Role of tcaA, a potential target as a ceftobiprole resistance breaker, in MRSA ß-lactam resistance.

OBJECTIVES: Using a random forest algorithm, we previously found that teicoplanin-associated gene A (tcaA) might play a role in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactams, which we have investigated further here. METHODS: Representative MRSA strains of prevalent clones were selected to identify the role of tcaA in the MRSA response to ß-lactams. tcaA genes were deleted by homologous recombination in the selected MRSA strains, and antibiotic susceptibility tests were applied to evaluate the effect of tcaA on the minimum inhibitory concentrations (MICs) of glycopeptides and ß-lactams. Scanning electron microscopy, RNA sequencing, and quantitative reverse transcription-polymerase chain reaction were performed to explore the mechanism of tcaA in MRSA resistance to ß-lactams. RESULTS: The MIC of penicillin plus clavulanate decreased from 3 mg/L to 0.064 mg/L and that of oxacillin decreased from 16 to 0.5 mg/L when tcaA was knocked out in the LAC strain. Compared with wild-type MRSA isolates, when tcaA was deleted, all selected strains were more susceptible to ß-lactams. Susceptibility to ceftobiprole was restored in the ceftobiprole-resistant strain when tcaA was deleted. tcaA knockout caused "log-like" abnormal division of MRSA, and tcaA deficiency mediated low expression of mecA, ponA, and murA2. CONCLUSIONS: Machine learning is a reliable tool for identifying drug resistance-related genes. tcaA may be involved in S. aureus cell division and may affect mecA, ponA, and murA2 expression. Furthermore, tcaA is a potential resistance breaker target for ß-lactams, including ceftobiprole, in MRSA.

Identification of the novel fosfomycin resistance gene fosSC in Staphylococcus capitis.

OBJECTIVES: Fosfomycin has regained attention for treating infections caused by methicillin-resistant Staphylococcus aureus and multidrug-resistant coagulase-negative staphylococci. In this research, our objective was to investigate the mechanisms underlying fosfomycin resistance in Staphylococcus capitis. METHODS: The minimum inhibitory concentrations (MICs) of fosfomycin were assessed in 109 clinical S. capitis isolates by the agar dilution method. By cloning the fos-like genes into the shuttle vector, pTSSCm-Pcap, and observing the change in fosfomycin MICs, the gene function was verified. Core genome multilocus sequence typing and comparative genomics analysis were conducted to determine the population characteristics of S. capitis isolates and analyse the genetic environment of the fos-like genes. RESULTS: We identified a novel fosfomycin resistance gene, fosSC, on the chromosome in 58 out of 109 (53.2%) S. capitis isolates. The deduced products of the fosSC genes shared 67.15-67.88% amino acid sequence identity with FosB. The RN-pT-fosSC transformants carrying fosSC showed a 512-fold increase in the fosfomycin MICs. The fosSC gene was embedded in a conserved genetic context, but IS431mec was located to the left of the fosSC gene in cluster L due to the insertion of staphylococcal cassette chromosome mec. CONCLUSIONS: The chromosomal fosSC genes in some lineages of S. capitis explained their high-level fosfomycin resistance. Ongoing surveillance is crucial for monitoring the potential threat of horizontal transfer, which could be facilitated by the presence of mobile genetic elements surrounding the fosSC gene.

Vancomycin heteroresistance caused by unstable tandem amplifications of the vanM gene cluster on linear conjugative plasmids in a clinical Enterococcus faecium.

Vancomycin heteroresistance is prone to missed detection and poses a risk of clinical treatment failure. We encountered one clinical Enterococcus faecium strain, SRR12, that carried a complete vanM gene cluster but was determined as susceptible to vancomycin using the broth microdilution method. However, distinct subcolonies appeared within the clear zone of inhibition in the E-test assay, one of which, named SRR12-v1, showed high-level resistance to vancomycin. SRR12 was confirmed as heteroresistant to vancomycin using population analysis profiling and displayed "revive" growth curves with a lengthy lag phase of over 13 hours when exposed to 2-32 mg/L vancomycin. The resistant subcolony SRR12-v1 was found to carry an identical vanM gene cluster to that of SRR12 but a significantly increased vanM copy number in the genome. Long-read whole genome sequencing revealed that a one-copy vanM gene cluster was located on a pELF1-like linear plasmid in SRR12. In comparison, tandem amplification of the vanM gene cluster jointed with IS1216E was seated on a linear plasmid in the genome of SRR12-v1. These amplifications of the vanM gene cluster were demonstrated as unstable and would decrease accompanied by fitness reversion after serial passaging for 50 generations under increasing vancomycin pressure or without antibiotic pressure but were relatively stable under constant vancomycin pressure. Further, vanM resistance in resistant variants was verified to be carried by conjugative plasmids with variable sizes using conjugation assays and S1-pulsed field gel electrophoresis blotting, suggesting the instability/flexibility of vanM cluster amplification in the genome and an increased risk of vanM resistance dissemination.

ß-Hemolysin, not agrA mutation, inhibits the hemolysis of α-hemolysin in Staphylococcus aureus laboratory and clinical strains.

Staphylococcus aureus produces various hemolysins regulated by the Agr-QS system, except ß-hemolysin encoded by the gene hlb. A classical laboratory S. aureus strain RN4220 displays only the ß-hemolysin phenotype. It was suspected that the 8A mutation at the end of its agrA gene delayed the expressions of hla and RNAIII, then failed to express α- and δ-hemolysins. However, hla gene expression was detected at the later culture time without α-hemolysin phenotype, the reason for such a phenotype has not been clearly understood. We created hlb knockout and complementary mutants via homologous recombination in RN4220 and NRS049, two strains that normally produce ß-hemolysin and carry agrA mutation. We found interestingly that the presence or absence of α-hemolysin phenotype in such strains depended on the expression of ß-hemolysin instead of agrA mutations, which only inhibited δ-hemolysin expression. The hemolysis phenotype was verified by the Christie-Atkinson-Munch-Peterson (CAMP) test. Quantitative reverse transcription PCR was carried out to evaluate the relative gene expressions of hlb, hla, and RNAIII. The construction of mutants did not affect the agrA mutation status. We demonstrate that the absence of α-hemolysin in S. aureus RN4220 and NRS049 strains is attributed to their production of ß-hemolysin instead of agrA mutation. Our findings broaden the understanding of the molecular mechanisms that control hemolysin expression in S. aureus that is crucial for the development of new therapeutic strategies to combat S. aureus infections. IMPORTANCE: α-Hemolysin is a critical virulence factor in Staphylococcus aureus and its expression is largely controlled by the Agr-QS system. Nonetheless, the hemolysis phenotype and the regulation of the Agr-QS system in S. aureus still hold many mysteries. Our study finds that it is the expression of ß- hemolysin rather than the agrA mutation that inhibits the function of the α-hemolysin in an important S. aureus strain RN4220 and a clinical strain presents a similar phenotype, which clarifies the misunderstood hemolytic phenotype and mechanism of S. aureus. Our findings highlight the interactions among different toxins and their biological roles, combined with QS system regulation, which is ultimately the true underlying cause of its virulence. This emphasizes the importance of considering the collaborative action of various factors in the infection process caused by this significant human pathogen.

Profiling daptomycin resistance among diverse methicillin-resistant Staphylococcus aureus lineages in China.

Daptomycin (DAP) is effective against methicillin-resistant Staphylococcus aureus (MRSA). However, reduced susceptibility to DAP in MRSA may lead to treatment failures. We aim to determine the distribution of DAP minimum inhibitory concentrations (MICs) and DAP heteroresistance (hDAP) among MRSA lineages in China. A total of 472 clinical MRSA isolates collected from 2015 to 2017 in China were examined for DAP susceptibility. All isolates (n = 472) were found to be DAP susceptible, but 35.17% (166/472) of them exhibited a high DAP MIC (MIC >0.5 µg/mL). The high DAP MIC group contained a larger proportion of isolates with a higher vancomycin or teicoplanin MIC (>1.5 µg/mL) than the low DAP MIC group (19.3% vs 7.8%, P < 0.001; 22.3% vs 8.2%, P < 0.001). We compared the clonal complex (CC) distributions and clinical characteristics in MRSA isolates stratified by DAP MIC. CC5 isolates were less susceptible to DAP (MIC50 = 1 µg/mL) than CC59 isolates (MIC50 = 0.5 µg/mL, P < 0.001). Population analysis profiling revealed that 5 of 10 ST5 and ST59 DAP-susceptible MRSA isolates investigated exhibited hDAP. The results also showed that CC5 MRSA with an agrA mutation (I238K) had a higher DAP MIC than those with a wild-type agrA (P < 0.001). The agrA-I238K mutation was found to be associated with agr dysfunction as indicated by the loss of δ-hemolysin production. In addition, agr/psmα defectiveness was associated with hDAP in MRSA. Whole-genome sequencing analysis revealed mutations in mprF and walR/walK in DAP-resistant subpopulations, and most DAP-resistant subpopulations (6/8, 75%) were stable. Our study suggests that the increased DAP resistance and hDAP in MRSA may threaten the effectiveness against MRSA infections.

Exploring the third-generation tetracycline resistance of multidrug-resistant livestock-associated methicillin-resistant Staphylococcus aureus ST9 across healthcare settings in China.

BACKGROUND: The overuse of antibiotics in livestock is contributing to the burden of antimicrobial resistance in humans, representing a One Health challenge. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has recently become a growing concern, and ST9 is the major LA-MRSA lineage in China and has emerged in clinical settings. METHODS: Antimicrobial susceptibility testing was used to evaluate the tetracycline resistance of ST9 MRSA collections, and gene cloning experiments were performed to explore the resistance mechanisms. Whole-genome sequencing and comparative genomics were used to analyse the genetic features of clinical ST9 isolates. A phylogenetic tree was constructed to investigate the relationship of human- and livestock-derived ST9 isolates. RESULTS: Clinical ST9 isolates were found to possess several types of resistance genes and resistance-related mutations and were multidrug-resistant. Notably, all clinical ST9 isolates were resistant to third-generation tetracyclines. Cloning experiments showed that both the acquisition of the tetracycline resistance gene tet(L)/tet(63) and a mutation in the rpsJ gene contributed to third-generation tetracycline resistance. Phylogenetic analysis showed that the ST9 isolates collected in healthcare systems were probably transmitted from livestock. The ST9 lineage underwent multiple interspecies recombination events and gained many resistance elements. Furthermore, the resistance to third-generation tetracyclines may have evolved under tetracycline pressure in livestock. CONCLUSIONS: The evolution of ST9 MRSA in livestock and transmission of this clone between humans and livestock highlight the importance of establishing control strategies with the One Health approach to reduce the burden of antibiotic resistance.

Epidemiology and Genetic Characteristics of Carbapenem-Resistant Escherichia coli in Chinese Intensive Care Unit Analyzed by Whole-Genome Sequencing: a Prospective Observational Study.

This 4-month-long prospective observational study investigated the epidemiological characteristics, genetic composition, transmission pattern, and infection control of carbapenem-resistant Escherichia coli (CREC) colonization in patients at an intensive care unit (ICU) in China. Phenotypic confirmation testing was performed on nonduplicated isolates from patients and their environments. Whole-genome sequencing was performed for all E. coli isolates, followed by multilocus sequence typing (MLST), and antimicrobial resistance genes and single nucleotide polymorphisms (SNPs) were screened. The colonization rates of CREC were 7.29% from the patient specimens and 0.39% from the environmental specimens. Among the 214 E. coli isolates tested, 16 were carbapenem resistant, with the blaNDM-5 gene identified as the dominant carbapenemase-encoding gene. Among the low-homology sporadic strains isolated in this study, the main sequence type (ST) of carbapenem-sensitive Escherichia coli (CSEC) was ST1193, whereas the majority of CREC isolates belonged to ST1656, followed by ST131. CREC isolates were more sensitive to disinfectants than were the carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates obtained in the same period, which may explain the lower separation rate. Therefore, effective interventions and active screening are beneficial to the prevention and control of CREC. IMPORTANCE CREC represents a public health threat worldwide, and its colonization precedes or occurs simultaneously with infection; once the colonization rate increases, the infection rate rises sharply. In our hospital, the colonization rate of CREC remained low, and almost all of the CREC isolates detected were ICU acquired. Contamination of the surrounding environment by CREC carrier patients shows a very limited spatiotemporal distribution. As the dominant ST of the CSEC isolates found, ST1193 CREC might be considered a strain of notable concern with potential to cause a future outbreak. ST1656 and ST131 also deserve attention, as they comprised the majority of the CREC isolates found, while blaNDM-5 gene screening should play an important role in medication guidance as the main carbapenem resistance gene identified. The disinfectant chlorhexidine, which is used commonly in the hospital, is effective for CREC rather than CRKP, possibly explaining the lower positivity rate for CREC than for CRKP.

Development of a novel core genome MLST scheme for tracing multidrug resistant Staphylococcus capitis.

Staphylococcus capitis, which causes bloodstream infections in neonatal intensive care units, is a common cause of healthcare-associated infections. Thus, a standardized high-resolution typing method to document the transmission and dissemination of multidrug-resistant S. capitis isolates is required. We aimed to establish a core genome multilocus sequence typing (cgMLST) scheme to surveil S. capitis. The cgMLST scheme was defined based on primary and validation genome sets and tested with outbreaks of linezolid-resistant isolates and a validation set. Phylogenetic analysis was performed to investigate the population structure and compare it with the result of cgMLST analysis. The S. capitis population consists of 1 dominant, NRCS-A, and 4 less common clones. In this work, a multidrug-resistant clone (L clone) with linezolid resistance is identified. With the features of type III SCCmec and multiple copies of mutations of G2576T and C2104T in the 23S rRNA, the L clone has been spreading silently across China.

Staphylococcal cassette chromosome mec amplification as a mechanism for ceftobiprole resistance in clinical methicillin-resistant Staphylococcus aureus isolates.

OBJECTIVES: In this study, we evaluated the ceftobiprole (BPR) susceptibilities of 472 methicillin-resistant Staphylococcus aureus (MRSA) isolates, and investigated the mechanisms underlying BPR resistance. METHODS: For all MRSA isolates, BPR MIC was determined by agar dilution. We sequenced the BPR-resistant isolates through Illumina short- and MinION long-read sequencing. We also selected MRSA isolates of ST5, ST59, and ST239, and exposed them to increasing BRP concentrations. The isolated mutants developing BPR resistance were sequenced. RESULTS: A total of 471 MRSA isolates were susceptible to BPR, with MICs ranging from 0.25 to 2 mg/L. Compared with HA-MRSA isolates (MIC50 = 2 mg/L; MIC90 = 2 mg/L), CA-MRSA isolates (MIC50 = 0.5; MIC90 = 2 mg/L) were more susceptible to BPR (p < 0.001). Compared with isolates with staphylococcal cassette chromosome mec (SCCmec) type II or III (MIC50 = 2 mg/L; MIC90 = 2 mg/L), isolates with SCCmec type IV (MIC50 = 1 mg/L; MIC90 = 1 mg/L) or V (MIC50 = 0.5 mg/L; MIC90 = 1 mg/L) were more susceptible to BPR (p < 0.001). Nanopore sequencing revealed two copies of SCCmec repeats in the BPR-resistant MRSA isolate. In addition, SCCmec amplification could be induced by BPR exposure in ST239 MRSA isolates; however, no amplification was observed in the other lineages. The induced BPR-resistant MRSA isolates also acquired mutations in mecA and other genes, such as guaA, guaB, relA, rpoA, and oatA, which were speculated as factors contributing to BPR-resistance development. DISCUSSION: BPR showed significant antibacterial activity against MRSA isolates in China; however, the emergence of a BPR-resistant isolate before its launch was a cause for concern. Multiple genes and pathways are potentially involved in the development of BPR resistance in MRSA, and our data demonstrated the role of nanopore-sequencing in revealing the tandem repeat-mediated resistance mechanism in MRSA.

The novel fosfomycin resistance gene fosY is present on a genomic island in CC1 methicillin-resistant Staphylococcus aureus.

Fosfomycin has gained attention as a combination therapy for methicillin-resistant Staphylococcus aureus infections. Hence, the detection of novel fosfomycin-resistance mechanisms in S. aureus is important. Here, the minimal inhibitory concentrations (MICs) of fosfomycin in CC1 methicillin-resistant S. aureus were determined. The pangenome analysis and comparative genomics were used to analyse CC1 MRSA. The gene function was confirmed by cloning the gene into pTXΔ. A phylogenetic tree was constructed to determine the clustering of the CC1 strains of S. aureus. We identified a novel gene, designated fosY, that confers fosfomycin resistance in S. aureus. The FosY protein is a putative bacillithiol transferase enzyme sharing 65.9-77.5% amino acid identity with FosB and FosD, respectively. The function of fosY in decreasing fosfomycin susceptibility was confirmed by cloning it into pTXΔ. The pTX-fosY transformant exhibited a 16-fold increase in fosfomycin MIC. The bioinformatic analysis showed that fosY is in a novel genomic island designated RIfosY (for "resistance island carrying fosY") that originated from other species. The global phylogenetic tree of ST1 MRSA displayed this fosY-positive ST1 clone, originating from different regions, in the same clade. The novel resistance gene in the fos family, fosY, and a genomic island, RIfosY, can promote cross-species gene transfer and confer resistance to CC1 MRSA causing the failure of clinical treatment. This emphasises the importance of genetic surveillance of resistance genes among MRSA isolates.

Genetic Characteristics of Multiple Copies of Tn1546-Like Elements in ermB-Positive Methicillin-Resistant Staphylococcus aureus From Mainland China.

Objective: To determine the genetic structure of ermB-positive Tn1546-like mobile elements in methicillin-resistant Staphylococcus aureus (MRSA) from mainland China. Methods: A total of 271 erythromycin-resistant MRSA isolates were isolated from Sir Run Run Shaw Hospital (SRRSH) from 2013 to 2015. Whole-genome sequencing was performed for the ermB-positive strains, and the genetic environment of the ermB genes was analyzed. Southern hybridization analysis and transformation tests were performed to confirm the location of the ermB gene. Results: A total of 64 isolates (64/271, 23.6%) were ermB-positive strains, with 62 strains (62/64, 96.9%) belonging to the CC59 clone. The other two strains, SR130 and SR231, belonging to CC5-ST965, both harbored 14,567 bp ermB-positive Tn1546-like elements and displayed multidrug-resistant profiles. PFGE followed by Southern blot demonstrated that the ermB genes were located on the plasmids of both SR130 and SR231, while two copies of ermB were located on the chromosome of SR231. Further sequencing demonstrated that SR231 carried one Tn1546-ermB elements in the plasmid and two identical copies integrated on the chromosome, which had 99.99% identity to the element in the plasmid of SR130. The Tn1546-ermB elements were highly similar (100% coverage, >99.9% identity) to the element Tn6636 reported in a previous study from Taiwan. The plasmids (pSR130 and pSR231) harboring ermB-positive Tn1546-like elements were also identical to the mosaic plasmid pNTUH_5066148. However, conjugation of ermB-carrying plasmids of SR130 and SR231 were failed after triple repeats. Conclusion: Multiple copies of ermB-positive Tn1546-like mobile elements were found in CC5-ST965 MRSA from mainland China, showing the wide dissemination of these Enterococcus faecium-originated ermB-positive Tn1546-like elements. Molecular epidemiological study of Tn1546-like elements is essential to avoid the spreading of resistant determinants.

Epidemiology, evolution and cryptic susceptibility of methicillin-resistant Staphylococcus aureus in China: a whole-genome-based survey.

OBJECTIVES: The aim of this study was to investigate the genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in China to identify predominant lineages and their associations with clinical data and antimicrobial resistance profiles. METHODS: We performed a national prevalence study of patients with S. aureus infections in 22 tertiary hospitals in China from 2015 to 2017. Clinical data from patients and the antimicrobial phenotypes were collected for each isolate. Genome sequencing was performed on a proportion of isolates and a phylogenetic analysis was undertaken. Genotypic and phenotypic ß-lactam susceptibilities were compared. RESULTS: A total of 1900 patients with S. aureus infections were included, of which 40% involved MRSA. Community-associated MRSA (CA-MRSA) infections were 24% of the total isolates. Genomic data showed that more than three-quarters of the MRSA were from three dominant lineages CC239 (25%, 116/471), CC5 (21%, 96/471) and CC59 (33%, 154/471) with CC59 accounting for more than half of the CA-MRSA isolates. Penicillin susceptibility genomic features were observed in 53% (251/470) of MRSA, including almost all of the CC59 (152/154) lineage, and 96% (242/251) of these isolates demonstrated in vitro susceptibility to penicillin or amoxicillin combined with clavulanic acid. Phylogenetic analysis indicated that the CC59 lineage can be divided into six lineages with all Asian CC59 isolates likely arising from an ancestral Mainland China lineage. CONCLUSIONS: This study showed a high prevalence of CA-MRSA in China, largely due to the widespread presence of CC59. As almost all isolates in this lineage possess genetic variants leading to increased ß-lactam susceptibility, we suggest that to improve antibiotic stewardship combinations of penicillins and ß-lactamase inhibitors should be included in the antibiotic susceptibility testing panels used to inform treatment decisions and research undertaken on this combination therapy.

The Role of mprF Mutations in Seesaw Effect of Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Isolates.

The emergence of daptomycin-resistant (DAP-R) Staphylococcus aureus strains has become a global problem. Point mutations in mprF are the main cause of daptomycin (DAP) treatment failure. However, the impact of these specific point mutations in methicillin-resistant S. aureus (MRSA) strains associated with DAP resistance and the "seesaw effect" of distinct beta-lactams remains unclear. In this study, we used three series of clinical MRSA strains with three distinct mutated mprF alleles from clone complexes (CC) 5 and 59 to explore the seesaw effect and the combined effect of DAP plus beta-lactams. Through construction of mprF deletion and complementation strains of SA268, we determined that mprF-S295A, mprF-S337L, and one novel mutation of mprF-I348del within the bifunctional domain lead to DAP resistance. Compared with wild-type mprF cloned from a DAP-susceptible (DAP-S) strain, these three mprF mutations conferred the seesaw effect to distinct beta-lactams in the SA268ΔmprF strains, and mutated mprF (I348del and S337L) did not alter the cell surface positive charge (P > 0.05). The susceptibility to beta-lactams increased significantly in DAP-R CC59 strains, and the seesaw effect was found to be associated with distinct mutated mprF alleles and the category of beta-lactams. The synergistic activity of DAP plus oxacillin was detected in all DAP-R MRSA strains. Continued progress in understanding the mechanism of restoring susceptibility to beta-lactam antibiotics mediated by the mprF mutation and its impact on beta-lactam combination therapy will provide fundamental insights into treatment of MRSA infections.

Transmission and microevolution of methicillin-resistant Staphylococcus aureus ST88 strain among patients, healthcare workers, and household contacts at a trauma and orthopedic ward.

Background: Surgical sites infections (SSIs) caused by Methicillin-resistant Staphylococcus aureus (MRSA) constitute a major clinical problem. Understanding the transmission mode of MRSA is important for its prevention and control. Aim: We investigated the transmission mode of a MRSA outbreak in a trauma and orthopedic hospital ward. Methods: Clinical data were collected from patients (n = 9) with MRSA infection in a trauma and orthopedic ward from January 1, 2015 to December 31, 2019. The wards (n = 18), patients (n = 48), medical staff (n = 23), and their households (n = 5) were screened for MRSA. The transmission mode of MRSA isolates was investigated using next-generation sequencing and phylogenetic analyses. The resistance genes, plasmids, and single-nucleotide variants of the isolates were analyzed to evaluate microevolution of MRSA isolates causing SSIs. The MRSA colonization-positive doctor was asked to suspend his medical activities to stop MRSA spread. Findings: Nine MRSA infected patients were investigated, of which three patients were diagnosed with SSI and had prolonged hospitalization due to the persistent MRSA infection. After screening, MRSA isolates were not detected in environmental samples. The surgeon in charge of the patients with SSI caused by MRSA and his son were positive for MRSA colonization. The MRSA from the son was closely related to the isolates detected in MRSA-induced SSIs patients with 8-9 single-nucleotide variants, while ST88-MRSA isolates with three different spa types were detected in the surgeon's nasal cavity. Comparative genomic analysis showed that ST88-MRSA isolates acquired mutations in genes related to cell wall synthesis, colonization, metabolism, and virulence during their transmission. Suspending the medical activity of this surgeon interrupted the spread of MRSA infection in this ward. Conclusion: Community-associated MRSA clones can invade hospitals and cause severe postoperative nosocomial infections. Further MRSA surveillance in the households of health workers may prevent the transition of MRSA from colonization to infection.

Distribution of erm genes among MRSA isolates with resistance to clindamycin in a Chinese teaching hospital.

The objective of this study was to analyze erythromycin and clindamycin resistance patterns among different MRSA lineages in China. Antimicrobial susceptibility testing, resistance determinant screening, plasmid electroporation and sequence comparisons were performed. High rates of clindamycin (92.5%, 270/292) and erythromycin (92.8%, 271/292) resistance were observed. Additionally, 88.2% (60/68) of the ST59 MRSA isolates and 78.9% (15/19) of the ST239 MRSA isolates had constitutive resistance to clindamycin, while 82.0% (123/150) of the ST5 MRSA isolates showed inducible clindamycin resistance. The ermB gene was identified in 80.9% (55/68) of the ST59 isolates but was not detected in ST5 and ST239 MRSA isolates. Detection rates of ermA were high in the ST5 (99.3%, 149/150) and ST239 (89.5%, 17/19) MRSA isolates, but no ermA-positive ST59 MRSA isolates were identified. The ermC gene, observed to be harbored on similar, transmissible plasmids ranging in size from 2402 to 2473 bp, were found in different MRSA lineages. Summarily, high erythromycin and clindamycin resistance rates were observed in MRSA isolates. ST59 and ST239 MRSA isolates primarily exhibited constitutive resistance, while ST5 MRSA isolates showed inducible resistance phenotypes. ermA and ermB genes were frequently carried by specific MRSA clones, while ermC gene was present within small transmissible plasmids in all lineages. Erythromycin and clindamycin resistance genes transfer between MRSA isolates in healthcare settings remains a problem, and infection control procedures should be applied.

A random forest model based on core genome allelic profiles of MRSA for penicillin plus potassium clavulanate susceptibility prediction.

Treatment failure of methicillin-resistant Staphylococcus aureus (MRSA) infections remains problematic in clinical practice because therapeutic options are limited. Penicillin plus potassium clavulanate combination (PENC) was shown to have potential for treating some MRSA infections. We investigated the susceptibility of MRSA isolates and constructed a drug susceptibility prediction model for the phenotype of the PENC. We determined the minimum inhibitory concentration of PENC for MRSA (n=284) in a teaching hospital (SRRSH-MRSA). PENC susceptibility genotypes were analysed using a published genotyping scheme based on the mecA sequence. mecA expression in MRSA isolates was analysed by qPCR. We established a random forest model for predicting PENC-susceptible phenotypes using core genome allelic profiles from cgMLST analysis. We identified S2-R isolates with susceptible mecA genotypes but PENC-resistant phenotypes; these isolates expressed mecA at higher levels than did S2 MRSA (2.61 vs 0.98, P<0.05), indicating the limitation of using a single factor for predicting drug susceptibility. Using the data of selected UK-sourced MRSA (n=74) and MRSA collected in a previous national survey (NA-MRSA, n=471) as a training set, we built a model with accuracies of 0.94 and 0.93 for SRRSH-MRSA and UK-sourced MRSA (n=287, NAM-MRSA) validation sets. The AUROC of this model for SRRSH-MRSA and NAM-MRSA was 0.96 and 0.97. Although the source of the training set data affects the scope of application of the prediction model, our data demonstrated the power of the machine learning approach in predicting susceptibility from cgMLST results.

Household Transmission of Community-Associated Methicillin-Resistant Staphylococcus Aureus.

Currently, the mechanism of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) transmission mechanism is unclear; however, it must be considered in conjunction with asymptomatic S. aureus strains colonization dynamics. This epidemiological study aimed to determine the role of the household in CA-MRSA transmission in China. Five patients with culture-confirmed CA-MRSA infection and five control patients were recruited from the Sir Run Run Shaw Hospital in Zhejiang, China, between December 2019 and January 2020. The household members of the patients, their pets, and environmental surfaces were sampled and screened for MRSA colonization. Mass spectrometry identification and antimicrobial susceptibility testing were performed on the MRSA isolates. Whole-genome sequencing and core genome multilocus sequence typing (cgMLST) were performed to determine the origin and transmission of the MRSA isolates in the households. Overall, 14 S. aureus-positive specimens (14.1%, 14/99) were obtained from the five households of patients with CA-MRSA infections, of which 12 (85.7%) were MRSA. The overall positivity of MRSA was 12.1% (12/99) among the samples from the CA-MRSA households, while no MRSA isolates were detected in the five control households. Most MRSA isolates belonged to epidemic CA-MRSA clones, such as ST59 (15/35, 42.9%) and ST508 (15/35, 42.9%). The cgMLST results confirmed that MRSA was transmitted among patients, contacts, and pets in the households and was present on environmental surfaces in the CA-MRSA patients' households. In conclusion, the study revealed that the home environment was an important MRSA reservoir. Therefore, focusing on MRSA decolonization in patients alone is not sufficient for infection control of CA-MRSA.

Prevalence, predictors, and mortality of bloodstream infections due to methicillin-resistant Staphylococcus aureus in patients with malignancy: systemic review and meta-analysis.

BACKGROUND: Cancer patients are more likely to develop and die of bloodstream infection (BSI) than noncancer patients. Methicillin-resistant Staphylococcus aureus (MRSA), which is associated with immense mortality and economic burden worldwide, is not covered by the recommended initial antibiotic therapy for cancer patients with BSI. This systemic review was performed to estimate the global methicillin-resistant Staphylococcus aureus (MRSA) prevalence among bacteremia in patients with malignancy, and further study the predictors and mortality of cancer patients with MRSA bacteremia. METHODS: The PubMed and EMBASE databases were searched for studies published from Jan. 2000 to Mar. 2020 that provided primary data on the prevalence, predictors, or mortality of MRSA bacteremia in cancer patients. A random-effects model meta-analysis was performed to estimate the pooled prevalence of MRSA with 95% confidence intervals (95% CIs). RESULTS: The pooled prevalence of MRSA was 3% (95% CI 2-5%) among all bloodstream infections (BSIs) and 44% (95% CI 32-57%) among S. aureus bacteremia in cancer patients. Based on geographical stratification, the pooled prevalence was 5% in Africa (95% CI 1-14%), 1% in Americas (95% CI 1-2%), 2% in Europe (95% CI 1-4%), 4% in Western Pacific (95% CI 2-7%), 8% in South-east Asia (95% CI 4-14%) and 0% in Eastern Mediterranean (95% CI 0-3%). No significant temporal change in MRSA rates was detected in this analysis (R2 = 0.06; P = 0.24). Predictors for MRSA BSIs among cancer patients were identified by comparison with their methicillin-susceptible counterparts, and they were mainly related to healthcare-associated infections and immunosuppression. Finally, the 60-day mortality in adult cancer patients with MRSA BSIs was reported to be 12%, and the 6-month overall mortality was 43.2%, with community-onset infection, secondary BSI, and vancomycin MIC≥2 g/mL being the risk factors for mortality. CONCLUSIONS: Although the prevalence of MRSA BSIs among cancer patients is relatively low, it did not decline over time as MRSA BSIs in the general hospital population and the high mortality rate was related to MRSA BSIs in patients with malignancy.