The impact of methicillin resistance on clinical outcome among patients with Staphylococcus aureus osteomyelitis: a retrospective cohort study of 482 cases.

This study was designed to evaluate the impact of methicillin resistance on the outcomes among patients with S. aureus osteomyelitis. We reviewed all extremity osteomyelitis patients treated in our clinic center between 2013 and 2020. All adult patients with S. aureus pathogen infection were included. Clinical outcome in terms of infection control, length of hospital stay, and complications were observed at the end of a 24-month follow-up and retrospectively analyzed between populations with/without methicillin resistance. In total, 482 osteomyelitis patients due to S. aureus were enrolled. The proportion of methicillin-resistant S. aureus (MRSA) was 17% (82) and 83% (400) of patients had Methicillin-sensitive S. aureus (MSSA). Of 482 patients, 13.7% (66) presented with infection persistence after initial debridement and antibiotic treatment (6 weeks), needed repeated debridement, 8.5% (41) had recurrence after all treatment end and a period infection cure, complications were observed in 17 (3.5%) patients (pathologic fracture; 4, nonunion; 5, amputation; 8) at final follow-up. Following multivariate analysis, we found patients with S. aureus osteomyelitis due to MRSA are more likely to develop a persistent infection (OR: 2.26; 95% CI 1.24-4.13) compared to patients with MSSA. Patients infected with MRSA also suffered more complications (8.5% vs. 2.5%, p = 0.015) and longer hospital stays (median: 32 vs. 23 days, p < 0.001). No statistically significant differences were found in recurrence. The data indicated Methicillin resistance had adverse clinical implication for infection persistence among patients with S. aureus osteomyelitis. These results will help for patients counsel and preparation for treatment.

Community acquired methicillin resistant Staphylococci (CA-MRS) in fecal matter of wild birds - A 'one health' point of concern.

BACKGROUND: Antibiotic resistance in Staphylococci, particularly methicillin resistance is a major public health concern. While this problem has been reported from the clinical settings, its presence in non-clinical settings also needs to be investigated. The role of wildlife in carrying and disseminating the resistant strains has been established in different studies but its role in Pakistani environment has not been explored yet. To evaluate this, we investigated the carriage of antibiotic resistant Staphylococci in wild birds from Islamabad region. METHODOLOGY: Birds fecal matter were collected during September 2016-August 2017 from eight different environmental settings of Islamabad. Prevalence of Staphylococci, their susceptibility profile against eight classes of antibiotics through disc diffusion method, their SCCmec types, co-resistance of macrolide and cefoxitin through PCR assay and biofilm formation through microtitre plate assay were studied. RESULTS: Out of 320 birds feces collected, 394 Staphylococci were isolated, where 165 (42%) were resistant to at least one or two classes of antibiotics. High resistance was found against erythromycin (40%) and tetracycline (21%) while cefoxitin resistance was 18% and vancomycin resistance was only in 2%. One hundred and three (26%) isolates exhibited multi-drug resistance (MDR) pattern. mecA gene was detected in 45/70 (64%) cefoxitin resistant isolates. Community acquired methicillin resistant Staphylococci (CA-MRS) were 87% while Hospital acquired methicillin resistant Staphylococci (HA-MRS) were 40%. In the MRS isolates showing co-resistance to macrolides, mefA (69%) and ermC (50%) genes were more prevalent. Strong biofilm formation was observed in 90% of the MRS, of which 48% were methicillin resistant Staphylococcus aureus (MRSA) isolates while 52% were methicillin resistant coagulase negative Staphylococci (MRCoNS). CONCLUSION: Occurrence of methicillin resistant strains of Staphylococci in wild birds suggests their role in the carriage and dissemination of resistant strains into the environment. The findings of the study strongly recommend the monitoring of resistant bacteria in wild birds and wildlife.

Untargeted proteomic differences between clinical strains of methicillin-sensitive and methicillin-resistant Staphylococcusaureus.

Staphylococcus aureus is a common disease-causing bacterium that has developed resistances to a wide variety of antibiotics. This increasing antibiotic resistance has made management of these infections difficult. A better understanding of the general differences among clinical S. aureus strains beyond the well characterized resistance mechanisms may help in identifying new anti-microbial targets. This study aimed to identify and compare the general differences in protein profiles among clinical strains of S. aureus sensitive and resistant to methicillin. The proteomic profiles of five methicillin sensitive (MSSA) and five methicillin resistant (MRSA) S. aureus strains were analyzed by ultra-performance liquid chromatography-mass spectrometry. Protein identification was done using Progenesis QI for Proteomics and the UniProt S. aureus database. Proteins that play roles in virulence, metabolism, and protein synthesis were found to be present at different abundances between MSSA and MRSA (Data available via ProteomeXchange with identifier PXD021629). This study shows differences in protein profiles between antibiotic sensitive and antibiotic resistant clinical strains of S. aureus that may affect the resistance mechanism. Further research on these differences may identify new drug targets against methicillin resistant S. aureus strains.

Decrease in vancomycin MICs and prevalence of hGISA in MRSA and MSSA isolates from a German pediatric tertiary care center.

PURPOSE: Resistance of Staphylococcus aureus to vancomycin includes a general increase of minimal inhibitory concentrations (MIC) within the susceptible range over time (Vancomycin MIC Creep) and the presence of a subset of the bacterial population that expresses resistance (heterogeneous glycopeptide-intermediate S. aureus; hGISA). Increased MICs have been associated with adverse clinical outcomes. However, the vancomycin MIC creep is not a uniform trend suggesting the importance of regional surveys. METHODS: We performed a retrospective analysis at a German pediatric tertiary care hospital. Isolates from 2002 to 2017 were selected which were newly identified methicillin-resistant S. aureus (MRSA) or samples from invasive methicillin-susceptible S. aureus (MSSA) or MRSA infections. Vancomycin and oxacillin MICs as well as GISA/hGISA were measured using MIC test strips and resistance was evaluated over time. RESULTS: A total of 540 samples were tested, 200 from the early (2002-2009) and 340 from the later period (2010-2017). All samples were vancomycin susceptible, but the MIC was higher for the earlier samples as compared to the later ones (1.11 vs 0.99; p < 0.001). 14% of the samples were hGISA, GISA strains were not detected. Again, vancomycin resistance decreased over time with 28 vs. 6% hGISA (p < 0.001). There was no significant difference between MRSA and MSSA samples with respect to vancomycin MIC and hGISA prevalence. CONCLUSION: This study shows a decreasing trend for both MIC values and presence of hGISA strains highlighting the importance of monitoring local susceptibilities. Vancomycin remains a first-line treatment option for suspected severe infection with Gram-positive cocci and proven infection with MRSA.

A signal-off Cas14a1-based platform for highly specific detection of methicillin-resistant Staphylococcus aureus.

Antibiotic usage has become very widespread in aquaculture, and the abuse or overuse of antibiotics has led to the evolution of antibiotic-resistance bacteria, which has adverse effects on aquatic products and ecosystems. Moreover, this evolution can potentially cause harm to human health. Thus, there is an urgent need for diagnostic tools for antibiotic-resistant microorganisms. Herein, we proposed a signal-off Cas14a1-based platform (SOCP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In this SOCP, we have designed single-stranded DNA (ssDNA) that not only can activate the trans-cleavage ability of dual Cas14a1-sgRNA complex but also can be used as the primers for the amplified methicilin-resistant gene (mecA). When MRSA is present, the primers can be transformed into products with amplification, leading to the signal decrease of trans-cleavage activity of Cas14a1. The SOCP showed high specificity and fair sensitivity for mecA gene and MRSA. In the detection of real samples, this platform also showed consistent results compared with qPCR. The SOCP could provide an alternative tool for the diagnosis of antibiotic-resistant bacteria in aquaculture, food industry and other fields.

Intra-wound versus systemic vancomycin for preventing surgical site infection induced by methicillin-resistant S. aureus after spinal implant surgery in a rat model.

BACKGROUND: Systemic vancomycin administration pre-operatively for the infection prophylaxis of spinal implant surgery remains unsatisfactory. This study aimed to explore the efficacy and dosage of local use of vancomycin powder (VP) in preventing surgical site infections after spinal implant surgery in a rat model. METHODS: Systemic vancomycin (SV; intraperitoneal injection, 88 mg/kg) or intraoperative intra-wound VP (VP0.5: 44 mg/kg, VP1.0: 88 mg/kg, VP2.0: 176 mg/kg) was applied after spinal implant surgery and methicillin-resistant S. aureus (MRSA; ATCC BAA-1026) inoculation in rats. General status, blood inflammatory biomarkers, microbiological and histopathological evaluation were performed during 2 weeks post-surgery. RESULTS: No post-surgical deaths, wound complications and obvious signs of vancomycin adverse effects were observed. Bacterial counts, blood and tissue inflammation were reduced in the VP groups compared with the SV group. VP2.0 group showed better outcomes in weight gain and tissue inflammation than the VP0.5 and VP1.0 group. Microbial counts indicated that no bacteria survived in the VP2.0 group, whereas MRSA was detected in VP0.5 and VP1.0 groups. CONCLUSIONS: Intra-wound VP may be more effective than systemic administration in preventing infection caused by MRSA (ATCC BAA-1026) after spinal implant surgery in a rat model.

An epidemiological study of the predictors of multidrug resistance and methicillin resistance among Staphylococcus spp. isolated from canine specimens submitted to a diagnostic laboratory in Tennessee, USA.

Background: Understanding drivers of multidrug resistance (MDR) and methicillin resistance, which have increased among canine staphylococcal isolates, is essential for guiding antimicrobial use practices. Therefore, the objective of this study was to identify predictors of MDR and methicillin resistance among Staphylococcus spp. commonly isolated from canine clinical specimens. Methods: This retrospective study used records of canine specimens submitted to the University of Tennessee College of Veterinary Medicine Clinical Bacteriology Laboratory for bacterial culture and antimicrobial susceptibility testing between 2006 and 2017. Records from 7,805 specimens positive for the following Staphylococcus species were included for analysis: Staphylococcus pseudintermedius, Staphylococcus aureus, Staphylococcus coagulans (formerly Staphylococcus schleiferi subspecies coagulans), and Staphylococcus schleiferi (formerly S. schleiferi subsp. schleiferi). Generalized linear regression models were fit using generalized estimating equations (GEE) to identify predictors of MDR (defined as resistance to three or more antimicrobial classes) and methicillin resistance among these isolates. Results: Multidrug resistance (42.1%) and methicillin resistance (31.8%) were relatively common. Isolates from skeletal (joint and bone) specimens had the highest levels of MDR (51.3%) and methicillin resistance (43.6%), followed by cutaneous specimens (45.8% multidrug-resistant, 37.1% methicillin resistant). Staphylococcus species, specimen site, and clinical setting were significant (p < 0.01) predictors of both outcomes. Compared to S. pseudintermedius, S. schleiferi had higher odds of methicillin resistance, while S. coagulans and S. schleiferi had lower odds of MDR. The odds of both MDR and methicillin resistance for isolates from hospital patient specimens were significantly higher than those from referral patients for urine/bladder and otic specimens. Odds of MDR among isolates from skeletal specimens of hospital patients were also higher than those of referral patients. Conclusions: Staphylococcus isolates in this study had substantial levels of MDR and methicillin resistance. Differences in the odds of these outcomes between referral and hospital patient isolates did not persist for all specimen sites, which may reflect differences in diagnostic testing and antimicrobial use practices with respect to body site or system. Judicious antimicrobial use, informed by culture and susceptibility testing, is important to limit treatment failures and curb selection pressure.

Evaluation of the performance of GeneSoC®, a novel rapid real-time PCR system, to detect Staphylococcus aureus and methicillin resistance in blood cultures.

Staphylococcus aureus bacteremia results in substantial mortality. Rapid identification and the determination of methicillin susceptibility are crucial for immediate treatment with appropriate antibiotics. In the present study, we aimed to evaluate the basic assay performance of GeneSoC®, a novel rapid quantitative polymerase chain reaction (qPCR) method, for the detection of methicillin-susceptible (MS) or -resistant (MR) S. aureus in blood culture (BC) bottles. qPCR pimers and probes were desinged for femA and mecA genes to diagnose S. aureus and its methicilline-resistance status. GeneSoC® system can detect target genes within 12 min per sample using microfludic thermal cycling. A total of 100 BC-positive samples, showing clusters of gram-positive cocci using microscopy, were tested. The analytical sensitivity was demonstrated for the target sequence of femA and mecA genes at 10 copies/µL, respectively. The detection limit of the MRSA bacterial burden using this system was 104 and 103 CFU/mL for femA and mecA, respectively. Compared with culture-based identification and susceptibility testing, the sensitivity and specificity for the detection of femA (+)/mecA (+) MRSA using GeneSoC® were 90.9 and 98.9%, respectively, whereas the sensitivity and specificity for detection of femA (+)/mecA (-) MSSA were 96.2% and 97.3%, respectively. In conclusion, although this was a small sample and pilot study, the GeneSoC® system is beneficial for rapid, reliable, and highly sensitive real-time testing of MRSA and MSSA in BC bottles.

Sensitivity of the PBP2a SA Culture Colony Test on shortly incubated subcultures of methicillin-resistant staphylococci from positive blood cultures.

The sensitivity of an immunochromatographic assay for detecting methicillin resistance (PBP2a SA Culture Colony Test, Alere-Abbott) on shortly incubated subcultures of staphylococci in blood cultures was evaluated. The assay is highly sensitive for the detection of methicillin-resistant Staphylococcus aureus after 4 hour-subculture but requires 6 hour-incubation for methicillin-resistant coagulase-negative staphylococci.

Virulent potential of methicillin-resistant and methicillin-susceptible Staphylococcus pseudintermedius in dogs.

Staphylococcus pseudintermedius is a zoonotic pathogen responsible for several infectious diseases in pet animals, yet its pathogenic potential is not fully understood. Thus, this study aims to unravel the virulence profile of S. pseudintermedius from canine origin. Methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains were isolated from different infection sites and their genotypic and phenotypic features were compared to determine the clinical implications of MRSP and MSSP strains. Bacterial identification was performed using MALDI-TOF and 16S-rDNA sequencing. In addition, we used multilocus sequence typing (MLST) for strains' sequence type (ST) determination and phylogenetic relationship. The strains were screened for toxin genes, including cytotoxins (lukS, lukF), exfoliative toxin (siet), enterotoxins (sea, seb, sec, secCanine, sel, sem, and seq) and toxic shock syndrome toxin (tst-1). In vitro phenotypic analyses assessing antimicrobial susceptibility profile, biofilm formation ability, and expression of extracellular matrix components were performed. The investigated S. pseudintermedius strains belong to 17 unique ST, most of which were classified as ST71. MSSP and MRSP strains shared siet, lukS, and lukF virulence markers. Our findings showed that some MSSP strains also harbored sel, seq, and sem enterotoxin genes, suggesting a more diverse virulence profile. All MRSP strains and 77% of MSSP strains were classified as multidrug resistant (MDR). Moreover, all investigated S. pseudintermedius strains showed strong biofilm formation ability. In summary, our findings highlight the wide spread of highly virulent and drug-resistant zoonotic S. pseudintermedius strains, being a potential concern for One Health issues.

Nasal microbiota profiles in shelter dogs with dermatological conditions carrying methicillin-resistant and methicillin-sensitive Staphylococcus species.

Dermatological conditions may be complicated by Staphylococcus spp. infections influencing skin and nasal microbiota. We investigated the associations between the resident nasal microbiota of shelter dogs with and without dermatological conditions carrying methicillin-resistant and -sensitive Staphylococcus spp. Nasal sampling of 16 dogs with and 52 without dermatological conditions were performed upon shelter admission (baseline), and then bi-weekly until discharge (follow-up). All samples were cultured for Staphylococcus spp., while 52 samples underwent microbiota analysis. Two elastic net logistic regression (ENR) models (Model 1-baseline samples; Model 2-follow-up samples) were developed to identify predictive associations between dermatological conditions and the variables: signalment, antimicrobial treatment, and nasal microbial genera. Follow-up nasal samples of dogs with dermatological conditions had decreased microbiota diversity and abundance compared to dogs without dermatological conditions. Our ENR models identified predictive differences in signalment and nasal microbial genera between baseline and follow-up samples. Co-occurrence networks showed nasal microbial genera were more dissimilar when comparing dogs with and without dermatological conditions at follow-up. Overall, this study is the first to investigate Staphylococcus spp. carriage effects on nasal microbial genera in a canine animal shelter population, and ultimately reveals the importance of investigating decolonisation and probiotic therapies for restoring nasal microbiota.

Nafcillin Augmentation of Daptomycin and Cathelicidin LL-37 Killing of Methicillin-resistant Staphylococcus epidermidis: Foundations of Successful Therapy of Endocarditis.

Methicillin-resistant Staphylococcus epidermidis (MRSE) endocarditis failing conventional therapy has been successfully treated with nafcillin plus daptomycin in the clinic. In vitro studies showed that nafcillin enhanced daptomycin killing of MRSE in both planktonic cells and biofilm. Nafcillin exposure also sensitized MRSE to killing by human neutrophils and cathelicidin antimicrobial peptide LL-37. Fluorescent microscopy showed increased daptomycin and LL-37 binding to the MRSE bacterial surface upon nafcillin treatment. Ceftaroline also increased MRSE killing by daptomycin in planktonic cultures and biofilms, as well as daptomycin and LL-37 binding on the bacterial surface. Nafcillin, ceftaroline, and possibly other ß-lactams, may serve an important role in the therapy of MRSE endocarditis through augmentation of cationic peptide, the innate immune system, and daptomycin killing. Clinical studies will be needed to determine how early these regimens should be deployed to optimize clinical outcome.

Occurrence of virulence genes and methicillin-resistance in Staphylococcus aureus isolates causing subclinical bovine mastitis in Tiaret area, Algeria.

Mastitis remains the most frequent and the most expensive disease of dairy breeding. The objective of the study was to study S. aureus isolates collected from subclinical bovine mastitis in the Tiaret region, Algeria, by determining their antimicrobial susceptibility and their virulence traits. Sixty-two S. aureus isolates collected from subclinical bovine mastitis were studied by determining their antimicrobial susceptibility according to CLSI guidelines, and nine genes encoding virulence factors and resistance to methicillin and penicillin were determined by PCR. Multi-drug resistance was observed in 19 (30.64%) isolates and five (8%) were methicillin-resistant S. aureus (MRSA), four of them harbored the mecA gene; however, the mecC gene was not detected. Out of 59 penicillin-resistant isolates, 14 harbored the blaZ gene; one of them co-harbored the mecA gene. The following virulence genes were detected: eta (n = 23; 37%), icaA (20; 32.2%), icaD (18; 29%), etb (16; 25.8%), luk E-D (14; 22.5%), and sea (6; 9.6%). The tsst-1, lukF/lukS, and luk-M genes were not detected. The occurrence of MRSA and multi-drug resistant (MDR) S. aureus isolates as well as genes encoding virulence factors playing an important role in the pathogenesis of subclinical bovine mastitis and of harmful potential to human is a cause for concern.

MRSA Isolates from Patients with Persistent Bacteremia Generate Nonstable Small Colony Variants In Vitro within Macrophages and Endothelial Cells during Prolonged Vancomycin Exposure.

Staphylococcus aureus (especially methicillin-resistant S. aureus [MRSA]) is frequently associated with persistent bacteremia (PB) during vancomycin therapy despite consistent susceptibility in vitro. Strategic comparisons of PB strains versus those from vancomycin-resolving bacteremia (RB) would yield important mechanistic insights into PB outcomes. Clinical PB versus RB isolates were assessed in vitro for intracellular replication and small colony variant (SCV) formation within macrophages and endothelial cells (ECs) in the presence or absence of exogenous vancomycin. In both macrophages and ECs, PB and RB isolates replicated within lysosome-associated membrane protein-1 (LAMP-1)-positive compartments. PB isolates formed nonstable small colony variants (nsSCVs) in vancomycin-exposed host cells at a significantly higher frequency than matched RB isolates (in granulocyte-macrophage colony-stimulating factor [GM-CSF], human macrophages PB versus RB, P < 0.0001 at 48 h; in ECs, PB versus RB, P < 0.0001 at 24 h). This phenotype could represent one potential basis for the unique ability of PB isolates to adaptively resist vancomycin therapy and cause PB in humans. Elucidating the molecular mechanism(s) by which PB strains form nsSCVs could facilitate the discovery of novel treatment strategies to mitigate PB due to MRSA.

Precise Photothermal Treatment of Methicillin-Resistant S. aureus Infection via Phage Lysin-Cell Binding Domain-Modified Gold Nanosheets.

The increasing spread of antibiotic resistance in bacterial pathogens poses a huge threat to global human health. Precise targeting of bacterial pathogens while avoiding collateral damage to healthy tissues has become the overriding goal for bacterial infection treatment. Inspired by the host specificity of bacteriophages, a biomimetic intelligent platform was designed for highly precise photothermal treatment herein. As proof-of-concept, the lysin cell-binding domain (CBD) from a newly discovered virulent methicillin-resistant S. aureus (MRSA) phage Z was applied to the functionalization of gold nanosheets. Our results demonstrated that the bionanocomposite gold particles (Au@PEG-CBDz) could be effectively delivered directly to MRSA and kill them effectively under near infrared irradiation in vitro, while displaying good in vivo biocompatibility. This work is the first to report the combination of phage lysin navigatory function with photothermal effect-induced bactericidal activity from Au nanosheets, providing a novel therapeutic mode for the precision treatment of antibiotic-resistant bacterial infections.

Novel cassette chromosome recombinases CcrA8B9 catalyse the excision and integration of the staphylococcal cassette chromosome mec element.

OBJECTIVES: A defining feature of MRSA is the SCCmec element. The excision and integration of SCCmec elements are catalysed by Ccr recombinases. Currently, seven ccrA, eight ccrB and two ccrC allotypes have been described. However, there have been no recent reports of a novel Ccr recombinase and thus this area should be explored. METHODS: According to the proposed criteria of the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) committee, novel ccr genes were explored by searching the genome of our laboratory staphylococcal strains, which were isolated from bovine mastitis in Northwest China. The biological activity of the novel Ccr recombinases to excise and integrate SCCmec elements was determined. The distribution of the novel ccr genes in staphylococci was conducted by querying the NCBI nr/nt database. RESULTS: We report a set of novel Ccr recombinases CcrA8B9, which share nucleotide identities of 46.6%-50.2% and 47.4%-52.8% with the ccrA and ccrB alleles, respectively. We used PCR to show that CcrA8B9 can excise and integrate the SCCmec element. Furthermore, using NCBI BLAST we showed that the ccrA8B9 genes exist in other staphylococcal strains. Unlike the common ccr genes, ccrA8B9 is located outside the SCCmec/SCC element. CONCLUSIONS: The novel Ccr recombinases CcrA8B9 can help excise and integrate SCCmec/SCC from the genome and provide a new way to facilitate the transmission of SCCmec/SCC elements among staphylococci.