Virulence, agr groups, antimicrobial resistance and epidemiology of Staphylococcus aureus isolated from bovine subclinical mastitis

Fifty-two Staphylococcus aureus recovered from papillary ostium and milk samples collected from cows with subclinical mastitis and milking environments in three small dairy herds located in southeastern Brazil were subjected to PCR identification based on the thermonuclease (nuc) gene. All the strains were submitted to in vitro antimicrobial susceptibility testing, and we investigated the sequence types (STs), agr groups (I-IV), virulence genes encoding for Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), biofilm-associated proteins, bi-component toxins, pyrogenic toxin superantigens, and enterotoxins. Screening for oxacillin resistance (2-6 μg/ml oxacillin), beta-lactamase activity assays, and PCR for the mecA/mecC genes detected 26 methicillin-susceptible S. aureus (MSSA) and 26 mec-independent oxacillin-nonsusceptible S. aureus (MIONSA). While MSSA isolates were found to be susceptible to all antimicrobial agents tested, or only resistant to penicillin and ampicillin, MIONSA isolates were multidrug-resistant. ST126-agr group II MSSA isolates were prevalent in milk (n=14) and carried a broad set of virulence genes (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla, and hlb), as well as the ST126-agr group II MIONSA isolated from milking liners (n=1), which also carried the eta gene. ST1-agr group III MIONSA isolates (n=4) were found in papillary ostium and milk, but most MIONSA isolates (n=21), which were identified in both papillary ostium and milking liners, were agr-negative and assigned to ST126. The agr-negative and agr group III lineages showed a low potential for virulence. Studies on the characterization of bovine-associated MSSA/MIONSA are essential to reduce S. aureus mastitis to prevent economic losses in dairy production and also to monitor the zoonotic potential of these pathogens associated with invasive infections and treatment failures in healthcare.

Molecular Epidemiology, Virulence Factors, Antibiotic Resistance and Risk Factors for Nasal Carriage of Staphylococcus aureus in A Teenage Student Population: High Prevalence of Oxacillin Susceptible MRSA Isolates

Background: Asymptomatic carriage of Staphylococcus aureus can lead to endogenous infections and cross-transmission to other individuals. Objectives: The prevalence, molecular epidemiology, antibiotic resistance, and risk factors for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) were studied in school children in Ardabil, Iran. Methods: Totally, 510 nasal samples were collected during 2017. Isolates were identified and subjected to antimicrobial susceptibility testing, identification of oxacillin resistance, and molecular typing. Results: Totally, 13.5% of volunteers were positive for methicillin-susceptible Staphylococcus aureus (MSSA) and 17.5% colonized with mecA positive S. aureus strains, including 6.07% oxacillin-resistant MRSA (OR-MRSA) and 11.56% oxacillin-susceptible MRSA (OS-MRSA). Excluding β-lactam antibiotics, high resistance rate was observed for erythromycin (71%), tetracycline (25.8%), clindamycin (35%) in our isolates. Surprisingly, 11% of the isolates [OR-MRSA (25.8%), OS-MRSA (10.1%), and MSSA (5.7%) isolates] were resistant to mupirocin. Moreover, 18 (58%), 29 (49%), and 29 (42%) of OR-MRSA, OS-MRSA, and MSSA isolates were multidrug-resistant (MDR), respectively. Overall, 97.48% of isolates carried ≥ 3 toxin encoding genes. The pvl gene was found in 46 (29%) isolates. In comparison, 25.50% of MRSA (9.60% OR-MRSA and 34% OS-MRSA) and 33% of MSSA isolates carried pvl gene. SCCmec type IV had the highest rate among OR-MRSA (87%) and OS-MRSA (74.5%) isolates, which indicates CA-MRSA phenotype. Eleven and 21 spa types were identified in OR-MRSA, and OS-MRSA isolates, respectively. The most common spa types were t11332 (14.3%) and t012 (11.4%) in OS-MRSA isolates. ERIC-PCR revealed high genetic diversity among isolates. The number of students in classroom and incomplete antibiotic course were associated with OS-MRSA nasal carriage. Conclusions: This study showed a high proportion of MDR CA-MRSA nasal carriage among Iranian healthy school children community.

194. Antimicrobial Susceptibility Data of Staphylococcus lugdunensis After the Implementation of Rapid Molecular Blood Culture Diagnostics (Verigene® Gram-Positive Blood Culture Nucleic Acid Test)

Background S. lugdunensis is a coagulase negative staphylococci (CoNS) demonstrating high level pathogenicity. In contrast to other CoNS, S. lugdunensis (SL) remains susceptible to most antibiotics. Prior to the implementation of Verigene®, SL was identified by provider request only. We sought to describe the susceptibility data of SL isolated from blood culture after the implementation of multiplex PCR, as well as to determine the correlation of the mecA gene provided by Verigene® and oxacillin resistance. Methods Retrospective review of all blood culture isolates positive for SL from two major hospital systems, Memorial Hermann Hospital System (14 hospitals) and HarrisHealth System (two acute care hospitals) identified on Verigene® PCR. Multiple isolates detected from the same patients were excluded from this analysis. Memorial Hermann utilized Microscan®, and HarrisHealth utilized BD Phoenix® for susceptibility testing. Results Between 2017 – 2021, 157 patients were identified with SL positive blood cultures. Of them, 141 isolates had susceptibility data collected, which is summarized in table 1. Resistance rates were highest amongst clindamycin 97/141 (68.8 %) susceptible, erythromycin 98/141 (69.5%) susceptible, and oxacillin 120/141 (85.1%) susceptible. 127/141 (90.1%) of isolates were tested for mecA on Verigene®. 13 of 21 oxacillin resistant isolates were from pure culture, of these isolates, none had mecA detected. Conclusion In our study, clindamycin and erythromycin demonstrated similar susceptibility compared to prior studies in the literature, however oxacillin susceptibility rate was lower than expected at 85.1%, compared to 95.3% in a prior large-scale United States based study in 2017. Absence of mecA gene detection on multiplex PCR did not correlate with oxacillin susceptibility suggesting that oxacillin susceptibility cannot be accurately predicted by the use of multiplex PCR systems, such as Verigene®, as demonstrated in Table 2. Our study also suggested that increased prevalence of oxacillin resistant SL isolates may be emerging. Disclosures All Authors: No reported disclosures

Antimicrobial Susceptibility Testing for Staphylococcus lugdunensis

Evaluation of penicillin and oxacillin susceptibility testing was conducted on two hundred Staphylococcus lugdunensis isolates. Disc diffusion with penicillin 1 IU (P1, EUCAST) and penicillin 10 IU (P10, CLSI) was compared with nitrocefin discs (Cefinase®) and automated broth microdilution (Vitek2®). Oxacillin susceptibility was extrapolated from cefoxitin 30μg disc diffusion (FOX) and compared with Vitek2®. Reference methods were blaZ and mecA PCR. Penicillin zone diameter and zone edge correlated with blaZ in all except two P10 susceptible isolates (VME; very major error) and one P1 resistant isolate (ME). One hundred and forty-eight isolates were blaZ -negative of which one hundred and forty-six and one hundred and forty-nine isolates were susceptible by P1 and P10 respectively. One hundred and twenty-seven isolates were penicillin susceptible by Vitek2®. Vitek2® overcalled resistance in twenty-one blaZ -negative, twenty P1 and twenty-two P10 susceptible isolates (Vitek2® ME rate, 14.2%). Two mecA -positive isolates were oxacillin resistant by FOX and Vitek2® (categorical agreement). However, eighteen FOX susceptible, mecA -negative isolates tested resistant by Vitek2®. In conclusion, Vitek2® over-estimated penicillin and oxacillin resistance compared with disc diffusion and PCR. Disc diffusion with zone edge interpretation was more accurate and specific than automated broth microdilution for S. lugdunensis in our study.

Impact of PrsA on membrane lipid composition during daptomycin-resistance-mediated β-lactam sensitization in clinical MRSA strains

Background The cyclic anionic lipopeptide daptomycin is used in the treatment of severe infections caused by Gram-positive pathogens, including MRSA. Daptomycin resistance, although rare, often results in treatment failure. Paradoxically, in MRSA, daptomycin resistance is usually accompanied by a concomitant decrease in β-lactam resistance in what is known as the ‘see-saw effect’. This resensitization is extensively used for the treatment of MRSA infections, by combining daptomycin and a β-lactam antibiotic, such as oxacillin. Objectives We aimed: (i) to investigate the combined effects of daptomycin and oxacillin on the lipid composition of the cellular membrane of both daptomycin-resistant and -susceptible MRSA strains; and (ii) to assess the involvement of the post-translocational protein PrsA, which plays an important role in oxacillin resistance in MRSA, in membrane lipid composition and remodelling during daptomycin resistance/β-lactam sensitization. Results The combination of microbiological and biochemical studies, with fluorescence microscopy using lipid probes, showed that the lipid composition and surface charge of the daptomycin-resistant cells exposed to daptomycin/oxacillin were dependent on antibiotic concentration and directly associated with PrsA, which influenced cardiolipin remodelling/relocation. Conclusions Our findings show that PrsA, in addition to its post-transcriptional role in the maturation of PBP 2a, is a key mediator of cell membrane remodelling connected to the see-saw effect and may have a key role in the resensitization of daptomycin-resistant strains to β-lactams, such as oxacillin.

Improved Detection of mecA-Mediated β-Lactam Resistance in Staphylococcus lugdunensis Using a New Oxacillin Salt Agar Screen

Oxacillin resistance mediated by mecA in Staphylococcus lugdunensis is emerging in some geographic areas. We evaluated cefoxitin disk diffusion (DD) and a new oxacillin agar (supplemented with 2 μg/ml oxacillin and 2% sodium chloride) screen for the detection of mecA-mediated resistance in S. lugdunensis. A total of 300 consecutive, non-duplicated clinical S. lugdunensis isolates from diverse sources in Hong Kong in 2019 were tested. The categorical agreement and errors obtained between cefoxitin DD test, oxacillin agar screen and mecA PCR were analyzed. Isolates with discordant results were further tested by MIC, penicillin binding protein 2a (PBP2a) assays, population analysis and molecular typing. PCR showed that 62 isolates were mecA-positive and 238 isolates were mecA-negative. For cefoxitin DD results interpreted using S. aureus/S. lugdunensis breakpoints, the categorical agreement (CA) for two brands of Muller-Hinton agars, MH-II (Becton Dickinson) and MH-E (bioMérieux) were both 96.0%; MEs were both 0%; and VMEs were 19.4 and 12.9%, respectively. The new oxacillin agar reliably differentiated mecA-positive and mecA-negative isolates (100% CA) without any ME or VME results. The 8 isolates with false susceptibility in the cefoxitin DD testing had cefoxitin and oxacillin MICs in the susceptible range. The isolates showed heterogeneous oxacillin resistance with resistant subpopulations at low frequencies. All had positive PBP2a results and were typed as sequence type 27/SCCmec V. The findings highlight the inability of cefoxitin DD and MIC tests for reliable detection of some mecA-positive S. lugdunensis isolates.

Molecular fingerprinting of bovine mastitis-associated Staphylococcus aureus isolates from India

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. Owing to the mostly backyard dairy practices, we hypothesized that genetic diversity among mastitis-associated S. aureus from India would be high, and investigated 166 isolates obtained mostly from the Southern State of Karnataka, but also from a few other states. The results revealed (a) 8 to 13 fragments in pulsed-field gel electrophoresis (PFGE), forming 31 distinct patterns, and (b) 34 spa types, of which three (t17680, t18314, and t18320) were newly identified. Multi-locus sequencing typing (MLST) identified 39 sequence types (STs), with ST2454 (34.4%) and ST2459 (24%) being the most commonly represented, which clustered to clonal complexes (CC) CC9 and CC97, respectively; 12 STs were newly identified. Thirty-four (20.5%) of the 166 isolates displayed oxacillin resistance. On the other hand, whereas none were mecC+, 44 (26.5%) isolates were mecA+, with a predominance of SCCmecIVb (26/32 isolates, others being untypeable); 24 isolates (14.46%) were oxacillin-susceptible methicillin-resistant S. aureus (OS-MRSA; mecA+ but OS). Integrated analysis revealed that CC9-ST2454- and CC97-ST2459-SCCmecIVb were the predominant MRSA, although the distribution of CC9 and CC97 was similar between methicillin-resistant and -susceptible isolates. By PCR, 56.25%, 28.75% and 47.5% of the 166 isolates were positive for hlg, tsst and pvl genes, respectively. Our results, for the first time describe the application of a combination of various molecular methods to bovine mastitis-associated S. aureus isolates from India, corroborate the worldwide distribution of CC97 and CC9, and suggest pathogenic potential of the isolates.

Nontargeted Metabolomics Analysis of Oxacillin Resistance in Staphylococcus Aureus

BackgroundStaphylococcus aureus has acquired resistance to antibiotics in the long-term struggle against antibiotics. Treatment of Staphylococcus aureus infection has become more difficult. In this study, based on nontargeted metabolic figure printing technique, the metabolome of a pair of isogenic methicillin-susceptible and resistant Staphylococcus aureus (MSSA and MRSA) strains treated with the sublethal dose of oxacillin was characterize to investigate the mechanism of antibiotic resistance.ResultsMassive alternations of metabolite expression were observed in both MSSA and MRSA treated with oxacillin. The results of accurate mass and mass fragmentation analysis showed that 7 and 29 metabolites of MRSA and MSSA have changed significantly after oxacillin treatment. The dysregulated metabolites suggested that CoA and fatty acids could help Staphylococcus aureus survive under antibiotic stress. Metabolic pathways engaged in antibiotic resistance were discovered through pathway enrichment analysis. The enriched pathways suggested that DNA repairing and flavin biosynthesis are universal pathways to help MSSA and MRSA survive under antibiotic stress. Compared with MSSA, MRSA systematically and effectively fight against oxacillin through precisely controlling energy producing, PBP2a substrate biosynthesis and antioxidant function. ConclusionsCoenzyme A and fatty acids help both MSSA and MRSA survive under the antibiotic stress. MSSA was susceptible to oxacillin and was forced to response. On the contrary, MRSA systematically and effectively fight against oxacillin. The different metabolome responses of MSSA and MRSA provide us with new insights into how Staphylococcus aureus develops antibiotics resistance.

Improved diagnostic prediction of the pathogenicity of bloodstream isolates of Staphylococcus epidermidis

With an estimated 440,000 active cases occurring each year, medical device associated infections pose a significant burden on the US healthcare system, costing about $9.8 billion in 2013. Staphylococcus epidermidis is the most common cause of these device-associated infections, which typically involve isolates that are multi-drug resistant and possess multiple virulence factors. S. epidermidis is also frequently a benign contaminant of otherwise sterile blood cultures. Therefore, tests that distinguish pathogenic from non-pathogenic isolates would improve the accuracy of diagnosis and prevent overuse/misuse of antibiotics. Attempts to use multi-locus sequence typing (MLST) with machine learning for this purpose had poor accuracy (~73%). In this study we sought to improve the diagnostic accuracy of predicting pathogenicity by focusing on phenotypic markers (i.e., antibiotic resistance, growth fitness in human plasma, and biofilm forming capacity) and the presence of specific virulence genes (i.e., mecA, ses1, and sdrF). Commensal isolates from healthy individuals (n = 23), blood culture contaminants (n = 21), and pathogenic isolates considered true bacteremia (n = 54) were used. Multiple machine learning approaches were applied to characterize strains as pathogenic vs non-pathogenic. The combination of phenotypic markers and virulence genes improved the diagnostic accuracy to 82.4% (sensitivity: 84.9% and specificity: 80.9%). Oxacillin resistance was the most important variable followed by growth rate in plasma. This work shows promise for the addition of phenotypic testing in clinical diagnostic applications.