Serotypes of carriage and invasive isolates of Streptococcus pneumoniae in Brazilian children in the era of pneumococcal vaccines

Nasopharyngeal carriage of Streptococcus pneumoniae is a key factor in the development of invasive disease and the spread of resistant strains within the community. A single nasopharyngeal swab was obtained from 648 unvaccinated children aged <5 years, either healthy or with acute respiratory tract infection or meningitis, during the winters of 2000 and 2001. The overall pneumococcal carriage rate was 35.8% (95% CI 32.1-39.6). The pneumococcal serotypes found most frequently in the nasopharynx were 14, 6B, 6A, 19F, 10A, 23F and 18C, which included five of the seven serotypes in the currently licensed seven-valent conjugate vaccine (PCV7); serotypes 4 and 9V were less common. Serotypes 1 and 5 were isolated rarely from the nasopharynx. A comparison of 222 nasopharyngeal isolates with 125 invasive isolates, matched for age and time to the carrier isolates, showed a similar prevalence of penicillin non-susceptible pneumococci (PNSp) (19.8% and 19.2%, respectively). PNSp serotypes were similar (6B, 14, 19F, 19 A, 23B and 23F) for carriage and invasive disease isolates. The coverage of PCV7 for carriage isolates (52.2%) and invasive isolates (62.4%) did not differ significantly (p 0.06); similarly, there was no significant difference in PCV7 coverage for carriage isolates (34.5%) and invasive isolates (28.2%) of PNSp. These data suggest that PCV7 has the potential to reduce pneumococcal carriage and the number of carriers of PNSp belonging to vaccine serotypes.     

Antimicrobial resistance of nasopharyngeal pneumococci from children from day-care centres and orphanages in Russia: results of a unique prospective multicentre study

This study assessed the antimicrobial resistance of nasopharyngeal pneumococci isolated from children aged < 5 years in day-care centres and orphanages throughout Russia during 2001-2002. Swabs were collected from 2484 children in 43 day-care centres and eight orphanages in 11 cities of European Russia, and from 1669 children in 37 day-care centres and three orphanages in eight cities of Asian Russia, with a total of 1144 and 912 Streptococcus pneumoniae isolates being recovered in European and Asian Russia, respectively. All macrolide-non-susceptible (MICs 0.5-128 mg/L) and fluoroquinolone-non-susceptible (ciprofloxacin MICs > or = 4 mg/L) isolates were tested for resistance mechanisms and clonal relatedness. Non-susceptibility rates, by CLSI criteria, were 19.3%, 0.9% and 0.4% for penicillin G, cefotaxime and amoxycillin-clavulanate, respectively. Resistance to macrolides and lincosamides was also relatively low, i.e., < 7% for clindamycin and 14- and 15-membered macrolides. The highest rates of non-susceptibility were for tetracycline and co-trimoxazole (52.0% and 64.5%, respectively). No clones resistant to ciprofloxacin (MICs > or = 8 mg/L) were found, but 1.7% of isolates were non-susceptible (MIC 4 mg/L). No resistance was found to levofloxacin, gemifloxacin, telithromycin or vancomycin. Pulsed-field gel electrophoresis analysis showed no relationship between ciprofloxacin- and macrolide-non-susceptible isolates in European and Asian Russia. Resistance among macrolide-resistant isolates resulted mostly from the presence of erm(B) and mef(A), and from changes in L4; additionally, L22 mutations were common in isolates from Asian Russia. Non-susceptibility to quinolones was associated with mutations in parC and parE among European isolates. Asian Russian isolates had mutations in parC and gyrA, and alterations in parE were more common. There were substantial differences in non-susceptibility and mechanisms of resistance between pneumococci from Asian and European Russia, with orphanages appearing to be 'hot-spots' of resistance.     

A simple and reliable method to screen isolates of Escherichia coli and Klebsiella pneumoniae for the production of TEM- and SHV-derived extended-spectrum β-lactamases

Objective: To evaluate which of 24 β-lactams used in susceptibility tests best discriminated between strains of Klebsiella pneumoniae and Escherichia coli that produce extended spectrum β-lactamases (ESBLs) from strains that produce older, more familiar, plasmid-mediated β-lactamases such as TEM-1 and SHV-1.
Methods: Susceptibility to the 24 β-lactam agents was determined by agar dilution and disk diffusion methodologies, using 27 strains of K. pneumoniae and E. coli that produced 22 different older plasmid-mediated β-lactamases and 28 strains that produced 17 different ESBLs.
Results: In general, strains that produced ESBLs were intermediate or resistant to cefpodoxime, whereas those that produced other β-lactamases were susceptible to this agent. The agar dilution test exhibited 96% sensitivity and 100% specificity in discriminating these two groups of organisms. The disk diffusion test exhibited 100% sensitivity and 96% specificity. All other β-lactam agents tested were inferior discriminators between the two groups of organisms.
Conclusions: Agar dilution and disk diffusion tests with cefpodoxime can be used to discriminate strains of K. pneumoniae and E. coli that produce ESBLs from those that produce older, plasmid-mediated β-lactamases.     

Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in a university hospital, Ankara, Turkey

Penicillin-resistant Streptococcus pneumoniae isolates (n = 76) from clinical samples of patients admitted to Hacettepe University Hospital between January 1997 and December 2001 were included in the study. MICs of penicillin G, erythromycin A, clindamycin, cefaclor, cefotaxime, vancomycin, chloramphenicol, tetracycline, ciprofloxacin and rifampicin were determined by agar dilution. The isolates were serogrouped on the basis of the Neufeld Quellung reaction and were typed by BOX-PCR. Genetic polymorphism of the penicillin resistance genes pbp2b and pbp2x was investigated by restriction fragment length polymorphism (RFLP) analysis. Of the 76 isolates tested, 64 (84.2%) showed intermediate resistance to penicillin, while 12 (15.8%) were resistant to higher levels of penicillin (MIC > or = 2 mg/L). The resistance patterns of the isolates revealed six different resistance profiles. There were 22 different serotypes, with c. 55% of the isolates belonging to serotypes 23B, 19A, 19F, 14, 6 A and 9V. Five distinct patterns for pbp2b and 12 distinct patterns for pbp2x were obtained by RFLP analysis of penicillin-binding protein genes. The combination of these patterns allowed isolates to be classified into 22 fingerprint subgroups. BOX-PCR analysis showed that the isolates fell into 14 distinct BOX genotypes, with 33 subtypes. Serotype 9V isolates with pbp genotype 2-6 and BOX-PCR type 4, 4.1 or 4.2 were related to the pandemic clone Spain(9V)-3. No relatedness to other international clones was detected among the other study strains, but genetic relatedness was observed among some of the serotype 19A and 23B isolates. Overall, the results demonstrated that most of the penicillin-resistant pneumococcal isolates in Turkey, other than those belonging to serotypes 9V, 19A and 23B, were derived from several independent clones, possibly resulting from multiple importation of strains originating from outside the country. Differences in pbp patterns, serotypes and resistance profiles among isolates that showed similar BOX-PCR patterns supported the hypothesis that horizontal transfer of capsular genes, pbp genes and other genetic determinants between S. pneumoniae and viridans group streptococci may have occurred.     

In vivo efficacy of cefotaxime and amoxicillin against penicillin-susceptible, penicillin-resistant and penicillin-cephalosporin-resistant strains of Streptococcus pneumoniae in a mouse pneumonia model

Objective: To compare cefotaxime (CTX) to amoxicillin (AMO) (usually considered the definitive therapy for penicillinsusceptible Streptococcus pneumoniae infections) in an immunocompromised mouse pneumonia model.
Methods: Three S. pneumoniae clinical isolates were used: two serotype 19 strains, a penicillin-susceptible (P^s) strain (penicillin MIC = 0.03 μ/mL) and a highly penicillin-resistant (P^r) strain (penicillin MIC = 4 μ/mL), and one serotype 23F strain, a penicillin-cephalosporin-resistant (CFTR) strain (CTX MIC = 4 μ/mL).
Results: CTX activity in this mouse model of pneumonia induced by the highly penicillin-resistant strain of S. pneumoniae was lower than expected from its low MIC against this organism. Furthermore, AMO had greater efficacy than CTX against a CFTR S. pneumoniae strain.
Conclusion: Our data suggest that there is no major difference in the in vivo efficacy of the two agents, cefotaxime and amoxicillin, against penicillin-resistant and penicillin-cephalosporin-resistant S. pneumoniae.     

柑桔黄龙病的直接荧光诊断法

试验证明,直接荧光诊断法是诊断柑桔黄龙病的一种快速、简便、准确的方法。 将健叶和黄龙病叶叶柄用保险刀片徒手切片,在透射式荧光显微镜下检查,可看到健叶叶柄的木质部导管细胞壁发黄色荧光,韧皮纤维细胞发绿色荧光,而病叶叶柄韧皮部中有1～多个鲜明的黄色或黄绿色荧光团块。这种特异性的荧光,在健叶柄韧皮部中是没有的,其它由病毒、类病毒、细菌、真菌等病原引起的病叶柄韧皮部中亦没有此种特异性荧光。     

探针ASPCR检测肺炎支原体 微量耐药突变A2063G方法的建立

[摘要]　目的　 建立能够特异性检测微量肺炎支原体（Mycoplasma pneumoniae, MP）A2063G耐药突变基因的特异性扩增等位基因的探针法实时定量PCR（probe-based allele-specific real-time PCR, 探针ASPCR）方法。方法?建立特异性检测A2063G耐药突变位点的探针ASPCR方法,并验证其灵敏度、特异度及准确度等性能。结果?特异性扩增2063G和非特异性扩增2063A/G的引物/探针组合分别扩增105拷贝野生基因型（2063A）模板的Ct值的差(△Ct)高达10.93,能够特异性检测A2063G突变。探针ASPCR方法检测2063G基因型占总MP的比例的准确度可低至1％;检测MP的灵敏度低至10拷贝,检测A2063G耐药突变比例的灵敏度低至0.01％。探针ASPCR方法与前期建立的染料ASPCR方法检测临床样本的MP感染结果一致,MP阳性检出率均为94.83%（55/58）,高于传统巣式PCR联合测序方法的检测结果（75.86%,44/58）;探针ASPCR和染料ASPCR 2种方法检测MP耐药率分别为63.64%（35/55）、70.91%（39/55）,高于传统巣式PCR联合测序方法检测结果59.09%（26/44）。结论?新建探针ASPCR方法是一种具有高特异度、准确度和灵敏度的快速检测MP微量A2063G耐药突变的方法;与染料ASPCR方法相比,探针ASPCR方法检测耐药MP的灵敏度略低,但其临床样本检测复查率也低于染料ASPCR方法,且其结果判读简单,更适合在临床中应用推广,能够为临床制定MP及耐药MP感染的治疗方案提供理论依据。     

内膜转运蛋白编码基因PEG-344在肺炎克雷伯菌毒力鉴别中的应用

 目的 评估新型标志物PEG-344在高毒力肺炎克雷伯菌以及经典肺炎克雷伯菌毒力鉴别中的应用价值。方法 收集2020—2021年某院血流感染患者血标本分离的肺炎克雷伯菌,采用大蜡螟毒力试验结果作为金标准进行分组,将菌株分为高毒力组和经典组;进一步进行黏液丝试验、wzi基因测序和毒力基因检测_p(rmpA、_prmpA2、PEG-1631、PEG-589、PEG-344)。结果 共收集肺炎克雷伯菌36株,根据大蜡螟毒力试验结果将其分为高毒力组16株,经典组20株;高毒力组黏液丝阳性菌株占87.50%(14株),经典组黏液丝阳性菌株占15.00%(3株);毒力基因PEG-344检测的灵敏度、特异度高于经典的独立标志物_prmpA、_prmpA2。除1株经典组wxwh3(wzi-2)菌株PEG-344阳性以外,其余菌株PEG-344结果与毒力分组完全一致。结论 内膜转运蛋白编码基因PEG-344能更准确地鉴别高毒力菌株,及时提醒临床合理用药,为临床医生优化诊疗方案提供依据。     

应用MALDI-TOF MS快速鉴定blaKPC-2基因型肺炎克雷伯菌

 目的 探讨基质辅助激光解析/电离飞行时间质谱(MALDI-TOF MS)对携带bla_KPC-2基因型肺炎克雷伯菌的快速鉴定能力。方法 收集2018年9月-2020年11月蚌埠医学院第一附属医院临床住院患者分离的肺炎克雷伯菌,采用纸片扩散法进行药敏试验。应用聚合酶链反应(PCR)方法筛选携带bla_KPC-2基因型肺炎克雷伯菌。MALDI-TOF MS鉴定菌株并收集携带bla_KPC-2基因型和碳青霉烯类敏感肺炎克雷伯菌(CSKP)的质谱图,各自选取其中70株菌株图谱,建立携带bla_KPC-2基因型和CSKP的超级图库(Super-Spectra)。采用超级图库鉴定除建库以外的肺炎克雷伯菌,根据耐药表型和PCR结果,判断鉴定结果是否准确。结果 共收集295株肺炎克雷伯菌,经耐药表型筛选耐碳青霉烯类肺炎克雷伯菌(CRKP)143株和CSKP 152株。CRKP中鉴定出140株携带碳青霉烯酶基因(134株检出bla_KPC-2基因,3株检出bla_KPC-18基因,2株检出bla_NDM-1基因,1株检出bla_IMP基因),3株不携带碳青霉烯酶基因;其中2株同时检出bla_KPC-2基因和bla_NDM-1基因。根据建库要求,建立携带bla_KPC-2基因型和CSKP的超级图库(Super-Spectra);设置error值<0.5,二者重合率达80%;对比图发现,4 154.4、8 310.7、10 880.8、3 579、10 079.3 m/z五个峰可作为区分携带bla_KPC-2基因肺炎克雷伯菌和CSKP的特征峰。选取除建库以外的155株肺炎克雷伯菌进行验证,准确率为92.90%(144/155);其中携带bla_KPC-2基因肺炎克雷伯菌准确率为94.52%(69/73),CSKP准确率为91.46%(75/82)。结论 通过MALDI-TOF MS建立Super-Spectra,可快速预测bla_KPC-2基因型肺炎克雷伯菌,为临床CRKP感染的诊疗及医院感染防控提供可靠的实验室依据。     

沈阳地区肺炎支原体分离株P1蛋白基因限制性酶切图谱分型研究

目的：根据肺炎支原体（MP）P1蛋白基因限制性酶切图谱分类MP临床分离株,调查沈阳地区肺炎支原体流行情况。方法：多聚酶链反应扩增MP FH标准株和MP临床分离株的P1基因片段,扩增产物用限制性内切酶HeaⅢ消化,1.5％琼脂糖凝胶电泳分析。结果：肺炎支牟体临床分离株和FH标准株酶切图谱相同。结论：沈阳地区肺炎支原体分离株属于Ⅱ型。