Methicillin-susceptible S. Aureus Strains Research Articles

Prevalence of Panton-Valentine leucocidin (pvl) and exfoliative toxin A (eta) gene within methicillin resistant and susceptible Staphylococcus aureus in an urban tertiary hospital: A molecular epidemiology pilot study

Background Staphylococcus aureus is well known to cause a multitude of clinical manifestations, from mild to severe bloodstream infections that could lead to death. Infections are common, either in community-acquired or hospital-acquired settings, and treatment remains a challenge due to methicillin-resistant Staphylococcus aureus (MRSA). The pathogenesis of S. aureus is mediated by several cell-surface and secreted virulence factors. The virulence factors discussed in this study are Panton-Valentine leucocidin (pvl) and exfoliative toxin A (eta). Identifying both pvl and eta gene may help in studying bacterial pathogenesis and biology thus creating possible therapeutic pathway or intervention. Our pilot study aimed to observe pvl and eta as virulence gene prevalence in a North Sumatera tertiary referral health center. Methods Our study was a descriptive-analytical observational study with a cross-sectional design in which we collected isolates over a single time period. The frequency of genes is reported as a percentage comparison between MRSA and methicillin-susceptible S. aureus (MSSA). Qualitative gene prevalence analysis was carried out using the polymerase chain reaction (PCR). Results Our results showed that from 38 MRSA sample isolates, 32 samples were found to be pvl-positive, or 84,3% of the total samples. From 40 MSSA sample isolates, one sample was found to be pvl-positive MSSA, or 97,5%. Regarding eta, from 38 MRSA sample isolates, 81,6% of the total sample did not have eta, while from 40 MSSA sample isolates, all samples were found to be positive for eta. We found that both pvl and eta were significantly more likely to be expressed in the MSSA strain. Conclusions Our study shows that pvl and eta are more likely expressed in MSSA strains than in MRSA strains in Indonesia.

Investigation of Antimicrobial, Antioxidant, and Cytotoxic Activities of Boesenbergia rotunda rhizome extract

In recent years, antimicrobial resistance (AMR) has become a global threat to public health. In Hawai’i, there is an increasing rate of staph infections of methicillin-resistant Staphylococcus aureus (MRSA), and, hence, a need for new agents to combat the increase of AMR bacteria. This study evaluates the antimicrobial, antioxidant, and cytotoxic activities of Hawaiian-grown Boesenbergia rotunda. Test bacteria included clinical isolates of Gram-positive MRSA and methicillin-susceptible S. aureus (MSSA) as well as Gram-negative Serratia marcescens and Escherichia coli. Five compounds–cardamonin, pinostrobin, pinocembrin, pinostrobin chalcone, and isopanduratin A–were isolated from the ethyl acetate extract of B. rotunda rhizome, and their structures were identified by NMR spectroscopy. These samples exhibited antimicrobial activity against MRSA and MSSA strains, with minimum inhibitory concentration (MIC) values between 128 and 2 µg/mL, with isopanduratin A giving MIC values as low as 2 µg/mL. The antioxidant potential of samples was examined using ferric-reducing antioxidant power (FRAP) assay. At 1 mg/mL of tested samples, FRAP values ranged between 8.74 to 17.76 µM/µg, with pinostrobin chalcone exhibiting the highest FRAP value (17.76 ± 0.65 μM/μg). Moreover, cytotoxicity was measured via sulforhodamine B (SRB) assay. Cardamonin (IC50 of 19.43 ± 0.33 μM) and isopanduratin A (IC50 of 26.84 ± 1.06 μM) exhibited effectiveness against the lung cancer cell line A549. Compounds from B. rotunda showed potent antimicrobial effect against MRSA and MSSA strains as well as antioxidant and cytotoxic activities, and may have the potential for further evaluation and development for pharmaceutical applications.

Accessory gene regulators and virulence genes associated with the pathogenicity of Staphylococcus aureus from clinical and community settings in Lagos, Nigeria

Staphylococcus aureus is a prominent pathogen that causes serious community and hospital-acquired infections globally. Its pathogenicity is attributed to a variety of secreted and cell surface associated proteins that are modulated by the quorum-sensing accessory gene regulator (agr) system. In this study, we investigated the presence of toxin genes and agr involved with S. aureus from clinical samples and apparently healthy individuals. Unequivocal identification of the isolates was obtained with the Vitek 2 system. We screened 70 clinical (CL) and 22 community (C) S. aureus strains for the methicillin resistance (mecA) gene, agr and superantigens (SAg) (enterotoxins and toxic shock syndrome toxin-1) using PCR techniques. A total of 12 clinical isolates were classified as methicillin-resistant S. aureus (MRSA); 89 isolates belonged to one of the four agr groups (agr1-4), and 3 isolates were non-typeable. Of the agr groups, agr1 was the most prominent and mostly consisted of isolates from pus/wounds. The methicillin-susceptible S. aureus (MSSA) isolates were distributed within the four agr groups while MRSA strains were restricted to agr1 and agr3. The most common enterotoxin gene, sei, was likewise more prevalent in MSSA strains than in MRSA strains, where sea predominated. The co-existence of two or more enterotoxins was confirmed in 40% of the isolates. sea occurred through all the agr groups except agr3 and sei was not found in agr1 and agr4. The toxic shock toxin (tst) gene was detected in six MSSA. These findings suggest that MSSA may cause more lethal infections than MRSA because of the increased frequency of toxic genotypes seen in MSSA strains. Significance: Isolates in the agr1,3 groups had more SAg toxin genes, whereas isolates in the agr4 groups possessed more tst genes. The MSSA isolates contained higher proportions of virulence genes than MRSA. The clinical implications of this discovery include that MSSA may cause more lethal infections than MRSA due to the greater number of toxigenic genotypes discovered.

Genetic alterations in methicillin-susceptible Staphylococcus aureus associated with high vancomycin minimum inhibitory concentration

There are many reports on gene mutations observed in methicillin-resistant Staphylococcus aureus (MRSA) showing reduced susceptibility to vancomycin. However, there are limited studies on the genetic alterations that contribute to high vancomycin minimum inhibitory concentrations (MICs) in methicillin-susceptible S. aureus (MSSA). This study aimed to compare MSSA strains with high vancomycin MICs with those with low MICs, and to identify specific genetic alterations associated with increased vancomycin MICs. In total, 124 MSSA strains were analysed, with 62 having vancomycin MICs of 1-2 mg/L (MS-HV) and the remaining 62 having MICs <1 mg/L (MS-LV) as control. Polymerase chain reaction amplification and sequencing were conducted to identify point mutations and amino acid changes in the vraSR, graRS and walRK operons and rpoB gene. The number of single nucleotide polymorphisms (SNPs) and specific mutations in the indicated gene were compared between the two groups. The MS-HV strains had a significantly higher median number of SNPs in studied genes than the MS-LV strains (5 vs 3; P < 0.0001), with higher frequency of SNPs in the graR and walK genes. The MS-HV strains also displayed a significantly higher prevalence of specific mutations in the graR gene (V135I, I136V and V136I) compared with the MS-LV strains. The odds of having a high vancomycin MIC was 5.54 times higher in strains with a mutation in the graR gene, and 5.32 times higher in strains with a mutation in the walK gene, compared with those without these mutations. Mutations in the graR and walK genes may contribute to reduced vancomycin susceptibility in MSSA. This study gives key insights into the mechanisms underlying this phenomenon.

Molecular Characteristics of Staphylococcus aureus Strains to Carry Panton-Valentine Leukocidin Genes Isolated from Hospitalized Patients in Tehran, Iran

Background: Staphylococcus aureus with concurrent resistance to antibacterial agents is emerging globally. This emergence might be due to the production of different virulence determinants, notably Panton-Valentine leukocidin (PVL). Objectives: This study aimed to investigate the genetic characteristics of PVL-positive S. aureus strains isolates from clinical samples. Methods: An epidemiological study was conducted on 65 S. aureus isolates carrying pvl genes. An antibiogram test by the disk diffusion and broth microdilution methods was conducted to assess antimicrobial resistance profiles. Results: All detected methicillin-resistant S. aureus (MRSA) isolates were confirmed by mecA polymerase chain reaction (PCR) assays. The PVL-positive isolates were characterized using multiplex PCR assay to detect staphylococcal cassette chromosome mec (SCCmec) and agr types. The PVL frequency was 19.5% and 17.6% in MRSA and methicillin-susceptible S. aureus (MSSA), respectively. Among the PVL-positive isolates, 66.2% and 33.8% were MRSA and MSSA, respectively. Multidrug resistance amounted to 84.6% of the isolates (MRSA: 61.5%, MSSA: 23.1%). Staphylococcal cassette chromosome mec III was dominated (55.8%; 24/43). The most commonly identified agr was type III (53.8%; 35/65). Resistance to vancomycin amounted to 12.3% of the isolates, and all belonged to agr type III and SCCmec type III. The frequency of inducible and constitutive clindamycin resistance among PVL-positive MRSA strains (12.3% and 26.1%) was higher than PVL-positive MSSA strains (7.7% and 15.4%). Most constitutive and inducible clindamycin resistance isolates belonged to agr type III (26.2% and 18.5%) and SCCmec type III (each 27.9%). In the present study, 32.3% of the isolates were confirmed as mupirocin resistant, and all were MRSA, 9 (42.9%) and 12 (57.1%) isolates of which exhibited high-level mupirocin resistant (HLMUPR) and low-level mupirocin resistant phenotypes. All HLMUPR MRSA isolates belonged to SCCmec III and recovered from wound samples. Conclusions: The emergence of vancomycin-resistant S. aureus strains among PVL-positive S. aureus strains in Iran is a serious alarm and seems to be becoming the greatest concern in the treatment of staphylococcal infections in the healthcare setting. The present study reinforces plausible direct transfers between community and nosocomial PVL-positive S. aureus types.

Investigation of nasal Staphylococcus aureus carriage by real-time PCR in patients receiving hemodialysis treatment

Objectives: Staphylococcus aureus, coagulase-negative staphylococci (CoNS), and methicillin-resistant S. aureus (MRSA), which are significant nosocomial pathogens, have become a growing global problem because their carriage and diseases have become resistant to many antibiotics. This study aimed to investigate and determine the rate of MRSA carriage among patients receiving hemodialysis treatment using molecular methods. Methods: In the 254 hemodialysis patients, the nasal carriage rates, susceptibility and resistance to S. aureus, CoNS and MRSA were examined using culture and real-time PCR methods. Nasal samples from hemodialysis patients were examined using real-time PCR. Microscopic examination was performed using the Gram staining method, and S. aureus was identified using catalase and coagulase. The strains were then tested for antibiotic susceptibility. Staphylococci was isolated from 231 of the 254 patients. Results: S. aureus carriage was detected in 50 patients, MRSA in 16, methicillin-susceptible S. aureus (MSSA) in 33, CoNS in 66, methicillin-resistant CoNS (MR-CoNS) in 38, and methicillin-susceptible CoNS (MS-CoNS) in 28. S. aureus and MRSA strains exhibited 100% susceptibility to nitrofurantoin, and vancomycin. MSSA strains showed the highest susceptibility to chloramphenicol, clindamycin (84.8%), and co-trimoxazole (36.4%). CoNS showed 100% susceptibility to vancomycin, and 16.7% susceptibility to ampicillin. Vancomycin was found to be the most effective antibiotic against S. aureus, CoNS, and MRSA pathogens identified in patients undergoing hemodialysis, whereas penicillin resistance was found. Conclusions: It can be concluded that one of the most effective ways to prevent the formation of antibiotic resistant strains is the hygiene of the hospital and hospital staff. Real-time PCR is very important for analyzing with high sensitivity.

Phenotypic and Molecular Detection of Biofilm Formation in Methicillin-Resistant Staphylococcus Aureus Isolated from Different Clinical Sources in Erbil City.

Staphylococcus aureus is an important causative pathogen. The production of biofilms is an important factor and makes these bacteria resistant to antimicrobial therapy. the current study aimed to assess the prevalence of resistance to antibacterial agents and to evaluate the phenotypic and genotypic characterization of biofilm formation among S. aureus strains. This study included 50 isolates of Methicillin-resistant S. aureus (MRSA) and Methicillin-Susceptible S. aureus (MSSA). S. aureus was identified by molecular and conventional methods, and antimicrobial resistance was tested with a disc diffusion method. The biofilm formation was performed through the Microtiter plate method. Strains were subjected to PCR to determine the presence of nuc, mecA, icaA, icaB, icaC, and icaD genes. Of the 50 S. aureus isolates, 32(64%) and 18(36%) were MRSA and MSSA, respectively. A large number of MRSA and MSSA isolates showed resistance to Penicillin and Azithromycin, and a lower number of MRSA and MSSA isolates showed resistance to Amikacin Gentamicin. None of the isolates was resistant to Vancomycin. The MRSA strains had significantly higher resistance against antibiotics than MSSA strains (P = 0.0154). All isolates (MRSA and MSSA) were able to produce biofilm with levels ranging from strong (31.25 %), (16.6%) to moderate (53.12%), (50%) to weak (15.6%), (33.3%) respectively. The MRSA strains had a significantly higher biofilm formation ability than the MSSA strains (P = 0.0079). The biofilm-encoding genes were detected among isolates with different frequencies. The majority of S. aureus isolates, 42 (84%), were positive for the icaA. The prevalence rates of the icaB, icaC and icaD genes were found to be 37 (74%), 40 (80%) and 41 (82%), respectively. The prevalence of biofilm encoding genes associated with multidrug resistance in S. aureus strains is high. Therefore, identifying epidemiology, molecular characteristics, and biofilm management of S. aureus infection would be helpful.

In Vivo Pathogenicity of Methicillin-Susceptible Staphylococcus aureus Strains Carrying Panton-Valentine Leukocidin Gene.

Toxin-producing Staphylococcus aureus strains posing a potential risk for public health have long been a topic of scientific research. Effects of Panton-Valentine leukocidin (PVL) on tissue destruction mechanisms and activities of inflammatory cells were presented in animal models of pneumonia and skin infections induced by PVL-producing S. aureus strains. This study aimed to demonstrate the in vivo pathogenicity of PVL-producing S. aureus strains isolated from some foodstuffs, which can be a potential risk to public health. PVL-positive methicillin-susceptible S. aureus (MSSA) strains M1 and YF1B-b isolated from different foodstuffs and a PVL-positive MSSA strain HT480 (positive control) were administered to New Zealand rabbits. Blood samples were harvested three and six hours after the intratracheal inoculation. Lung tissue samples were collected for gross and microscopic exams and immunohistochemical (IHC) demonstration of IL-6, IL8, IL-10, and TNF-α expressions. Serum cytokine levels were also measured by ELISA. The strains isolated from lung tissue samples were confirmed by pulsed-field gel electrophoresis. The development of acute necrotising pneumonia and a significant elevation in IL-6, IL-8, IL-10, and TNF-α expressions demonstrated the significance of foodborne PVL-positive MSSA strains in public health for the first time.

High prevalence and expression of antiseptic resistance genes among infectious t037/ST239 methicillin-resistant Staphylococcus aureus (MRSA) strains in North Khorasan Province, Iran.

Objective(s): Staphylococcus aureus is an important infectious agent and the majority of methicillin-resistant S. aureus (MRSA) infections are of nosocomial origin. To define the level and distribution of antiseptic resistance among infectious S. aureus strains we studied MRSA and methicillin-susceptible S. aureus (MSSA) isolates collected from different infection sites in an assortment of patients.Materials and Methods: S. aureus isolates were investigated for in vitro susceptibility to antiseptic agents and detection of qacA/B, smr, vanA, and mecA genes. Results:Among the S. aureus isolates we studied, 25 and 41 were MRSA and MSSA, respectively. The mean of minimum inhibitory concentrations (MICs) for benzethonium chloride (BTC) among MRSA was statistically significantly higher than for MSSA (26 µg/ml versus 11.7 µg/ml, P=0.003) while there was no significant difference between MRSA and MSSA for benzalkonium chloride (BKC) and chlorhexidine digluconate (CHG). The qacA/B genes were carried in 68% of the MRSA and 58.2% of MSSA (P=0.601), while smr was carried in 39% of MRSA and 29.3% of MSSA strains (P=1.000). In 15 out of 25 cases, MRSA ST239 with spa types t037, t030, and t7688 was isolated from the infection site with 86.6% of them carrying a resistance gene (qacA/B or qacA/B + smr). Conclusion:The frequent presence of antiseptic resistance genes and a consequently elevated MIC against antiseptics among ST239 MRSA emphasizes the importance of mandatorily monitoring MRSA for effective infection control.

Association of PVL Gene in MSSA and MRSA Strains among Diabetic Ulcer Patients from Odisha, India.

Staphylococcus aureus has emerged as an important pathogen among diabetic foot ulcers in patients with diabetes. Infections with S. aureus in diabetic ulcers need surveillance of resistant microbial profile to provide the basis for empirical therapy for the reduction of lower extremities amputation. Panton valentine leucocidin (PVL) is considered as one of the major virulence gene of S. aureus which is responsible for destruction of white blood cells and tissue necrosis. This pore forming cytotoxin gene is carried out by both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains. The present study described the prevalence of PVL gene in MSSA and MRSA strains isolated from diabetic ulcer patients treated during November, 2019 to January, 2021 from a tertiary care hospital, Odisha. Infected tissue and blood samples from these patients were collected aseptically and sub-cultured using different media and standard techniques. The isolated genomic DNA of MSSA and MRSA strains were subjected to PCR assay for the detection of PVL gene. Two hundred ten S. aureus out of 402 diabetic ulcer patients were isolated having 59.52% MSSA and 40.47% MRSA strains. Wagner's grade III and grade IV ulcers were most prevalent in these ulcer patients. The prevalence of PVL gene in MSSA strains was more in comparison to MRSA strains. Forty five resistance patterns were observed from the antibiogram profiles of S. aureus. The present study highlighted that PVL gene could not be a marker for the detection of MRSA and MSSA strains in diabetic ulcer patients.

Efficacy of antimicrobial peptide LL-37 against biofilm forming Staphylococcus aureus strains obtained from chronic wound infections

The antimicrobial peptide LL-37 showed inhibitory effects against Staphylococcus aureus strains, which often responsible for wound infections. Understanding the molecular mechanisms of biofilm-containing wound infections is important. Thus, this study aimed to investigate both the antimicrobial and biofilm efficacy of LL-37 against biofilm-positive methicillin-susceptible S. aureus (MSSA) strains and biofilm-positive methicillin-resistant S. aureus (MRSA) strains obtained from chronic wound infections and its effect on different quorum sensing and virulence genes at suboptimal concentrations. Fifteen biofilm-forming MRSA and 15 biofilm-forming MSSA strains were included in this study. The minimum inhibitory concentration (MIC) values and biofilm formation were tested by microdilution methods. Real-time PCR was performed to determine gene expression levels. MIC values for LL-37 were 89.6 mg/L and 132.3 mg/L for MSSA and MRSA strains, respectively. No statistically significant difference was found between MRSA and MSSA strains in terms of the effect of LL-37 on biofilm formation. A statistically significant difference was found between MRSA and MSSA strains for atlA, RNAIII, and agrA gene expression levels following exposure to a suboptimal concentration of LL-37. Ultimately, the required LL-37 antimicrobial concentration was quite high; however, LL-37 antibiofilm concentration may be acceptable for use in humans against biofilm-forming MRSA and MSSA strains. This is the first study to investigate to effect of a suboptimal LL-37 concentration on gene expression levels of biofilm-forming MSSA and MRSA strains. LL-37 affected quorum sensing and biofilm producing mechanisms, even at suboptimal MIC concentrations.

High prevalence of spa type t790, coa type III and the emergence of spa types t309, t571 and t127 in community-acquired methicillin-susceptible Staphylococcus aureus isolated from wound, Tehran-Iran

A significant rise in the prevalence of methicillin-susceptible S. aureus (MSSA) strains associated with skin and soft tissue infections (SSTIs) has been reported worldwide. There have been few studies focused on this concern. In the present study, 70 community-acquired (CA)-MSSA strains recovered from wound investigated by the phenotypic resistance pattern, staphylocoagulase (SC) typing and S. aureus protein A locus (spa) typing. The MSSA strains consisted of 8 spa types and 3 clonal complexes. CC22/t790, CC22/t005, CC398/t571, CC8/t030, CC8/t037, CC8/t008, CC22/t309, and CC1/t127 were the main MSSA spa types which accounted for 44.3%, 11.4%, 11.4%, 10%, 8.6%, 7.1%, 4.3%, and 2.9% respectively. Coagulase type III was the most prevalent type (47.2%). All tst -positive isolates (14.3%) belonged to CC22/t790. The pvl gene was present in 51.4% of all MSSA strains belonged to CC8/t008 (11.4), CC22 (40%). Out of four strains with intermediate resistance to vancomycin, two isolates belonged to CC22/t790 and two isolates to CC8/t030 while vancomycin-resistant S. aureus strain observed in CC22/t790. Our findings indicated low heterogeneity of MSSA strains. Circulation of the CC22 CA-MSSA strains highlighted the possibility of transmission of this type between from community to hospital.

Swine as reservoirs of zoonotic borderline oxacillin-resistant Staphylococcus aureus ST398

Methicillin resistance mediated by the mecA gene in Staphylococcus aureus, also known as “true MRSA”, is typically associated with high oxacillin MIC values (≥8 mg/L). Because non-mecA-mediated oxacillin resistant S. aureus phenotypes can also cause hard-to-treat diseases in humans, their misidentification as methicillin-susceptible S. aureus strains (MSSA) can compromise the efficiency of the antimicrobial therapy. These strains have been refereed as Borderline Oxacillin-Resistant S. aureus (BORSA) but their characterization and role in clinical microbiology have been neglected. Considering the increasing importance of livestock-associated methicillin-resistant S. aureus ST398 (LA-MRSA) as an emerging zoonotic pathogen worldwide, this study aimed to report the genomic context of oxacillin resistance in porcine S. aureus ST398 strains. S. aureus isolates were recovered from asymptomatic pigs from three herds. Oxacillin MIC values ranged from 4 to 32 mg/L. MALDI-TOF-confirmed isolates were screened for mecA and mecC by PCR and genotyped by means of PFGE and Rep-PCR. Seven isolates were whole genome sequenced. None of the isolates harbored the mecA gene or its variants. Although all seven sequenced isolates belonged to one sequence type (ST398), two different spa types (t571 and t1471) were identified. All isolates harbored conserved blaZ gene operon and no mutations on genes encoding for penicillin-binding-proteins were detected. Genes conferring resistance against other drugs such as aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin (MLS), tetracycline and trimethoprim were also detected. Isolates also harbored virulence genes encoding for adhesins (icaA; icaB; icaC; icaD; icaR), toxins (hlgA; hlgB; hlgC; luk-PV) and protease (aur). Pigs can serve as reservoirs of non-mecA-mediated oxacillin-resistant ST398 strains potentially pathogenic to humans. Considering that mecA has been the main target to screen methicillin-resistant staphylococci, the occurrence of BORSA phenotypes is probably underestimated in livestock.

Environmental Surveillance and Characterization of Antibiotic Resistant Staphylococcus aureus at Coastal Beaches and Rivers on the Island of Hawai'i.

Staphylococcus aureus are human facultative pathogenic bacteria and can be found as contaminants in the environment. The aim of our study was to determine whether methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolated from coastal beach and river waters, anchialine pools, sand, and wastewater on the island of Hawaiʻi, Hawaiʻi, are a potential health risk. Samples were collected from three regions on Hawaiʻi Island from July to December 2020 during the COVID-19 pandemic and were characterized using whole-genome sequencing (WGS). From WGS data, multilocus sequence typing (MLST), SCCmec type, antimicrobial resistance genes, virulence factors, and plasmids were identified. Of the 361 samples, 98.1% were positive for Staphylococcus spp. and 7.2% were S. aureus positive (n = 26); nine MRSA and 27 MSSA strains were characterized; multiple isolates were chosen from the same sample in two sand and seven coastal beach water samples. The nine MRSA isolates were multi-drug resistant (6–9 genes) sequence type (ST) 8, clonal complex (CC) 8, SCCmec type IVa (USA300 clone), and were clonally related (0–16 SNP differences), and carried 16–19 virulence factors. The 27 MSSA isolates were grouped into eight CCs and 12 STs. Seventy-eight percent of the MSSA isolates carried 1–5 different antibiotic resistance genes and carried 5–19 virulence factors. We found S. aureus in coastal beach and river waters, anchialine pools, and sand at locations with limited human activity on the island of Hawaiʻi. This may be a public health hazard.

Nationwide Surveillance on Antimicrobial Resistance Profiles of Staphylococcus aureus Isolated from Major Food Animal Carcasses in South Korea During 2010-2018.

Contamination of meat with antimicrobial-resistant bacteria represents a major public health threat worldwide. In this study, we determined the antimicrobial resistance profiles and resistance trends of Staphylococcus aureus isolated from major food animal carcasses (408 cattle, 1196 pig, and 1312 chicken carcass isolates) in Korea from 2010 to 2018. Approximately 75%, 92%, and 77% of cattle, pig, and chicken carcass isolates, respectively, were resistant to at least one antimicrobial agent. Resistance to penicillin (62.1%) was the highest, followed by resistance to tetracycline (42.1%) and erythromycin (28.2%). About 30% of pig and chicken isolates were resistant to ciprofloxacin. We observed linezolid resistance only in pig isolates (2.3%). However, all S. aureus isolates were sensitive to rifampin and vancomycin. We noted an increasing but fluctuating trend of kanamycin and penicillin resistance in cattle isolates. Similarly, the chloramphenicol, ciprofloxacin, tetracycline, and trimethoprim resistance rates were increased but fluctuated through time in pig isolates. Methicillin-resistant S. aureus (MRSA) accounted for 5%, 8%, and 9% of the cattle, pig, and chicken isolates, respectively. The MRSA strains exhibited significantly high resistance rates to most of the tested antimicrobials, including ciprofloxacin, erythromycin, and tetracycline compared with methicillin-susceptible S. aureus (MSSA) strains. Notably, a relatively high percentage of MRSA strains (5.2%) recovered from pig carcasses were resistant to linezolid compared with MSSA strains (2.1%). In addition, almost 37% of the isolates were multi-drug resistant. S. aureus isolates recovered from major food animal carcasses in Korea exhibited resistance to clinically important antimicrobials, posing a public health risk.

[Multirresistência e fatores de virulência de Staphylococcus aureus isolados de suínos]

ABSTRACT The emergence of livestock-associated methicillin-resistant Staphylococcus aureus strains (LA-MRSA) and the potential role of pigs in the evolution of these strains has led to increased interest in research of these microorganisms. However, this has contributed to a lack of research in the isolation and characterization of methicillin-susceptible S. aureus strains (MSSA). In this study, the prevalence of S. aureus in pigs in the nursery and finishing stages were analyzed. The susceptibility profiles to antibiotics, tolerance to heavy metals, and biofilm production of the isolates were evaluated using phenotypic and genotypic techniques. A total of 1,250 colonies suggestive of Staphylococcus spp. were isolated from 128 pigs, of which 63.6% (n = 795) belonged to this microbial genus. Sixty-seven colonies isolated from 34 animals (26.5%) were confirmed as S. aureus (8.4%). No strains resistant to copper, zinc, or methicillin were detected; however, all strains presented a resistance profile to at least three different classes of antimicrobials and 21 produced biofilms. These data are of concern, as they indicate the need for increased surveillance in the use of antimicrobials as well as reinforce the importance of studies on MSSA strains.

Genomic analysis revealed a novel genotype of methicillin-susceptible Staphylococcus aureus isolated from a fatal sepsis case in dengue patient

Staphylococcus aureus (S. aureus) is an opportunistic pathogen capable of causing serious health implications in susceptible individuals once it invades the host’s protective barriers. Methicillin-susceptible S. aureus (MSSA) often receives lesser attention although it has been frequently associated with serious infections in human. We aim to investigate the genomic features of a highly virulent yet pan susceptible MSSA strain (coded as HS-MSSA) which caused concurrent bacteraemia in a dengue patient, ultimately resulted in sepsis death of the patient. Whole genome sequence analysis was performed. The draft genome of HS-MSSA is approximately 2.78 Mb (GC content = 32.7%) comprising of 2637 predicted coding sequences. In silico genotyping of the HS-MSSA strain revealed a novel combined genotype (t091/ST2990). The HS-MSSA carries a SaPIn1-like pathogenicity island that harbours the staphylococcal enterotoxin and enterotoxin-like genes (sec3 and selL). The strain-specific β-lactamase (blaZ)-bearing plasmid region was identified in HS-MSSA. Core genome phylogeny showed that the HS-MSSA strain shared a common ancestry with the European MRSA clone. We report herein the genomic features of an MSSA lineage with novel genotype previously not reported elsewhere.

Evaluation of the Susceptibility of Staphylococcus aureus Strains to Various Antibiotics

Objective: Staphylococcus aureus continues to be a major health problem due to widespread methicillin resistance. The aim of this study was to determine the susceptibility to various antibiotics of 100 methicillin-resistant S. aureus (MRSA) and 100 methicillin-susceptible S. aureus (MSSA) strains isolated from various clinical samples between January 2016 and November 2019. Methods: Gradient test strips were used to test the susceptibilities of isolates to vancomycin, teicoplanin, and linezolid. The susceptibilities to other antibiotics were determined by automated system and evaluated based on EUCAST recommendations. Results: Seventy five percent of MRSA strains and 66% of MSSA strains included in the study were isolated from inpatients and 40% of MRSA and 21% of MSSA strains were isolated from intensive care patients. Isolates were obtained from tissue-abscess, blood, sterile body fluid, deep tracheal aspirate and sputum, urine, and catheter tip cultures, respectively. We did not detect S. aureus resistance to vancomycin, teicoplanin and linezolid in our study. Vancomycin, teicoplanin and linezolid MIC50/MIC90 values for MSSA strains were found to be 1/1.5 µg/ml, 0.75/1.5 µg/mL, 0.75/1 µg/mL and 1.5/2 µg/mL, 1.5/2 µg/mL, 0.75/1.5 µg/mL for MRSA strains, respectively. Based on MIC90 values, linezolid was 1.3 times more effective than vancomycin and 2 times more effective than teicoplanin in MRSA strains; linezolid was 1.5 times more effective than vancomycin and teicoplanin in MSSA strains. MRSA strains showed high resistance rates to all antibiotics, except daptomycin, tigecycline, cotrimoxazole and fusidic acid. All antibiotics were found to be highly effective in MSSA strains. Conclusions: MIC values of glycopeptides in MSSA isolates were found to be close to MIC values of MRSA isolates. Regarding the MIC90 values, linezolid was found to be more effective than glycopeptides. All other antibiotics were highly effective in MSSA strains

The rapid and visual detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus using multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor

BackgroundStaphylococcus aureus (S. aureus), including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), is an eminent human pathogen that can colonize the human host and cause severe life-threatening infections. The development of a reliable, simple and rapid assay for detecting S. aureus and identifying MRSA is important for diagnosis and follow-up treatment.MethodsA novel molecular diagnosis technique, named multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor (m-LAMP-LFB), was applied to detect all S. aureus species and identify MRSA. Two sets of primers were designed based on the femA gene (S. aureus-specific gene) and the mecA gene (encoding penicillin-binding protein 2a), and the multiple-LAMP products were analyzed using LFB. The m-LAMP-LFB amplification conditions, including the target DNA concentration, reaction temperature and time, were optimized. The sensitivity and specificity of the m-LAMP-LFB method were tested in the current study, and the multiple-LAMP-LFB technology was applied to detect the MSSA and MRSA strains from clinical samples.ResultsThe S. aureus- and MRSA-specific primers based on the femA and mecA genes allowed the multiple-LAMP technology to detect S. aureus and MRSA, respectively. The multiple-LAMP conditions were optimized at 63 °C for 40 min. The full process, including genomic DNA template preparation, LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the multiple-LAMP assay for femA and mecA detection was 100 fg of genomic DNA template per reaction. The specificity of m-LAMP-LFB detection was 100 %, and no cross-reactions to non-S. aureus strains were observed.ConclusionThe multiple-LAMP-LFB technique developed in the current study is a reliable, simple, rapid, specific and sensitive method to identify MSSA and MRSA infections for appropriate antibiotic therapy.

Synergistic antimicrobial activity of melittin with clindamycin on the expression of encoding exfoliative toxin in Staphylococcus aureus

Staphylococcus aureus is an opportunistic human pathogens, with the ability to produce a series of virulence factors that contribute to the severity of infections. Exfoliative toxins (ETs) are one of the important virulence factors that participating in staphylococcal scalded skin syndrome. Melittin has different biological activities, comprising of antiviral, broad spectrum antibacterial, antiprotozoal, antifungal and anti-inflammatory effects. Twelve clinical isolates of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) were obtained from wound infection in the burn patients. The MIC plus three sub-inhibitory concentrations (I, II and III) of clindamycin and melittin were tested. Next, the synergistic effects of melittin and clindamycin were evaluated using the broth microdilution checkerboard assay. The detection of exfoliative toxin A and B genes were examined by PCR method. Then the effects of sub-MIC melittin on the expression levels of eta and etb were assessed by quantitative real-time PCR (qRT-PCR) assay. Melittin MIC values against MRSA and MSSA planktonic cells were 0.25–0.5 and 0.25–1 μg/ml, respectively. The clindamycin MIC values against MRSA and MSSA were between 0.5 and 8 μg/ml and 0.5–2 μg/ml, respectively. The results of the time-kill kinetics assay (3.5log10 and 3log10) against MSSA and MRSA planktonic cells were determined within 24 h using melittin. The mean expression of eta in MRSA and MSSA was significantly downregulated to approximately 3.5 and 4 fold, respectively. Moreover, the mean expression of etb in MRSA and MSSA were significantly downregulated to approximately 2.5 and 3 fold, respectively. Hemolytic assay showed that the extracted melittin indicates a strong hemolytic activity (HD50 = 2 μg/ml). Melittin at 0.5 μg/ml induced cell lysis and stimulated the formation of vesicles in S. aureus strains. Melittin could reduce the expression of eta and etb as encoding exfoliative toxin A and B genes. This component appears to be a good candidate for the treatment of MRSA and MSSA strains. So, melittin in combination with clindamycin can be classified as a complementary treatment of wound infections in burn patients.