Jizhen Yuan

Molecular and phenotypic evidence for the spread of three major methicillin-resistant Staphylococcus aureus clones associated with two characteristic antimicrobial resistance profiles in China

OBJECTIVES The distribution of methicillin-resistant Staphylococcus aureus (MRSA) clones is dynamic and geographically unique. To understand the changing epidemiology of MRSA infections in China, we performed a prospective, multicity surveillance study with molecular typing and phenotypic analysis to determine the association of major prevalent clones with their antimicrobial resistance profiles. METHODS A total of 517 S. aureus isolates collected between January 2009 and March 2012 from six cities in China were subjected to antibiogram analysis and molecular typing, including staphylococcal cassette chromosome mec typing, multilocus sequence typing, staphylococcal protein A gene typing and PFGE typing. RESULTS Among the isolates collected, 309 were characterized as MRSA, with a prevalence of 59.8%. Three major clones were found to be prevalent in China: ST239-MRSA-III-t030, ST239-MRSA-III-t037 and ST5-MRSA-II-t002. These three clones were associated with two characteristic resistance profiles, namely, gentamicin/ciprofloxacin/rifampicin/levofloxacin for the first clone and gentamicin/ciprofloxacin/clindamycin/erythromycin/tetracycline/levofloxacin/trimethoprim/sulfamethoxazole for the latter two. Several geographically unique minor clones were also identified. CONCLUSIONS The predominant MRSA clones in China were associated with characteristic antimicrobial resistance profiles. Antibiotics for treating patients with MRSA infections can be selected based on the strain typing data.

Cell wall thickening is associated with adaptive resistance to amikacin in methicillin-resistant Staphylococcus aureus clinical isolates

OBJECTIVES Methicillin-resistant Staphylococcus aureus (MRSA) infection is increasing and causing global concern. The mechanism of MRSA resistance to amikacin is poorly understood. We report on the first matched-pair study to reveal that the phenotypic cell wall thickening of MRSA is associated with adaptive resistance to amikacin. METHODS Two MRSA strains (CY001 and CY002) were isolated from blood and synovial fluid samples, respectively, from a 12-year-old male patient with osteomyelitis. The strains were subjected to a matched-pair study, including antimicrobial agent susceptibility determination, molecular typing, morphological observation and in vitro resistance induction. RESULTS Both strains are Panton-Valentine leucocidin-positive, multilocus sequence type 59, staphylococcal cassette chromosome mec type IV and spa type 437 MRSA with identical PFGE profiles. The drug susceptibility spectra of the two isolates are similar. However, CY001 is resistant to amikacin (CY001-AMI(R); MIC = 64 mg/L), contrary to the susceptible CY002 (CY002-AMI(S); MIC = 8 mg/L). CY001-AMI(R) may have developed adaptive resistance, because it lacks aminoglycoside-modifying enzymes and has an altered growth curve. Interestingly, CY001-AMI(R) has a thicker cell wall (36.43 ± 4.25 nm) than CY002-AMI(S) (18.15 ± 3.74 nm) in the presence of amikacin at its MIC. The thickened cell wall can also be observed in an in vitro-induced strain (CY002-AMI(R)) in the presence of amikacin at its MIC (36.78 ± 3.41 nm); this strain was obtained by gradually increasing the amount of amikacin. However, the cell wall-thickened strains cultured in the presence of amikacin are still susceptible to vancomycin. CONCLUSIONS Cell wall thickening is associated with adaptive resistance in MRSA and alternative antibiotics can be used to treat patients when adaptive resistance to amikacin has developed.

Panton-Valentine Leukocidin (PVL)-Positive Health Care-Associated Methicillin-Resistant Staphylococcus aureus Isolates Are Associated with Skin and Soft Tissue Infections and Colonized Mainly by Infective PVL-Encoding Bacteriophages

ABSTRACT The emergence of Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) is a public health concern worldwide. PVL is associated with community-associated MRSA and is linked to skin and soft tissue infections (SSTIs). However, PVL genes have also been detected in health care-associated (HA) MRSA isolates. The diseases associated with PVL-positive HA-MRSA isolates and the distributions of PVL-encoding bacteriophages in HA-MRSA have not been determined. In this study, a total of 259 HA-MRSA strains isolated between 2009 and 2012 in China from inpatients with SSTIs, pneumonia, and bacteremia were selected for molecular typing, including staphylococcal cassette chromosome mec typing, multilocus sequence typing, and staphylococcal protein A gene typing. The PVL genes and PVL bacteriophages in the MRSA isolates were characterized by PCR. Among the tested MRSA isolates, 28.6% (74/259) were PVL positive. The high prevalence of PVL-carrying HA-MRSA was observed to be associated with SSTIs but not with pneumonia or bacteremia. The PVL-positive HA-MRSA isolates were colonized mainly by infective PVL phages, namely, Φ7247PVL, ΦSLT, and ΦSa2958. The distribution of PVL-carrying bacteriophages differed geographically. Our study highlights the potential risk of the emergence of multidrug-resistant HA-MRSA strains with increased virulence.

First report of a sequence type 239 vancomycin-intermediate Staphylococcus aureus isolate in Mainland China.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that causes a wide range of both hospital- and community-acquired infections. The high prevalence of MRSA and the extensive use of vancomycin in Mainland China may lead to the emergence of vancomycin-intermediate S. aureus (VISA) isolates. In this case, we report a VISA isolate from a 34-year-old male patient with steam burn. The isolate was determined to be sequence type 239 staphylococcal cassette chromosome mec type III, the most prevalent MRSA clone in Mainland China.

Superficial vimentin mediates DENV-2 infection of vascular endothelial cells.

Damage to vascular endothelial cells (VECs) is a critical hallmark of hemorrhagic diseases caused by dengue virus (DENV). However, the precise molecular event involved in DENV binding and infection of VECs has yet to be clarified. In this study, vimentin (55 kDa) was identified to be involved in DENV-2 adsorption into VECs. This protein is located on the surface of VECs and interacts with DENV-2 envelope protein domain III (EDIII). The expression level of the superficial vimentin on VECs was not affected by viral infection or siRNA interference, indicating that the protein exists in a particular mode. Furthermore, the rod domain of the vimentin protein mainly functions in DENV-2 adsorption into VECs. Molecular docking results predicted several residues in vimentin rod and DENV EDIII; these residues may be responsible for cell–virus interactions. We propose that the superficial vimentin could be a novel molecule involved in DENV binding and infection of VECs. DENV EDIII directly interacts with the rod domain of vimentin on the VEC surface and thus mediates the infection.

Comparative Fitness and Determinants for the Characteristic Drug Resistance of ST239-MRSA-III-t030 and ST239-MRSA-III-t037 Strains Isolated in China.

Sequence type (ST) 239 with SCCmec type III methicillin-resistant Staphylococcus aureus (ST239-MRSA-III) is the most predominant multidrug-resistant clone in China. The subclone ST239-MRSA-III-t037 has been gradually replaced with ST239-MRSA-III-t030 since 2000. Subclones are characterized by drug resistance profiles. However, the mechanisms of the clonal dynamics and determinants of distinct drug resistance remain poorly understood. In the present study, 12 ST239-MRSA-III-t030 and 12 ST239-MRSA-III-t037 strains were collected from Chongqing, Guangzhou, and Shanghai; these strains were selected and investigated in terms of t030/t037 strain pairs. Independent growth curve assay revealed that the ST239-MRSA-III-t030 strains grew more rapidly, with significantly shorter doubling times, than the ST239-MRSA-III-t037 strains (p < 0.001). The ST239-MRSA-III-t037 strains exhibited slightly to moderately higher (3-13%) fitness cost than the ST239-MRSA-III-t030 strains in a competition assay in vitro. The ST239-MRSA-III-t037 strains yielded lower bacterial loads in the kidneys of the infected mice than the ST239-MRSA-III-t030 rivals in a coinfection assay (p < 0.05). The ST239-MRSA-III-t030 strains were resistant to rifampicin but susceptible to trimethoprim/sulfamethoxazole (SXT). In contrast, the ST239-MRSA-III-t037 strains were susceptible to rifampicin but resistant to SXT. The genetic determinants of the resistance to rifampicin and SXT in the MRSA strains were determined. Our results suggest that the relatively low fitness cost and characteristic drug resistance phenotype can help explain the current predominance of these ST239-MRSA-III-t030 strains in Chinese hospitals.

A novel SigB(Q225P) mutation in Staphylococcus aureus retains virulence but promotes biofilm formation

Staphylococcus aureus is an important pathogen that produces abundant virulence factors, which cause various diseases that burden human health worldwide. The stress response regulon called sigma factor B (SigB) is a well-characterized global regulator that is involved in the regulation of S. aureus virulence, pigmentation, and biofilm formation. However, the regulatory network upon SigB in S. aureus is incompletely described. Here, we identified a novel substitution mutation, SigB(Q225P), which contributed the nonpigmented phenotype of S. aureus. The S. aureus mutant carrying SigB(Q225P) substitution lacks staphyloxanthin, a key virulence factor in protecting bacteria from host-oxidant killing, but retains bacterial pathogenicity with pleiotropic alterations in virulence factors, resulting in similar lethality and abscess formation ability in animal models. We also reported the SigB(Q225P) promotion of biofilm formation in S. aureus. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed that the expression of nuc gene, which encodes thermonuclease, was significantly downregulated, resulting in accumulation of eDNA in the biofilm of SigB(Q225P) mutant strain. LacZ reporter assay showed that SigB(Q225P) influenced the activity of nuc promoter. Furthermore, electrophoretic mobility shift assay (EMSA) and Bio-layer interferometry (BLI) assay revealed that both SigB and SigB(Q225P) proteins could directly bind to nuc gene promoter; however, the binding activity decreased for SigB(Q225P). Our data renewed the understanding of the relationship between S. aureus golden pigment and its virulence and suggested that a single substitution mutation in SigB might enhance the biofilm formation of S. aureus by directly downregulating nuc expression.

WalK(S221P), a naturally occurring mutation, confers vancomycin resistance in VISA strain XN108

Objectives Vancomycin-intermediate Staphylococcus aureus (VISA) strains have spread globally. We previously isolated an ST239 VISA (XN108) with a vancomycin MIC of 12 mg/L. The mechanism for XN108 resistance to vancomycin was investigated in this study. Methods Genome comparison was performed to characterize mutations that might contribute to the XN108 resistance phenotype. The novel mutation WalK(S221P) was identified and investigated using allelic replacement experiments. Vancomycin susceptibilities, autolytic activities and morphologies of the strains were examined. Autophosphorylation activities of WalK and the WalK(S221P) mutant were determined in vitro with [λ- 32 P]ATP, and binding activity of WalK(S221P)-activated WalR to the promoter region of its target gene lytM was determined by electrophoretic mobility shift assay. Results Genome comparison revealed three mutations, GraS(T136I), RpoB(H481N) and WalK(S221P), which might be responsible for vancomycin resistance in XN108. The introduction of WalK(S221P) to the vancomycin-susceptible strain N315 increased its vancomycin MIC from 1.5 to 8 mg/L, whereas the allelic replacement of WalK(S221P) with the native N315 WalK allele in XN108 decreased its vancomycin MIC from 12 to 4 mg/L. The VISA strains have thickened cell walls and decreased autolysis, consistent with observed changes in the expression of genes involved in cell wall metabolism and virulence regulation. WalK(S221P) exhibited reduced autophosphorylation, which may lead to reduced phosphorylation of WalR. WalK(S221P)-phosphorylated WalR also exhibited a reduced capacity to bind to the lytM promoter. Conclusions The naturally occurring WalK(S221P) mutation plays a key role in vancomycin resistance in XN108.

Positive Feedback Cycle of TNFα Promotes Staphylococcal Enterotoxin B-Induced THP-1 Cell Apoptosis

Staphylococcal enterotoxin B (SEB) has been demonstrated to be of importance in Staphylococcus aureus related diseases, such as atopic dermatitis (AD). Dysregulated apoptosis in AD is remarkable, and SEB can induce apoptosis of various cell types. However, the mechanisms by which SEB induces apoptosis and influences disease processes remain unclear. In this study, the recombinant SEB-induced THP-1 monocyte apoptosis was demonstrated in the absence of preliminary cell activation in a time- and dose-dependent manner. SEB could up-regulate the expression of tumor necrosis factor alpha (TNFα) in THP-1 cells and induce apoptosis via an extrinsic pathway. TNFα could in turn increase the expression of HLA-DRa, the SEB receptor on the cell surface. As a result, a positive feedback cycle of TNFα was established. TNFα expression and SEB-induced apoptosis were decreased by knocking down the expression of either HLA-DRa or TNFR1. Therefore, the feedback cycle of TNFα is crucial for SEB functions. This work provides insights into the mechanisms of SEB-induced monocyte apoptosis and emphasizes the major role of TNFα in future related studies.

β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections worldwide. MRSA resists nearly all β-lactam antibiotics that have a bactericidal activity and a signal inducer effect. However, studies have yet to clarify whether the inducer effect of empirically used β-lactams stimulates MRSA pathogenicity in vivo. Here, we showed that a new cluster of tandem lipoprotein genes (tlpps) was upregulated in MRSA in response to the subinhibitory concentrations of β-lactam induction. The increased Tlpps significantly altered immune responses by macrophages with high IL-6 and TNFα levels. The deletion of the tlpps mutant (N315Δtlpps) significantly decreased the proinflammatory cytokine levels in vitro and in vivo. The bacterial loads of N315Δtlpps in the mouse kidney were also reduced compared with those of the wild type N315. The β-lactam-treated MRSA exacerbated cutaneous infections with increased lesion size, extended illness, and flake-like abscess-formation compared with those of the nontreatment. The β-lactam antibiotics that promoted the MRSA pathogenicity were SarA dependent, and the increasing expression of tlpps after β-lactam treatment was directly controlled by the global regulator SarA. Overall, our findings suggested that β-lactams should be used carefully because it might lead to a worse outcome of MRSA infection than inaction in the treatment. Author summary β-lactams are widely used in practice to treat infectious diseases, however, β-lactams worsening the outcome of a certain disease is poorly understood. In this study, we have identified a new cluster of tandem lipoprotein genes (tlpps) that is upregulated in the major clinically prevalent MRSA clones in response to the subinhibitory concentrations of β-lactams induction. The major highlight in this work is that β-lactams induce SarA expression, and then SarA directly binds to the tlpp cluster promoter region and upregulates the tlpp expression in MRSA. Moreover, the β-lactam stimulated Tlpps are important virulence factors that enhance MRSA pathogenicity. The deletion of the tlpps mutant significantly decreases the proinflammatory cytokine levels in vitro and in vivo. The β-lactam induced Tlpps enhance the host inflammatory responses by triggering the expression of IL-6 and TNFα, thereby promoting bacterial colonization and abscess formation. These data elucidate that β-lactams can worsen the outcome of MRSA infection through the induction of tlpps that are controlled by the global regulator SarA.