The rise and fall of bacterial clones: Streptococcus pneumoniae

Globally spreading bacterial strains belong to clonal types that have the capacity to colonize, spread and cause disease in the community. Recent comparative genomic analyses of well-defined clinical isolates have led to the identification of bacterial properties that are required for the successful spread of bacterial clones. In this Review, we discuss the evolution of bacterial clones, the importance of recombination versus mutations for evolution of clones, common methods used to study clonal relationships among bacteria, factors that may contribute to the clonal spread of bacteria and the potential relevance of bacterial clones to clinical disease. We focus on the common pathogen Streptococcus pneumoniae, although other bacteria are also briefly discussed, such as Helicobacter pylori, Staphylococcus aureus and Mycobacterium tuberculosis.     

Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis

To find potential targets of novel antimicrobial agents, we identified essential genes of Streptococcus pneumoniae using comparative genomics and allelic replacement mutagenesis. We compared the genome of S. pneumoniae R6 with those of Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, and selected 693 candidate target genes with > 40% amino acid sequence identity to the corresponding genes in at least two of the other species. The 693 genes were disrupted and 133 were found to be essential for growth. Of these, 32 encoded proteins of unknown function, and we were able to identify orthologues of 22 of these genes by genomic comparisons. The experimental method used in this study is easy to perform, rapid and efficient for identifying essential genes of bacterial pathogens.     

Multilocus sequence typing

Multilocus sequence typing (MLST) provides a new approach to molecular epidemiology that can identify and track the global spread of virulent or antibiotic-resistant isolates of bacterial pathogens using the Internet. MLST databases, together with interrogation software, are available for Neisseria meningitidis and Streptococcus pneumoniae and databases for Streptococcus pyogenes and Staphylococcus aureus will be released shortly.     

Population biology of Gram-positive pathogens: high-risk clones for dissemination of antibiotic resistance

Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium are well-known pathogens of hospitalized patients, frequently linked with resistance against multiple antibiotics, compromising effective therapy. Streptococcus pneumoniae and Streptococcus pyogenes are important pathogens in the community and S. aureus has recently emerged as an important community-acquired pathogen. Population genetic studies reveal that recombination prevails as a driving force of genetic diversity in E. faecium, E. faecalis, S. pneumoniae and S. pyogenes, and thus, these species are weakly clonal. Although recombination has a relatively modest role driving the genetic variation of the core genome of S. aureus, the horizontal acquisition of resistance and virulence genes plays a key role in the emergence of new clinically relevant clones in this species. In this review, we discuss the population genetics of E. faecium, E. faecalis, S. pneumoniae, S. pyogenes and S. aureus. Knowledge of the population structure of these pathogens is not only highly relevant for (molecular) epidemiological research but also for identifying the genetic variation that underlies changes in clinical behaviour, to improve our understanding of the pathogenic behaviour of particular clones and to identify novel targets for vaccines or immunotherapy.     

Antimicrobial-resistant bacteria in the community setting

Over the past decade, antimicrobial resistance has emerged as a major public-health crisis. Common bacterial pathogens in the community such as Streptococcus pneumoniae have become progressively more resistant to traditional antibiotics. Salmonella strains are beginning to show resistance to crucial fluoroquinolone drugs. Community outbreaks caused by a resistant form of Staphylococcus aureus, known as community-associated meticillin (formerly methicillin)-resistant Staphylococcus aureus, have caused serious morbidity and even deaths in previously healthy children and adults. To decrease the spread of such antimicrobial-resistant pathogens in the community, a greater understanding of their means of emergence and survival is needed.     

小儿细菌性肺炎痰培养物病原菌构成及耐药性分析

目的 分析肺炎患儿痰培养物中细菌构成及其耐药情况,为小儿细菌性肺炎治疗提供合理使用抗感染药物的依据。 方法 对2019年11月至2020年12月济宁市第一人民医院临床送检的肺炎患儿痰液标本进行病原菌的分离鉴定和药敏试验,收集相关资料,并对小儿肺炎病原菌在临床上的分布特点及其耐药情况进一步分析。 结果 痰液培养阳性患儿183例,其中多重细菌感染患儿16例,共培养出199株细菌(剔除同一患儿的重复菌株),其中革兰阴性菌128株(64.32%),革兰阳性菌71株(35.68%)。主流细菌为肺炎链球菌(19.60%)、金黄色葡萄球菌(16.08%)、卡他莫拉菌(13.57%)、肺炎克雷伯菌(11.06%)和大肠埃希菌(10.05%)。多重细菌感染主要以金黄色葡萄球菌+肺炎克雷伯菌、金黄色葡萄球菌+大肠埃希菌混合为主(共5例,31.25%)。肺炎链球菌和金黄色葡萄球菌对利奈唑胺、万古霉素绝对敏感。卡他莫拉菌对头孢噻肟、头孢曲松、米诺环素等绝对敏感。肠杆菌科中肺炎克雷伯菌和大肠埃希菌药敏结果相似,对阿米卡星、厄他培南、头孢替坦敏感率在95%~100%,对氨苄西林耐药率在95%~100%。肺炎链球菌、金黄色葡萄球菌、卡他莫拉菌对左氧氟沙星敏感率均大于90%,大肠埃希菌、肺炎克雷伯菌对左氧氟沙星不够敏感,但中敏率达到75%以上。 结论 肺炎链球菌、金黄色葡萄球菌、卡他莫拉菌、肺炎克雷伯菌、大肠埃希菌感染是本地肺炎患儿痰液中常见菌,抗生素耐药状况比较严重,亚胺培南对革兰阴性菌混合感染的效果较好,利奈唑胺对阳性菌效果较好;左氧氟沙星可用于革兰阴性和阳性菌混合感染,合理使用抗生素应注意针对性和用药时间,杀灭致病菌的同时尽可能维护正常菌群的平衡。     

Enolases from Gram-positive bacterial pathogens and commensal lactobacilli share functional similarity in virulence-associated traits

Enolase occurs as a cytoplasmic and a surface-associated protein in bacteria. Enolases of the bacterial pathogens Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus, as well as of the commensal lactic acid bacteria, Lactobacillus crispatus and Lactobacillus johnsonii, were purified as His(6)-fusion proteins from recombinant Escherichia coli. The fusion proteins were compared for putative virulence-associated functions, i.e., binding of human plasminogen, enhancement of plasminogen activation by human plasminogen activators, as well as binding to immobilized laminin, fibronectin and collagens. The individual enolases showed varying efficiencies in these functions. In particular, highly and equally effective interactions with plasminogen and laminin were seen with lactobacillar and staphylococcal enolases.     

Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. II. Immunological mechanisms involved in thrombocytolysis.

Immunological mechanisms are involved in the thrombocytolytic activity of peptidoglycan of Group A streptococcus, Streptococcus pneumoniae and Staphylococcus aureus. Inactivation of particular components of complement (heating of blood serum to 56 degrees C,incubation with zymosan or NH4OH) inhibited the thrombocytolytic activity of group A streptococcus peptidoglycan. So did preincubation of Group A streptococcus peptidoglycan with homologous antipeptidoglycan antibody. On the other hand, antibody to Group A streptococcus peptidoglycan did not inhibit the thrombocytolytic effect of Streptococcus pneumoniae or Staphylococcus aureus peptidoglycan. Human platelets are resistant to peptidoglycans. They remain resistant in the presence of rabbit serum although rabbit platelets are highly sensitive to peptidoglycans. This suggests that, for the expression of the thrombocytolytic activity of bacterial peptidoglycan, specific receptors on the surface of platelets must be present in addition to serum factors.     

Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens

A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens.     

Use of a DNA microarray for detection and identification of bacterial pathogens associated with fishery products

We established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes of Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica and the 16S-23S rRNA gene internal transcribed spacer (ITS) region of Proteus mirabilis and Proteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.     

重症肺炎新生儿支气管肺泡灌洗液病原菌分布及耐药性

目的 研究重症肺炎新生儿支气管肺泡灌洗液的病原菌分布和耐药性。方法 选择2016年4月至2018年4月在本院呼吸科治疗的新生儿268例,其中符合重症肺炎诊断标准的患儿142例,归为重症肺炎组;不符合重症肺炎诊断标准的患儿126例,归为对照组。检测患儿肺泡灌洗液病原菌分布情况和耐药情况。结果 重症肺炎组患儿肺炎克雷伯菌、流感嗜血菌、铜绿假单胞菌、阴沟肠杆菌、大肠埃希菌、金黄葡萄球菌、溶血葡萄球菌、表皮葡萄球菌、肺炎链球菌、草绿链球菌检出率明显高于对照组。肺炎克雷伯菌对亚胺培南,美罗培南的耐药性为0.0%,大肠埃希菌对亚胺培南,美罗培南,阿米卡星的耐药性为0.0%,阴沟肠杆菌对亚胺培南,美罗培南,左氧氟沙星的耐药性为0.0%,肺炎链球菌对万古霉素的耐药性为0.0%,金黄葡萄球菌对万古霉素的耐药性为0.0%。结论 新生儿重症肺炎患者病原菌以革兰阴性菌为主,亚胺培南、美罗培南、万古霉素可以用于治疗新生儿重症肺炎,但由于其毒副作用较大,应严格把握适应症。     

奶牛产后感染性子宫疾病对子宫和卵巢的影响研究进展

微生物入侵是引起奶牛产后子宫疾病的主要因素,产后子宫能检出丰富的微生物种群,主要包括公认的致病菌如大肠杆菌、隐秘脓杆菌、坏死梭杆菌等,机会病原菌如产气荚膜梭菌、肺炎克雷伯菌、微球菌等和潜在致病菌如消化链球菌、金黄色葡萄球菌等。近年来,运用分子微生态技术发现子宫中的微生物属于变形菌门、梭杆菌门、厚壁菌门、拟杆菌门和软壁菌门5个已知的门和一类未被培养的种群,其中拟杆菌属、梭菌属等种群与子宫疾病密切相关。细菌侵入子宫后,以大肠杆菌为代表的革兰氏阴性菌和以化脓隐秘杆菌为代表的革兰氏阳性菌可被子宫内膜细胞上的Toll样受体识别引起炎症反应,改变子宫前列腺素分泌类型,影响卵泡发育、黄体大小,降低血清中雌激素和孕激素浓度,造成奶牛不发情、不排卵,导致产犊间隔延长、产奶量和产犊数量下降,严重影响奶业经济效益。本文从产后奶牛子宫内主要病原菌的种类及其与子宫健康状态的关系、子宫内膜对病原菌的识别与先天免疫、子宫疾病对子宫和卵巢功能的影响等方面对国内外研究进展进行了综述。     

A Novel Computational Method Identifies Intra- and Inter-Species Recombination Events in Staphylococcus aureus and Streptococcus pneumoniae

Advances in high-throughput DNA sequencing technologies have determined an explosion in the number of sequenced bacterial genomes. Comparative sequence analysis frequently reveals evidences of homologous recombination occurring with different mechanisms and rates in different species, but the large-scale use of computational methods to identify recombination events is hampered by their high computational costs. Here, we propose a new method to identify recombination events in large datasets of whole genome sequences. Using a filtering procedure of the gene conservation profiles of a test genome against a panel of strains, this algorithm identifies sets of contiguous genes acquired by homologous recombination. The locations of the recombination breakpoints are determined using a statistical test that is able to account for the differences in the natural rate of evolution between different genes. The algorithm was tested on a dataset of 75 genomes of Staphylococcus aureus and 50 genomes comprising different streptococcal species, and was able to detect intra-species recombination events in S. aureus and in Streptococcus pneumoniae. Furthermore, we found evidences of an inter-species exchange of genetic material between S. pneumoniae and Streptococcus mitis, a closely related commensal species that colonizes the same ecological niche. The method has been implemented in an R package, Reco, which is freely available from supplementary material, and provides a rapid screening tool to investigate recombination on a genome-wide scale from sequence data.     

Organism-specific neutrophil-endothelial cell interactions in response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus

The recruitment of polymorphonuclear leukocytes (PMNs) from the vascular space into the lung interstitium and airspace is an early step in the host innate immune response to bacterial invasion of these sites. To determine the ability of intact bacteria to directly elicit PMN migration across an endothelial monolayer, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli, as well as to purified E. coli LPS. Bacterial induction of PMN migration was dose dependent and elicited by > or =10(4) bacteria/ml of each of the species tested. Pretreatment of PMNs with blocking Abs to CD18 significantly inhibited migration of PMN in response to all stimuli tested, but had the most profound effect on migration to S. pneumoniae and S. aureus. Intact E. coli were 10 times more potent in inducing transmigration of PMNs than a corresponding amount of purified LPS. Bacterial induction of PMN migration did not correlate with up-regulation of surface endothelial ICAM-1 expression (purified LPS > intact E. coli > S. aureus and S. pneumoniae) nor up-regulation of VCAM-1 and E-selectin. Neutralizing Ab to ICAM-1 had no effect on PMN migration to any of the bacteria or to purified LPS. These findings demonstrate that diverse bacterial pathogens induce PMN migration across a pulmonary microvascular endothelial cell monolayer in a fashion that appears to be organism specific. In addition, intact bacteria elicit PMN-endothelial cell interactions distinct from those seen when purified bacterial products are used as agonists.     

Universal primers for detection of common bacterial pathogens causing prosthetic joint infection

The diagnosis of low grade prosthetic joint infection is difficult and time consuming. Nested-PCR for universal bacterial DNA segments detection of "orthopaedic" bacteria was tested in a laboratory setting. This method is based on amplification of the 16S bacterial ribosomal RNA coding sequences. 11 species of the most frequent bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens) involved in prosthetic joint infections were studied. All could be detected rapidly and sensitively by this method.     

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2)

We describe the identification of Rex, a novel redox-sensing repressor that appears to be widespread among Gram-positive bacteria. In Streptomyces coelicolor Rex binds to operator (ROP) sites located upstream of several respiratory genes, including the cydABCD and rex-hemACD operons. The DNA-binding activity of Rex appears to be controlled by the redox poise of the NADH/NAD+ pool. Using electromobility shift and surface plasmon resonance assays we show that NADH, but not NAD+, inhibits the DNA-binding activity of Rex. However, NAD+ competes with NADH for Rex binding, allowing Rex to sense redox poise over a range of NAD(H) concentrations. Rex is predicted to include a pyridine nucleotide-binding domain (Rossmann fold), and residues that might play key structural and nucleotide binding roles are highly conserved. In support of this, the central glycine in the signature motif (GlyXGlyXXGly) is shown to be essential for redox sensing. Rex homologues exist in most Gram-positive bacteria, including human pathogens such as Staphylococcus aureus, Listeria monocytogenes and Streptococcus pneumoniae.     

闭环式护理对小儿细菌性肺炎症状和呼吸道菌群的影响

目的 探究细菌性肺炎患儿实行闭环式护理管理模式后患儿临床症状、呼吸道菌群及疾病复发的情况。方法 选取2016年5月至2018年11月我院收治的113例细菌性肺炎患儿为研究对象,随机分为观察组（n=55）和对照组（n=58）。两组患儿均给予抗生素治疗及常规护理。观察组患儿在常规护理基础上应用闭环式护理。观察两组患儿各主要临床症状缓解时间及住院时间、治疗有效率、治疗前及治疗1周后呼吸道菌群分布情况及治疗结束后6个月内细菌性肺炎复发率。结果 观察组患儿体温恢复时间、咳嗽及咳痰缓解时间及住院时间均短于对照组（均P0.05）。观察组患儿治疗有效率为89.09%（49/55）,高于对照组的72.41%（42/58）。结论 细菌性肺炎患儿呼吸道菌群以肺炎链球菌、金黄色葡萄球菌、肺炎克雷伯菌及大肠埃希菌为主。应用闭环式护理管理模式后,可明显改善细菌性肺炎患儿各临床症状,缩短症状缓解时间,抑制呼吸道革兰阳性菌群生长,但对治疗后复发率影响不大。     

How do microbes evade neutrophil killing?

Many microbial pathogens evolved to circumvent the attack of neutrophils, which are essential effector cells of the innate immune system. Here we review six major strategies that pathogenic bacteria and fungi use to evade neutrophil defences: (i) turning on survival and stress responses, (ii) avoiding contact, (iii) preventing phagocytosis, (iv) surviving intracellularly, (v) inducing cell death and (vi) evading killing by neutrophil extracellular traps. For each category we give examples and further focus on one particular pathogenic microbe in more detail. Pathogens include Candida albicans, Cryptococcus neoformans, Yersinia ssp., Helicobacter pylori, Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae.     

Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria

We deleted a subset of 27 open reading frames (ORFs) from Escherichia coli which encode previously uncharacterized, probably soluble gene products homologous to proteins from a broad spectrum of bacterial pathogens such as Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis and only distantly related to eukaryotic proteins. Six novel bacteria-specific genes essential for growth in complex medium could be identified through a combination of bioinformatics-based and experimental approaches. We also compared our data to published results of gene inactivation projects with Mycoplasma genitalium and Bacillus subtilis and looked for homologs in all known prokaryotic genomes. Such analyses highlight the enormous metabolic flexibility of prokaryotes. Six of 27 studied genes have been functionally characterized up to now, amongst these four of the essential genes. The gene products YgbP, YgbB and YchB are involved in the non-mevalonate pathway of isoprenoid biosynthesis. KdtB is characterized as the posphopantetheine adenylyltransferase CoaD. There are indications that the other two essential gene products YjeE and YqgF, which we have identified, also possess enzymatic functions. These findings demonstrate the potential of such proteins to be used in screening of large chemical libraries for inhibitors which could be further developed to novel broad-spectrum antibiotics.     

Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.