mupA Gene Research Articles

Molecular diversity in fusidic acid-resistant Methicillin Susceptible Staphylococcus aureus.

The recent emergence of fusidic acid (FA)-resistant Staphylococcus aureus has underscored the importance of active surveillance in isolating these strains. The molecular basis of fusidic acid resistance and the carriage of virulence factors in four borderline oxacillin-resistant Staphylococcus aureus (BORSA) clinical strains was assessed through phenotypical and genotypical methods. All S. aureus clinical strains were obtained from various hospital units in Sicily. In vitro antibiotic susceptibility testing was conducted. WGS was performed using the Illumina MiSeq Platform, and data analysis was carried out to determine ST, resistome and virulome profiles. Genotypic characterization revealed that the strains belong to four STs: ST630, ST8, ST15, and ST1. FA resistance was associated with mutations in the fusA gene or fusB and fusC genes. Additionally, one case exhibited resistance to mupirocin, related to the presence of the mupA gene. Borderline MIC values were observed for cefoxitin in three out of four cases, leading to their categorization as BORSA. Virulence gene content was complex and diversified, with one testing positive for the lukS/F genes, coding for PVL toxin. Resistance to FA is multifactorial, involving point mutations in chromosomal genes or association with mobile genetic elements. Monitoring the resistance to these antibiotics might help to manage and eradicate mupirocin- and FA-resistant S. aureus strains, which are also known to be important carriers of virulence determinants.

Assessing Mupirocin Resistance in MRSA Isolates in Hospitals in Cleveland, OH and Detroit, MI

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen responsible for nosocomial and community-acquired infections with high morbidity and mortality1. MRSA nasal colonization is a major risk factor for developing infection in the hospital setting2,3. Decolonization of MRSA carriers is a strategy to decrease recurrence or to prevent new MRSA infections3,4. Decolonization with nasal mupirocin 2% and chlorhexidine baths has been shown to decrease the risk of MRSA infection after hospital discharge3. Mupirocin is an RNA synthetase inhibitor with activity against MRSA5. Resistance of MRSA isolates to mupirocin has been described previously6. As topical disinfectants play a crucial role in prevention of MRSA infection in a variety of settings, it is important to monitor the emergence of resistance. The goal of this study was to determine the prevalence of mupirocin resistance among MRSA samples isolated from two different regions in the United States (U.S). Methods: Our study had a total of 474 MRSA samples that were obtained from hospitals in Detroit, MI (287 samples) and Cleveland, OH (187 samples). After whole genome sequencing using NextSeq (Illumina Inc., CA) platform the data was analyzed using ResFinder 4.1, to identify antimicrobial resistance which can be either acquired or chromosomally mediated mutations. To visualize the presence of genes of interest the resistance genes were tallied on a spread sheet. Results: Mupirocin resistance gene was detected in five of 287 (1.74%) MRSA samples from the Detroit hospitals, all of which were associated with the mupA gene. Samples collected from the Cleveland area hospital demonstrated mupirocin resistance in seven samples of 187 (3.74%), all associated again with the mupA gene. One sample from the Detroit group showed resistance to both mupirocin and chlorhexidine. Conclusions: Prevalence of mupirocin resistance gene varied between the two hospital locations. Resistance to mupirocin has been documented in association with mutations in the mupA gene as well as chromosomal point mutations that can lead to either low or high-level resistance7,8. Although the mechanisms are not fully clear, mupA gene has been associated with high-level resistance9. Mupirocin resistance among MRSA isolates has increased over time9. MRSA infections remain an important etiology of nosocomial and community-acquired infections and common practice to combat this issue is universal decolonization with mupirocin10. It is critical to understand and monitor for development of mupirocin resistance as mupirocin remains one of the most effective tools to prevent invasive infection with MRSA in many patient populations.

Investigation and control of an outbreak of methicillin-susceptible Staphylococcus aureus skin and soft tissue infections in a level IV NICU

Background: Neonatal intensive care units (NICU) outbreaks caused by methicillin-susceptible Staphylococcus aureus (MSSA) are less commonly reported than outbreaks caused by methicillin-resistant S. aureus. We report an unusual outbreak of MSSA skin and soft tissue infections (SSTIs) in a level IV NICU investigated by whole genome sequencing (WGS) and molecular typing. Methods: An investigation was initiated in a 56-single-bed NICU after four patients developed MSSA SSTIs in Week 1. Case-patients had positive MSSA cultures identified by clinical cultures or surveillance sampling (bilateral nares, axillae, umbilicus, and groin), and antibiotic susceptibility testing was performed. WGS and assessment of isolate relatedness through mutation event analysis and multi-locus sequence typing (MLST) was performed by the NYS DOH. Demographic and isolate characteristics were compared using Wilcoxon rank sum test, Fisher’s exact test and Pearson’s Chi-squared test, as appropriate. Results: From Week 2 to Week 32, 9 rounds of surveillance for MSSA colonization were conducted. In all, 30 case-patients had MSSA colonization and 16 infants developed infections including impetigo (n=7), pustules (n=5), staphylococcal scalded skin syndrome (SSSS, n=2), abscess (n=1), and bacteremia (n=1). All SSTI cases presented on infants’ faces, all of whom were on non-invasive respiratory support. MLST identified 4 distinct types including MLST 121 (n=12), MLST 398 (n=10), MLST 30 (n=6), and MLST 15 (n=6). Eight isolates were unrelated to other isolates. MLST 398 and MLST 30 included isolates not closely related (>9 mutation events). The 12 MLST 121 isolates were closely related (≤9 mutational events between all isolates), harbored the mupA gene, and were mupirocin-resistant (MIC>1024 ug/ml). Clinical infection and mupirocin resistance were associated with MLST 121 (Table 1). Multiple infection control measures were implemented, including increased availability of alcohol-based hand sanitizers, introducing bare-below-the-elbows practice for staff, contact precautions for case-patients, decolonization with mupirocin and chlorhexidine baths, environmental cleaning/disinfection, and removing excess equipment and supplies. No new cases of mupirocin-resistant or MLST 121 SSTIs occurred after Week 25. Conclusion: We report a MSSA outbreak associated with multiple MLST types and a predominant mupirocin-resistant strain. This report highlights the ability of molecular typing to characterize strains causing infections versus colonization and the potential loss of mupirocin as a control measure when outbreaks are caused by mupirocin-resistant strains. WGS analysis allows for increased discrimination of mutation events allowing for improved resolution of case relatedness compared to other typing methods. Successful control of this outbreak was achieved with a multitude of infection prevention and control.

Chronicity of high and low level mupirocin resistance in Staphylococcus aureus from 30 Indian hospitals

Mupirocin is one of the most effective topically used antibiotic for the treatment of dermatitis, nasal carriage, decolonization of methicillin susceptible Staphylococcus aureus and eradication of methicillin resistant Staphylococcus aureus. Extensive use of this antibiotic has resulted in mupirocin resistance in Staphylococcus aureus which is a matter of concern. This study was conducted to evaluate the high and low level of mupirocin resistance in Staphylococcus aureus collected from various Indian hospitals. A total of 600 samples, of which 436 were pus specimens and 164 wound site swabs were collected from 30 Indian hospitals. Disc diffusion and agar dilution methods were used to test mupirocin susceptibility in methicillin resistant Staphylococcus aureus. Out of 600 Staphylococcus aureus isolates, 176 isolates (29.33%) were found to be methicillin resistant Staphylococcus aureus (MRSA). Out of 176 non-duplicate MRSA strains, 138 isolates were found to be mupirocin sensitive, 21 isolates had high level resistance whereas 17 isolates had low level resistance to mupirocin, which contributed 78.41%, 11.93% and 9.66% respectively. Multidrug resistant susceptibility was tested for all the MRSA with Cefuroxime, Cotrimoxazole and Vancomycin antibiotics. All the high and low level resistant strain were subjected to genome screening for mupA ileS gene respectively. mupA gene was found positive in all the high level resistant strain and out of 17 low level resistant strain, 16 strain were found point mutation in V588F of ileS gene. Overall, high rate of mupirocin resistance was found in the studied samples which might be a result of indiscriminate use of mupirocin in the population of studied region. This data emphasizes the urgent need for formulation of a well-defined and regulated guidelines for mupirocin use. Moreover, continuous surveillance is needed for the use of mupirocin and routine test should be performed to detect MRSA in patients and health care personnel to prevent MRSA infections.

Baseline prevalence of antimicrobial resistance in patients who develop a surgical site infection in hip and knee replacements: A brief report

Prior to clean surgeries, decolonization with topical antimicrobials may lead to an increase in antimicrobial resistance. To provide a baseline prevalence of resistance to topical antimicrobials, in Alberta, specimens were collected from surgical site infections following hip and knee replacements. Among 81 samples with complex surgical site infections, in 43 specimens Staphylococcus species were isolated. Only coagulase-negative staphylococci isolates carried resistance genes with 10 carrying the gene qac and 6 carrying the MupA gene.

Sequence types 8, 59, and 45 methicillin resistant Staphylococcus aureus as the predominant strains causing skin and soft tissue infections in Taiwan's prisons and jails.

Methicillin-resistant Staphylococcus aureus (MRSA) is the predominant cause of skin and soft tissue infections (SSTIs), which is a problem in prisons and jails. We conducted this study to understand MRSA molecular characteristics among inmates with SSTIs, and we chose MRSA isolates from a community hospital as a comparison. A total of 219 MRSA isolates from three custodial facilities and 134 isolates from a community hospital in Taiwan were collected in the 2017 calendar year. MRSA isolates were investigated molecularly by staphylococcal chromosome cassette mec (SCCmec) type, mupirocin, and chlorhexidine genotypical resistance, and multi-locus sequence typing (ST). Of the 219 MRSA isolates from custodial facilities, SCCmec IV was the most prevalent type (65.3%), followed by type VT (32.4%) and type V (1.8%). Regarding sequence types, ST59 (36.4%), 8 (35.3%), and 45 (17.9%) were the leading three predominant types out of 184 selected MRSA isolates, and ST45 MRSA was more prevalent in custodial facilities (p=0.019). The antimicrobial resistance rates varied for different MRSA strains, with ST45 MRSA having the lowest rates of resistance to most antimicrobials. Overall, 91.5% of isolates carried mupA gene and 25.8% were positive for qacA/B gene, this was independent of the MRSA sequence types. ST59, ST8, and ST45 MRSA are the leading three MRSA strains causing SSTIs in Taiwan, 2017, but the molecular distribution varied distinctly between the custodial facilities and hospital settings. The genotypical mupirocin resistance rate is quite high in this study. The frequency of chlorhexidine resistance gene is relatively low, especially in MRSA isolates from custodial facilities.

Distribution of sasX, qacA/B and mupA genes and determination of genetic relatedness of methicillin-resistant Staphylococcus aureus among clinical isolates and nasal swab samples from the same patients in a hospital in Malaysia.

This study determined the distribution of sasX, qacA/B and mupA genes from methicillin-resistant Staphylococcus aureus (MRSA) isolated from clinical samples and nasal swab samples of the same patients and analysed their genetic relatedness. Polymerase chain reaction was used to detect the presence of sasX, qacA/B and mupA genes from 47 paired MRSA isolates. A paired isolate was defined as one nasal swab (colonising) isolate and clinical isolate that caused infection in the same patient. 22 selected paired isolates were subjected to multilocus sequence typing (MLST). The genetic relatedness among the isolates and association between the putative genes with epidemic sequence types (STs) were investigated. 7 (14.9%, n = 14) paired isolates were positive for the sasX gene. qacA/B genes were positive in 7.4% (n = 7) of the isolates, from three paired isolates and one clinical isolate whose paired colonising isolate was negative. The paired sample of three patients were positive for both genes. The mupA gene was not detected in all the isolates. MLST revealed two epidemic STs, ST22 and ST239, and a novel ST4649. sasX and qacA/B genes were found in ST239 in 29.5% (n = 13) and 13.6% (n = 6) of cases, respectively. Gene co-existence occurred in 13.6% (n = 6) of MRSA ST239 and 2.3% (n = 1) of MRSA ST4649. sasX and qacA/B genes were present in the MRSA isolates, while the mupA gene was undetected. ST22 and ST239 were the major MRSA clones. The circulating MRSA genotypes conferred different virulence and resistance determinants in our healthcare settings.

First Report of a Methicillin-Resistant, High-Level Mupirocin-Resistant Staphylococcus argenteus.

We describe the identification of a methicillin-resistant, high-level mupirocin-resistant Staphylococcus argenteus. The isolate (1801221) was characterized as t6675-ST2250-SCCmecIVc, and whole-genome sequencing revealed that the isolate possessed two plasmids. One plasmid (34,870 bp), designated p1_1801221 with rep23, harboured the mupirocin resistance (mupA) gene. The second plasmid (20,644 bp), assigned as p2_1801221 with rep5a and rep16, carried the resistance determinants for penicillin (blaZ) and cadmium (cadD). Phylogenetic analysis revealed that the isolate clustered with the European ST2250 lineage. The overall high similarity of both plasmids in S. argenteus with published DNA sequences of Staphylococcus aureus plasmids strongly suggests an interspecies transfer. The pathogenic potential, community and nosocomial spread, and acquisition of antibiotic resistance gene determinants, including the mupA gene by S. argenteus, highlight its clinical significance and the need for its correct identification.

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.

Prevalence of mupirocin resistant (mupA) gene in S. aureus isolates from two tertiary care hospitals of Punjab

Prevalence of mupirocin resistant (mupA) gene in S. aureus isolates from two tertiary care hospitals of Punjab

Molecular characterization of invasive Staphylococcus aureus strains isolated from patients with diabetes in Iran: USA300 emerges as the major type.

There have been few studies focused on the molecular characterization of invasive Staphylococcus aureus strains in patients with diabetes in Iran. In the present study, 20 invasive S. aureus strains recovered from the patients with diabetes characterized by the virulence and resistance analysis, biofilm formation, staphylocoagulase (SC) typing, S. aureus protein A locus (spa) typing staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing (MLST). Virulence gene detection indicated a high prevalence of strains encoding the pvl genes (50%), a low prevalence of the tst and seg gene (each of them was 5%) and a markedly high prevalence of fnbB (95%), fnbA (85%), icaD (75%), icaA (65%). A total of 3 coagulase types (III, 85%; II, 10%; V, 5%), 2 agr types (I, 90%; II 10%) and 2 SCCmec types (IV, 65%; III, 35%) and four different clones namely ST8-MRSA-IV/t008 (50%) (USA300), ST239-MRSA-III/t030 (35%), ST5-MRSA-IV/t002 (10%), and ST45-MRSA-IV/t038 (5%) were detected in this study. Eighty-five percent of the isolates were biofilm producers. All the 4 high-level mupirocin resistance (HLMUPR) strains belonged to CC/ST8-MRSA-IV/t008 clone and carried mupA gene. Fusidic acid-resistant isolate belonged to ST239-SCCmec III/t030 clone. One vancomycin-intermediate resistance isolates was detected in our study, which belonged to ST5-MRSA-IVt002. Circulating clone in MRSA strains (USA300) isolated from the patients with diabetes highlighting the possibility of transmission of these microorganisms' clones between hospital, community, and environments. However, further studies require providing critical insights into the importance of continued controlling and treatment of S. aureus infections in patients with diabetes.

Increasing Mupirocin Resistance Among MRSA Nasal Surveillance Isolates in the Chicago Area

Background: In 2005, our healthcare system began universal admission screening for nasal colonization with MRSA and decolonization of MRSA positive patients with mupirocin. In 2010–2012, we studied the impact of nasal MRSA decolonization and concluded that it does not add benefit when contact precautions are used; plus, it resulted in increased rates of mupirocin resistance up to 9.4% in 2012. In September 2012 routine decolonization of hospitalized patients was discontinued. In the 2 years following discontinuation of mupirocin use for decolonization of MRSA carriers, the rate of mupirocin resistance gradually declined. We undertook a contemporary review of mupirocin resistance rates to ensure that the rates were stable. Methods: NorthShore University HealthSystem, Illinois, consists of 4 hospitals in the northern suburbs of Chicago, with 750 beds and 60,000 annual admissions. Admission nasal swab samples were collected from at-risk hospitalized patients based on a risk-adjusted algorithm. Nasal swabs were tested using the BD MAX MRSA assay. Positive samples were cultured onto BD BBL CHROMagar MRSA to recover the organism and were tested for the mupA gene, which confers high-level mupirocin resistance using an in-house PCR test. Data for mupirocin orders were provided by the pharmacy. Results: Mupirocin resistance rates and prescription orders are shown in Figs. 1 and 2. Conclusions: Mupirocin resistance rates plateaued between 2012 and 2014 and then increased from 9.1% in 2015 to 18.1% in 2019, despite discontinuation of routine decolonization of hospitalized patients. The reason for the increase is unclear; inpatient mupirocin orders were stable from 2015 to 2017.Funding: NoneDisclosures: None

Localization and Characterization of MupA Gene in High and Low-Level Mupirocin Resistant Methicillin Resistant Staphylococcus Aureus

Background: Two types of mupirocin resistance among methicillin resistant Staphylococcus aureus (MRSA) have been reported; low-level mupirocin resistance (LL-MR), and high-level mupirocin resistance (HL-MR). The mupA gene is typically located on mobile genetic elements which facilitate the resistance dissemination. Objective: The aim of this work was to identify the mupA gene location, as well as the restriction fragment length polymorphism (RFLP) patterns in high and low-level mupirocin resistant MRSA. Methodology: This study was conducted on 100 MRSA isolates; seven of them were mupirocin resistant. The E test was used to identify high and low level mupirocin resistance. Amplification of mupA gene in total and plasmid DNA was performed. We also detected the spacer region (trsLM–IS257-like–mupA) in the 7 isolates by PCR then we investigated its RFLP patterns. Results: Four MR MRSA isolates had low level resistance, their MupA gene was located on chromosomal DNA, whereas, three isolates showed high level MR, their MupA gene was located on plasmid DNA. Four types of different RFLP patterns of the spacer region were identified; type-1 included two LL-MR isolates, each of type-2 and 3 included both HL-MR and LL-MR isolates, and type-4 included one HL-MR isolate. Conclusions: Staphylococcus aureus mupA gene responsible for LL-MR is located on the chromosome while that responsible for HL-MR is plasmid-mediated. The spacer region variations appear to occur in both chromosomal and plasmid-located mupA gene regardless the type of mupirocin resistance.

Screening of antibiotic resistance genes and virulence determinants of Staphylococcus aureus from skin infections

BackgroundVirulent strains of Staphylococcus aureus (S. aureus) express a series of virulence factors which cause severe infections such as skin and soft tissue infections which can be life-threatening. Additionally, extensive antibiotic resistance among nosocomial pathogens has left limited choices for their eradication. Our objective was investigation of antibiotic resistance and virulence determinants of S. aureus from skin infections. Materials and methodsTwo-hundred non-duplicate S. aureus isolates were collected from skin infections. Antibiotic susceptibility profile was evaluated using disc diffusion and methicillin resistance was confirmed. Biofilm formation was assessed by microtiter tissue plate assay. Determinants of virulence factors including hlA-α, tsst-1, pvl, PSM's, eta and etb genes and also the mupirocin resistance mup A gene and class I integron were detected by PCR. MLST was performed for isolates containing the pvl gene. ResultsThe age range of patients included 12–76 years (mean ± SD = 56.34 ± 5.43). MRSA (70/200) were isolated significantly higher among ages>50 years (p < .001) but not significantly different between both genders (p = .112). Prior antibiotic consumption and hospitalization were significantly associated with MRSA (81.42%) and MDR (84.28%) isolation. The existence of int1 gene (44.5%) was significantly higher in multidrug-resistant (MDR) isolates (p < .001). Thirty-six (18%) MDR isolates carried the mupA gene. The existence of mupA gene was significantly associated with prior hospitalization (n = 33, 91.66%, p < .0001) and antibiotic consumption (n = 30, 83.33%, p < .001). Predominant virulence determinant included PSMα (61.5%), followed by tsst-1 (17.5%), hla-α (9.5%), pvl (2.5%), eta (2.5%) and etb (1%). The rate of strong biofilm producers (totally 25%) was not significantly different between MRSA and MSSA. The existence of PSM-α gene was significantly higher among strong biofilm producers compared to biofilm non-producers (p = .002). PFGE exhibited no genetic relation among strains. ConclusionVirulence factors of S. aureus from skin infections contributed in severity of infection and biofilm formation. MDR phenotype was more common among older patients with history of hospitalization and prior antibiotic consumption. Mupirocin resistance has emerged among MDR and MRSA isolates. Hence suitable control strategies must be performed to inhibit the spread of these strains in healthcare and community settings.

Genetic Diversity Analysis of Methicillin-resistant Staphylococcus aureus Strains Isolated from Intensive Care Unit in Iran.

ObjectivesStaphylococcus aureus has emerged as a major public health concern. It is a common pathogen in high-risk hospital intensive care units (ICUs). We analyzed the molecular characteristics on the SCCmec and spa genes of S. aureus isolates gathered from ICUs. The antibiotic resistance patterns and carriage of resistance and virulence determinants were also identified.MethodsIn this cross-sectional study, 84 non-duplicated S. aureus strains isolated from ICU patients in were genotyped using SCCmec and spa typing. The Kirby-Bauer disk diffusion and micro-broth dilution methods were used to determine resistance patterns. Virulence and resistance gene profiling were also determined using the polymerase chain reaction technique.ResultsAll isolates were methicillin-resistant S. aureus and belonged to seven spa types: t388 (36.9%), t852 (14.3%), t924 (13.1%), t790 (11.9%), t064 (10.7%), t037 (9.5%), and t084 (3.6%). They differed in the carriage of resistance and toxin genes. The most common SCCmec type was III detected in 50 isolates (59.5%), followed by type IV in 34 isolates (40.5%). The pvl gene was detected in 14.3% (n = 12) of isolates, of which 66.7% (n = 8) belonged to t852 and 33.3% (n = 4) belonged to t790. Among the tested strains, 9.5% (n = 8) carried the mupA gene and belonged to the t064 spa type.ConclusionsThe data revealed a high resistance rate to antibiotics, which could be a threat to ICU patients. It is necessary to detect antimicrobial resistance and resistance and toxin-encoding of gene profiles in different molecular types.

Design of Melting Curve Analysis (MCA) by Real-Time Polymerase Chain Reaction Assay for Rapid Distinction of Staphylococci and Antibiotic Resistance

Background: Staphylococci are important pathogenic bacteria responsible for a range of diseases in humans. Hence, detection of Staphylococcus aureus from coagulase-negative staphylococci (CoNS) is essential in various infections. Objectives: The aim of this study was to design a melting curve analysis (MCA) assay based on Multiplex Real-Time PCR for rapid detection of staphylococci and antibiotic resistance. Methods: The current study used standard strains of positive and negative coagulase staphylococci. As the first step, serial dilutions were prepared with ratios of 108 to 101 cfu/mL based on standard bacterial concentration with 0.5 McFarland and the results were illustrated in dedicated melting curves. Results: All melting curves of gene amplification had an equal melting point. In all dilutions, the observed melting temperatures shown in the melting curves of gene amplification were equal to 83.79°C for ITS-gene, 74.2°C for phop gene, 76.49°C for sap-gene, 78.2°C for mvaA gene, 79.57°C for 16srRNA-gene, 74.83°C for mupA gene, and 76.6°C for mecA gene. Conclusions: The MCA based on real time-PCR for identifying staphylococcal species and antibiotic resistance is a highly effective method with high sensitivity and specificity. © 2019, Archives of Clinical Infectious Diseases.

Phenotypic and genotypic determinants of mupirocin resistance among &lt;em&gt;Staphylococcus aureus&lt;/em&gt; isolates recovered from clinical samples of children: an Iranian hospital-based study

BackgroundsThe aim of this study was to evaluate both phenotypic and genotypic determinants of mupirocin resistance among methicillin-resistant Staphylococcus aureus (MRSA) and methicillin susceptible S. aureus (MSSA) strains recovered from different clinical samples of children who were admitted to the Children’s Medical Center (CMC) Hospital, Tehran, Iran.Materials and methodsA total of 120 clinical isolates of S. aureus were collected from the microbiology laboratory of CMC Hospital. Antimicrobial susceptibility of the isolates to different antimicrobial agents was determined by disk diffusion method. The methicillin resistance phenotype (MRSA) was identified using a 30 µg cefoxitin disk. The minimum inhibitory concentration (MIC) of mupirocin was determined by broth microdilution method. Strains with mupirocin MIC between 8 and 256 µg/mL were considered as low-level mupirocin resistant (LLMR), and strains with an MIC≥512 µg/mL were considered as high-level mupirocin resistant (HLMR). The presence of genes encoding HLMR (ie, mupA and mupB genes) was evaluated by PCR method.ResultsFour out of 120 isolates (3%) had mupirocin MIC≥512 µg/mL and were HLMR; however, no LLMR isolate was detected. Fifty-two isolates (43%) were MRSA, and there were no differences in the distribution of mupirocin resistance among MRSA and MSSA isolates (P>0.05). The PCR method identified mupA gene in two out of four HLMR isolates, and mupB gene was not detected in any HLMR isolates.ConclusionBecause of discrepancies between the phenotypic and genotypic patterns of mupirocin resistance and due to the avoidance of false-negative results, it is better to determine the mupirocin resistance by both antibiotic susceptibility tests and PCR method. Considering the increasing need of mupirocin for the control of S. aureus infections, continuous checking of its susceptibility status is necessary.

Genetic Diversity Analysis of Mupirocin-Resistant Staphylococcus aureus Clinical Isolates in Tehran Hospitals, Iran.

Few studies describe the molecular characterization of mupirocin resistance in Staphylococcus aureus strains. In this study, we evaluated the characteristics of mupirocin resistance in S. aureus isolates for 1-year period in Tehran, Iran. In a cross-sectional study, we collected 51 unique mupirocin-resistant strains obtained from 648 S. aureus isolates. High- and low-level resistance were determined by minimum inhibitory concentration with broth microdilution method. Presence of the genes for resistance to antibiotics and toxins was detected by polymerase chain reaction assays. Genotyping was performed by S. aureus protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) typing, whereas clones were determined by multilocus sequence typing. Among mupirocin-resistant isolates, methicillin-resistant S. aureus (MRSA) 23 (45.1%) and 28 (54.9%) isolates were classified as high- and low-level mupirocin resistance, respectively. Among the 51 tested strains 25 (49%) isolates were positive for mupA genes. Seven different clones were detected in this study. High-level mupirocin-resistant (HLMUPR) strains belonged to ST8-SCCmec IV/t064 (n = 10, 19.6%), ST5-SCCmec IV/t002 (n = 5, 9.8%), ST8-SCCmec IV/t008 (n = 4, 7.8%), and ST239-SCCmec III/t631 (n = 4, 7.8%) clones, while low-level mupirocin-resistant (LLMUPR) strains belonged to ST22-SCCmec IV/t790 (n = 11, 21.6%), ST239-SCCmec III/t860 (n = 9, 17.7%), and ST15-SCCmec IV/t084 (n = 8, 15.7%) clones. Two strains belonged to ST22-SCCmec IV/t790 clone, despite carrying mupA gene, and demonstrated LLMUPR phenotype. One ST8-SCCmecIV/t008 strain with HLMUPR was confirmed as Vancomycin-intermediate S. aureus strain. Our data demonstrated the need for thorough epidemiological monitoring and a routine mupirocin testing program to prevent and detect mupirocin resistance in MRSA.

1218. Retapamulin as a Potential Decolonizing Agent: Activity against Mupirocin-Resistant Strains From Pediatric Patients With Methicillin-Resistant Staphylococcus aureus Infection

BackgroundControlling methicillin-resistant Staphylococcus aureus (MRSA) colonization is a common strategy to prevent transmission and recurrent infection. Standard decolonization regimens include nasal application of mupirocin ointment; however, increasing rates of mupirocin-resistance (Mup-R) have been noted globally. At our institution there has been an increase in community-acquired MRSA (CA-MRSA) infections among children living in Brooklyn, New York. A genotypic geographic cluster of an outbreak clone of the CA-MRSA strain USA 300 with a high rate (>85%) of mupirocin resistance, mediated by the plasmid borne mupA gene, was identified prompting investigation into an alternative decolonizing agent. We sought to investigate retapamulin, a topical pleuromutilin antibiotic, which has been shown to be effective against S. aureus with in vitro and in vivo activity against MRSA and a low propensity to develop resistance.MethodsBroth microdilution was used to determine the minimum inhibitory concentrations (MIC) of retapamulin against 53 Mup-R MRSA isolates collected from pediatric patients (aged 9 months–17 years) presenting to our institution over an 18 month period with clinical MRSA infection. Susceptibility defined as ≤0.5 mg/L susceptible (EUCAST). Whole genome sequence data were analyzed for the presence of rplC and cfr gene mutations known to confer resistance to retapamulin.ResultsAll 53 isolates were susceptible to retapamulin. 49/53 (92%) strains were inhibited at MIC 0.25 mg/L, 2/53 (4%) at MIC 0.125 mg/L, and 2/53 (4%) at MIC 0.5 mg/L. DNA sequence analysis showed that one isolate had a first-step mutation in the rplC gene, but it was not associated with reduced phenotypic susceptibility to retapamulin, as the MIC of that isolate was 0.25 mg/L.ConclusionRetapamulin demonstrated excellent in vitro activity against a genotypic cluster of Mup-R isolates from pediatric patients presenting to our institution with MRSA infection. These data suggest that retapamulin may be a promising alternative decolonization therapy for MRSA and a viable option to prevent the spread of mupirocin-resistant MRSA clones. Further research includes an ongoing randomized, placebo-controlled trial testing the in vivo efficacy of retapamulin as a nasal and perirectal decolonizing agent in children.Disclosures A. Patel, Aqua Pharmaceuticals: Investigator inititiated grant, Research grant. J. Lighter-Fisher, Aqua Pharmaceuticals: Investigator Initiated Grant, Research grant.

Molecular Characterization of Vancomycin, Mupirocin and Antiseptic Resistant Staphylococcus aureus Strains.

BackgroundStaphylococcus aureus is a common cause of nosocomial infections leading to a broad spectrum of diseases. Increasing antibiotic resistance among S. aureus strains, particularly methicillin-resistant S. aureus (MRSA), is a serious concern. In addition, the emergence of antiseptics resistance in MRSA helps the organism to persist and spread in healthcare environments easily. The aim of this study was to determine the molecular characteristics of vancomycin, mupirocin, and antiseptic resistant S. aureus strains.Materials and MethodsThis cross-sectional study was performed on a total of 120 MRSA isolates collected from two major hospitals in Shiraz, Iran. Minimum inhibitory concentrations (MICs) of vancomycin and mupirocin were determined by E-test method according to CLSI and Eucast guidelines. Presence of resistance genes was investigated by PCR method.ResultsAntibacterial susceptibility tests for MRSA isolates showed that three isolates (2.5%) were vancomycin-intermediate S. aureus (VISA), seven isolates (5.8%) were vancomycin-resistant S. aureus (VRSA), and 15 isolates (12.5%) were high-level mupirocin-resistant (MuH). None of the isolates had vancomycin resistance gene (vanA), but the frequency of mupirocin resistance gene was significant, and 55 (45.8%) isolates carried the mupA gene. Moreover, norA, smr and qacA/B genes were detected in 110 (91.7%), 55 (45.8%) and 36 (30%) strains, respectively.ConclusionThis study showed the existence of VISA and VRSA strains in our region, and we also found a high frequency of mupirocin and biocide resistance genes among them.