耐喹诺酮类铜绿假单胞菌的耐药性分析及血清学分型

目的了解广州地区喹诺酮类耐药铜绿假单胞菌的耐药性及泵抑制剂对其耐药水平降低的作用,并调查血清型分布情况。方法用法国生物梅里埃公司的微生物鉴定和药敏分析系统VITEK-2对127株铜绿假单胞菌进行鉴定和药敏检测,并采用羰酰氰基-对-氯苯胺(CCCP)与环丙沙星共同作用,以琼脂稀释法测定耐药菌的最低抑菌浓度(M IC)的变化,同时用玻片凝集法对耐药株进行血清学分型。结果环丙沙星耐药菌对哌拉西林/他唑巴坦(65.5%)的敏感率最高,只有阿米卡星(64.4%)、哌拉西林(51.7%)和妥布霉素(50.6%)的敏感率大于50.0%,而敏感菌对美罗培南(97.5%)及左氧氟沙星(97.5%)的敏感率最高,妥布霉素(95.0%)次之,对临床常用的13种抗生素,耐药菌较敏感菌的敏感性明显降低(P值均<0.001);耐药菌受泵抑制CCCP作用,M IC降低1~4个稀释度;血清分型率为93.1%,耐药菌的血清型以B型(20.7%)和L型(19.5%)为主。结论耐喹诺酮类铜绿假单胞菌对临床常用抗生素的敏感性降低,并呈多重耐药,使用抗生素 泵抑制剂可提高药物对铜绿假单胞菌的敏感性;血清学分型可以快速简单地监测铜绿假单胞菌在医院内的流行情况。     

Experience with epidemiologic studies of pyocyaneus infections. I. Feasibility of using serotypes and antibiotic resistance as a epidemiologic label for clinical strains]

The authors carried out serological typing of 98 Pseudomonas aeruginosa strains, isolated from patients of burn department of the Sklifosovsky First Aid Institute in January-July, 1974, and of 215 strains obtained from other sources; their sensitivity to 13 antibiotics was determined. Pseudomonas aeruginosa cultures isolated from the patients were typed with O-sera of 10 serological types. The presence of several hospital strains of Pseudomonas aeruginosa was found by means of serological typing; along with these there were revealed cultures of this causative agent sporadically appearing in the department. Sensitivity to some antibiotics could serve as an additional criterion for differentiation of Pseudomonas aeruginosa strains of the same serological type.     

Characterization by pyocine typing and serotyping of oral and sputum strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients

Oral and sputum isolates of Pseudomonas aeruginosa in patients with cystic fibrosis were investigated. Of the 17 patients studied, 12 patients (71%) yielded both mucoid and nonmucoid variants of Pseudomonas aeruginosa from sputum and (or) various oral ecological sites, such as buccal mucosa, tongue dorsum, dental plaques, and saliva. A total of 51 strains of mucoid and nonmucoid Pseudomonas aeruginosa were isolated from these patients and were phenotypically characterized by both pyocine typing and serotyping. Five patients (42%) were colonized or infected by a single strain of Pseudomonas aeruginosa, whereas 7 patients (58%) were cocolonized or coinfected by two or more phenotypically different strains of Pseudomonas aeruginosa. To understand the mechanisms involved in Pseudomonas aeruginosa colonization, it may be necessary to identify multiple isolates of Pseudomonas aeruginosa not only from the sputum but also from the various oral ecological sites and to further explore the role of the oral cavity in this colonization.     

Bacteriocin (Marcescin) Typing of Clinical Isolates of Serratia marcescens

A simple, reproducible technique is described for bacteriocin typing of clinical isolates of Serratia marcescens. With 10 marcescin-producer strains, a total of 46 of 50 isolates (92%) of S. marcescens could be typed and categorized into 16 provisional types according to their sensitivity or tolerance to marcescins. The potential significance of this procedure with regard to delineation of outbreaks of nosocomial infection is discussed on the basis of preliminary epidemiological data.     

Pyocin production by bacillus pyocyaneus and sensitivity of strains of different origin to them

Using the method proposed by Gillies and Govan and their indicator strains, 342 P. aeruginosa strains isolated from the patients were studied in respect to their pyocinogenicity and typed according to the production of different types of pyocins. Besides, in 206 cultures the pyocin sensitivity of 16 standard P. aeruginosa strains (5 strains obtained from Govan and 11 strains provided by the authors) was determined. All the tested cultures fell into 23 pyocin types; of these, types I and X occured most frequently, 56 strains identified by means of indicators could not be typed due to the fact that the corresponding pyocin types were absent in Govan's scheme. The cultures isolated from the patients and the environmental objects during the outbreak of P. aeruginosa in a hospital were proved to belong to the same pyocin type (III). The double typing of the cultures, according to pyocin production and pyocin sensitivity, allowed to determine individual characteristics of 75% of the tested cultures.     

Production of cytotoxin by clinical strains of Pseudomonas aeruginosa

Presence of cytotoxin was studied in extracts of 57 strains of Pseudomonas aeruginosa (46 bacteremia, 4 environmental, and 7 Fisher immunotype), 10 Pseudomonas species, and 7 nonpseudomonas isolates. Cytotoxin was identified by Western immunoblot in extracts of all P. aeruginosa isolates. None of the Pseudomonas species or nonpseudomonas isolates were shown to produce this protein. No immunologic cross-reactivity was observed between cytotoxin antibody and P. aeruginosa alkaline protease, toxin A, or elastase. In partially purified extracts of two bacteremia strains and PA 158 (parent strain for cytotoxin production), detection of cytotoxin by Western immunoblot was correlated with biological activity, as measured by the cell swelling assay. Cytotoxin appears to be produced by all strains of P. aeruginosa and biological activity can be demonstrated in extracts of the strains tested. This biological activity is neutralized by specific antibody. Because of its known marked cytotoxic effect on most eukaryotic cells, P. aeruginosa cytotoxin might be an important factor in the pathogenesis of P. aeruginosa infections.     

Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant.

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.     

Epidemiology of Pseudomonas aeruginosa infections investigated by pyocin typing.

All strains of Pseudomonas aeruginosa isolated in a large Canadian hospital over a 3-year period were typed by their pyocin production. Smaller collections of P. aeruginosa from other hospitals were also typed. Almost 3000 strains were examined. The typing method did not require use of complex reagents and was successful in subdividing P. aeruginosa into numerous types. No single type was restricted to infections of one particular kind. Infections of all kinds were associated with a wide variety of pyocin types. Extensive crossinfection with one particular pyocin type was observed only in urinary infection of patients with urologic disorders. The four pyocin types that were most frequent in our entire series have been reported as the commonest types causing infections in many other parts of the world.     

Siderophore synthesis by mucoid Pseudomonas aeruginosa strains isolated from cystic fibrosis patients.

Nonmucoid Pseudomonas aeruginosa responds to iron deprivation by synthesizing the siderophores pyochelin and pyoverdine. When grown in iron-deficient medium, six mucoid P. aeruginosa strains isolated from cystic fibrosis patients synthesized copious amounts of the exopolysaccharide alginate. A procedure that eliminated the interference of alginate was developed so that siderophores could be extracted from the growth medium. All six isolates were then noted to produce both pyoverdine and pyochelin. This report thus confirms that mucoid P. aeruginosa, like its nonmucoid counterparts, elicits the siderophores commonly cited as those of the microbe.     

临床分离铜绿假单胞菌对氟喹诺酮类抗菌药物的耐药性检测

目的了解临床分离铜绿假单胞菌对喹诺酮类等抗菌药物的耐药性。方法琼脂稀释法测定86株铜绿假单胞菌对5种氟喹诺酮类抗菌药物以及头孢吡肟、美罗培南的耐药性。结果铜绿假单胞菌对诺氟沙星、氧氟沙星、左氧氟沙星、环丙沙星、加替沙星的耐药率分别为50%、61.6%、51.2%、48.8%、51.2%;对头孢吡肟和美罗培南的耐药率分别为30.2%、23.2%。结论铜绿假单胞菌对氟喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Modulation of Pseudomonas aeruginosa adherence to collagen type I and type II by carbohydrates

Abstract This study was undertaken to examine if receptor recognizing saccharides may be involved in the adherence of Pseudomonas aeruginosa to collagen type I and type II. We performed an adherence inhibition assay: cells of individual P. aeruginosa isolates attached to immobilized collagen type I or type II in the presence of monosaccharides, which could serve as blockers of bacterial receptors. Bacterial binding to collagen type I molecules was inhibited to the highest degree by sugar composition d -galactose/ d -mannose/ N -acetylneuraminic acid (5:5:1), whereas attachment of P. aeruginosa to collagen type II was inhibited by composition d -glucose/ d -galactose (1:1). The same strains which were sensitive to inhibition of binding to collagen type II by both collagen types, were also sensitive to blocking by composition d -glucose/ d -galactose. It suggests that saccharides play a role in adherence of P. aeruginosa to collagen type I and type II, and a common receptor for both types of collagen may be available on the surface of P. aeruginosa cells.     

Agricultural Plants and Soil as a Reservoir for Pseudomonas aeruginosa

Pseudomonas aeruginosa was detected in 24% of the soil samples but in only 0.13% of the vegetable samples from various agricultural areas of California. The distribution of pyocin types of soil and vegetable isolates was similar to that of clinical strains, and three of the soil isolates were resistant to carbenicillin. Pseudomonas aeruginosa multiplied in lettuce and bean under conditions of high temperature and high relative humidity (27 C and 80-95% relative humidity) but declined when the temperature and humidity were lowered (16 C, 55-75% relative humidity). The results suggest that soil is a reservior for P. aeruginosa and that the bacterium has the capacity to colonize plants during favorable conditions of temperature and moisture.     

铜绿假单胞菌氨基糖苷16SrRNA甲基化酶基因分析

目的调查215株湖州地区临床分离铜绿假单胞菌对氨基糖苷类抗生素的耐药性和16S rRNA甲基化酶基因分布情况。方法收集2011年1月至2012年12月湖州地区临床分离铜绿假单胞菌215株,琼脂稀释法测定5种氨基糖苷类抗菌药物（庆大霉素、阿米卡星、妥布霉素、伊帕米星、奈替米星）的MIC值;PCR检测armA、rmtA、rmtB、rmtC、rmtD和npmA六种氨基糖苷类16S rRN甲基化酶基因,序列分析明确基因型。测定产16S rRNA甲基化酶菌株对常见抗菌的敏感性,并检测碳青霉烯耐药株产碳青霉烯酶情况。结果铜绿假单胞菌对异帕米星敏感率最高为81.4%,对5种氨基糖苷类抗生素全部耐药的22株菌株中,17株检出armA基因;未发现其他16S rRNA甲基化酶基因阳性菌株。17株armA阳性菌株对碳青霉烯类抗生素耐药5株（耐药率为29.4%）,对头孢他啶、头孢吡肟、哌拉西林/他唑巴坦、环丙沙星耐药率均超过40%。5株碳青霉烯耐药菌株中检测到2株产VIM-2型金属碳青霉烯酶。结论铜绿假单胞菌对氨基糖苷类抗生素耐药率高,检测到16S rRNA甲基化酶基因armA。产16S rRNA甲基化酶铜绿假单胞菌耐药性强,部分菌株同时产金属碳青霉烯酶,给临床抗感染治疗及院内感染控制带来挑战。     

Antibiotic resistance and virulence properties of Pseudomonas aeruginosa strains from mechanically ventilated patients with pneumonia in intensive care units: comparison with imipenem-resistant extra-respiratory tract isolates from uninfected patients

We investigated the epidemiology of antibiotic resistance and virulence properties among Pseudomonas aeruginosa clinical isolates collected in 1999 from patients hospitalized in the intensive care units of the centre hospitalier d'Orléans, in France. We compared the totality of the strains from mechanically ventilated patients with pneumonia (33 non-duplicate isolates, group 1) to 15 randomly chosen, imipenem-resistant, extra-respiratory tract isolates, collected from non-infected patients hospitalized in the same units (group 2). The isolates were serotyped, typed by random amplified polymorphic DNA (RAPD), and screened for their pneumocyte cell adherence, cytotoxicity, and antibiotic resistance. A total of 35 RAPD profiles were found, and only two profiles were encountered in both groups, demonstrating a high genetic diversity. 84.8% of the group 1 and 93.3% of the group 2 isolates adhered to A549 cells. Three non-exclusive adhesive patterns were observed: a diffuse adhesion in 38 isolates, a localized adhesion in 14 isolates, and an aggregative adhesion in seven isolates. 78.8% of the group 1 and 93.3% of the group 2 isolates were cytotoxic. Considering all 48 isolates, there was a strong and statistically significant correlation between cytotoxicity and adherence. Among the three dominant serotypes, O:12 isolates were in majority avirulent, but the great majority of O:1 and all the O:11 isolates were found adherent and cytotoxic. Gentamicin was the least active antibiotic for both groups, and ceftazidime was the most active antibiotic for group 1 and amikacin for group 2. The penicillinase production phenotype was significantly correlated with a decrease in P. aeruginosa virulence.     

铜绿假单胞菌的院内感染分布及耐药性的调查

目的了解铜绿假单胞菌（PA）在医院内感染的分布特点及其耐药性,指导临床合理用药,降低医院感染率。方法回顾分析2005年1月至2008年11月我院临床分离的铜绿假单胞菌152株,按WHO推荐的NC-CLS标准方法（K—B法）进行药敏试验,分析菌株的临床分布特征及体外药敏结果。结果铜绿假单胞菌在临床上主要以呼吸道感染为主。在17种临床常用抗生素中,多表现为多重耐药。美洛培南、亚胺培南及左旋氧氟沙星抗PA效果最好,耐药率分别只有14．47％、14．47和5．26％。结论多重耐药铜绿假单胞菌在临床广泛存在,应当根据临床体外药敏结果合理选择抗菌药物进行治疗,以取得良好临床效果并减少耐药菌产生;同时规范院内感染控制措施,减少耐药菌株的播散。     

Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegradative properties irrespective of their origin

Virulence properties of pathogenic bacteria, as well as resistance to antibiotics, are thought to arise through a specialization process favoured by the strong selection pressure imposed in clinical treatments. Nevertheless, in the case of opportunistic pathogens, it is unclear whether strains can be classified into virulent and non-virulent isolates. Clones of the opportunistic pathogen Pseudomonas aeruginosa do not seem to be associated to a particular biovar or pathovar, which suggests that virulence characteristics in opportunistic pathogens may already be present in environmental (non-clinical) isolates. We have explored this possibility, studying environmental isolates (mainly from oil-contaminated soils) and clinical isolates (from bacteraemia and cystic fibrosis patients) of P. aeruginosa . All environmental strains were found to actively efflux quinolones, which are synthetic antibiotics not expected to be present in the environment. These strains contained multidrug resistance determinants, were capable of invading epithelial cells and presented genes from the quorum-sensing and type III secretion systems. Some of them expressed either haemolytic or proteolytic activities or both, characteristics considered to be typical of virulent strains. All the strains tested, of clinical or environmental origin, could use alkanes (oil hydrocarbons) as a carbon source. Our results suggest that clinical and non-clinical P. aeruginosa strains might be functionally equivalent in several traits relevant for their virulence or environmental properties. Selection of clinically relevant traits, such as antibiotic resistance or cellular invasiveness, in opportunistic pathogens present in soil ecosystems is discussed.     

圈养林麝铜绿假单胞菌的分离鉴定及耐药性和病原特性分析

【背景】铜绿假单胞菌感染所致的化脓性疾病是困扰林麝驯养的重要因素,是一类较难防治的细菌性疾病,目前尚无疫苗可用来预防该病。【目的】研究林麝源铜绿假单胞菌的感染现状和分子流行病学规律。【方法】对2014年10月至2015年10月四川宝兴和陕西镇坪两个林麝养殖中心发病林麝中铜绿假单胞菌进行分离鉴定,并对其耐药情况进行分析,利用脉冲场凝胶电泳(PFGE)分型技术研究分离菌的PFGE指纹图谱,探究其流行病学趋势,并对部分菌株的致病性进行分析。【结果】分离得到60株铜绿假单胞菌,其中34株来自镇坪,26株来自宝兴。耐药结果表明:60株林麝源铜绿假单胞菌对17种抗菌药物呈现不同程度耐药性,不同地区和不同样本源间分离的铜绿假单胞菌对17种抗菌药物的耐药性总体趋于一致,多重耐药情况严重,以5耐、6耐为主。分型结果显示:60株铜绿假单胞菌PFGE图谱的相似性为49.1%-100%。经聚类分析得到A-O共15种基因簇,其中优势基因簇为C、E、G、J。致病性结果表明,流行菌株的致病力强弱依次为:动物源菌株环境源菌株,且主要流行菌株(基因簇E、F、J)的致病力大于其余菌群。【结论】铜绿假单胞菌在两地区具有水平传播的途径,本研究可为跨地区林麝化脓性炎症的防控提供理论依据。     

Diversity of infectious pancreatic necrosis virus strains isolated from fish, shellfish, and other reservoirs in Northwestern Spain

A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.     

Pathogenic Escherichia coli found in sewage treatment plants and environmental waters

We previously demonstrated that some Escherichia coli strains with uropathogenic properties survived treatment stages of sewage treatment plants (STPs), suggesting that they may be released into the environment. We investigated the presence of such strains in the surrounding environmental waters of four STPs from which these persistent strains were isolated. In all, 264 E. coli isolates were collected from 129 receiving water sites in a 20-km radius surrounding STPs. We also included 93 E. coli strains collected from 18 animal species for comparison. Isolates were typed using a high-resolution biochemical fingerprinting method (the PhPlate system), and grouped into common (C) types. One hundred forty-seven (56%) environmental isolates were identical to strains found in STPs' final effluents. Of these, 140 (95%) carried virulence genes (VGs) associated with intestinal pathogenic E. coli (IPEC) or uropathogenic E. coli (UPEC) and were found in a variety of sites within areas sampled. Of the remaining 117 environmental strains not identical to STP strains, 105 belonged to 18 C types and 102 of them carried VGs found among IPEC or UPEC strains. These strains belonged mainly to phylogenetic groups A (A0 and A1) and B1 and to a lesser extent B2(2), B2(3), D1, and D2. Eight of 18 environmental C types, comprising 50 isolates, were also identical to bird strains. The presence of a high percentage of environmental E. coli in waters near STPs carrying VGs associated with IPEC and UPEC suggests that they may have derived from STP effluents and other nonpoint sources.     

Pseudomonas aeruginosa Exhibits Frequent Recombination, but Only a Limited Association between Genotype and Ecological Setting

Pseudomonas aeruginosa is an opportunistic pathogen and an important cause of infection, particularly amongst cystic fibrosis (CF) patients. While specific strains capable of patient-to-patient transmission are known, many infections appear to be caused by unique and unrelated strains. There is a need to understand the relationship between strains capable of colonising the CF lung and the broader set of P. aeruginosa isolates found in natural environments. Here we report the results of a multilocus sequence typing (MLST)-based study designed to understand the genetic diversity and population structure of an extensive regional sample of P. aeruginosa isolates from South East Queensland, Australia. The analysis is based on 501 P. aeruginosa isolates obtained from environmental, animal and human (CF and non-CF) sources with particular emphasis on isolates from the Lower Brisbane River and isolates from CF patients obtained from the same geographical region. Overall, MLST identified 274 different sequence types, of which 53 were shared between one or more ecological settings. Our analysis revealed a limited association between genotype and environment and evidence of frequent recombination. We also found that genetic diversity of P. aeruginosa in Queensland, Australia was indistinguishable from that of the global P. aeruginosa population. Several CF strains were encountered frequently in multiple ecological settings; however, the most frequently encountered CF strains were confined to CF patients. Overall, our data confirm a non-clonal epidemic structure and indicate that most CF strains are a random sample of the broader P. aeruginosa population. The increased abundance of some CF strains in different geographical regions is a likely product of chance colonisation events followed by adaptation to the CF lung and horizontal transmission among patients.