解脲脲原体生物被膜形成与耐药性的相关性研究

目的 研究解脲脲原体(Ureaplasma urealyticum,Uu)标准株及临床分离株体外形成生物被膜的能力及游离状态与形成生物被膜后药物敏感性的差异.方法 对Uu标准株3、8血清型(Uu3、Uu8)及从女性患者宫颈中分离鉴定的21株Uu临床株进行体外培养后,扫描电镜、激光共聚焦显微镜鉴定生物被膜形成,并在生物被膜形成前后进行约敏测定(四环素、红霉素、环丙沙星).配对秩和检验及x2检验分别比较Uu游离状态及形成生物被膜后最低抑菌浓度间及耐药率间的差异.结果 Uu3、Uu8及21株Uu临床株均具有体外形成生物被膜的能力.Uu形成牛物被膜后对四环素、红霉素及环丙沙星的最低抑菌浓度较游离状态明显增高(P<0.001).Uu形成生物被膜后对红霉素及环丙沙星的耐药率增高具有统计学意义(P值分别为<0.001及0.035),但对四环素的耐药率增高无统计学意义(P=0.293).结论 Uu标准株及临床株均具有体外形成生物被膜的能力,Uu形成生物被膜后对抗菌素的抵抗力增加,出现了多重耐药现象.     

解脲脲原体生物被膜形成与耐药性的相关性研究

目的 研究解脲脲原体(Ureaplasma urealyticum,Uu)标准株及临床分离株体外形成生物被膜的能力及游离状态与形成生物被膜后药物敏感性的差异.方法 对Uu标准株3、8血清型(Uu3、Uu8)及从女性患者宫颈中分离鉴定的21株Uu临床株进行体外培养后,扫描电镜、激光共聚焦显微镜鉴定生物被膜形成,并在生物被膜形成前后进行约敏测定(四环素、红霉素、环丙沙星).配对秩和检验及x2检验分别比较Uu游离状态及形成生物被膜后最低抑菌浓度间及耐药率间的差异.结果 Uu3、Uu8及21株Uu临床株均具有体外形成生物被膜的能力.Uu形成牛物被膜后对四环素、红霉素及环丙沙星的最低抑菌浓度较游离状态明显增高(P<0.001).Uu形成生物被膜后对红霉素及环丙沙星的耐药率增高具有统计学意义(P值分别为<0.001及0.035),但对四环素的耐药率增高无统计学意义(P=0.293).结论 Uu标准株及临床株均具有体外形成生物被膜的能力,Uu形成生物被膜后对抗菌素的抵抗力增加,出现了多重耐药现象.     

嗜肺军团菌生物膜的形成与毒力基因表达量分析

目的 初步探讨生物膜相关军团菌独特的毒力基因表达特征.方法 在富营养条件下建立静止状态的单细胞嗜肺军团菌生物膜模型.运用反转录real-time PCR技术,比较和分析浮游和生物膜状态下嗜肺军团菌部分毒力基因的表达,毒力基因包括mip、flaA和fliA基因.结果 对数生长后期与对数生长期浮游菌的mip、flaA和fliA基因表达量比值分别为0.53、4.45、3.67,对数生长期浮游菌显示复制态军团菌的毒力基凶表达特征,对数生长后期表达转移态军团菌特点.生物膜相关军团菌与对数生长期浮游菌的mip、flaA和fliA基因表达量比值为4.42、5.24、16.21;与对数生长后期浮游菌的比值为8.39、1.18、4.43,生物膜相关军团菌同时表达复制态和转移态军团菌毒力基因特征.结论 生物膜相关军团菌毒力基因的表达具有其特殊性,有待进一步研究.

Abstract：

Objective To investigate the physiological state of L. pneumophila in biofilm. Methods Genes previously identified as good markers for the transmissive and replicative phases of the L. pneumophila life cycle during growth in Acanthamoeba castellanii were examined for their expression fold change in the sessile cells as compared to planktonic cells using real-time RT PCR. Results Mature L. pneumophila biofilms were formed at 37t in 75 cm2 cell culture treated flasks for 18 days. The ratio of gene (mip, flaA and fliA) expression in post-exponential cells compared to exponential cells is 0. 53, 4. 45 and 3. 67. The exponential phase cultures display replicative traits and post-exponential bacteria express transmissive factors. The ratio of gene (mip, flaA and fliA) expression in sessile cells compared to exponential cells is 4.42, 5.24 and 16.21, while the sessile cells compared to exponential cells is 8.39, 1. 18 and 4. 43, respectively. Conclusion The violence gene expression of L pneumophila in biofilm is unique.     

泛耐药肺炎克雷伯菌株的医院内流行特性的研究

目的 了解华山医院泛耐药肺炎克雷伯菌株及其流行的特点.方法 收集2006年8月-2009年12月对CLSI推荐常规检测药物均耐药的肺炎克雷们菌临床分离株,共57株.所有菌株都进行药物敏感试验、超广谱β-内酰胺酶(ESBLs)初筛及表型确证试验、改良Hodge试验、等电聚焦电泳,聚合酶链反应及其产物测序、接合试验、肠杆菌基因间重复共有序列PCR(ERIC-PCR)和多位点序列分型(MLST).结果 所有菌株都携带blaKPC-2、blaCTX-M-14、blaSHV12和blaTEM-1及qnrB和aac(6')-I b-cr基因.57株细菌中ST423型5株,MIST ST11型52株.ST423型散发,而ST11型呈医院内流行.57株细菌都对替加环素耐药,对多黏菌素、米诺环素和多西环素部分敏感.结论 本次泛耐药肺炎克雷们流行主要为ST11型菌株;不同的肺炎克雷伯菌株,播散能力不同;检出泛耐药肺炎克雷伯菌时应增加检测药物的种类.

Abstract：

Objective To understand the epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae occurred between 2006 and 2009 in a university hospital of Shanghai, China. Methods A total of 57 panresistance K. pneumoniae isolates were collected from August 2006 to December 2009.Antibiotic susceptibility of the isolates were determined by Kirby-Bauer disc diffusion method and microbroth dilution (MBD). ESBLs-producing initial screen test and phenotypic confirmatory test and carbapenemase-producing modified Hodge test ( MHT) were performed to detect the resistance phenotype of the isolates. Be-ta-lactamases were studied by IEF, PCR and the product sequencing. While conjugation assay were conducted to understand the transferability of these genes. The genetic relationship between isolates was established by ERIC-PCR and multilocus sequence typing (MLST). Except for the antibiotics recommended by CLSI guideline in the routine test, the other antibiotics were added to find out the effective drugs to treat the infection. Results All 57 isolates were highly resistant to all examined antibiotics. All isolates produced ESBLs and carbapenemase. IEF revealed that each isolate produced four beta-lactamases. All isolates carried blaKPC-2,blaCTX-M-14,blaSHV12,blaTEM-1,qnrB and aac(6') - I b-cr. Forty-four of the 57 (77.2% ) isolates were successful to transfer their resistance genes to E. coli recipient J53 by conjugation assay. By RAPD, all 57 isolates were grouped into two genotypes that were further identified as members of MUST types 423 and 11.Sequence types 423(ST423) only occurred before May 2008 and ST11 occurred (52 isolates) after May 2008. Most of isolates of the outbreak were ST11 (91. 2% ). A part of isolates were susceptive to added antibiotics. Conclusion The outbreak of panresistance K.pneumoniae was caused by those isolates which carried multiple resistant genes. There is a different ability of dissemination between different ST types K. pneumoniae isolate. It was necessary to add the antibiotics to find out the effective drugs to treat the infection.     

铜绿假单胞菌噬菌体的分离鉴定及其控制生物被膜的应用研究

目的 分离鉴定铜绿假单胞菌烈性噬菌体,研究噬菌体控制宿主菌形成牛物被膜的效率.方法 以铜绿假单胞菌临床菌株为指示菌,从不同环境样品中分离噬菌体;采用限制性内切酶图谱分析和宿主范围测定方法,对分离的噬菌体进行分类;利用透射电子显微镜对分离噬菌体进行形态学研究;以TJC729为指示菌,开展噬菌体控制牛物被膜形成的应用研究.结果 分别以14株铜绿假单胞菌临床分离菌株为指示菌,共分离得到13株烈性噬菌体,命名为C1～C13.利用限制性内切酶EcoR Ⅰ分析结果表明,13株噬菌体基因组均为双链DNA,并可被分成8组;宿主谱测定结果显示,c1和C13、C6和C7、C9和C11分别具有相同的宿主范围,其余7株噬菌体的宿主范围各不相同.随机挑选噬菌体C1进行形态学研究,发现噬菌体C1头部具有二十面体结构,尾部较长且无收缩性尾鞘,属于长尾噬菌体科.生物被膜控制实验结果显示,混合噬菌体能够较好地抑制TJC729生物被膜的形成.进一步实验结果显示,噬菌体C1、C10和C12分别与牛物被膜混合培养24 h后,牛物被膜的量分别下降到初始量的32.7%、57.6%、32.8%.结论 分离了13株铜绿假单胞菌烈性噬菌体,它们能够显著抑制宿主菌生物被膜的形成,并对生物被膜造成一定程度的破坏,为控制铜绿假单胞菌引起的感染提供了一个新方法.

Abstract：

Objective To isolate and classigy the bacteriophages specific to Pseudomonas aetuginosa and to investigate biofilm control efficaey of the isolated virulent phages.Methods With P. aeruginosa clinical strains as indicators.bacteriophages were isolated by screening difierent environmental samples.Classification of the isolated phages was done with the methods of restriction fragment analysis of phage genome and host range analysis.Transmission electron microscopy(TEM)was used in phage morphology study.In biogilm control tests,TJC729 was used as the jndicator strain to study the biofilm control efficacy of the isolated phages.Results Total 13 lytic phages specific to P.aeruginosa strains were isolated and named as C1-C13.According to the result of restriction fragment analysis.all 13 phages were double-stranded DNA viruses and could be divided into eight groups.Host range experiments were conducted with 5 laboratory strains and 12 clinical strains of P. aeruginosa.The same infection profiles were observed among phage C1 and C13,C6 and C7,and C9 and C11,respectively.While the remaining 7 phages each had different unique infection profile.Phage C1 was selected randomly to study its morphology.The obtained images showed that phage C1 had an icosahedral head with a non-contractile tail,belonging to the Siphoviridae family.Compared with the single phage,phage cocktail had the best effect on biofilm control.Further experiment results showed that phage C1.C10 and C12 can destroy biofilm after treatment of the biofilm for 24 h.The biofilm amounts were deceased to 32.7%,57.6%and 32.8%of the initial values,respectively.Conclusion Thirteen virulent phages specific to P. aeruginosa had been isolated.The phages could significantly inhibit the biofilm formation and had a certain degree of damage on the biofilm.The results suggested an alternative method for the treatments of P.aeruginosa infections.     

Molecular mechanism of the qnrA genemediated quionlone resistance in Gram-negative bacteria

To explore the prevalence of the plasmid-mediated quinolone resistance gene qnrA in Gramnegative bacteria and to investigate its molecular genetic background and resistance profile in isolates harboring this gene, a total of 629 nalidixic acid-resistant isolates of non-repetitive Gram-negative bacteria were collected from clinical specimens between April 2004 and April 2006 and these isolates were screened for qnrA gene by PCR using specific primers combined with DNA sequencing. The extended spectrum β-1actamase （ESBL） or AmpC-producing isolates were distinguished by the phenotypic confirmatory test combined with DNA sequencing, and the antibiotics susceptibility test for qnrA-positive isolates was carried out by Kirby-Bauer and E-test method. To detect the location of the qnrA gene, plasmid conjugation and Southern hybridization were performed and the integron structure containing the qnrA gene was cloned by PCR strategy and sequenced by primer walking. It was demonstrated that the incidence of the qnrA-positive strains in nalidixic acid-resistant bacteria was 1.9% （12/629）, in which the detection rates for Klebiesiella pneumoniae. Enterobacter cloacae, Enterobacter aerogenes, Citrobacterfreundii and Salmonella choeraesuis were 2.2% （3/138）, 17. 1% （6/35）, 9. 1% （1/11）, 12.5% （1/8）, and 14.3% （1/7）, respectively. The qnrA gene was found to be embedded in the complex sull-type integron located on plasmids with varied size （80-180 kb）. Among them, 4 qnrA-positive isolates carried integron In37 and 8 isolates carried a novel integron, temporarily desig- nated as InX. All the qnrA-positive isolates were ESBL-producing and transferable for the multi-drug resistance. It is concluded that the plasmid-mediated drug-resistance mechanism exists in the quinolone resistant strains of isolates from hospitals in Guangdong area, but the incidence was rather low. Nevertheless, it is still possible that the horizontal transfer of the resistant qnrA gene might lead to the spreading of drug-resista     

Analysis on drug-resistance and molecular epidemiology of Acinetobacter baumannii isolated from the clinical samples in two Chinese hospitals

In the present study, the drug-resistance genes encodingβ-lactamases, aminoglycoside modifying enzymes, DNA topoisomerases and integron as well as their molecular epidemiology were investigated by means of analyzing the drug-resistance and molecular epidemiology of Acinebacter bau mannii isolated from the clinical samples in two hospitals in Qiangzhou and Huzhou city of Jiangsu and Zhejiang province from July 2000 to March 2005. The minimal inhibitory concentrations (MICs) of these 307 isolates were detected by automatic microbiological system, and 35 strains against 5-fluoro-quinolones were performed by agar dilution assay. Meanwhile, the resistant genes in 80 isolates were amplified by PCR with identification by DNA sequencer. It was found that most of the 307 isolates of A . baumannii were resistant to multiple antibiotics tested, in which the resistance rates of the isolates against piperacillin, piperacillin/tazobactam, amoxacillin/clavulanic acid, cefotaxime, ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, chloramphenicol and sulfamethoxazole/trimethoprim were all above 35% , but those of imipenem and meropenem were quite low, ranged only 2.6% and 3.3%. In addition, it was also demonstrated that the positive rates of TEM and SHVβ-lactamase genes accounted for 93.8% and 22.5% respectively, and those of the aminoglycoside-modifying enzyme genes including aacC1, aacC2, aacC3, aacC4, aacC4A, aphA6, ant(2")-I and ant(3") I were 58.8%, 8.8%, 7.5%, 28.8%, 45.0%, 2.5%, 28.8% and 65.0% respectively. The mutations in the quinolone-resistant determining region (QRDR) of gyrA and parC genes indicated that substitution in Ser-83 residue of GyrA protein was most frequently occurred among strains with MIC for ciprofloxacin of more than 4μg/ml, whereas a double mutation at Ser-83 residue of gyrA and Ser-80 of parC was found in strains with MIC of ciprofloxacin of more than 8μg/ml. As to the positive rates of class 1 integron (Int I -1) and qacE△1-sul-1, it was found to be 60.0% and 77.5% respectively, and the rates of resistant genes of strains isolated in these two hospitals varied considerably. The results obtained in the present study indicate the presence of the multiple resistant genes in strains of A. baumannii , and great measures should be taken to control the spread of the resistant strains carrying the resistant genes.     

Correlation of the antiseptic resistance in Acinetobacter baumannii and the antiseptic resistance gene qacE Δ 1 located in class Ⅰ integron

In the past decade, uses of antiseptics and disinfectants in hospitals and other health care centers are rather common, but the chance to develop resistance to antiseptics and disinfectants is also increased. Acinetobacter baumannii is one of the opportunistic bacteria involving in the nosocomial infection . In the present study, the correlation of the antiseptic resistance in A . baumannii and the antiseptic resistance gene qacEΔ1 was investigated by means of determination of MICs. Meanwhile, the MICs of glutaraldehyde, chlorhexidine, benzalkonium bromide, iodophor and trichloroisocyanurate to 80 clinical isolates of A. baumannii were detected by tube dilution assay and the resistance genes intI1 and qacEΔ1 in these isolates were amplified by PCR and verified by DNA sequencer. It was found that the MIC50 for these 5 antiseptics tested were 32, 8, 8, 4 and 1μg/ml respectively, and the detection rates of intI1 and qacEΔ1 gene were 60.0% and 77.6% respectively. In addition, 55% of the 80 isolates simultaneously possessed both intll and qacEΔ1 gene, and the percentage of antiseptic resistance of A . baumannii carring both genes to benzalkonium bromide were higher than that without these two genes, however, there was no significant difference between intll and qacEΔ1 gene. The result in bactericidal efficiency assay indicated that chlorhexidine could still produce rapid and strong bactericidal effect at concentration of 1 MIC after 10 min exposure. These results suggest that the antiseptic resistance of A . baumannii to various antiseptics is correlated with the presence of the antiseptic resistance genes qacEΔ1 in bacteria, thus warning that the increase of the antiseptic resistance should not be ignored and the relative high concentration or prolonged application time is required to achieve a sufficient bactericidal effect.     

Construction and Verification of LuxS-negative Mutants of Streptococcus Mutans and the Effect of the Absence of LuxS Gene on the Acid Tolerance

Objective： To knock out the entire Luxs gene of Streptococcus mutans （S mutans） UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the acid resistance of S. mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods： Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S. mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. mutans transformants, which was identified by polymerase chain reaction, V. harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period. Terminal growth situation was compared. Firstly acidized in pH 5.5 BHI liquid, the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared. Results ： Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxsdeleted status of S. mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S. mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S. mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S. mutans standard strain and LuxS mutant strain. The acid resistance of standard strain was stronger than that of LuxS mutant strain. The two strains both displayed the capability of acid tolerance responses. Conclusion：The S. mutans gene allelic exchange plasmid is constructed correctively and a Luxs-negative mutants of S. mutans is constructed, which can help to further study the role of Luxs in the pathogenesis of S. mutans. LuxS mutant strain is more sensitive to acid inactivation, but the capability of acid tolerance responses exist still.     

Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol (EMB) resistance of the clinical isolates of tuberculous patients in China was investigated by reverse dot blot hybridization (RDBH) in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates (65.9%) showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation (38/91, 41.8%) and the ATA mutation (16/91, 17.6%) were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing for one wide-type probe and 5 probes for specific mutations was 100% . It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.     

Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials

Failure to treat and eradicate prosthetic hip infection with systemic antibiotic regimens is usually due to the fact that the infection is associated with biofilm formation and that bacterial cells growing within a biofilm exhibit increased resistance to antimicrobial agents. In this in vitro study, we investigated the susceptibility of prosthetic hip Propionibacterium acnes and Staphylococcus spp. isolates growing within biofilms on polymethylmethacrylate (PMMA) bone cement to a range of antibiotics. All P. acnes isolates in the biofilm mode of growth demonstrated considerably greater resistance to cefamandole, ciprofloxacin and vancomycin. In contrast, only four of the eight P. acnes isolates demonstrated an increase in resistance to gentamicin. All ten Staphylococcus spp. isolates in the biofilm mode of growth exhibited large increases in resistance to gentamicin and cefamandole with eight of the ten isolates also exhibiting an increase in resistance to vancomycin. However, only three of the ten Staphylococcus spp. isolates exhibited an increase in resistance to ciprofloxacin. Biofilms were also formed on three different titanium alloys and on PMMA bone cement using P. acnes, Staphylococcus epidermidis and Staphylococcus aureus strains to determine if the underlying biomaterial surface had an effect on biofilm formation and the antimicrobial susceptibility of the bacteria growing within biofilms. Although differences in the rate at which the three strains adhered to the different biomaterials were apparent, no differences in biofilm antibiotic resistance between the biomaterials were observed. In the light of these results, it is important that the efficacy of other antibiotics against P. acnes and Staphylococcus spp. prosthetic hip isolates growing within biofilms on orthopaedic biomaterials be determined to ensure optimal treatment of orthopaedic implant infection.     

Comparative antimicrobial susceptibility of biofilm versus planktonic forms of Salmonella enterica strains isolated from children with gastroenteritis

In the present study, 194 Salmonella enterica strains, isolated from infected children and belonging to various serotypes, were investigated for their ability to form biofilms and the biofilm forms of the isolated strains were compared to their corresponding planktonic forms with respect to the antimicrobial susceptibility. For the biofilm-forming strains, the minimum inhibitory concentration for bacterial regrowth (MICBR) from the biofilm of nine clinically applicable antimicrobial agents was determined, and the results were compared to the respective MIC values of the planktonic forms. One hundred and nine S. enterica strains out of 194 (56%) belonging to 13 serotypes were biofilm-forming. The biofilm forms showed increased antimicrobial resistance compared to the planktonic bacteria. The highest resistance rates of the biofilm bacteria were observed with respect to gentamicin (89.9%) and ampicillin (84.4%), and the lowest rates with respect to ciprofloxacin and moxifloxacin (2.8% for both). A remarkable shift of the MICBR₅₀ and MICBR₉₀ toward resistance was observed in the biofilm forms as compared to the respective planktonic forms. The development of new consensus methods for the determination of the antimicrobial susceptibility of biofilm forms seems to be a major research challenge. Further studies are required in order to elucidate the biofilm antimicrobial resistance mechanisms of the bacterial biofilms and their contribution to therapeutic failure in infections with in vitro susceptible bacteria.     

解脲脲原体对红霉素体外耐药性与ermB基因相关性研究

目的 探讨解脲脲原体(Uu)临床株对红霉素体外耐药性与ermB耐药基因之间的关系.方法 采用微量肉汤稀释法体外测定143株临床分离的Uu对红霉素的最低抑菌浓度(MIC),以MIC≥8μg/ml为耐药判读标准;设计引物扩增ermB基因,并以多条带抗原基因为靶位设计引物对Uu进行生物学分群.结果 Uu对红霉素MIC范围为≤0.125μg/至≥128μg/ml,MIC50为16 μg/ml,MIC90≥128 μg/ml,耐药率为64.38%.ermB基凶总的阳性率为27.97%,主要分布在MIC≥8 μg/ml的菌株.两生物群之间不存在红霉素耐药性及ermB基因阳性率的差异.结论 ermB基因可能是介导Uu对红霉素耐药的基因,两生物群在对红霉素耐药性机制的差异需进一步研究.     

Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors

Propionibacterium acnes plays an important role in the pathogenesis of acne vulgaris, a common disorder of the pilosebaceous follicles. Recently, it was suggested that P. acnes cells residing within the follicles grow as a biofilm. In the present study, we tested the biofilm-forming ability of several P. acnes strains in a microtiter plate model. We also evaluated the resistance of biofilm-grown P. acnes towards antimicrobial agents commonly used in the treatment of acne and the production of putative virulence factors. Our results indicate that P. acnes can form biofilms in vitro. The results also show that sessile P. acnes cells are more resistant to various commonly used antimicrobial agents than planktonic cells. In addition, sessile cells produce more extracellular lipases as well as significant amounts of the quorum-sensing molecule autoinducer-2.     

Interleukin-1beta-induced growth enhancement of Staphylococcus aureus occurs in biofilm but not planktonic cultures

Staphylococcus aureus causes recalcitrant infections and forms resistant biofilms. Mechanisms of biofilm resistance to host defenses may include changes in gene expression that confer responsiveness to chemical mediators. In earlier studies fresh clinical isolates responded to inflammatory cytokines, but responsiveness was lost after multiple in vitro passages [Meduri et al. Cytokines IL-1beta, IL-6, and TNF-alpha enhance the In vitro growth of bacteria. Am J Respir Crit Care Med 1999;160:961-7]. Since biofilms more closely resemble in vivo growth and are implicated in recalcitrant infections, we hypothesized that biofilms, but not planktonic cells, would respond to cytokines. Biofilms were induced by ethanol in S. aureus ATCC 12600. Biofilms treated with 2 ng/mL interleukin-1beta (IL-1beta) for 6 h contained 2.5-fold more cells than untreated biofilms, but no growth-enhancement occurred in planktonic cultures. As determined by flow cytometry, IL-beta bound to 63.1% of biofilm cells, but only 11.2% of planktonic cells. Our results provide evidence of a differential response of biofilm and planktonic bacteria to chemical mediators, and suggest that biofilm bacteria may evade host defenses by growing more rapidly in response to the inflammatory mediators released by activated host defense cells.     

gyrA和parE基因检测在脲原体基因分型中的应用

目的 研究gyrA和parE基因检测在脲原体基因分型中的作用.方法 脲原体培养与药敏分析用Mycoplasma IST检测试剂盒;在脲原体培养阳性者中选取对喹诺酬耐药标本60份,PCR扩增gyrA和parE基因,扩增产物经测序分析后与基因库中的脲原体各血清型进行比对.结果 gyrA扩增片段在血清型1、3、6、14之间核苷酸序列相似性为100%,在血清型2,4、5、7～13之间核苷酸序列相似性100%,两组之间核苷酸序列相似性91%.parE扩增片段在血清型1、3、6、14之间的核苷酸序列相似性为98%～99%;pare扩增片段在血清犁2、5、7、8、11之间核苷酸序列相似性100%,在血清型4、12、13之间核苷酸序列相似性100%,两组之间核苷酸序列相似性为90%.60份标本中微小脲原体(Ureaplasma parvum,Up)占68.3%(41/60),解脲脲原体(Ureaplasma urealyticum,Uu)占21.7%(13/60),两型混合感染占10%(6/60).在Up中,血清型3占48.8%(20/41).结论 gyrA检测可把脲原体分为微小脲原体和解脲脲原体两个基因型,parE检测可把微小脲原体分为4个亚型,分别与血清型1、3、6、14完全一致,其中血清型3感染最常见.     

Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade‐offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well‐defined collection of 42 CF Pseudomonas aeruginosa isolates. MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10^?8) than in biofilm (9.9 × 10^?9) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10^?9 in the planktonic mode to 6 × 10^?8 in biofilm, whereas normomutators (from 9.4 × 10^?8 to 5.3 × 10^?8) and hypermutators (from 1.6 × 10^?6 to 7.7 × 10^?7) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa, leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.     

Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and ATCC 10145 biofilms

Pseudomonas aeruginosa is a relevant opportunistic pathogen involved in nosocomial infections that frequently shows low antibiotic susceptibility. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the isolation and characterization of lytic phages capable of infecting antibiotic-resistant P. aeruginosa strains. In addition, characterization of P. aeruginosa biofilms and the potential of newly isolated phages for planktonic and biofilm control was accessed. According to the results, the isolated phages showed different spectra of activity and efficiency of lysis. Four broad lytic phages were selected for infection of planktonic cells; however, despite their broad range of activity, two of the selected phages failed to efficiently control planktonic cultures. Therefore, only two phages (phiIBB-PAA2 and phiIBB-PAP21), highly capable of causing strong biomass reduction of planktonic cells, were tested against 24 h biofilms using a m.o.i. of 1. Both phages reduced approximately 1-2 log the biofilm population after 2 h of infection and reduction was further enhanced after 6 h of biofilm infection. However, biofilm cells of P. aeruginosa PAO1 acquired resistance to phiIBB-PAP21; consequently, an increase in the number of cells after 24 h of treatment was observed. Conversely, phage phiIB-PAA2 for P. aeruginosa ATCC10145 continued to destroy biofilm cells, even after 24 h of infection. In these biofilms, phages caused a 3 log reduction in the number of viable counts of biofilm cells.