Fusidic Acid-resistant Isolates Research Articles

First Report of the Emergence of mecC Gene and CC8/ST239 Tigecycline-Resistant Staphylococcus aureus Clonal Lineage Isolated from Chronic Suppurative Otitis Media

Background: Staphylococcus aureus is one of the most significant bacteria involved in ear infections. However, insights into the molecular attributes of S. aureus collected from patients with chronic otitis media have yet to be reported in Iran. Objectives: The objective of this study was to assess the molecular characteristics of S. aureus isolated from patients with chronic otitis media. Methods: A total of 55 S. aureus strains retrieved from patients with chronic otitis media were analyzed by the disk diffusion method and polymerase chain reaction (PCR) to identify the nucA gene. Isolates were genetically classified using the coagulase typing method. S. aureus protein A (spa) typing, staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing (MLST) were performed on isolates with resistance to specific antibiotics. Results: Overall, out of 55 S. aureus isolates, resistance to mupirocin, fusidic acid, and tigecycline was identified in 12.7%, 5.4%, and 3.6% of isolates, respectively. Fusidic acid-resistant isolates belonged to ST5-SCCmecII/t002/coaII. Two tigecycline-resistant isolates belonged to CC8/ST239-SCCmecIII/t234/coaVIII. One positive mecC isolate belonged to the CC/ST130-SCCmecXI/t843/coaIII clone. Isolates with the iMLSB phenotype belonged to CC/ST80-SCCmecIV/t044/coaII (4 isolates), CC8/ST239-SCCmecIII/t388/coaVI (3 isolates), and CC8/ST8-SCCmecIV/t008/coaIII (1 isolate). Conclusions: Our results indicated that S. aureus isolated from patients with chronic otitis media possesses a unique molecular profile with a high percentage of resistance to multiple medications. These findings suggest that resuming the molecular analysis to improve the control and prevention of ear infections related to S. aureus is necessary.

Prevalence and genetic characteristics of fusidic acid resistant Staphylococcus aureus clinical isolates: Emergence of t030 strains carrying fusB in Tehran, Iran.

The literature on fusidic acid resistant Staphylococcus aureus strains is scarce in Iran, although the emergence of these strains in health care settings is increasing. This descriptive cross-sectional study was conducted on 68 fusidic acid resistant S. aureus strains to learn about the molecular characteristics and antimicrobial resistance of strains isolated from hospitalized patients. In the present study, the prevalence of resistance to fusidic acid in S. aureus isolates was 15.1%. Fusidic acid resistance determinative factors (fusB, fusC and fusD) were identified by multiplex PCR assay. To detect the existence of fusA and fusE determinants and their mutation status, amplifications and sequencing were performed. Molecular characterization of fusidic acid resistant isolates was investigated by SCCmec and spa typing methods. All strains were MRSA and multi drug resistant. Two (2.9%) and 31 (45.6%) isolates were resistant to vancomycin and mupirocin respectively. The SCCmec type IV was highly prevalent representing 50% followed by types III (51.5%), and SCCmec types II (13.2%). fusB, was the most predominant acquired gene (66.2%) followed by fusC (19.1%), and fusA (14.7%). The mutations in fusA were present in 10 isolates with 5 (50%) having L461K mutation showing fusidic acid MIC values of ≥256 μg ml-1 followed by H457Y (40%), and H457Q (10%) showing fusidic acid MIC values of 128 and 64 μg ml-1 respectively. Isolates were allocated to ten particular t030 (22.1%), t037 (14.6%), t408 (11.8%), t064 (11.8%), t008 (10.3%), t002 (8.8%), t005 (5.9%), t790 (5.9%), t318 (4.4%), and t018 (4.4%) spa types. fusA positive isolates were assigned to particular spa types t002 (60%), and t005 (40%). There may be be a spreading of fusidic acid resistance among MRSA, creating worrying public concern. This research notes the importance of adequate data of local prevalence of FA-resistant MRSA in Iran for taking appropriate measures to treat, control and reduce the incidence of these isolates.

Profiles of Non-aureus Staphylococci in Retail Pork and Slaughterhouse Carcasses: Prevalence, Antimicrobial Resistance, and Genetic Determinant of Fusidic Acid Resistance.

As commensal colonizers in livestock, there has been little attention on staphylococci, especially non-aureus staphylococci (NAS), contaminating meat production chain. To assess prevalence of staphylococci in retail pork and slaughterhouse carcass samples in Korea, we collected 578 samples from Korean slaughterhouses (n=311) and retail markets (n=267) for isolation of staphylococci and determined antimicrobial resistance phenotypes in all the isolates. The presence of and prevalence of fusB-family genes (fusB, fusC, fusD, and fusF) and mutations in fusA genes were examined in fusidic acid resistant isolates. A total of 47 staphylococcal isolates of 4 different species (Staphylococcus aureus, n=4; S. hyicus, n=1; S. epidermidis, n=10; Mammaliicoccus sciuri, n=32) were isolated. Fusidic acid resistance were confirmed in 9/10 S. epidermidis and all of the 32 M. sciuri (previously S. sciuri) isolates. Acquired fusidic acid resistance genes were detected in all the resistant strains; fusB and fusC in S. epidermidis and fusB/C in M. sciuri. Multi-locus sequence type analysis revealed that ST63 (n=10, 31%) and ST30 (n=8, 25%) genotypes were most prevalent among fusidic acid resistant M. sciuri isolates. In conclusion, the high prevalence of fusB-family genes in S. epidermidis and M. sciuri strains isolated from pork indicated that NAS might act as a reservoir for fusidic acid resistance gene transmissions in pork production chains.

Mupirocin-Resistant Staphylococcus aureus in Iran: A Biofilm Production and Genetic Characteristics.

The spread of mupirocin-resistant Staphylococcus aureus strains in hospitals and communities is a universal challenge. Limited data is available on the genetic features of high-level mupirocin resistant- (HLMUPR-) S. aureus isolates in Tehran. In the present research, we investigated 48 high-level mupirocin resistance S. aureus by antimicrobial activity, virulence analysis, biofilm formation, multilocus sequence typing (MLST), and staphylocoagulase (SC) typing. All the HLMUPR strains were positive for mupA gene. The frequency of multidrug resistance was 97.9%. Twenty-one (43.8%) were toxinogenic with 14 producing pvl (29.2%), 5 tst (10.4%), and two eta (4.2%). Among the HLMUPR isolates, biofilm production was detected in 45 (89.6%) isolates with complete dominance clfB, clfA genes, and a noticeably high frequency fnbA (95.8%), followed by fnbB (93.8%), eno and icaD (each 83.3%), sdrC (81.3%), ebps (79.2%), icaA (75%), sdrD (66.7%), fib (60.4%), sdrE (50%), cna (41.7%), and bap (4.2%). Coagulase typing distinguished isolates into four genotypic patterns including III (50%), II (27.1%), and type IVa (22.9%). A total of three clonal complexes (CCs) and 4 sequence types (STs) including CC/ST22 as the most prevalent (52.1%), CC8/ST239 (20.8%), CC/ST8 (16.7%), and CC/ST5 (10.4%) were identified in current work. According to our analysis, nonbiofilm producer isolates belonged to CC8/ST239 (6.3%) and CC/ST8 (4.2%). Fusidic acid-resistant isolates belonged to CC/ST45 (n = 3) and CC8/ST239 (n = 1). Observations highlighted the circulation of the CC/ST22 HLMUPR S. aureus strains with strong biofilm-production ability in our hospitals, indicating the possibility of transmission of this type between community and hospital.

A Detailed Study of Antimicrobial Sensitivity Pattern of Penton Valentine Leucocidin Gene Positive and Negative Staphylococcus aureus from Pus Samples

Introduction: Staphylococcus aureus harboring Panton Valentine Leucocidin gene are emerging and spreading worldwide. PVL gene was first identified by Noel Panton and Francis Valentine in 1932 who IC Pakistan only limited data is available regarding the effect of PVL gene on sensitivity pattern of Staphylococcus aureus. Therefore, this study was conducted to understand the antimicrobial sensitivity pattern of both PVL positive and negative Staphylococcus aureus isolates. Aims & Objectives: This study was conducted to understand the antimicrobial sensitivity pattern of both PVL positive and PVL negative Staphylococcus aureus isolated from pus samples received from various indoor and outdoor departments of a tertiary care hospital of Lahore. Place and duration of study: Microbiology and Molecular Biology Laboratory Shaikh Zayed Hospital Lahore. Duration of study is one year after the approval of research topic. Material & Methods: A total of 384 Staphylococcus aureus isolates from skin and soft tissue infections were identified and selected. Their antimicrobial sensitivity testing was done by Kirby disc diffusion method using Muller Hinton agar. Results: Frequencies of PVL gene in MRSA and MSSA were 51% and 44% respectively. Frequency of PVL gene was also found to be high in Ciprofloxacin sensitive, Gentamicin sensitive, Erythromycin resistant and fusidic acid resistant isolates. Conclusion: Almost half of Staphylococcus aureus isolates were found PVL positive. They were mostly multidrug resistant. The PVL positive Staphylococcus aureus isolates showed high resistance against antibiotics than PVL negative isolates

Identification of fusidic acid resistance in clinical isolates of Staphylococcus pseudintermedius from dogs in Korea.

Staphylococcus pseudintermedius is a major bacterial species associated with canine pyoderma and otitis. Fusidic acid is used to treat skin infections caused by Gram-positive bacteria. The incidence of resistance to fusidic acid in S.pseudintermedius has importance in terms of limiting treatment options for bacterial infections. To evaluate the occurrence and mechanisms of fusidic acid resistance in clinical isolates of S.pseudintermedius. Fifty-two S.pseudintermedius isolates were collected from dogs with pyoderma (n=36) or otitis (n=16). The disk diffusion method determined that isolates <24mm were resistant to fusidic acid. Minimum inhibitory concentrations (MICs) were measured by the E-test in those with confirmed resistance to fusidic acid by the disk diffusion method. Phenotypically fusidic acid resistant isolates were subjected to PCR to detect the presence of resistance-related genes (fusA, fusB, fusC and fusD) and fusA was further sequenced to identify point mutations. Fourteen of 52 (27%) S.pseudintermedius isolates were resistant to fusidic acid and all of these showed low-level resistance. Among fusidic acid resistant isolates, fusA point mutations were confirmed in 11 isolates and amino acid substitutions were found in five. fusC was detected in seven isolates, but neither fusB nor fusD was detected. This study demonstrates the occurrence and resistance mechanisms to fusidic acid in clinical isolates of S.pseudintermedius. Continuous monitoring for fusidic acid resistance is recommended.

Spread of the epidemic European fusidic acid-resistant impetigo clone (EEFIC) in general practice patients in the south of The Netherlands

We evaluated the susceptibility to fusidic acid, mupirocin and retapamulin of Staphylococcus aureus isolated from nasal and wound swabs. The susceptibility to the three agents of S. aureus isolated from general patients in the south of The Netherlands with a skin or soft tissue infection was determined between January 2007 and December 2008. Fusidic acid-resistant isolates were tested for the presence of fusidic acid-resistant genes and compared with the epidemic European fusidic acid-resistant impetigo clone (EEFIC). Fusidic acid resistance was found in 23% of the nasal and 35% of the wound isolates, the majority (~90%) being fusB positive. Most of the isolates belonged to spa type t171 and were isolated from younger patients. One isolate was retapamulin resistant (MIC 8 mg/L) and two were mupirocin resistant. The EEFIC clone was relatively highly prevalent among the isolated S. aureus. The usefulness of fusidic acid as first-line agent for the treatment of impetigo is questionable. As mupirocin is used in The Netherlands for eradication of methicillin-resistant S. aureus, it is not an alternative; retapamulin might be useful, but further in vivo studies are warranted.

Occurrence and molecular characterization of fusidic acid resistance mechanisms among Staphylococcus spp. from European countries (2008)

To determine fusidic acid resistance rates (MICs of > or = 2 mg/L) and the prevalence of fusidic acid resistance mechanisms among Staphylococcus spp. collected from European countries (2008). Staphylococcal isolates (3134) collected from Europe were tested by CLSI broth microdilution. Isolates displaying a fusidic acid MIC of > or = 2 mg/L (non-susceptible; European Committee on Antimicrobial Susceptibility Testing breakpoint) were tested for fusB, fusC and fusD. The fusidic acid target sites fusA and fusE were then sequenced, and a method for the detection of the fusA mutation L461K was developed. Selected isolates were typed by PFGE. Fusidic acid resistance rates were higher among coagulase-negative staphylococci (CoNS) compared with Staphylococcus aureus. Acquired fusidic acid resistance genes were detected in 64.9% of the samples; fusB and fusC were detected among 10.1% and 16.9% of the fusidic acid-resistant S. aureus and among 26.5% and 11.3% of the CoNS, respectively. Ireland and Greece showed the highest S. aureus fusidic acid resistance levels, with low rates of acquired fusidic acid resistance genes. Isolates from these countries displayed MIC values of > or = 512 mg/L, the presence of the elongation factor G L461K alteration and clonal occurrences. Low S. aureus fusidic acid resistance rates (1%-3%) were observed in Germany, Israel, Italy, Poland, Spain and Sweden. Isolates with MIC values < or = 64 mg/L showed a great diversity of acquired fusidic acid resistance mechanisms. Acquired fusidic acid resistance genes were detected in the majority of fusidic acid-resistant isolates from Belgium, France, Switzerland, Sweden, Italy and the UK (72.2%-92.9%), and were slightly less frequent in Germany, Spain and Israel (61.3%-66.7%). This contemporary study showed that acquired fusidic acid resistance genes were prevalent among fusidic acid-non-susceptible European staphylococcal isolates.

Antimicrobial Resistance inStaphylococcus pseudintermediusin the Norwegian Dog Population

The occurrence of antimicrobial resistance in Staphylococcus pseudintermedius from skin and ear infections in a representative sample of dogs unexposed to antimicrobial treatment before sampling was examined. The obtained isolates were further examined for genetic polymorphism and genetic background of resistance. A total of 59 isolates of S. pseudintermedius originating from 96 samples of 91 dogs in five different regions in Norway were included in this study. Susceptibility testing was performed using a broth dilution method. Resistant isolates were subjected to polymerase chain reaction for detection of resistance genes. All isolates were subjected to pulsed-field gel electrophoresis to examine the genetic polymorphism. In total, 19% of the isolates were susceptible to all antimicrobial agents included. Resistance to penicillin was most prevalent (70%), followed by resistance to fusidic acid (49%) and oxytetracycline (42%). Resistance to quinolones or cephalosporins was not observed. Resistance to penicillin, tetracycline, and erythromycin was mediated by the blaZ beta-lactamase gene, the tetM gene, and the ermB gene, respectively. One of the fusidic acid-resistant isolates harbored a fusC gene, whereas the mechanisms involved in resistance in the other fusidic acid-resistant isolates remained unknown. Pulsed-field gel electrophoresis showed a high genetic polymorphism of S. pseudintermedius. This study indicates that the occurrence of antimicrobial resistance is common among S. pseudintermedius from dogs unexposed to antimicrobial treatment before sampling, and that there is a high genetic polymorphism among S. pseudintermedius.

Presence of the epidemic European fusidic acid-resistant impetigo clone (EEFIC) of Staphylococcus aureus in France

Sir, Denton et al. recently reported the results of the EPISA study, which compared the susceptibility of Staphylococcus aureus causing community-acquired skin and soft tissue infections (SSTIs) in three European countries. They highlighted in particular that the resistance rate to fusidic acid was significantly lower in French (6.3%) than in Irish (31.4%) or British (24.4%) isolates. This difference was attributed by the authors to the likely spread of a fusidic acid-resistant clone, named the epidemic European fusidic acid-resistant impetigo clone (EEFIC) in the latter two countries. This clone, initially described in Scandinavia, had so far been reported sporadically in the UK. It is characterized by: (i) agr allelic group 4, spa type t171 [or single-locus variants (t408, t659, t874, t875)], sequence type (ST) 123, clonal complex 121; (ii) the presence of the eta (exfoliative toxin A) gene with the majority of strains also carrying the etb (exfoliative toxin B) gene; (iii) resistance to fusidic acid (MIC of 2–8 mg/L) related to the expression of the fusB gene; and (iv) strong association with impetigo. In the same issue, Larsen et al. reported the molecular characterization of all these fusidic acid-resistant isolates (n 1⁄4 136) collected during the EPISA study. The results confirmed that the EEFIC represented 63% (34/54) of fusidic acid-resistant strains in the UK and 49% (34/69) in Ireland, but was not detected in France (0%, 0/13). These results seemed to indicate that the EEFIC was not present in France. We were puzzled by this unexpected and surprising French epidemiology and decided to screen a part of the collection of the French national reference centre for staphylococci (Lyon, France) for the presence of the EEFIC. First, between December 2003 and August 2004, corresponding to the appropriate period of the EPISA study, we identified 19 isolates harbouring agr allelic group 4 and the eta gene, characteristics matching the EEFIC. We then performed a fusidic acid susceptibility test using disc diffusion (using the breakpoint from the BSAC) and fusB PCR on these isolates. Four strains (21%, 4/19) appeared to be resistant and PCR-positive, strongly suggesting that they belonged to the EEFIC. Second, we applied the same screening to all S. aureus isolates collected from October 2000 to November 2003 (before the EPISA study) and from September 2004 to December 2007 (after the EPISA study). The agr allelic group 4 and eta positive strains represented a total of 256 isolates. Forty-four of these isolates were randomly selected to undergo the fusidic acid susceptibility test and fusB PCR. Six isolates (14%, 6/44) were detected as resistant to fusidic acid and positive for fusB PCR. Finally, the fusidic acid MICs (determined by Etest) and spa types were determined for the 10 isolates thus screened. All revealed MICs between 4 and 8 mg/L and belonged to spa type t171 or a related spa type (t171, n 1⁄4 8; t408, n 1⁄4 2) corresponding to ST123, which definitively confirmed that they belonged to the EEFIC. The 10 isolates were collected from eight different hospitals and over a long period (2000–07), confirming the geographical and temporal dissemination of the EEFIC in France. As expected, the clinical data for the 10 patients indicated that impetigo or its generalized form were the motives for consultation. Even if we are not presently able to establish the proportion of the EEFIC within fusidic acid-resistant S. aureus in our collection, because of the design of our screening procedure, these data definitively demonstrated that the EEFIC is present in France and likely represents 15% to 20% of all French S. aureus isolates harbouring exfoliative toxin A. In the EPISA study, the low rate of impetigo in the French patients (12.2%) compared with British patients (33.3%) could partly explain the underrepresentation of the EEFIC in the French data. The low number in the series is probably due to the French practice of consulting a specialist (paediatrician or dermatologist) or hospital emergency wards for impetigo, rather than a general practitioner as would tend to be the case in the UK and Ireland. So, the fact that the EPISA inclusion criterion was ‘patient attending a general practice clinic in the community with SSTIs’, probably introduced a recruitment bias in the French isolates implicated in impetigo and more generally in severe SSTIs. Conversely, the French national reference centre for staphylococci collects S. aureus strains of clinical interest with recruitment from hospitals as well as non-hospital community laboratories, thereby avoiding the bias found in the EPISA study and allowing us to detect the presence of the EEFIC in France in the 2000–07 period.

High prevalence of resistance to fusidic acid in clinical isolates of Staphylococcus epidermidis

To determine the prevalence and mechanisms of resistance to fusidic acid in clinical isolates of Staphylococcus epidermidis. MICs of fusidic acid were determined for S. epidermidis isolates collected from the Leeds General Infirmary and from around Europe. Fusidic acid-resistant isolates were probed for the presence of the horizontally acquired resistance determinants fusB and fusC by a novel multiplex PCR assay. Mutations in the gene encoding the drug target (fusA) were detected by PCR and DNA sequencing. Resistant isolates were subjected to typing using the repeat region of the aap gene. Of 50 S. epidermidis isolates screened, 23 (46%) exhibited resistance to fusidic acid. The most common resistance determinant was fusB, found in 18 of the 23 isolates. Of the remaining isolates, two harboured fusC and three carried an identical mutation in fusA, leading to the substitution L(461)K in the target protein, elongation factor G. Molecular typing showed that this collection of isolates was genetically diverse. This study suggests a high prevalence of resistance to fusidic acid in clinical isolates of S. epidermidis. As in Staphylococcus aureus, resistance to fusidic acid in S. epidermidis is commonly associated with the fusB determinant.

Compensatory Adaptation to the Loss of Biological Fitness Associated with Acquisition of Fusidic Acid Resistance in Staphylococcus aureus

Recent studies have shown that individual amino acid exchanges within elongation factor G (EF-G) cause fusidic acid resistance in Staphylococcus aureus. The data from the present study illustrate that the fusidic acid resistance-mediating amino acid substitutions P406L and H457Y are associated with a marked impairment of the biological fitness of S. aureus. In particular, strains producing EF-G derivatives with these mutations showed reduced growth, decreased plasma coagulase activity, and an impaired capability to compete with the isogenic wild-type strain. Second-site mutations within EF-G, such as A67T and S416F, that have been encountered in clinical fusidic acid-resistant isolates containing the amino acid exchanges P406L and H457Y, respectively, were shown not to contribute to resistance. Furthermore, the substitution A67T had no impact on the biological fitness in vitro. The exchange S416F, however, was found to function as a fitness-compensating mutation in S. aureus carrying the substitution H457Y in EF-G. In conclusion, the data presented in this report provide evidence at the molecular level that the deleterious effects of fusidic acid resistance-mediating exchanges within EF-G of S. aureus can be reduced considerably by specific compensating mutations in this target protein. This compensatory adaptation most likely plays a significant role in the stabilization of resistant bacteria within a given population.

Genetic characterization of the fusidic acid and cadmium resistance determinants of Staphylococcus aureus plasmid pUB101.

We report the cloning of the fusidic acid and cadmium resistance determinants from Staphylococcus aureus plasmid pUB101. The pUB101 fusidic acid resistance determinant was located on a 2.9 kb HindIII fragment. Sequencing of this fragment revealed three putative open reading frames (ORFs) of 213 (far1), 152 (orf152) and 170 amino acids (orf170), which are flanked by the right-hand end of insertion sequence IS431/257 (IS431/257RH) and a partial ORF. Far1 and Orf152 demonstrated homology with a chromosomally encoded fibronectin-binding protein of Listeria monocytogenes and the putative protein YosT, found on the SP beta c2 prophage of Bacillus subtilis, respectively. Transformation of S. aureus with a construct containing a 949 bp far1-specific amplicon led to the isolation of a fusidic acid-resistant transformant, thereby identifying the pUB101 fusidic acid resistance structural gene. Between orf152 and far1 we identified a unique 113 bp symmetrical element and other repeat elements that may be involved with the control of orf152 and/or far1 expression. Hybridization of Southern blots revealed that far1 was not located on the chromosome or plasmid content of a limited number of Australian, UK and Hong Kong fusidic acid-resistant isolates. The pUB101 cadmium resistance determinant was located on a 3.6 kb HindIII fragment that carried a cadDX operon, remnants of two putative plasmid replication protein genes and IS431/257RH. Sequence analysis also demonstrated the presence of a single-stranded origin of replication, normally found on rolling circle replicating plasmids, within the putative promoter region of the cadDX operon.

Disk diffusion interpretive criteria for fusidic acid susceptibility testing of Staphylococci by the National Committee for Clinical Laboratory Standards method

Fusidic acid is used in many countries for the treatment of multiresistant staphylococcal infection, especially multiresistant Staphylococcus aureus infection (MRSA). We collected consecutive fusidic acid-resistant isolates of staphylococci from the routine laboratory over several years, and compared these strains with fusidic acid-susceptible staphylococci to establish interpretive criteria for disk diffusion testing by National Committee for Clinical Laboratory Standards (NCCLS) methods. The minimum inhibitory concentrations (MICs) and zone diameters for strains of S. aureus ( n = 102), including MRSA, S. saprophyticus ( n = 20) and other coagulase-negative staphylococci ( n = 115) were determined by NCCLS agar dilution and disk diffusion tests using a 2.5-μg disk of fusidic acid. MICs were bimodally distributed. No isolates had MICs of 0.5 or 1 μg/ml; thus, we chose these values to define strains of intermediate susceptibility. The error-rate-bounded method was used to determine interpretive zone diameters for disk testing. Interpretive zone diameter criteria were found to be: susceptible ⩾22 mm, intermediate 18–21 mm, and resistant ⩽17 mm. All S. saprophyticus were intrinsically resistant to fusidic acid (MIC ⩾2 μg/ml).