Electrotransformation of the stable L-form of Proteus mirabilis

Abstract To improve the transformability of stable protoplast type L-forms of Proteus mirabilis for recombinant plasmid DNA, conditions for efficient electrotransformation were explored. Exposing cells from the exponential phase of growth at a density of 6−8 × 10⁹/ml in electrotransformation buffer having a conductivity of 1.4 mS/cm to a field strength of 6.5 kV/cm for a mean pulse duration time of 1.2 ms reproducibly yielded transformation efficiencies in the order of 5 × 10⁴ transformants per μg of DNA. Compared to the polyethylene glycol method for transformation, electrotransformation appeared to be the method of choice for introduction of plasmid DNA into L-form cells.     

Fimbriae of uropathogenic Proteus mirabilis

Proteus mirabilis is a common causative agent of cystitis and pyelonephritis in patients with urinary catheters or structural abnormalities of the urinary tract. Several types of fimbriae, which are potentially involved in adhesion to the uroepithelium, can be expressed simultaneously by P. mirabilis: mannose-resistant/Proteus-like (MR/P) fimbriae, P. mirabilis fimbriae (PMF), uroepithelial cell adhesin (UCA), renamed by some authors nonagglutinating fimbriae (NAF), and ambient-temperature fimbriae (ATF). Proteus mirabilis is a common cause of biofilm formation on catheter material and MR/P fimbriae are involved in this process. The considerable serious pathology caused by P. mirabilis in the urinary tract warrants the development of a prophylactic vaccine, and several studies have pointed to MR/P fimbriae as a potential target for immunization. This article reviews P. mirabilis fimbriae with regard to their participation in uropathogenesis, biofilm formation and as vaccine targets.     

Studies on the accumulation of cadmium by a strain of Proteus mirabilis

Abstract A bacterium isolated from a wastewater plant sludge, identified as Proteus mirabilis , was tested for cadmium tolerance and accumulation capacity. The organism was able to grow in the presence of Cd²⁺ up to 300 mg l⁻¹.
Accumulation of cadmium is reported for growing and non-growing cells of the organism. In non-growing cultures a 70% removal of cadmium was observed when the initial concentration of Cd²⁺ was 1 mg l⁻¹ whereas at the same concentration the removal by growing cells was only 22%. The metal was shown to be associated with the cell envelope (80%) and accumulated in the cytoplasm (20%).     

尿液中奇异变形杆菌耐药性变迁及监测分析

目的了解尿液中奇异变形杆菌临床分布及耐药性变迁情况,为临床医生合理选用抗生素提供理论依据。方法收集2008年至2012年住院及门诊患者尿液标本中分离的215株奇异变形杆菌,采用VITEK2 Compact全自动微生物分析仪进行鉴定及药敏试验,采用WH0NET 5.4软件进行统计分析。结果2008、2009、2010、2011和2012年分离奇异变形杆菌分别为26株（占12. 2% ）、26株（占12.2% ）、36株（占16. 9% ）、55株（占25. 8% ）和72株（占33. 8% ）,总计215株。奇异变形杆菌对呋喃妥因的耐药率最高,均〉90%,对丁胺卡那霉素和美洛培南的耐药率最低,均为0%,对左旋氧氟沙星的耐药率在2008？2011年均〉50% ,2012年为28.1% ;对哌拉西林/他唑巴坦的耐药率在2008 - 2009年均〉20%,而2010-2012年均〈5% ;头孢替坦、舒普深耐药率均〈7%。结论奇异变形杆菌的分离率从2008 -2012年呈日益增长趋势,提醒我们随着临床上大量使用抗生素,医院的感染率亦不断上升。耐药率从2008-2011年基本呈上升趋势,而在2012年有所下降,这可能与近几年本院临床严格执行抗菌药物的合理应用有关。     

同株奇异变形杆菌存在不同生长状态

揭示同株奇异变形杆菌存在多种不同的生长状态。通过环形接种、点种传代、革兰染色和鞭毛染色等方法观察同株奇异变形杆菌的形态学变化。同株奇异变形杆菌的生长速度、迁徙能力、生长形态及鞭毛形态发生变化。同株奇异变形杆菌存在不同的生长形态。     

First-time characterization of viable but non-culturable Proteus mirabilis: Induction and resuscitation

Pathogenic bacteria can enter into a viable but non-culturable (VBNC) state under unfavourable conditions. Proteus mirabilis is responsible for dire clinical consequences including septicaemia, urinary tract infections and pneumonia, but is not a species previously known to enter VBNC state. We suggested that stress-induced P. mirabilis can enter a VBNC state in which it retains virulence. P. mirabilis isolates were incubated in extreme osmotic pressure, starvation, low temperature and low pH to induce a VBNC state. Resuscitation was induced by temperature upshift and inoculation in tryptone soy broth with Tween 20 and brain heart infusion broth. Cellular ultrastructure and gene expression were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR), respectively. High osmotic pressure and low acidity caused rapid entry into VBNC state. Temperature upshift caused the highest percentage of resuscitation (93%) under different induction conditions. In the VBNC state, cells showed aberrant and dwarf morphology, virulence genes and stress response genes (envZ and rpoS) were expressed (levels varied depending on strain and inducing factors). This is the first-time characterization of VBNC P. mirabilis. The ability of P. mirabilis pathogenic strains to enter a stress-induced VBNC state can be a serious public health threat.     

Structure of Proteus mirabilis biofilms grown in artificial urine and standard laboratory media

Proteus mirabilis is a urinary pathogen that can differentiate from a swimmer cell into a swarmer cell morphotype and can form biofilms on the surfaces of urinary catheters. These biofilms block these catheters due to crystals trapped within these structures. The effect of encrustation on biofilm formation and structure has not been studied using confocal scanning laser microscopy (CSLM). Therefore, a comparison of biofilm structure in artificial urine (AU) and laboratory media was undertaken. We compared the structure of P. mirabilis biofilms in AU and Luria-Bertani broth using CSLM and 3D imaging. Biofilms grown in Luria-Bertani broth formed mushroom structures at 24 h and contained nutrient channels. AU biofilms were observed to form a different structure at 24 h. AU biofilm structure was observed to be a flat layer, almost devoid of nutrient channels. Swarmer cells were observed protruding out of the biofilm into the bulk fluid. This could be due to nutrient depravation within the biofilm or a means of further colonizing the surface. This study has demonstrated that two markedly different biofilm structures are formed, depending on the growth media utilized.     

奇异变形杆菌耐药性的4年监测及碳青霉烯类耐药株的耐药机制研究

目的了解本地区2007年到2010年奇异变形杆菌的临床分布与常用抗菌药物的耐药情况,了解碳青霉烯类耐药菌株可能存在的机制。方法回顾分析2007年到2010年临床分离奇异变形杆菌的资料及整体耐药情况;对保存的耐亚胺培南（IPM）、美罗培南（MEM）或厄他培南（ETP）的菌株进行复苏,并做Hoage试验进行产碳青霉烯酶的确认,同时对试验菌株进行耐药基因的PCR扩增检测。结果2007年到2010年,奇异变形杆菌在临床各送检样本中以痰液分离率最高：51．1％、34．4％、22．1％和35．4％,其次为尿液：14．3％、28．O％、34．9％和33．6％;耐药监测分析显示,4年间对喹诺酮类、青霉素类、头孢菌素类及氨基糖苷类耐药率相对较高且较为稳定;对碳青霉烯类耐药最低但增加明显,亚胺培南从2007年的1．8％升到2010年的16．1％,美罗培南从2007年的1．7％升到2010年的16．8％。15株耐碳青霉烯类菌株中,Hoage试验阳性7株,6fn。基因阳性11株,blaCTX-M基因阳性13株。结论本地区奇异变形杆菌对临床常用的抗菌药物均有较高的耐药性,对碳青霉烯类药物的耐药率最低,但增加明显。位于质粒上的blaKPc基因所产生的碳青霉烯酶和6如cTx-M基因所产生的超广谱β-内酰胺酶是本菌对β-内酰胺类抗菌药物耐药的主要原因,临床应引起高度重视。     

奇异变形杆菌耐药性及亚胺培南不敏感株感染的临床特点研究

目的研究奇异变形杆菌的临床分布和耐药情况、亚胺培南不敏感奇异变形杆菌感染的临床特点。方法分析浙江大学医学院附属第一医院2013年1月至2013年12月分离的非重复奇异变形杆菌的药物敏感性、临床分布,回顾性分析亚胺培南不敏感奇异变形杆菌感染患者的临床资料、治疗及预后情况。结果2013年该院共分离107株奇异变形杆菌,以分离自尿液最多,其次为痰液;来源最多的是外科病房和重症监护病房。体外药敏显示：奇异变形杆菌对美罗培南、厄他培南、头孢吡肟、氨曲南、哌拉西林/他唑巴坦、头孢他啶、头孢哌酮/舒巴坦、阿米卡星等抗菌药物敏感性良好,敏感率达85%以上;对亚胺培南敏感率为80.4%;对头孢呋辛、环丙沙星、氨苄西林、头孢曲松、庆大霉素耐药率较高,超过30%;对呋喃妥因耐药率为99%。其中21株亚胺培南不敏感奇异变形杆菌对包括美罗培南、厄他培南在内的其他各类抗菌药物耐药率与亚胺培南敏感株基本相仿。亚胺培南不敏感奇异变形杆菌引起院内获得性感染主要发生在入住ICU、外科术后、广谱抗菌药物使用后、留置各类置管和梗阻性尿路疾病的患者,可引起泌尿系统、皮肤创面、腹腔、血流、生殖道等部位感染,表现为全身炎症反应及局部感染症状。选择敏感抗菌药物治疗后该部分患者预后良好。结论奇异变形杆菌对三、四代头孢菌素,β-内酰胺酶抑制剂合剂等抗生素敏感性良好。亚胺培南不敏感奇异变形杆菌对其他碳青酶烯类抗生素仍保持较高的敏感性。亚胺培南不敏感奇异变形杆菌所引起院内获得性感染主要发生在入住ICU、外科术后、广谱抗菌药物使用后、留置各类置管和梗阻性尿路疾病的患者,预后良好。     

Microbial decolorization of azo dyes by Proteus mirabilis

A bacterium identified as Proteus mirabilis was isolated from acclimated sludge from a dyeing wastewater treatment plant. This strain rapidly decolorized a deep red azo dye solution (RED RBN). Features of the decolorizing process related to biodegradation and biosorption were also studied. Although P. mirabilis displayed good growth in shake culture, color removal was best in anoxic static cultures. For color removal, the optimal pH and temperature were 6.5–7.5 and 30–35°C, respectively. The organism exhibited a remarkable color removal capability, even at a high concentration of azo dye. More than 95% of azo dye was reduced within 20 h at a dye concentration of 1.0 g L⁻¹. Decolorization appears to proceed primarily by enzymatic reduction associated with a minor portion, 13–17%, of biosorption to inactivated microbial cells. Received 06 January 1999/ Accepted in revised form 22 April 1999     

132株奇异变形杆菌的临床分布及药敏分析

目的了解宁波市妇女儿童医院奇异变形杆菌的临床分布和耐药情况,为临床合理用药提供依据。方法对该院2009年6月1日至2011年5月31日期间分离的132株奇异变形杆菌进行分析,菌株鉴定采用法国生物梅里埃公司的VITEK 60分析仪,药敏试验采用K-B法,用参考菌株作质量控制。结果该院分离的奇异变形杆菌主要来自尿液（51.50%）,其次是创口分泌物（21.21%）,再次是阴道分泌物（19.70%）,其他（7.59%）。对奇异变形杆菌敏感率较高的抗生素是：丁胺卡那霉素、氨曲南、美罗培南、亚胺培南、头孢唑林、头孢呋辛酯、头孢曲松、头孢他啶、头孢噻肟、马斯平、头孢西丁、罗红霉素、氨苄西林/舒巴坦、哌拉西林/他唑巴坦和头孢哌酮/舒巴坦。结论加强奇异变形杆菌的分离鉴定及耐药性的测定,是临床合理应用抗生素的重要依据。     

Acidic environments induce differentiation of Proteus mirabilis into swarmer morphotypes

Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced-induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen-ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease-producing bacteria, as well as in some animals with acidic or alkaline urine.     

Linkage of formate hydrogenlyase with anaerobic respiration in Proteus mirabilis

The linkage between the enzyme system catalysing formate hydrogenlyase and reductases involved in anaerobic respiration in intact cells of anaerobically grown Proteus mirabilis was studied. Reduction of nitrate and fumarate by molecular hydrogen or formate was possible under all growth conditions; reduction of tetrathionate and thiosulphate occurred only in cells harvested at late growth phase from a pH-regulated batch culture and not in cells harvested at early growth phase or in cells grown in pH-auxostat culture. Under all conditions, cells possessed the enzyme tetrathionate reductase. We conclude that linkage between tetrathionate reductase (catalysing also reduction of thiosulphate) and the formate hydrogenlyase chain is dependent on growth conditions. During reduction of high-potential oxidants such as fumarate, tetrathionate (when possible) or the artificial electron acceptor methylene blue by formate, there was no simultaneous H₂ evolution due to the formate hydrogenlyase reaction. H₂ production started only after complete reduction of methylene blue or fumarate, in the case of methylene blue after a lag phase without gas production. In preparations with a low fumarate reduction activity this was accompanied by an acceleration in CO₂ production. During reduction of thiosulphate (a low-potential oxidant) or of tetrathionate in the presence of benzyl viologen (a low-potential mediator) by formate, H₂ was evolved simultaneously. From this we conclude that formate hydrogenlyase is regulated by a factor that responds to the redox state of any electron acceptor couple present such that lyase activity is blocked when the acceptor couple is oxidised to too great an extent.     

2010年CLSI头孢菌素折点改变对产ESBLs奇异变形杆菌药物敏感性结果评估和分析

目的评估2009年CLSI M100-S19及2010年CLSI M100-S20文件中头孢他啶（CAZ）、头孢噻肟（CTX）和头孢唑啉（CFZ）最低抑菌浓度新旧折点变化对产ESBLs奇异变形杆菌药物敏感性试验结果的影响。方法对临床分离33株奇异变形杆菌进行产ESBLs菌株的确证试验,琼脂稀释法检测最低抑菌浓度（MIC）,根据药物敏感性结果分别对产ESBLs奇异变形杆菌和非产ESBLs奇异变形杆菌在S19和S20新旧折点下CAZ、CTX和CFZ三种药物的敏感性以及ESBLs阳性菌株分布率进行界定。结果 33株奇异变形杆菌中,产ESBLs菌株6株（18.2%）,由旧折点下耐药率50%（CAZ）、50%（CTX）和66.7%（CFZ）分别上升为新折点下66.7%（CAZ）、100%（CTX）和100%（CFZ）,敏感率由旧折点下33.3%（CAZ）和50%（CTX）分别下降为新折点下16.7%（CAZ）和0%（CTX）,CFZ在新旧折点下均为0,新旧折点标准下药敏结果分布率的差异均有统计学意义。结论对CTX、CFZ,按照CLSI S20新折点琼脂稀释法MICs折点标准判读药敏结果与ESBLs表型检测结果具有高度一致性,临床可以根据药敏结果选择用药,无需进行产ESBLs检测。但对于CAZ,尚需要进一步深入研究和评价。     

Evaluation of Proteus mirabilis structural fimbrial proteins as antigens against urinary tract infections

Proteus mirabilis is a common cause of urinary tract infection (UTI) and produce several types of different fimbriae, including mannose-resistant/Proteus-like fimbriae, uroepithelial cell adhesin (UCA), and P. mirabilis fimbriae (PMF). Different authors have related these fimbriae with different aspects of P. mirabilis pathogenesis, although the precise role of fimbriae in UTI has not yet been elucidated. In this work we expressed and purified recombinant structural fimbrial proteins of these fimbriae (MrpA, UcaA, and PmfA) and assessed their role as protective antigens using an ascending and a haematogenous model of UTI in the mouse. MrpA protected subcutaneously immunised mice in both models, suggesting that it could be taken into account as a promising vaccine candidate against P. mirabilis UTI. UcaA could also be an interesting subunit to be studied although it only protected mice that were challenged intravenously. All subunits elicited a strong specific serum IgG response but there was no significant correlation between antibody levels and protection. Only PmfA-immunised mice elicited a significant urinary antibody response but this protein was unable to confer protection against P. mirabilis experimental challenges. These results may contribute to the development of vaccines against P. mirabilis, an important cause of complicated UTI.     

Aggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cells

Proteus mirabilis is an important cause of urinary tract infection (UTI) in patients with complicated urinary tracts. Thirty-five strains of P. mirabilis isolated from UTI were examined for the adherence capacity to epithelial cells. All isolates displayed the aggregative adherence (AA) to HEp-2 cells, a phenotype similarly presented in LLC-MK(2) cells. Biofilm formation on polystyrene was also observed in all strains. The mannose-resistant Proteus-like fimbriae (MR/P), Type I fimbriae and AAF/I, II and III fimbriae of enteroaggregative Escherichia coli were searched by the presence of their respective adhesin-encoding genes. Only the MR/P fimbrial subunits encoding genes mrpA and mrpH were detected in all isolates, as well as MR/P expression. A mutation in mrpA demonstrated that MR/P is involved in aggregative adherence to HEp-2 cells, as well as in biofilm formation. However, these phenotypes are multifactorial, because the mrpA mutation reduced but did not abolish both phenotypes. The present results reinforce the importance of MR/P as a virulence factor in P. mirabilis due to its association with AA and biofilm formation, which is an important step for the establishment of UTI in catheterized patients.     

The XerC recombinase of Proteus mirabilis: characterization and interaction with other tyrosine recombinases

XerC and XerD are two site-specific recombinases, which act on different sites to maintain replicons in a monomeric state. This system, which was first discovered and studied in Escherichia coli, is present in several species including Proteus mirabilis, where the XerD recombinase was previously characterized by our laboratory. In this paper, we report the presence of the xerC gene in P. mirabilis. Using in vitro reactions, we show that the two P. mirabilis recombinases display binding and cleavage activity on the E. coli dif site and the ColE1 cer site, together or in collaboration with E. coli recombinases. In vivo, P. mirabilis XerC and XerD are able to resolve and monomerize a plasmid containing two cer sites, increasing its stability. However, P. mirabilis XerC, in combination with E. coli XerD, is unable to perform these functions.