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1.
IntroductionStreptococcus pneumoniae with a mucoid-type capsule is associated with invasive pneumococcal diseases (IPDs). Despite the introduction of pneumococcal vaccines, IPDs caused by mucoid-type isolates are still prevalent. The present study aimed to characterize mucoid-type S. pneumoniae isolated from IPD patients throughout Japan in 2017 (post-vaccination era).MethodsA total of 225 mucoid-type isolates were collected. The serotype, antimicrobial susceptibility, and multilocus sequence type of these isolates were determined.ResultsThe prevalence of IPDs caused by mucoid-type isolates was high in adults, especially in the elderly (≥65 years of age), and prognosis in these patients was significantly poor. Of the mucoid-type isolates, the predominant serotype was serotype 3 (84.4%), and the remaining were serotypes 37 (15.1%) and 8 (0.4%). Antimicrobial susceptibility showed that most mucoid isolates exhibited the penicillin-intermediate resistant S. pneumoniae genotype (gPISP). However, the serotype 3 isolate exhibited the penicillin-resistant S. pneumoniae genotype (gPRSP). This gPRSP isolate was classified into ST166, which is related to serotypes 9 V and 11 strains. Sequence analysis of the capsule-coding regions and its flanking regions indicated that recombination occurred upstream and downstream of the capsule-coding region, suggesting that gPRSP (serotype 9 V/ST166) obtaining the type-3 capsule gene cluster resulted in the emergence of gPRSP (serotype 3/ST166).ConclusionsOur findings indicated that IPDs caused by mucoid-type S. pneumoniae are still a serious concern and mucoid-type S. pneumoniae with novel phenotype could emerge via capsular switching in response to environmental changes such as introduction of vaccines and improper use of antimicrobial agents.  相似文献   

2.
We investigated the change in clones and serotypes of Streptococcus pneumoniae isolates in a Korean tertiary-care hospital. Serotypes of S. pneumoniae isolates were determined by the capsular quellung method, and in vitro susceptibility testing was performed by broth microdilution method. Multilocus sequence typing was performed to determine the genotypes of the S. pneumoniae isolates. The erm(B) and mef(A) genes in erythromycin-resistant isolates were also detected using the duplex polymerase chain reaction method. During the 2 periods assayed (1998–2000 and 2007), 7-valent pneumococcal conjugate vaccine (PCV7) serotypes decreased significantly from 58.3% to 30.9% (P = 0.001). Especially, serotypes 19F and 23F decreased significantly from 31.7% to 8.5% (P < 0.0001) and from 20.0% to 7.4% (P = 0.021), respectively. In contrast to the other PCV7 serotypes, serotype 14 coupled with CC554 emerged in 2007, which may indicate no effect of PCV7 against serotype 14 isolates from Korea and the possibility of a different subtype. Of the non- PCV7 serotypes, serotype 19A increased from 8.3% to 14.9% (P = 0.227) and serotype 15 increased from 0% to 8.5% (P = 0.023). The increase of serotype 19A was due to the expansion of a preexisting clone with serotype 19A, ST320. However, S. pneumoniae isolates of serotype 15 showed diverse STs. Our data may provide helpful information in local vaccine serotype expansion or replacement in Korea.  相似文献   

3.
We report the first case of a teenage patient with chromosome 22q11.2 deletion syndrome who died of overwhelming postsplenectomy infection (OPSI) by Streptococcus pneumoniae despite appropriate prevention by pneumococcal vaccine. He had congenital heart disease and underwent several surgeries. Immunodeficiency had not been noticed clinically. Two years prior to death, splenectomy was performed for a drug-resistant idiopathic thrombocytopenic purpura and he was immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23) 4 months after splenectomy. He died suddenly after a mild flu-like symptom. Autopsy was performed and OPSI was diagnosed. Blood culture was positive for S. pneumoniae. This isolated S. pneumoniae strain was serotypically un-typable by polyvalent serum agglutination test. On the contrary, multilocus sequence typing followed by DNA sequencing indicated the molecular serotype as 10A. Additional testing using monovalent and factor-specific sera confirmed the strain as serotype 10A. Ultrastructural observation of this S. pneumoniae strain showed that the polysaccharide capsule was thin and sparse. We speculate that the abnormal morphology of the capsule may have accounted for the polyvalent serum agglutination failure and may possibly be associated with severity of OPSI observed in this case. Chromosome 22q11.2 deletion syndrome is associated with certain immunodeficiency, especially susceptible to S. pneumoniae infections; however, fatal OPSI has not been reported. In addition to vaccination, prophylactic antibiotics may be necessary for these patients who are at risk of immunodeficiency.  相似文献   

4.
The 7-valent pneumococcal conjugate vaccine (PCV7) and Haemophilus influenzae type b (Hib) vaccine reduce nasopharyngeal carriage of vaccine-type bacteria, which may in turn influence the presence of other nasopharyngeal bacterial pathogens. To investigate this possibility, nasopharyngeal carriage of potential pathogens was examined before and after official financial support was provided to offer the PCV7 and Hib vaccines in healthy children attending a day care centre in Japan during 2011–2012. Despite a virtual disappearance of PCV7 serotypes over time, the overall pneumococcal carriage rate remained unchanged. Although others have reported an increase in PCV13 serotypes following PCV7 vaccination, only non-PCV13 serotypes were observed to have increased in this study. The majority of H. influenzae isolates were non-typeable and Hib was not found. Our data identified an unexpected pattern of pneumococcal serotype replacement following PCV7. Continuous monitoring of pneumococcal carriage is important for decisions regarding the future of national vaccination policy in Japan.  相似文献   

5.
IntroductionStreptococcus pneumoniae is a commensal bacterium of the human nasopharynx and a major causative pathogen of bacterial diseases worldwide. Pilus of S. pneumoniae is one of the virulence factors which enhance the adhesion to the host epitherial cells in the upper respiratory tract.MethodsWe analyzed the serotype distribution and presence of pilus genes, rrgC and sipA, among 785 S. pneumoniae isolates from specimens of patients with invasive or non-invasive disease in a regional Japanese hospital between October 2014 and August 2018. We next performed multilocus sequence typing and penicillin-resistant genotyping for 86 isolates of serotype 35B.ResultsSerotype 35B was the most frequent serotype which accounted for 11.0% of total isolates and had pilus genes at high rate (80.2%). Clonal complex (CC) 558 isolates accounted for 77.9% of serotype 35B and were highly positive for rrgC and gPRSP (98.5%). In contrast, all CC2755 isolates (19.8%) were rrgC-negative and gPISP.ConclusionsOur results suggest that CC558 may assist the prevalence of serotype 35B after the introduction of vaccines, as that clone has pili as adhesins in addition to non-susceptibility against penicillin. These results may be useful information for development of optimal preventive strategies. Continuous studies on serotype distribution and virulence factors of S. pneumoniae are necessary.  相似文献   

6.
A study was conducted to determine the prevalence of the 2 newly described types, 6C and 6D, among pneumococcal isolates collected in Hong Kong before availability of the 7-valent pneumococcal conjugate vaccine. A total of 154 serogroup 6 isolates obtained from nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2) during 1995 to 2001 were analyzed by polymerase chain reaction typing. Five nasopharyngeal and 2 sputum isolates were found to belong to 6C and 6D, respectively. The isolates were genetically diverse, but one 6C and two 6D isolates exhibited some clonal relationship. Phylogenetic analysis of the wchA-wciN(β)-wciO nucleotide sequences showed that the Hong Kong 6C/6D isolates had 2 allelic profiles, which were more closely related to 6C/6D isolates from Fijian and Korea than were those from Brazil and the United States. However, all of the wciP gene sequences for both Hong Kong and non-Hong Kong isolates clustered together: 6C isolates with the wciP-9 allele and 6D isolates with the wciP-5 allele. In conclusion, the prevalence of the 2 newly described serotypes was low before the era of the pneumococcal conjugate vaccine. Nonetheless, results from the molecular studies indicated that the evolution of the capsular genes have involved complex pathways.  相似文献   

7.
Chlorhexidine is an antiseptic agent used for hand hygiene worldwide. To evaluate the susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) to chlorhexidine, this study determined MICs of chlorhexidine and another 12 antimicrobial agents, carriage of the Panton–Valentine leukocidin, qacA/B, and smr genes, genetic relatedness by multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec element type for 206 MRSA clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance program III and IV (years 2002 and 2004) from 26 hospitals. Using MLST, we respectively identified 102 (49.5%), 68 (33.0%), 13 (6.3%), 5 (2.4%), 5 (2.4%), and 13 (6.3%) isolates as ST239, ST59, ST5, ST241, ST573, and other types. The MIC50 and MIC90 of chlorhexidine for all 206 isolates were 2 and 8 μg/mL, respectively. Seventy-three (35.4%) isolates carried qacA/B gene, but none carried smr. For the 72 (35.0%) MRSA isolates with chlorhexidine MIC ≥4 μg/mL, 53 were ST239 (49 of them carried qacA gene), 12 were ST5 (all carried qacB gene), 5 were ST241 (4 carried qacA gene), 1 was ST338 (and carried qacA gene), and 1 was ST573 (and carried qacA gene). Compared with other sequence-type MRSA isolates, ST239 MRSA isolates were the most resistant to both chlorhexidine and other antimicrobial agents. Methicillin-resistant S. aureus strains with disinfectant resistance qacA/B genes are common in Taiwan. High frequency of qacA/B genes among specific sequence types (ST239, ST5, and ST241) resulted in low susceptibility to chlorhexidine. Periodic surveillance of antiseptic susceptibility among MRSA isolates is important for the control of nosocomial hospital-acquired infections.  相似文献   

8.
This study assessed the changes in serotype distribution and antibiotic resistance of Streptococcus pneumoniae isolates in children before and after introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in Hong Kong. Nasopharyngeal specimens were collected from 1978 and 2211 children (ages, 2 to 6 years) attending day care centers or kindergartens in period 1 (1999-2000) and period 2 (2009-2010), respectively. Carriage of PCV7 serotypes decreased from 12.8% to 8.6% (P < 0.01). The relative contribution of PCV7 serotypes 14 and 18C had decreased, whereas that for non-PCV7 serotypes 19A, 6A, 6C, 23A, and 15B had increased. In period 2, PCV7 penetration rate (at least 1 dose) for children aged 2, 3, 4, and 5 years was 43%, 35.7%, 26.7%, and 20.4%, respectively. In multivariate analysis, PCV7 use was the only independent variable associated with fewer PCV7 serotype carriages (odds ratio 0.5; P = 0.001). In period 2, high rates of dual penicillin/erythromycin nonsusceptibility were found in serotypes 6B (77.3%), 14 (100%), 19F (100%), 23F (78%), 19A (75%), 6A (87.8%), 6C (59.3%), and 23A (78.9%).  相似文献   

9.
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), which is often positive for Panton-Valentine leucocidin (PVL), is increasingly noted as an emerging pathogen worldwide. In Japan, PVLpositive CA-MRSA belonging to multilocus sequence type (ST) 30 has spread and caused, for example, pediatric death due to community-acquired pneumonia and severe pelvic abscesses in an athlete. In this study, we investigated a new rapid screening method for PVL-positive ST30 CA-MRSA and its related clone by a combination of multiplex polymerase chain reaction (M-PCR) and pulsed-field gel electrophoresis (PFGE). For M-PCR, the targets of the assay were the five genes for PVL, collagen adhesin, bone sialoprotein adhesin, methicillin resistance, and S. aureus-specifi c thermostable nuclease. Only PVL-positive ST30 CA-MRSA strains produced all five bands in M-PCR. With PFGE, Japanese strains and most foreign strains of PVLpositive ST30 CA-MRSA shared the same pattern. Moreover, PFGE distinguished current PVL-positive CA-MRSA ST30/spa19 strains from previous PVL-positive MRSA ST30/spa43 strains (which were isolated at the time of nosocomial MRSA outbreaks in the late 1980s and early 1990s) in Japan. Thus, the M-PCR assay rapidly, and the M-PCR/PFGE combination assay more precisely, discriminated between PVL-positive ST30 CA-MRSA (or its related clone) and PVL-positive CA-MRSA belonging to other ST types such as ST1, 8, 59, and 80, PVL-negative CA-MRSA, hospital-acquired MRSA, methicillin-susceptible S. aureus, or coagulase-negative staphylococci (CNS), including MRCNS. This screening method is more useful than genotyping for routine work in many clinical laboratories.  相似文献   

10.
Active surveillance on nasopharygeal carriage of Streptococcus pneumoniae in children was conducted in 5581 children under 16 years old admitted with respiratory illness to the pediatric wards in a Hong Kong teaching hospital during 2008–2010. The isolation rate of S. pneumoniae was 14.5%. The most common serotypes/groups from 911 isolates were 19F, 6B, 23F, 14, 6C, 6A, and 3. Considering only children under 2 years old, the percentage serotype belonging to that of the 7-, 10- and 13-valent conjugate pneumococcal vaccines in S. pneumoniae were 56.0% (115/205), 57% (117/205), and 80.5% (165/205), respectively. The prevalence of penicillin-nonsusceptibility (MIC ≥4.0 μg/mL) was 9.1% and for cefotaxime (MIC ≥2.0 μg/mL) was 14.7%. A high prevalence of non-6B serotype, including 6A, 6C, and 6D was noted after the introduction of PCV7 conjugate pneumococcal vaccines in Hong Kong.  相似文献   

11.
ObjectiveWe aimed to determine novel alleles and their prevalence in Streptococcus canis M-like protein (SCM) and to elucidate association of their alleles with sequence types (STs)/clonal complexes (CCs) and antimicrobial resistance (AMR) phenotypes/genotypes.MethodsWe amplified and sequenced scm, by using primers reported by Pinho recently, for 40 isolates in 2015 and 2017, in which the sequences could not be determined with conventional primers. Isolates, for which SCM alleles, STs, and AMR phenotypes/genotypes were previously determined, were included as controls. A phylogenetic tree of SCM amino acid sequences was constructed. Alleles, based on the tree positions with their prevalence, as well as STs/CCs and AMR phenotypes/genotypes were characterized.ResultsAlthough one isolate possessed SCM allele type 1, 39 isolates had novel allele types 10–15, based on cluster analysis. The 11 and 12 allele types were firstly found in this study. We designated novel allele types as group II and non-novel allele types as group I. Prevalence of group II alleles was 29.9% and 16.2% in 2015 and 2017. Prevalent group II types were allele 10 (10.3%), allele 11 (2.7%), and allele 15 (3.3%) through both periods. There was a significant difference in distribution of STs/CCs between groups I/II SCM populations. We found significant differences in distribution of macrolide/lincosamide AMR genotype (7.7% vs. 26.8%) and AMR rates of fluoroquinolone (0% vs. 12.5%) between the two populations.ConclusionOur study presents group II scm sequences and their prevalence among diseased companion animals in Japan, with association of their alleles with STs.  相似文献   

12.
脑钠肽和内皮素对充血性心力衰竭诊断及预后判断的比较   总被引:2,自引:0,他引:2  
目的探讨脑钠肽(BNP)和内皮素(ET)对充血性心力衰竭(CHF)诊断及预后判断的价值。方法56例CHF患者人院和出院时测定血浆BNP和ET浓度,同时用心脏彩色多普勒测定其左室射血分数(LVEF);分析CHF患者血浆BNP和ET与LVEF的相关性;并以同期住院治疗的无CHF患者32例为对照。随访CHF患者出院后6个月心脏病意外事件的发生情况。结果CHF患者血浆BNP和ET浓度明显高于非CHF患者(P〈0.01);BNP和ET浓度随CHF加重而升高(P〈0.01),但ET浓度心功能Ⅲ级和Ⅱ级比较差异却无统计学意义(P〉0.05);CHF患者BNP和ET与LVEF呈负相关(P〈0.01)。血浆BNP和ET诊断CHF的敏感性分别为93.6%和80.7%,特异性分别为90.8%和75.6%,阴性预测值分别为95.2%和85.3%。发生心脏病意外事件组血浆BNP浓度明显高于未发生事件组,而ET浓度两组比较差异却无统计学意义(P〉0.05)。结论血浆BNP对CHF诊断和预后的判断价值均高于血浆ET。  相似文献   

13.
Studies have shown that pneumococcal vaccination reduces the incidence of Streptococcus pneumoniae infections but does not change the prevalence of S. pneumoniae nasopharyngeal colonization. To comprehensively and longitudinally assess the epidemiology of S. pneumoniae after the introduction of pneumococcal vaccination, we monitored the prevalence and antimicrobial susceptibility of S. pneumoniae, irrespective of its serotypes or pathogenicity, by analyzing specimens collected from a large number of patients at Jikei University Hospitals from 2009 to 2017. A total of 5763 S. pneumoniae isolates were identified out of 375,435 specimens from various sources of patients in different age groups. The prevalence of S. pneumoniae isolated only from patients <5 years old was significantly reduced with the widespread use of pneumococcal vaccines, although this reduction differed by areas where patients resided. The incidence of pneumococcal infections, including bacteremia and otitis media, clearly decreased among patients <5 years old after the introduction of pneumococcal vaccination, while the prevalence of S. pneumoniae isolated from blood specimens of patients 15–64 years old increased, suggesting the involvement of non-vaccine serotypes in the incidence of invasive pneumococcal infections. The antimicrobial susceptibility of S. pneumoniae improved after the introduction of pneumococcal vaccination. Our results show that pneumococcal vaccination has a suppressive effect on the prevalence of S. pneumoniae and the incidence of pneumococcal infections, at least for children <5 years old, in association with an improvement in the antimicrobial susceptibility of S. pneumoniae. However, further measures will be needed to control invasive pneumococcal infections caused by non-vaccine serotypes.  相似文献   

14.
IntroductionB(A) is a relatively rare ABO subtype. The frequency of B(A) in European Caucasians is low, but is higher in China. B(A) is common in northern China, gradually decreasing from north to south. The frequency of B(A) has been researched in northern China, but not in southern China. This study aimed to investigate the serological characteristics and molecular mechanism of the B(A) subtype in Hunan.MethodsThe phenotypes of ABO blood group were analyzed by blood group serology for the patients in the Third Xiangya Hospital from 2016 to 2020; Exons 1–7 of the ABO gene were screened by PCR-SSP and sequencing.ResultsNine cases were suspected as belonging to the B(A) subtype by serological tests. The activity of D-galactosyltransferase in these patients' serum was significantly higher than that of group B plasma; meanwhile, there was no activity of Nacetylgalactosyltransferase. The ABO genotype was ABO*B.01/O.01.01 or ABO*B.01/O.01.02, and the gene sequencing results confirmed the phenotype as B(A), whose gene frequence was 1/26,000. Allele ABO*BA.02 (1/38,000) had the highest frequency, followed by ABO*BA.04 (1/77,000).ConclusionAmong the patients in Hunan, the most common allele encoding the B(A) phenotype was ABO*BA.02. The identification of the B(A) blood group required serological methods combined with genotyping.  相似文献   

15.
Understanding genetically encoded inherited differences in drug metabolism and drug targets offers the promise of minimizing adverse drug reactions and improving therapies. We have compared two real-time PCR-based methods, the TaqMan and LightCycler for the pharmacogenetic evaluation of CYP2B6 516G>T polymorphism. Both methods were found to be suitable for pharmacogenetic testing of CYP2B6 516G>T in the meaning of time consumption, accurate genotype calling, flexible reaction format, simple data analysis, and low cost per assay. The genotype success rate was similar, but the LightCycler procedure was less expensive in terms of cost per sample and shorter running time.  相似文献   

16.
PurposeIn Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains.Materials and methodsPneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to β-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling.ResultsIn PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations.ConclusionsNVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.  相似文献   

17.
Panton Valentine Leukocidin (PVL) is one of the many toxins produced by Staphylococcus aureus. In Japan, PVL-positive S. aureus strains are mainly methicillin-resistant S. aureus (MRSA). Data regarding PVL-positive methicillin-sensitive S. aureus (MSSA) are scarce. In this report, we describe a case of severe infection by PVL-positive MSSA. A 12-year-old healthy girl was admitted with high fever and pain in the lower back. Computed tomography revealed a diagnosis of psoitis and multiple venous thromboses. Blood cultures obtained after admission revealed infection with MSSA. Her fever continued despite adequate antibiotic therapy. On the fifth hospitalization day, she developed bladder dysfunction, and an abscess was noted near the third lumbar vertebra. She underwent an emergency operation and recovered. Bacterial analyses revealed that the causative MSSA was a PVL-producing single variant of ST8 (related to USA300clone), of sequence type 2149. PVL is known to cause platelet activation. This case demonstrates the need for detailed analyses of the causative strain of bacteria in cases of S. aureus infection with deep vein thrombosis, even in cases of known MSSA infection.  相似文献   

18.
目的探讨磷脂酶A2(PLA2)和白细胞介素6(IL-6)在婴幼儿先天性心脏病体外循环(CPB)术后发生急性肺损伤(ALI)的变化及意义。方法采用酶联免疫吸附试验(ELISA)方法对10例先天性心脏病体外循环术后急性肺损伤的婴幼儿、10例先天性心脏病体外循环术后未发生急性肺损伤的婴幼儿和10例来我院门诊健康体检婴幼儿进行白介素6检测;同时对上述3组研究对象应用酶底物显色法检测磷脂酶A2活性。结果先天性心脏病体外循环术后急性肺损伤患儿的磷脂酶A2及白介素6均较手术前及体外循环术后无肺损伤的患儿以及健康儿童明显升高,呈显著性差异,P〈0.01。结论磷脂酶如及白介素6在婴幼儿先天性心脏病体外循环术后急性肺损伤中升高,并呈正相关。在婴幼儿先天性心脏病体外循环术后急性肺损伤中,如果能及早应用磷脂酶A:抑制剂或早期干预细胞因子释放,对于阻止肺损伤的进一步发展,以免发生急性呼吸窘迫综合征(ARDS)具有重要意义。  相似文献   

19.
Staphylococcus aureus (S. aureus) sequence type 398 (ST398) has aroused great concern for its spread throughout the world. ST398 community-acquired MRSA (CA-MRSA) has been given greater emphasis because of its high virulence and high probability of treatment failure. Herein, A 22-year-old male was admitted to our hospital with a history of fever, chest pain and dyspnea for 2 days. A chest CT scan showed infiltrative and nodular shadows. The sequence type of the isolates from blood culture was ST398, the virulence genes detected was PVL gene (lukS-PV and lukF-PV). Despite resuscitation efforts, he died of multiple organ failure on admission 3rd day. This is the first described case of severe pneumonia and sepsis due to hematogenous spread of scalp furuncles caused by Panton–Valentine leukocidin (PVL) positive community-acquired methicillin-susceptible S. aureus (CA-MSSA) ST398 strains in an immunocompentent adult in mainland China. This report highlight the emergence CA-PVL-MSSA ST398 infection and its association with life-threatening infections. Early decolonization and identification of ST398 is critical. Severe skin and soft tissue infections should be suspected for ST398 PVL-MSSA.  相似文献   

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