Sequence types diversity of Legionella pneumophila isolates from environmental water sources in Guangzhou and Jiangmen,China

In this study, 159 Legionella pneumophila strains isolated from various natural and artificial water sources in Guangzhou and Jiangmen, China, were subjected to genotyping by the sequence-based typing (SBT) scheme. These isolates were assigned into 53 sequence types (STs) (50 STs with seven loci data and three unidentified STs with incomplete loci profiles) with ST1 as the dominant one (14.5%), and the index of diversity (IOD) was 0.950. Eight new alleles and 34 new STs were reported here. Notably, most of the newly identified STs with seven loci data (24/34) contained no new allele, implying frequent recombination events in L. pneumophila. Five intragenic recombination events were identified in the concatenated sequences of seven loci. The diversity of STs in natural environmental isolates (41 STs, IOD = 0.956) is higher than that of artificial environmental ones (17 STs, IOD = 0.824). The ST patterns varied in isolates from these two sources: the most common STs from artificial water sources, ST1 and ST752 (39.2% and 13.7%), were only occasionally isolated from natural water sources (2.9% and 3.8%, respectively); while the predominant STs from natural water sources, ST1048, ST739 and ST1267 (15.2%, 6.7% and 6.7%), were less frequently seen in artificial environments (2.0%, 0% and 0%, respectively). We also found out that Legionnaires’ disease associated STs might be more frequently isolated in artificial environments than in natural ones. Our data revealed remarkable genetic diversity of L. pneumophila isolates from environmental water systems of Guangzhou and Jiangmen, and the different ST distribution patterns between natural water and artificial water sources as well.     

三种分子分型技术在嗜肺军团菌分型中的应用与评价

目的对脉冲场凝胶电泳方法 （PFGE）、基因序列分型（SBT）和mip基因分型方法在嗜肺军团菌分型研究中的分辨力和潜在价值进行比较和论述。方法采用PFGE、SBT和mip基因分型方法对29株嗜肺军团菌和1株标准菌株ATCC33153进行分型比较。结果 PFGE可将30株嗜肺军团菌分为5大类群,19个PFGE型,分辨力为0.9586;SBT可分为22个ST型,分辨力为0.9609;mip基因分型可分为9个型别,分辨力为0.8344。血清型LP1和LP14菌株具有相同的PFGE和mip基因型别,相同或相近的SBT型别。结论 SBT较PFGE具有稍高的分辨力,适用于全球数据库比对和进化亲缘关系的研究,结合PFGE和SBT分型方法有利于流行病学溯源分析。     

工业循环冷却塔水嗜肺军团菌分离株分子生学特性研究

目的 了解钢铁企业工业循环冷却塔水中的嗜肺军团菌污染状况及菌株分子生物学特性.方法 于2011年3月-2012年9月对邯郸某钢铁厂相对固定的车间冷却塔水进行连续监测,进行嗜肺军团菌的定量分离培养,对分离到的菌株进行血清分型、脉冲场凝胶电泳分型、基因序列分型、毒力基因(lvh、rtxA)检测.结果 共检测117份水样,其中20份水样检出嗜肺军团菌,检出率为17.1％;菌落总数为100～50000 CFU/L,中位数为12000 CFU/L;共分离到嗜肺军团菌23株,血清型包括Lp1、Lp3、Lp5、Lp6、Lp8,并以Lp1为主(占36.1％,9/23).23株嗜肺军团菌PFGE分型得到19种不同带型,其中15种带型为国内首次发现.23株嗜肺军团菌分成20个SBT型别,有12个型别为本研究新发现.23株菌中,lvh、rtxA基因阳性的分别有20、22株,两种毒力基因全部阳性的有19株.结论 钢铁企业的冷却塔水存在嗜肺军团菌污染,菌型呈现基因多态性,同时具有独特的基因组成,并普遍具有毒力.     

Cytopathogenicity and molecular subtyping of Legionella pneumophila environmental isolates from 17 hospitals

The cytopathogenicity of 22 Legionella pneumophila isolates from 17 hospitals was determined by assessing the dose of bacteria necessary to produce 50% cytopathic effect (CPED50) in U937 human-derived macrophages. All isolates were able to infect and grow in macrophage-like cells (range log10 CPED50: 2.67-6.73 c.f.u./ml). Five groups were established and related to the serogroup, the number of PFGE patterns coexisting in the same hospital water distribution system, and the possible reporting of hospital-acquired Legionnaires' disease cases. L. pneumophila serogroup 1 isolates had the highest cytopathogenicity (P=0.003). Moreover, a trend to more cytopathogenic groups (groups 1-3) in hospitals with more than one PFGE pattern of L. pneumophila in the water distribution system (60% vs. 17%) and in hospitals reporting cases of hospital-acquired Legionnaires' disease (36.3% vs. 16.6%) was observed. We conclude that the cytopathogenicty of environmental L. pneumophila should be taken into account in evaluating the risk of a contaminated water reservoir in a hospital and hospital acquisition of Legionnaires' disease.     

Legionellae isolated from clinical and environmental samples in Spain (1983-90): monoclonal typing of Legionella pneumophila serogroup 1 isolates.

Legionella isolates recovered in 21 different Spanish provinces over 8 years from both clinical (67 isolates) and environmental (181) samples, mostly from case-associated buildings, are described; 92.5% of clinical isolates were L. pneumophila serogroup 1 (SG1), only five isolates belonging to other species or serogroups: two L. pneumophila SG6, two SG8 and one L. bozemanii SG1 not clearly related with clinical infection. L. pneumophila SG1 accounted for 53.6% of isolates from the environment, followed by SG8 (27.6%), SG3 (9.4%) and SG6 (7.2%). Three isolates were labelled as SG8/10. Subtyping of L. pneumophila SG1 by the standardized panel of monoclonal antibodies revealed 90.3% of clinical and 78.3% of environmental isolates as belonging to Pontiac subgroup. Pontiac isolates were further divided into 55.3% Philadelphia 1 or Allentown 1, 21.9% Benidorm 030E and 20.4% Knoxville 1. Characterization of samples from four outbreaks in which both clinical and environmental isolates had been recovered permitted the recognition of three Philadelphia 1 or Allentown 1 and one Knoxville 1 strains as the aetiological agents.     

环境水中嗜肺军团菌的分离培养法和巢式PCR法比较

目的应用传统分离培养法和巢式PCR法检测环境水中嗜肺军团菌污染状况,并比较两种方法的检测效果。方法于2012年7-10月,采集北京市丰台区商场和超市、宾馆和饭店、综合性医院三类场所的中央空调冷却水、自来水、淋浴水、景观水,分别使用培养法及巢式PCR法检测嗜肺军团菌。结果共检测187件水样,传统分离培养法对嗜肺军团菌的检出率为2.67%(5/187),低于巢式PCR法[19.79%(37/187)],差异有统计学意义(χ~2=27.465,P0.01)。结论北京丰台区多种环境水体存在嗜肺军团菌污染;巢式PCR法对环境水中嗜肺军团菌的灵敏度优于传统分离培养法。     

Clinical and environmental distribution of Legionella pneumophila in a university hospital in Italy: efficacy of ultraviolet disinfection

The molecular epidemiology of Legionella pneumophila in the 'V. Monaldi' University Hospital was studied. Seven cases of nosocomial Legionnaires' disease were diagnosed between 1999 and 2003. Two clinical legionella strains obtained from two patients in the adult cardiac surgery unit (CSU) and 30 environmental legionella strains from the paediatric and adult CSUs, neonatal intensive care unit (NICU) and the cardiorespiratory intensive care unit (CR-ICU) were serotyped and genotyped. L. pneumophila serogroup 1/Philadelphia with an identical pulsed-field gel electrophoresis (PFGE) profile A was isolated from two patients in the adult CSU, and from three and one water samples taken in the adult CSU and the paediatric CSU, respectively, from 2001 to 2002. Furthermore, L. pneumophila serogroup 3 with an identical PFGE profile B was identified in 20 environmental strains from all wards, L. pneumophila serogroup 3 with PFGE profile C was identified in a single environmental strain from the CR-ICU, and non-pneumophila Legionella with identical PFGE profile D was identified in five environmental strains from the adult CSU, paediatric CSU and NICU. Ultraviolet irradiation was effective in disinfection of the hospital water supplies in the adult and paediatric CSUs contaminated by L. pneumophila clone associated with nosocomial Legionnaires' disease. In conclusion, these data demonstrate that two cases of nosocomial legionellosis were caused by the persistence of a single clone of L. pneumophila serogroup 1/Philadelphia in the hospital environment, and that disinfection by ultraviolet irradiation may represent an effective measure to prevent nosocomial Legionnaires' disease.     

我国九省（市、区）82株嗜肺军团菌血清1型菌株的序列分型

目的 了解我国环境水中嗜肺军团菌血清1型（Lp1）菌株的序列分型特征,并初步建立我国军团菌序列分型数据库。方法 采用序列分型(SBT)方法,对我国9个省（市、自治区）2005-2008年间环境水中分离的82株Lp1菌株进行分型,同时采用脉冲场凝胶电泳(PFGE)分型方法对这些菌株进行分型,并采用BioNumerics 5.1软件对2种方法的分型结果进行聚类分析和比较。结果 82株Lp1菌株分为22种序列(ST)型,其中17种ST型是新序列型,新发现1个等位基因;ST-1型为主要的序列型,在8个省（市、自治区）均有发现,该型菌株占所有菌株的46.3％ (38/82);ST-1、ST-150、ST-154、ST-159、ST-160和ST-630出现于2个以上的分离地点,5个分离位点(B4、B5、B6、S3和S8)发现2种以上ST型;通过聚类分析,15种ST型被分为3个序列群（ST-1序列群、ST-154序列群和ST-149序列群）,其余7种ST型没有序列群归类。采用PFGE分型方法,这些菌株可被分为46种带型。SBT和PFGE两种方法结合可将82株菌株分为54种分子型别。两种方法的聚类分析结果具有良好的一致性。结论 我国环境水中Lp1菌株具有独特的SBT分布;通过研究,初步建立了我国军团菌序列分型数据库。     

杭州市2002-2008年甲型副伤寒沙门菌分子分型研究

目的研究2002-2008年杭州市甲型副伤寒疫情分离株的亲缘性,并探讨脉冲场凝胶电泳（PFGE）、多位点序列分型（MLST）在甲型副伤寒沙门菌分子分型中的应用。方法对404株甲型副伤寒沙门菌运用脉冲场凝胶电泳（PFGE）进行分子分型,并用K-B法测定菌株抗生素敏感性。挑选9株甲型副伤寒沙门菌进行多位点序列分型（MLST）以及稳定性试验。结果 404株甲型副伤寒沙门菌可分为6个PFGE型和4个耐药型,P1型和P2型属于同一个克隆系,该克隆系菌株占试验菌株数的99%,分离自临安市的310株菌均为P1型,分离自杭州市西湖区的主要菌型是P2型和P1型。9株甲型副伤寒沙门菌根据SucA位点的差异可分为2个MLST型。结论杭州市2002-2008年的甲型副伤寒疫情由同一克隆系的菌株主导。PFGE、MLST分型方法各有侧重,可互为补充。     

2010-2012年南宁市职业暴露人群血清学和环境高致病性禽流感监测分析

目的了解南宁市职业暴露人群高致病性H5N1禽流感病毒的接触感染状况和南宁市及周边地区活禽市场、养殖厂等环境禽流感病毒H5、H7、H9亚型污染状况,为防治人禽流感提供科学依据。方法在2010-2012年采集从事家禽养殖、屠宰等人群血清标本用红细胞凝集抑制试验（HI）检测H5N1抗体;对在城乡活禽市场外环境采集的笼具涂抹、粪便和污水标本用荧光定量PCR法检测禽流感病毒FluA、HS、H7、H9核酸。结果共采集职业暴露人群80人份血清H5N1抗体均为阴性;环境标本175份禽流感病毒核酸监测阳性标本为47份,阳性率为26．86％。A型禽流感病毒核酸阳性标本分布于6个城区（县）的9个活禽农贸市场,各个季节采集的标本的阳性率有差异。3种类型的标本中,水的阳性率最高,其次是笼具涂抹标本,粪便的阳性率最低。结论禽流感H5亚型在南宁市职业暴露人群中未发现隐性感染;活禽市场环境普遍存在高致病性禽流感病毒的污染,H5和H9亚型是主要的病原体。     

Lineage and virulence of Streptococcus suis serotype 2 isolates from North America

We performed multilocus sequence typing of 64 North American Streptococcus suis serotype 2 porcine isolates. Strains were sequence type (ST) 28 (51%), ST25 (44%), and ST1 (5%). We identified nonrandom associations between STs and expression of the virulence markers suilysin (SLY), muramidase-relased protein (MRP), and extracellular factor (EF). Expression of pili encoded by the srtF and srtG pilus clusters was also nonrandomly associated with STs. ST1 strains were SLY+ EF+ MRP+ srtF pilus+ srtG pilus-. ST25 strains were SLY- EF- MRP- srtF pilus- srtG pilus+, and most ST28 strains were SLY- MRP+ EF- srtF pilus+ srtG pilus+. ST28 isolates proved essentially nonvirulent in a mouse infection model; ST25 strains showed moderate virulence and ST1 isolates were highly virulent. ST1 is responsible for a high proportion of S. suis disease in humans worldwide. Its presence in North America indicates that potential zoonotic S. suis outbreaks in this continent cannot be disregarded.     

Clonal comparison of Staphylococcus aureus isolates from healthy pig farmers, human controls, and pigs

Pig farming is a risk factor for increased nasal Staphylococcus aureus colonization. Using sequence typing and phylogenetic comparisons, we showed that overcolonization of farmers was caused by a few bacterial strains that were not present in nonfarmers but often caused swine infections. This finding suggests a high rate of strain exchange between pigs and farmers.     

Genotype analysis of Mycobacterium tuberculosis isolates from a sentinel surveillance population

As part of the National Tuberculosis and Genotyping Surveillance Network, isolates obtained from all new cases of tuberculosis occurring in seven geographically separate surveillance sites from 1996 through 2000 were genotyped. A total of 10883 isolates were fingerprinted by the IS6110-restriction fragment length polymorphism method, yielding 6128 distinct patterns. Low-copy isolates (those with six or fewer bands) were also spoligotyped. The distribution of specific genotype clusters was examined. Databases were also examined for families of related genotypes. Analysis of IS6110 patterns showed 497 patterns related to the W-Beijing family; these patterns represent 946 (9%) of all isolates in the study. Six new sets of related fingerprint patterns were also proposed for isolates containing 6-15 copies of IS6110. These fingerprint sets contain up to 251 patterns and 414 isolates; together, they contain 21% of isolates in this copy number range. These sets of fingerprints may represent endemic strains distributed across the United States.     

Detection of Leishmania infantum cryptic infection in asymptomatic blood donors living in an endemic area (Eivissa, Balearic Islands, Spain) by different diagnostic methods

The extent of cryptic leishmaniasis in blood donors from a Spanish endemic area, (Eivissa Island) was studied using various immunological and parasitological methods. Sera from 656 blood donors were analysed: 16 (2.4%) were positive by ELISA and 50 (7.6%) by Western blot. Peripheral blood mononuclear cells (PBMC) and buffy coat (BC) samples, were analyzed by culture and nested-PCR. DNA of L. infantum was amplified in 27 (22.1%) of 122 PBMC. Parasites were isolated in 3 (4.5%) of 67 BC cultures and the strains were identified as L. infantum zymodeme MON-28. No parasites were isolated in PBMC culture. After 12 months, a second blood sample was obtained from 18 blood donors who were positive by nested-PCR in the first extraction; nine of them remained positive. Delayed type hypersensitivity (DTH) tests on 15/67 donors (22.3%) were positive. Comparison of results obtained by ELISA, WB and DTH; ELISA, WB and nested-PCR and nested-PCR and BC culture showed a significant association (Pearson test, P < 0.05). L. infantum zyodeme MON-28 was identified in three strains isolated from asymptomatic donors, which suggests a low virulence capacity of these strains. The detection of Leishmania DNA in a high number of asymptomatic subjects supports the need to monitor it in blood donors endemic areas.     

Rapid genotyping of Legionella pneumophila serogroup 1 strains by a novel DNA microarray-based assay during the outbreak investigation in Warstein,Germany 2013

Between 1 August and 6 September 2013, an outbreak of Legionnaires’ disease (LD) with 78 cases confirmed by positive urinary antigen tests occurred in Warstein, North Rhine-Westphalia, Germany. Legionella (L.) pneumophila, serogroup (Sg) 1, monoclonal antibody (mAb) subgroup Knoxville, sequence type (ST) 345, was identified as the epidemic strain. This strain was isolated from seven patients. To detect the source of the infection, epidemiological typing of clinical and environmental strains was performed in two consecutive steps. First, strains were typed by monoclonal antibodies. Indistinguishable strains were further subtyped by sequence-based typing (SBT) which is the internationally recognized standard method for epidemiological genotyping of L. pneumophila. In an early stage of the outbreak investigation, many environmental isolates were found to belong to the mAb subgroup Knoxville, but to two different STs, namely to ST 345, the epidemic strain, and to ST 600. A majority of environmental isolates belonged to ST 600 whereas the epidemic ST 345 strain was less common in environmental samples. To rapidly distinguish both Knoxville strains, we applied a novel typing method based on DNA-hybridization on glass chips. The new assay can easily and rapidly discriminate L. pneumophila Sg 1 strains. Thus, we were able to quickly identify the sources harboring the epidemic strain, i.e., two cooling towers of different companies, the waste water treatment plants (WWTP) of the city and one company as well as water samples of the river Wester and its branches.     

The molecular epidemiology of serial Candida tropicalis isolates from ICU patients as revealed by multilocus sequence typing and pulsed-field gel electrophoresis

The genetic profiles of 50 Candida tropicalis isolates serially collected from 14 patients during a prospective surveillance study in adult intensive care units (ICUs) were characterized by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) of NaeI restriction fragments. A total of 21 diploid sequence types (DSTs) and 43 genotypes were differentiated by MLST and PFGE, respectively. Significant correlations were found between PFGE genotypes and DST types (P < 0.05). Dendrograms generated by either MLST or PFGE-NaeI showed that most isolates from the same patient co-clustered with high similarity regardless of the anatomical source of isolation. Maintenance, microvariation or replacement of C. tropicalis isolates could be observed within the individual patients by further analysis of variations in MLST sequence data. Antifungal susceptibility testing revealed that 17 (34%) of 50 isolates presented high MICs to flucytosine (MIC ≥ 8 μg/mL). Sixteen (94%) of these isolates belonged to DST 164, and these were collected from four patients with different PFGE genotypes. Isolates sharing the same DST may represent a common clone that underwent extensive mutation over time to cope with drug selection pressure, different hosts or different geographic environments.     

Preliminary studies on bacteriophage typing of Mycobacterium tuberculosis strains isolated in Salamanca (Spain)

A phage typing of 202 strains of Mycobacterium tuberculosis isolated in Central and Northwest Spain was carried out. The commonest phage type was A (64%) and within this type a₀ (45%). This was followed in frequency by phage type B (26%) and in last place type I (10%). No relationship was observed between the phage type and the geographical or anatomical origin of the strains.Corresponding author.     

陕西省婴幼儿食品中分离的132株蜡样芽胞杆菌呕吐型基因的检测

目的 了解2012-2016年陕西省市售婴幼儿食品中蜡样芽胞杆菌的呕吐型基因的携带情况及携带呕吐基因菌株的分子分型。方法 采用PCR 方法对2012-2016年陕西省不同地区市售婴幼儿食品中收集到的132株蜡样芽胞杆菌的呕吐型基因进行检测,用多位点序列分型(MLST)方法对携带呕吐型基因的蜡样芽胞杆菌进行分子分型。结果 132株蜡样芽胞杆菌中携带呕吐型基因的共计9株,携带率682%。这9株菌分为4个序列类型(Sequence Type,ST),分别为ST-100(1111%,1/9)、ST-164(1111%,1/9)、ST-246(1111%,1/9)、ST-26(6667%,6/9)。结论 陕西省市售婴幼儿食品中的蜡样芽胞杆菌携带有呕吐型基因,携带率682%,以ST-26为主要流行株,基因型多样,地区间有地区特征性差异。     

Anemia in a population from an endemic area of malaria, Rond?nia (Brazil)]

With the purpose of describing the prevalence rate of anaemia among inhabitants of a malaria endemic area--Candeias district, a periurban locality near Porto Velho, in Rond?nia State, Brazilian Amazon Basin--a random population sample comprehending 1,068 individuals of all age groups (14.1% of the total population) was screened for anaemia (measurement of blood haemoglobin concentration) and malaria (Giemsa-stained thick-smear microscopy). Two-hundred and ninety-nine individuals (28.0% of the sample) were found to be anaemic, using the cut-off haemoglobin values proposed by the World Health Organization for each age group. Highest prevalence rates were found among children with ages varying from 6 months to 1 year (70.0%) and from 1 to 6 years (38.4%), as well as in pregnant women (41.2%, 7/17) and malaria patients (44.4%, 8/18). Parasitological stoll examinations were made on a voluntary sample of 476 individuals (44.6% of the sample population); of these, 118 (26.8%) were positive. Eggs of Ascaris lumbricoides, the most frequent intestinal parasite in this population sample, was detected in 67 stool samples (14.1%); only 27 patients (5.7%) eliminated Ancylostomidae eggs. In this voluntary sample, no significant difference in anaemia prevalence rates between parasite carriers and non-parasited individuals was detected. On the other hand, the more recent the last malarial episode referred to by the patients, the lighter prevalence rate of anaemia in individuals above the age of 14 years. The role played by malaria as an underlying cause of anaemia in Candeias district inhabitants, particularly in the economically active age group, is further discussed.