target site as a major resistance mechanism selected by azithromycin treatment. Further studies are needed to characterize the evolution of antibiotic resistance during antibiotic treatment with respect to SCV formation and mutation of the antibiotic target sites under specific circumstances as crucial mechanisms.

Footnotes

Acknowledgment

Supported by grant No. NT12395-5/2011 from the Internal Grant Agency (IGA) of the Ministry of Health of the Czech Republic.

Disclosure Statement

No competing financial interests exist.

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Characterization of Staphylococcus aureus Strains Isolated from Czech Cystic Fibrosis Patients: High Rate of Ribosomal Mutation Conferring Resistance to MLS B Antibiotics as a Result of Long-Term and Low-Dose Azithromycin Treatment

Abstract

Introduction

Methods

Patients and isolates

Susceptibility testing

Polymerase chain reaction detection of MLSB, aminoglycoside, and methicillin resistance genes

Detection of mutations conferring resistance to MLSB

spa, MLST, and SCCmec typing

Virulence gene detection

SCV auxotrophy tests

Statistical analyses

Results

Patients

Susceptibility to antibiotics

Resistance determinants

Small-colony variant

Typing

Virulence genes

Discussion

Footnotes

Acknowledgment

Disclosure Statement

References

Polymerase chain reaction detection of MLS_B, aminoglycoside, and methicillin resistance genes

Detection of mutations conferring resistance to MLS_B