Unexpected finding of the "tropical" bacterial pathogen<Emphasis Type="Italic"> Plesiomonas shigelloides</Emphasis> from lake water north of the Polar Circle

The occurrence of Plesiomonas shigelloides in fresh water from six lakes located north of the Polar Circle in the northern part of Sweden was investigated. Bacteriological analysis of the water samples revealed the presence of nine isolates of P. shigelloides from one of the six lakes. All nine isolates were tested for biochemical characterisation, and serological and genomic typing. The biochemical profiles of all nine isolates were identical. Two strains were non-agglutinating for the O and H antigens. The other seven displayed an O19 serotype, whereas H antigen showed non-agglutinating properties. The genotyping results showed that the O19 isolates presented the same pattern, while the two non-agglutinating strains presented patterns that were different to that of the seven others. The results show that P. shigelloides can occur in aquatic environments where the temperature conditions are extremely low for long periods. This could have ecological and clinical importance for the aquatic and terrestrial fauna at these latitudes.     

Plesiomonas shigelloides Infection in Southeast China

Background

Plesiomonas shigelloides can cause gastroenteritis and extra-intestinal diseases in humans. However, the prevalence of P. shigelloides infections has not been investigated in China.

Methods

Consecutive fecal specimens from outpatients with acute diarrhea and non-diarrheal patients at nine sentinel hospitals in southeast China were collected from March 2010 to May 2012. Bacterial pathogens were detected by culture, and P. shigelloides isolates were subjected to antimicrobial susceptibility testing. We also retrospectively reviewed the hospital microbiology laboratory and infection-control databases for all P. shigelloides isolates identified from 2001–2012 at our institution in addition to data on the patients'' clinical and demographic characteristics.

Results

A total of 3,536 outpatients with acute diarrhea were enrolled in the study. P. shigelloides was isolated from 104 (2.9%) patients and accounted for 7.3% of bacterial isolates. Single-pathogen infections with P. shigelloides were present in 76 (73.1%) patients. No strain of P. shigelloides was isolated from the 478 non-diarrheal patients. Based on 444,684 nonfecal specimens, eight patients developed P. shigelloides-related extra-intestinal infections over the 12-year period. All eight patients had underlying diseases, including four with biliary tract diseases and three with liver diseases. Six cases were classified as nosocomial, and five cases were polymicrobial. P. shigelloides was sensitive to most antimicrobial drugs, except ampicillin.

Conclusions

In southeast China, P. shigelloides has significant clinical relevance, although the isolation rate is low.     

Incidence of Non-01 Vibrio cholerae and Aeromonas spp. in Fresh Water in Araraquara, Brazil

The occurrence of Aeromonas spp., Vibrio cholerae, and Plesiomonas shigelloides in fresh water from various sources in Araraquara, State of S?o Paulo, Brazil was determined. Samples from ten distinct irrigation systems used in vegetable cultivation, from five distinct streams, from two reservoirs, from one artificial lake, and from three distinct springs were analyzed. All isolates were serotyped and tested for hemolysin, cytotoxin, heat-stable (ST) and heat-labile (LT) enterotoxins production; presence of plasmid; autoagglutination and drug resistance. V. cholerae isolates were also tested for cholera enterotoxin (CT) production, and Aeromonas isolates for suicide phenomenon. No P. shigelloides was found. V. cholerae non 01 was found in five irrigation water samples and in three stream samples. Aeromonas sp. were isolated in two samples of irrigation water, in three streams, and in one reservoir. All the V. cholerae and Aeromonas isolates were positive for β-hemolysin production, and all Aeromonas isolates were positive for suicide phenomenon; cytotoxic activities were observed in two Aeromonas strains. Cholera enterotoxin was not found in eight V. cholerae non-01 isolates tested by the Y-1 mouse adrenal cell. All isolates were also negative for the other virulence markers. V. cholerae isolates were found to be sensitive to the majority of drugs tested, while Aeromonas strains presented multiple drug resistance. Received: 4 November 1997 / Accepted: 23 January 1998     

New O and H antigens of the international antigenic scheme forPlesiomonas shigelloides

A revised update of the International antigenic scheme forPlesiomonas shigelloides is presented. Twenty-six new O (O77–O102) and 10 new H (H42–H50 and H1a1d) antigens have been described since 1994. The sources of antigens are mostly human clinical strains, isolates from warmblooded animals and a few environmental cultures.     

High genetic diversity in the coat protein and 3′ untranslated regions among geographical isolates of<Emphasis Type="Italic">Cardamom mosaic virus</Emphasis> from south India

A survey was conducted to study the biological and genetic diversity ofCardamom mosaic virus (CdMV) that causes the most widespread disease in the cardamom growing area in the Western Ghats of south India. Six distinct subgroups were derived based on their symptomatology and host range from the sixty isolates collected. The serological variability between the virus isolates was analysed by ELISA and Western blotting. The 3′ terminal region consisting of the coat protein (CP) coding sequence and 3′ untranslated region (3′UTR) was cloned and sequenced from seven isolates. Sequence comparisons revealed considerable genetic diversity among the isolates in their CP and 3′UTR, making CdMV one of the highly variable members ofPotyviridae. The possible occurrence of recombination between the isolates and the movement of the virus in the cardamom tract of south India are discussed.     

Properties, relationships and transmission of a strain of raspberry ringspot virus infecting raspberry cultivars immune to the common Scottish strain*

Plants of Lloyd George and Seedling M raspberry (Rubus idaeus L.) were found in eastern Scotland infected with raspberry ringspot (RRV), a virus to which these varieties were previously considered immune. Most RRV isolates from affected plants caused milder symptoms in herbaceous test plants than did the type isolates of the common Scottish and English strains. In graft-transmission tests the Lloyd George strain of RRV infected all the raspberry cultivars tested, including those immune to the common Scottish strain. No consistent differences were found between isolates of the two strains in in vitro properties or serological behaviour. Both strains were transmitted in seed of Stellaria media and in soil containing Longidorus elongatus. Possible reasons why the new strain is uncommon in Scotland are discussed.     

Molecular evidence of <Emphasis Type="Italic">Plesiomonas shigelloides</Emphasis> as a possible zoonotic agent

The most frequently used method for establishing epidemiological relationships between Plesiomonas shigelloides strains is O:H serotyping. However, a number of strains are not serotypeable and isolates from diverse sources can display the same serovar. Moreover, since the zoonotic nature of Plesiomonas has been suggested and this hypothesis is based on the identical serovars found in animals and humans, we intend to use four DNA-based techniques: random amplified polymorphic DNA-PCR, enterobacterial repetitive intergenic consensus-PCR, repetitive extragenic palindromic-PCR, and pulsed field gel electrophoresis in order to screen 24 strains belonging to nine O:H serovars isolated from humans, animals, and the environment. In general, P. shigelloides showed a high genetic heterogeneity. Three pairs of strains, each containing a human and an animal isolate, displayed similar genotypes. This is the first report that provides molecular evidence that P. shigelloides may be zoonotic.     

Plum Pox Virus in Japanese Plum from Argentina: Serological Detection and Molecular Characterization of an Isolate from cv. Red Beauty

A Plum pox virus (PPV) isolate detected in a Japanese plum orchard in Pocito (San Juan, Argentina) was transmitted mechanically to Prunus tomentosa and Nicotiana benthamiana. DAS‐ELISA and DASI‐ELISA indicated the virus presence and serological relationship with D‐strain isolates; IC‐RT‐PCR amplified a 1.2‐kb fragment of the virus genome encoding the CP‐3′ nc region. The analysis of the sequence showed the presence of the DAG motif at the 5′ end of the capsid protein and the Rsa I and Alu I sites at the 3′ end. The phylogenetic relationships and multiple alignment with PPV isolates from NCBI database indicated greatest (+98%) homology with the D strain and close identity with MNAT1 ( AF360579 ) USA peach isolate. The sequence analysed showed two amino acid mutations towards the 5′ N‐terminus of CP (the most variable region) with respect to a consensus of PPV D‐strain isolates. This is the first molecular characterization of 3′terminal genome region of PPV isolate to confirm D strain in a Japanese plum from Argentina.     

Isolates of cucumber mosaic virus from spontaneously infected plants ofChelidonium majus andImpatiens parviflora

Cucumber mosaic virus (CMV) isolates obtained from spontaneously infected (1)Chelidonium majus L. and (2)Impatiens parviflora DC. plants growing in the same part of a scree forest in central Bohemia showed differences in ten test plants, but not in further 26 plant species tested and in TIP. Slight quantitative differences between both isolates were obtained in serological tests. Properties of both isolates did not suit precisely any proposed CMV strain classification.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

Molecular typing and antimicrobial resistance of <Emphasis Type="Italic">Salmonella</Emphasis> Enteritidis isolated from poultry,food, and humans in Serbia

Molecular typing and resistotyping coupled with gyrA single nucleotide polymorphism (SNP) of 60 Salmonella Enteritidis (SE) isolates originated from poultry, food, and humans in Serbia is described. Molecular fingerprinting was performed by randomly amplified polymorphic DNA (RAPD) using four primers, and the diversity index (D) was 0.688. In combination with resistotyping and gyrA SNP, D increased to 0.828. A total of 23 genetic groups were obtained. When four RAPD primers were combined, epidemic isolates from a fast-food restaurant outbreak were clustered in a distinctive genetic group. Among 60 SE strains, three had multiple resistances to three or more antibiotics. Nine strains were resistant to nalidixic acid (NAL; a non-fluorinated quinolone). The mutations in quinolone resistance-determining region (QRDR) found in NAL-resistant strains were attributed to Asp⁸⁷ → Asn in six strains, Asp⁸⁷ → Gly in one strain, and Ser⁸³ → Phe in one strain. One NAL-resistant strain had no mutations in QRDR, suggesting another mechanism of resistance.     

Antibiotic resistance and molecular characterization of clinical isolates of methicillin-resistant coagulase-negative staphylococci isolated from bacteremic patients in oncohematology

Polymerase chain reaction (PCR) amplification of antibiotic resistance genes as well as staphylococcal cassette chromosome mec (SCCmec) typing and pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA were used to characterize 45 methicillin-resistant coagulase-negative staphylococci (MRCoNS) isolates responsible of bacteremia recovered in patients at the Bone Marrow Transplant Centre of Tunisia in 1998–2007. Among the 45 MRCoNS isolates, Staphylococcus epidermidis was the most prevalent species (75.6%) followed by Staphylococcus haemolyticus (22.2%) and Staphylococcus hominis (2.2%). Extended susceptibility profiles were generated for MRCoNS against 16 antimicrobial agents. Out of 45 mecA-positive strains, 43 (95.6%) were phenotypically methicillin-resistant and two (4.4%) were methicillin-susceptible. The msr(A) was the most prevalent gene (13 isolates; 48.1%) among erythromycin-resistant isolates. The erm(C) was found alone in seven (25.9%) or in combination with both erm(A) and erm(B) in two (7.4%) isolates. The aac(6′)-Ie-aph(2″)-Ia was the most prevalent gene among aminoglycoside-resistant isolates, detected alone in 14 isolates (33.3%) isolates, in combination with ant(4′)-Ia in 18 (42.8%) isolates, in combination with aph(3′)-IIIa in four (9.5%) or with both ant(4′)-Ia and aph(3′)-IIIa in two (4.7%) isolates. The ant(4′)-Ia was detected in three (7.1%) isolates and the aph(3′)-IIIa in one (2.4%) isolate. Among tetracycline-resistant isolates, six (85.7%) strains harbored the tet(K) gene and one (14.3%) strain carried tet(K) and tet(M) genes. SCCmec types IV (31%) and III (24.5%), the most prevalent types detected, were found to be more resistant to non-β-lactam antibiotics. A wide diversity of isolates was observed by PFGE among MRCoNS.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.     

A distinct strain of lucerne Australian latent virus in white clover in New Zealand

A strain of lucerne Australian latent virus (LALV) was widespread in Trifolium repens (white clover) in Northland, New Zealand, occurring in 20–100% of plants tested on 23 of 26 farms. Infected plants showed chlorotic line patterns or were symptomless. In agar gel double diffusion serological tests, white clover isolates of LALV differed from lucerne isolates by two or three twofold dilution steps of antisera. In mechanical inoculation tests, white clover isolates did not infect lucerne and lucerne isolates did not systemically infect white clover. White clover and lucerne isolates also differed in their reactions in some other hosts. Six LALV isolates from white clover were indistinguishable in double diffusion serological tests and by enzyme-linked immunosorbent assay, although the isolates could be differentiated by some reactions in test plants. Purified LALV particles of lucerne and white clover isolates contained two nucleoprotein components and two RNA species of molecular weight 2.82 × 10⁶ (8150 nucleotides) and 2.46 × 10⁶ (7100 nucleotides) (estimated under denaturing conditions).     

Studies of Polymorphic DNA Fingerprinting and Lipid Pattern of Mycobacterium tuberculosis Patient Isolates in Japan

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L -rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.     

Serological grouping of indigenous Bradyrhizobium sp. (Arachis) isolated from various soils of Thailand

ELISA and antibody adsorption tests were applied to determine the minimal somatic antigen constitution of 243 strains of Bradyrhizobium sp. (Arachis) using 12 antisera. The 243 indigenous bradyrhizobial isolates were from 15 sites in four regions of Thailand. A total of 29 serogroups were identified. Most (80%) of the isolates tested had at least one heat-stable antigen in common with strain 280A, forming a so-called 280A serocluster. At 11 of 15 sites tested, 53 to 100% of the isolates fell into one or two predominant serogroups. The serological properties of the indigenous bradyrhizobia were not related to the cropping history of the cultivated fields from which they were isolated.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; No. 3608-E, 1992 series.     

一株类志贺邻单胞菌噬菌体生物学特性及全基因组分析

[目的]多重耐药菌株的出现给食品安全带来严重威胁.噬菌体是不同于抗生素的一类重要杀菌因子,对其生物学特性及基因组的研究和分析可为噬菌体的抗菌应用提供依据.[方法]对噬菌体phiP4-7的生物学特性、基因组学、分类学进行研究.[结果]经透射电子显微镜观察,确定phiP4-7头部直径为(50.59±1.68) nm,尾部长...     

Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity

More than 1,000 Spiroplasma isolates have been obtained from horse flies and deer flies (Diptera:Tabanidae) in the United States and Canada. However, the spiroplasma biota of Central America is poorly known. In August of 1995 and 1998, 13 isolates were obtained in 14 attempts from horse flies of a single species, Poeciloderas quadripunctatus, taken in the Costa Rican highlands (1,100–2,000 m). The majority of the “isolates” proved to be mixtures of two or more Spiroplasma species, but after filter cloning, single strains emerged that were designated as representatives of the 13 accessions. Six distinct spiroplasma serogroups were identified from these isolations. Three of the strains are putative new species with no serological relationship to any other Spiroplasma species. A fourth strain is a putative new species that may be distantly related to S. helicoides, a southeastern U.S. species. These four strains are accorded herein status as representatives of new serogroups: strain BARC 4886 (group XXXV); strain BARC 4900 (group XXXVI); strain BARC 4908 (group XXXVII); and GSU5450 (group XXXVIII). A fifth Spiroplasma species was very closely related to S. lineolae, known previously only from the Georgia (U.S.) coast. The sixth was most closely related to subgroup VIII-3, known from Texas and the southeastern U.S. Discovery of six spiroplasma species in only 13 attempted isolations reflects diversity seldom equaled in southeast Georgia, and never elsewhere in the U.S. These results are consistent with a hypothesis that spiroplasma diversity increases from north (Nova Scotia) to south (Georgia and Costa Rica). The discovery of significant affinity between some spiroplasmas of the southeastern U.S. and the Costa Rican highlands was unexpected, but may reflect a climatically complex Pleistocene history.

<Emphasis Type="Italic">Ectothiorhodospira magna</Emphasis> sp. nov., a new large alkaliphilic purple sulfur bacterium

Two strains of purple sulfur bacteria of the family Ectothiorhodospiraceae were isolated from moderately saline steppe lakes (with pH above 9.0) of the Transbaikal region (strain B7-7) and Mongolia (strain M10). The cells of the novel strains were spiral-shaped, 2.0–3.2 × 9.6–20.0 μm, motile due to a polar tuft of flagella. Photosynthetic pigments were represented by bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Photosynthetic membranes were represented by long strands of lamellae distributed throughout the whole cell; unlike most Ectothiorhodospiraceae species, the membranes were not packed into regular stacks. Bacteria were capable of weak growth on sulfide and slow grow on hydrogen under photoautotrophic conditions. The best growth was noted on sulfide in the presence of acetate and bicarbonate. Thiosulfate did not stimulate phototrophic growth, even in the presence of organic substrates. The new isolates were alkaliphiles growing at a pH optimum of 9–10. Growth was possible within a salinity range of 0–80 g/l NaCl, with an optimum at 5–15 g/l NaCl. The morphology, the structure of the photosynthetic apparatus (strands of lamellae), and the physiology of the new strains were similar to those of Thiorhodospira sibirica. However, analysis of the 16S rRNA gene sequences demonstrated that the studied isolates were closely related to the type strain Ectothiorhodospira shaposhnikovii (99% similarity) of the family Ectothiorhodospiraceae, whereas the level of similarity between the new strains and Thiorhodospira sibirica was only 94–95%. According to the results of DNA-DNA hybridization, the DNA-DNA homology level between the tested strains was almost 100%; the similarity between the new isolates and the type strain Ectothiorhodospira shaposhnikovii was only 58%. The isolates differed from other representatives of the genus Ectothiorhodospira in the structure of the gene encoding the key enzyme of autotrophic CO₂ fixation, ribulose-1,5-bisphosphate carboxylase (RuBisCo), which was similar to the RuBisCo genes of members of another family of sulfur bacteria, Chromatiaceae. The new isolates of purple bacteria were described as a new species of the genus Ectothiorhodospira, Ect. magna sp. nov. with the type strain B7-7^T (= VKM B-2537 = DSM 22250).     

<Emphasis Type="Italic">Aphanomyces sinensis</Emphasis> sp. nov., isolated from juvenile soft-shelled turtle, <Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, in Japan

A species of Aphanomyces was isolated from juvenile soft-shelled turtles, Pelodiscus sinensis, cultured in Japan. Typically, an infected turtle showed small whitish maculae on the carapace. Many hyphae were observed in the epidermis. The hyphae were isolated using glucose–yeast (GY) agar plates. The morphological characteristics were very similar to those of Aphanomyces laevis, but a clear nuclear spot was observed in the center of the oospore in the strains isolated from the soft-shelled turtles. The optimal growth temperature for the isolates was 25–30°C and the optimum pH was 6–9. Experimental infection tests with isolates produced small whitish maculae on the carapace, and soft-shelled turtles artificially infected with the zoospores showed high mortality, especially in the high-dose group. Phylogenetic analysis based on the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) indicated that the isolates from the soft-shelled turtles were unidentified species of Aphanomyces. As a result, the strain was described as a new species, Aphanomyces sinensis.