Biochemical properties of Shigella flexneri and their practical significance

As the result of the study of 921 S. flexneri strains 1-6 and 4 (IV: 7,8), isolated in 31 regions of the USSR in 1975-1984, their biochemical characterization by 33 tests was made. All the strains under study proved to be typical in most of their constant signs, only some of strains 2a showed deviations in mannitol and some of strains 4a, in acetate. In strains of serovar 6, circulating in the USSR, specific features with respect to dulcitol and xylose were noted. The possibility of the biochemical subserovar typing of S. flexneri 1-5, X- and Y-var., with respect to maltose, arabinose, sorbitol and rhamnose was confirmed.     

Bactericidal properties of hemo-cytolysin from Vibrio cholerae non O1 P-11702 strain in a panel of indicator cultures for detection of vibriocins

The influence of the preparation of hemo-cytolysin, obtained from V. cholerae non O1 strain P-11702 and inducing lysis of both red blood cells and V. cholerae cultures using a panel of indicator cultures for the detection of vibriocins, was studied. The set of indicator cultures contained 2 Shigella flexneri strains, 1 S. dysenteriae strain, 3 S. sonnei strains, 3 Escherichia coli strains and 2 V. cholerae strains, one of them being atypical. Hemo-cytolysin exhibited lytic activity with respect to S. dysenteriae, S. sonnei strains and 1 V. cholerae strain. i.e. to 4 out of 11 indicator strains. V. cholerae atypical strain proved to be resistant to the preparation in contrast to 33 V. cholerae typical strains, studied previously.     

福氏痢疾菌膜成分与细菌抗原性和毒力的关系

应用免疫转移技术,用从感染福氏痢疾菌的病人获取的恢复期血清分析了福氏痢疾菌膜成分与细菌抗原性和毒力的关一。发现福氏痢疾菌的膜蛋白67kD和63kD均含有两种成分,一种和膜蛋白60kD都可能是保护性抗原,而另一种与膜蛋白78kD和35kD一样与福氏痢疾菌的毒力有关。采用微细胞同位素掺入示踪显示这些与病人恢复期血清反应的膜蛋白由质粒编码。     

Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan

We conducted a prospective comparative study of community-onset (CO) and healthcare-associated (HA) methicillin-resistant Staphylococcus aureus(MRSA) strains between 2000 and 2001 at Tokyo Women's Medical University Hospital (1,500 beds) in Japan. Of the 172 consecutive MRSA isolates analyzed, 13 (8%) were categorized as CO-MRSA. The mean age of patients with CO-MRSA was significantly younger than that of patients with HA-MRSA. Most CO-MRSA strains were isolated from skin and more likely to be susceptible to erythromycin, clindamycin, tetracycline, levofloxacin, and spectinomycin compared to HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) analysis, staphylococcal cassette chromosome mec(SCCmec) typing, and multi-locus sequence typing (MLST) revealed that CO-MRSA strains were divided into the following multi-clones: 3 clone A: II: ST5 (PFGE type: SCCmec type: MLST sequence type); 1 L: II: ST5; 1 H: IV: ST1; 1 I: IV: ST81; 2 D: IV: ST8; 1 B: IV: ST89; 1 B: IV: ST379; and 3 B: IV: ST91. Of the 159 HAMRSA strains, 124 (78%) belonged to a single clone (PFGE clone A: SCCmec type II: tst and sec positive: coagulase type II: multi-drug resistance). Four CO-MRSA strains belonging to PFGE clone B: SCCmec type IV: MLST clonal complex 509 (ST89, 91, 379) had the exfoliative toxin B (etb) genes, but all CO-MRSA and HA-MRSA strains did not possess the Panton-Valentine leukocidin (pvl) genes. These results demonstrate that multiple lineages of CO-MRSA have the potential for dissemination in the community in Japan.     

Atypical Class 1 Integron Coexists with Class 1 and Class 2 Integrons in Multi-Drug Resistant Shigella flexneri Isolates from China

The antimicrobial resistance and the character of integrons were determined in 58 Shigella flexneri strains isolated from China. All isolates were multi-drug resistant and found to carry integrons of class 1 (94.8%), class 2 (100%), or both (94.8%). No intI3 was detected. The typical class 1 integrons were found in conjugative plasmids and could be transferred to the recipient E. coli DH5α. The gene cassettes of typical class 1 integrons dfrA17-aadA5 and dfrA12-orfF-aadA2 were detected in 54 strains (93.1%) and 1 strain, respectively. Atypical class 1 integrons located on the chromosome with gene cassettes bla (oxa-30)-aadA1 were detected in 55 isolates (94.8%). All the intI2 positive isolates carried gene cassettes dfrA1-sat1-aadA1. To our knowledge, this is the first report that atypical and typical class 1 integrons coexisted with class 2 integron in multi-drug resistant S. flexneri strains.     

Differentiation of Shigella strains by plasmid profile analysis, serotyping and phage typing

Ninety-one Shigella flexneri and 29 Shigella sonnei strains isolated during 1994 from sporadic cases of shigellosis and healthy carriers were analyzed for plasmid profile in order to compare the discriminating ability of this method with that of serotyping and phage typing. Our study revealed 10 plasmid profiles (PP) among S. sonnei strains. A total of 26 out of 29 (89%) S. sonnei isolates could be placed into two phage types (type 1 and 20) comprising four PP for phage type 1 and seven PP for type 20, respectively. Twenty-three different PP were identified among S. flexneri strains. Each serotype was associated with a specific predominant plasmid profile, except serotype 2a. This serotype, the most frequently isolated in Romania, was still rather homogeneous: 33 out of 39 isolates belonged to phage type 125, 27 of which could be placed into two related PP (F10 and F17). Comparison of plasmid patterns of epidemiologically independent S. flexneri serotype 2a isolates with those exhibited by 45 serotype 2a isolates associated to six independent outbreaks revealed the same homogeneity. Thirty-eight strains, representing 4 of 6 outbreaks, had F10 and F17 plasmid patterns. The discrimination indices (D) for plasmid profile analysis alone (D = 0.890) and for the combination of serotyping and phage typing (D = 0.841) indicate that both typing systems have a nearly similar ability of discriminating among S. flexneri strains. By combining the results of the three typing methods, a total of 42 types are distinguished and the D value is 0.942. Our data suggest that plasmid profile analysis can complement phenotyping methods resulting in a degree of discrimination that cannot be achieved by either system alone.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80-100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

Genetic characteristics and trends in the spread of a new Shigella flexneri subserovar with the antigenic formula IV:7,8

As the result of experiments with the conjugation of S. flexneri strains 4 belonging to an unusual subserovar (IV: 7,8) with Escherichia coli donor strains K12 Hfr C and Hfr H, as well as experiments with converting phages IV and 7,8, this new subserovar of S. flexneri 4, similarly to other S. flexneri subserovars, was proved to be the Y-variant of shigellae rendered lysogenic by the two above phages. The experiments also revealed that 97.9% of all S. flexneri strains 4 (IV: 7,8) under study possessed invasive properties and were capable of inducing specific keratoconjunctivitis in guinea pigs. Observations on the isolation of S. flexneri strains 4 belonging to the new serovar (IV: 7,8), carried out by the All-Union Shigelloses Center on its basal territories in 1980-1984, made it possible to establish the tendency towards a wider circulation of this infective agent in the USSR.     

Serological properties of Abiotrophia and Granulicatella species (nutritionally variant streptococci)

Serological variations were examined among 12 type or reference strains and 91 oral isolates of vitamin B6-dependent Abiotrophia and Granulicatella spp. Rabbits were immunized with whole cells of 12 selected strains and 10 typing antisera were obtained, which were unreactive with the Lancefield group A to G antigen preparations. The reactivity of the antisera and autoclaved cell surface antigen extracts was tested by double diffusion in agar gel and a capillary precipitin test. These typing antisera categorized all Abiotrophia defectiva strains, all except one Granulicatella elegans strain, three-quarters of the Granulicatella adiacens, and half of the Granulicatella paraadiacens into 8 serotypes and 2 subserotypes. The Granulicatella balaenopterae type strain was unserotypable. All A. defectiva strains were serotype I, some of which were divided into subserotype I-1 and/or I-5. The G. adiacens strains generally belonged to serotype II or III, and the G. paraadiacens strains to serotype IV, V or VI. All G. adiacens or G. paraadiacens serotype II strains were also subserotype I-5. The G. elegans strains were serotype VII or VIII. These Abiotrophia and Granulicatella serotypes were undetectable among 33 strains of the other 11 species including the bacteriolytic enzyme-producing but vitamin B6-independent strains of Streptococcus, Enterococcus, Dolosigranulum and Aerococcus. The proposed serotyping system for Abiotrophia and Granulicatella spp. would be helpful in the identification and classification of these unique coccal isolates in ecological and epidemiological studies.     

Unusual serovar of the causative agent of Flexner's dysentery. I. Its distribution and biochemical properties

The data on the circulation of some S. flexneri strains in the USSR are presented. The antigenic structure of these strains (type antigen 4 and group antigens 7, 8) is not characteristic of known serological subvariants 4a, 4b or var. X. The time course of changes in their spreading on certain base territories controlled by the All-Union Shigellosis Center in 1980-1983 is shown. The biochemical characteristics of S. flexneri strains IV:7,8 under study, isolated in the USSR and abroad (Czechoslovakia, London) from humans, from a monkey and from the environment, are given. Similarities in the biochemical reactions of these strains with respect to indole, rhamnose, saccharose, maltose, arabinose and raffinose have been revealed. The strains isolated in the USSR have shown differences in the spectra of their resistance to antibiotics, depending on their territorial origin.     

Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes.

The potential utility of Shigella flexneri aroD vaccine candidates for the development of bi- or multivalent vaccines has been explored by the introduction of the genetic determinants rfp and rfb for heterologous O antigen polysaccharide from Shigella dysenteriae serotype 1. The serotype Y vaccine strain SFL124 expressed the heterologous antigen qualitatively and quantitatively well, qualitatively in the sense of the O antigen polysaccharide being correctly linked to the S. flexneri lipopolysaccharide R3 core oligosaccharide and quantitatively in the sense that typical yields were obtained, with ratios of homologous to heterologous O antigen being 4:1 for one construct and 1:1 for another. Moreover, both polysaccharide chains were shown to be linked to position O-4 of the subterminal D-glucose residue of the R3 core. In contrast to the hybrid serotype Y SFL124 derivatives, analogous derivatives of serotype 2a vaccine strain SFL1070 did not elaborate a complete heterologous O antigen. Such derivatives, and analogous derivatives of rough, O antigen-negative mutants of SFL1070, formed instead a hybrid lipopolysaccharide molecule consisting of the S. flexneri lipid A R3 core with a single repeat unit of the S. dysenteriae type 1 O antigen. Introduction of the determinants for the S. dysenteriae type 1 O antigen into a second serotype 2a strain and into strains representing other serotypes of S. flexneri, revealed the following for the expression of the heterologous O antigen: serotypes 1a, 1b, 2a, and 5a did not produce the heterologous O antigen, whereas serotypes 2b, 3a, 3b, 4a, 4b, 5b, and X did.     

Bacteriophage types represented by lactose-fermenting Salmonella agona strains

Bacteriophage typing was performed on 1911 S. agona, lactose-fermenting strains. These strains were isolated from hospitalised newborns and neonates patients. Out of 1911 strains 98.8% were typable by means of phage set prepared for strains differentiation of Salmonella agona showing typical biochemical properties. It was shown that in 16 provinces from which the strains were obtained in 1983-1985 type V (49.5%) and type XI (25.4%) prevailed. Subtypes VA and VB were distinguished within type V. Altogether 20.3% of strains were classified as belonging to these subtypes. Their lytic reaction was weaker with phages 3, 4, and 9 with the characteristic range of phage type V strains. Among tested strains types I, XIII, and XVI were also represented composing 2, 6, 0, 9, and 0.3% of total number of strains respectively. 1.5% of strains were nontypable and 0.2% showed lytic reactions different from that included in up to now used scheme of typing. It can be concluded that lactose-fermenting S. agona strains show susceptibility to lowered number of phages than typical for Salmonella species strains. It seems that differentiation of this atypical biochemical variant of S. agona with, the use of phage set used up to now may be also usefull in practice as it is the case in respect to strains with typical biochemical properties.     

A numerical method for allocating microbial isolates to strain types when characterized by typing methods that are not 100% reproducible

Many methods for typing microbial strains are not 100% reproducible.This can create problems when deciding whether different groupsof isolates are really distinct or represent typing errors orvariation of a single strain. Neither hierarchical clusteringnor iterative partitioning methods are suited for analysingsuch data. A novel iterative partitioning method is describedwhich allows for the uncertainty of the typing method in use.Before grouping strains, the maximum dimension of the groupsis set based on a previous knowledge of the typing method'sreproducibility. Isolates are only allocated to a group if theydiffer from that group's typical strain type by less than thenumber of reaction differences required to distinguish betweentwo strains. In a series of Monte Carlo studies the accuracyof strain allocation was found to be very good, even when thetwo groups were situated close to each other.     

Interrelation of virulent and avirulent (vaccinal) strains of Shigella flexneri 2A in long-term bacterial carriage in gnotobiotic rats

The ability of S. flexneri 2a virulent and avirulent (vaccine) strains No. 516 and No. 516 m to displace one another during long-term carrier state in germ-free rats has been studied. The long-term persistence of the vaccine strain in the intestine of the rats has been shown to produce colonization resistance to subsequent infection with the virulent culture of these bacteria. During carrier state in rats progressive S--R dissociation of the bacteria occurs, type II antigen is lost with simultaneous retention of group 3, 4 antigens and the resulting transformation of S. flexneri, serotype 2a, into variant y; the virulence of Shigella S-forms is also lost.     

Interaction between bacteriophage Sf6 and Shigella flexner.

The Shigella flexneri phage Sf6 has an isometric head with hexagonal symmetry 53nm in diameter. The noncontractile tails in 16 nm long and terminates with a base plate containing six spikes. Sf6 is typical of the C phages in the morphological classification of Bradley. Phage Sf6 processes alpha-1,3-endorhamnosidase activity as demonstrated by methylation and reducing end group sugar analyses of the products obtained on interaction with the O-polysaccharide chain of S.flexneri strains which have the O-group 3,4 antigen. The major end product was an octasaccharide with the following structure: Rha III-GlcNAc-Rha I-Rha II-Rha III-GlcNAc-Rha I-Rha II. Acetylation of 0-2 of rhamnose III of the O-polysaccharide chain, either brought about by Sf6 lysogenization or found in wild-type S. flexneri (3b) strains, prevented enzymatic hydrolysis. O-deacetylation of the polysaccharide chain again made it susceptible to the S6f endorhamnosidase.     

Identification and typing of miso and soy sauce fermentation yeasts, Candida etchellsii and C. versatilis, based on sequence analyses of the D1D2 domain of the 26S ribosomal RNA gene, and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.     

Molecular characterization of methicillin-resistant Staphylococcus aureus in hospitals in Niigata, Japan: divergence and transmission

The major methicillin-resistant Staphylococcus aureus(MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCC mec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCC mec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst(+) sec(+) strains represented a majority of 86.5% and 9.4% were tst(-) sec(-). SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi-drug resistance with high virulence gene content. The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCC mec IV and seemed to originate in the community, while ST8 strains exhibited SCC mec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.     

1型CRISPR位点间区序列分析在嗜热链球菌菌株分型中的应用

为了建立适用于嗜热链球菌菌株资源多样性调查的菌株分型方法,尝试将1型CRISPR位点间区序列分析用于嗜热链球菌的菌株分型,并与常用ERIC-PCR指纹图谱方法进行了比较。结果表明,1型CRISPR位点间区序列分析可以把30株从三个不同样品中分离的嗜热链球菌分成三种差异明显的类型:不同类型菌株之间没有相同的间区序列;而同一类型菌株之间,间区序列的组成和排列则基本一致,并且上述分型的结果与用ERIC-PCR指纹图谱技术获得的结果完全一致。因此,1型CRISPR位点间区序列分析是嗜热链球菌分型鉴定的可靠方法,并适用于大量菌株的分型鉴定和多样性调查。     

临床分离耐甲氧西林溶血性葡萄球菌的SCCmec分型及同源性分析

目的了解临床分离耐甲氧西林溶血性葡萄球菌（MRSH）的SCCmec基因型别及相同SCCmec型别菌株的同源性。方法多重PCR进行SCCmec分型,ERIC-PCR法对相同SCCmec型别菌株进行同源性分析。结果83株临床分离MRSH菌株中,SCCmecI型有23株（27．7％）,SCCmecⅡ型有10株（12．1％）,SCCmecm型有24株（28．9％）,SCCmecIV型有1株（1．2％）,I、Ⅱ混合型有8株（9．6％）,I、Ⅲ混合型有6株（7．2％）,Ⅱ、11混合型有5株（6．0％）,I、Ⅱ、Ⅲ混合型有3株（3．6％）,未分型3株（3．6％）。ERIC—PCR结果显示,23株SCCmecI型分为11型,其中A型5株,B型5株,C型3株,其余8株各为1型,2株未分型;10株SCCmecⅡ型分为6型,其中D型4株,E型2株,3株各为1型,1株未分型;24株SCCmecm型分为9型,其中F型11株,G型2株,H型2株,I型2株,5株各为1型,2株未分型。结论临床分离MRSH中,SCCmecI、Ⅲ型为多,部分菌株呈混合型别;相同SCCmec型别的部分菌株之间可能存在克隆传播。     

Multiplex PCRs for assignment of Staphylocoagulase types and subtypes of type VI Staphylocoagulase

Staphylocoagulases (SCs) have been classified by the differences in antigenicity using a serological method. We have developed a system to classify them based on the nucleotide differences in SC genes (coa). The system was composed of three multiplex PCRs (M-PCRs): M-PCR:A, identifying types III, IV, VII, and VIII; M-PCR:B, identifying types I, II, V, and VI; M-PCR:C, identifying three subtypes of type VI. In this study, we found that coa genes of the serotype VI were not identical, but classified into three subtypes based on the nucleotide differences, especially in D2 and the central region: VIa, the coa gene carried by stp12 from human; and VIb and VIc, the coa genes carried by strains IFH556 and IFH514 isolated from bovine raw milk. The primer pair used in M-PCR:B was designed to identify all three subtypes of type VI coa. The results showed that coa types of 154 out of 155 Staphylococcus aureus strains from various origins assigned by M-PCR:A and B were identical to those obtained by serological methods, leaving a serotype IV strain unclassifiable. All 73 type VI strains were classified into one of three subtypes by M-PCR:C. Furthermore, we found that type VIa and VIb strains carried characteristic pyrogenic toxin superantigen genes, while no toxin genes were identified in type VIc strains, suggesting the correlation between the subtype of type VI coa gene and the carriage of genomic islands. Our results showed that these M-PCRs are convenient methods for SC typing that might be useful for epidemiological studies.