Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics

ABSTRACT

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.     

Fluorescent amplified fragment length polymorphism genotyping of Salmonella Enteritidis: a method suitable for rapid outbreak recognition

Objective   To perform fluorescent amplified fragment length polymorphism (FAFLP) analysis on phage type (PT) reference strains of Salmonella enterica subsp. enterica serotype Enteritidis ( S. Enteritidis), and S . Enteritidis PT 6 and 6a recent clinical isolates to determine its usefulness for primary characterization of clinical S. Enteritidis isolates, and then to determine whether FAFLP is suitable for rapid characterization of strains in an outbreak situation.
Methods   Twenty-five PT reference strains of S. Enteritidis and 20  S . Enteritidis PT 6 and 6a clinical isolates were subjected to FAFLP analysis using the selective primer combinations Eco  + 0– Mse  + T and Eco  + 0– Mse  + TA.
Results   FAFLP successfully separated each one of the 25 S . Enteritidis PT strains into distinct profiles, while macrorestriction and PFGE using Xba I identified 20 pulsed-field profiles. FAFLP also resolved cases and outbreaks due to S . Enteritidis PTs 6 and 6a.
Conclusions   The resolving power of FAFLP was higher than that of PFGE. FAFLP is a highly discriminatory genotyping method and, in conjunction with phage typing for primary subdivision of S . Enteritidis, provides a rapid and powerful tool for strain differentiation, both for outbreak investigation and for epidemiologic surveillance.     

PCR ribotyping and arbitrarily primed PCR for the comparison of enterotoxigenic Bacteroides fragilis strains from two Polish university hospitals

Objective: To study the clinical incidence and possible clonal relatedness of enterotoxigenic strains of Bacteroides fragilis among pediatric and adult patients in two Polish university hospitals.
Method: Fecal samples from 201 adults and 131 infants (with or without diarrhea) and vaginal samples from 100 pregnant women nursed in two Polish university hospitals were analyzed with respect to carriage of enterotoxin-producing Bacteroides fragilis (ETBF). This putative pathogen was identified by cultivation and subsequent cytopathogenicity testing of culture supernatants on HT/29 C1 cells.
Results and discussion: Two ETBF strains were isolated from childrens' feces; two additional strains were isolated from adults, and from the vaginal samples only a single strain was isolated. One strain (W2) was isolated from a child with diarrhea. These incidence figures, the fact that all ETBF isolates were shown to produce strongly differing amounts of the cytotoxin, and the genetic unrelatedness of the strains as demonstrated by two different PCR-mediated DNA typing procedures, indicates that clonal spread of ETBF is presently not a clinical problem in these hospitals. It was shown that PCR-mediated ribotyping and arbitrarily primed PCR can be applied with success to study the epidemiology of ETBF.     

A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90?fg/μl or 10² CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.     

Analysis of United Kingdom wild-type strains of varicella-zoster virus: Differentiation from the Oka vaccine strain

In Japan and the United States, where vaccination against varicella-zoster virus (VZV) infection with the live attenuated Oka strain of varicella is routine, cases of chickenpox or shingles occurring in vaccinees can be caused by either wild-type or vaccine virus. Differentiating such cases is important epidemiologically and can be achieved only using molecular typing methods. In the United Kingdom, the Oka vaccine is being considered for use in groups at risk of severe primary varicella, such as seronegative immunocompromised patients and women who may be considering pregnancy. In addition, seronegative health workers who may be occupationally exposed to VZV infection might also be offered vaccination. We analysed 249 U.K. wild-type VZV strains, 105 from cases of chickenpox and 144 from shingles cases, to determine whether they could be distinguished from Oka by the genotyping systems used in Japan and the United States. Four polymorphic loci were examined, a Pst 1 restriction site in gene 38, a Bgl 1 restriction site in gene 54, the R5 repeat region, and the R2 repeat region. The results suggest that U.K. strains of VZV are more similar to U.S. strains than to Japanese strains. All the U.K. wild-type viruses were positive for the Pst 1-1 restriction site, unlike Oka, which is negative. However, one of thirty strains was indistinguishable from Oka at all other loci. J. Med. Virol. 53:60–62, 1997. © 1997 Wiley-Liss, Inc.     

A comprehensive method to scan for point mutations of the glucose 6 phosphate dehydrogenase gene

Summary We have developed a fast and comprehensive method to scan for point mutations in a gene on X chromosome. A target region of the gene is first amplified. Then, using the amplified product as a template, PCR is carried out with multiple short-length forward primers arrayed in tandem in the scanned region, and a common reverse primer. The absence of amplified product defines the site of a mutation within a narrow region of the primer recognition site. To evaluate our method, point mutations in exon 12 of the human glucose-6-phosphate dehydrogenase (G6PD) gene were used as a model system. Out of 12 Singaporean G6PD-deficient patients, 6 cases were shown by the method to have a nucleotide change in this exon. Sequence analysis confirmed the presence of a nucleotide change in the region identified by our scanning. Thus, our method is accurate in localizing mutations within a narrow region, and allows large numbers of samples to be handled simultaneously.     

O148 Shiga toxin-producing Escherichia coli outbreak: microbiological investigation as a useful complement to epidemiological investigation

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O148 infection occurred among wedding attendees in France in June 2002. A retrospective cohort study was performed and ten cases were identified, including two adults with haemolytic uraemic syndrome (HUS). The analytical study revealed that > 80% of affected individuals had eaten lightly roasted mutton and poultry paté, but only the consumption of paté tended to be associated with illness (relative risk 3.4; 95% CI 0.8-14.4). Left-overs (cooked mutton and raw offal) and processed foods (paté) from the same batches as served at the party were sampled. Human, food and environmental samples were examined for the Shiga toxin (stx) gene and virulence traits by PCR. Stx-positive samples were cultured for STEC. HUS cases were tested for serum antibodies against 26 major STEC serogroups. An STEC O26 strain (stx1, eae, ehxA) was isolated from one case with diarrhoea, and an STEC O148 strain (stx2c) from one case of HUS. Serum antibodies against O26 were not detected in either of these patients; antibodies against O148 were not tested. Three STEC strains were isolated from the mutton and the offal (stx2c, O148), and two from the paté (stx2c, O-X and O-Y). The isolates from the mutton were indistinguishable from the human stx2c isolate, whereas the paté isolates differed. Although four different STEC strains were identified in patients and foods, the results of molecular subtyping, in conjunction with analysis of food consumption patterns, strongly suggested that this outbreak was caused by mutton contaminated with STEC O148.     

食物中毒中分离的副溶血性弧菌的分子分型研究

目的运用脉冲场凝胶电泳（PFGE）技术研究深圳市罗湖区2005-2007年食物中毒事件中分离获得的49株副溶血性弧菌的分子流行情况。方法样品采用荧光PCR与传统分离培养同时检测,对分离获得的副溶血性弧菌阳性菌株进行脉冲场凝胶电泳（PFGE）,BioNumerics软件对图谱进行聚类分析。结果 49株副溶血性弧菌中,有12株不能分型,其余36株副溶血性弧菌分离株大致可分为Ⅰ、Ⅱ、Ⅲ3个群,相同血清型的菌株其PFGE型大致相同,同一起食物中毒的菌株间有较高的相关性。结论罗湖区副溶血性弧菌食物中毒存在流行克隆株,PFGE分子分型技术在食物中毒追踪溯源方面有重要提示性作用。     

Application of microsatellite typing for the investigation of a cluster of cases of Candida albicans candidaemia

A cluster of cases of Candida albicans candidaemia in a surgical intensive care unit was investigated. The probability of such a cluster during a single month was highly significant compared with the frequency of candidaemia in the previous year. A molecular typing method, based on length analysis of three (EF3, CDC3, HIS3) microsatellite-containing regions, was used to investigate isolates from patients in and outside the ward. This demonstrated the involvement of different strains, indicating the absence of cross-transmission among patients. Results of microsatellite typing can be obtained almost in real-time, which is particularly useful in an outbreak context.     

Molecular typing for fungi—a critical review of the possibilities and limitations of currently and future methods

Invasive fungal infections represent an increasing problem in patients with inherited and acquired immunodeficiencies. Molecular biotyping techniques, such as DNA fingerprinting, are useful tools to increase our knowledge of the pathogenic organisms that cause them, and thus to improve their treatment and develop prevention strategies. In the present review, we evaluate and discuss the possibilities and limitations of the methods currently used for biotyping strains of fungal species. These include techniques based on restriction fragment length polymorphism (RFLP) with or without hybridization to probes (Southern), PCR-based techniques, electrophoretic karyotyping (EK), and multilocus enzyme electrophoresis (MLEE). Additionally, we discuss newer techniques that are being developed for the fingerprinting of fungal strains. Among them, we review conformation-based polymorphism scanning methods, such as single-strand conformation polymorphism analysis (SSCP) and heteroduplex mobility assays, sequencing strategies such as multilocus sequence typing (MLST) and DNA microarrays.     

Evaluation of the discriminatory power of pulsed-field gel electrophoresis and PCR fingerprinting for epidemiologic typing of Candida species

Objective: To evaluate the discriminatory power of genotyping methods (PCR fingerprinting and pulsed-field gel electrophoresis) validated for Candida albicans in other Candida species.
Methods: Molecular typing methods are increasingly being applied for studies where the interpretation of data essentially relies on the typing results rather than epidemiologic data. In this situation, the discriminatory power (ability to identify differences among epidemiologically unrelated strains) of the typing method is important in allowing one to draw valid conclusions. By applying PCR fingerprinting, electrophoretic karyotyping, and restriction fragment endo-nuclease analysis using standard restriction enzymes and primers proven to be useful in previous studies, we evaluated whether the use of multiple genotyping methods is sufficient to delineate known unrelated strains among seven Candida species.
Results: All three methods identified individual genotypes for each of the seven Candida species studied. However, optimal strain delineation required the combined use of all three typing methods and was observed only within the small number of C. albicans and C. tropicalis isolates tested in this study.
Conclusion: Typing assays that are able to delineate a certain Candida species may not be used blindly for other species of that genus. Regarding the limited number of strains tested, further validation of the discriminative power of genotyping methods (including in C. tropicalis ) should be done.     

Assessment of detection of Candida mannoproteinemia as a method to differentiate central venous catheter-related candidemia from invasive disease.

The proper management of candidemic patients is controversial because of the difficulties of an early differentiation of central venous catheter (CVC)-related candidemia from deep-seated invasive Candida infection. In particular, more information on possible markers of invasive disease is needed. We performed a retrospective, pilot investigation to assess the diagnostic potential of a dot immunobinding assay for Candida mannoprotein antigen in serial serum samples from 31 candidemic patients in the setting of hematologic malignancy. Mannoproteinemia (antigenemia) was detected in 1 of 14 (7.1%) patients with transient or CVC-related candidemia and in 13 of 17 (76.5%) patients with non-CVC-related persistent candidemia. Of the 11 subjects of this latter group with documented tissue invasion, 10 (91%) were antigenemic. The patients belonging to the different categories did not significantly differ in the duration of candidemia, nor was there any significant difference among the different groups of subjects either in the number of serum samples examined or in their collection time during candidemia. The day of the first antigenemic sample during candidemia greatly varied among subjects with invasive infection, although on average mannoproteinemia was detectable by the first week of candidemia. In summary, our data demonstrate a correlation between mannoproteinemia and tissue invasion by Candida spp. in candidemic patients and suggest that mannoprotein detection by our method has a potential for the diagnosis of invasive candidiasis in these subjects.     

CD34+细胞的心肌细胞分化潜能研究

目的:了解粒细胞集落刺激因子(G-CSF)动员的CD34+细胞的心肌细胞分化潜能。方法:用异丙肾上腺素(ISO)复制急性心肌梗死大鼠动物模型,于3 h后用G-CSF动员骨髓造血干细胞进行心肌梗死动物模型的“自身干细胞移植”,用免疫组化和HE染色方法检测动物模型心梗区的CD34⁺细胞浸润以及心肌细胞再生情况。结果:用ISO后24 h,G-CSF处理组大鼠心梗区可见大量CD34⁺单个核细胞浸润,并有CD34⁺的新生心肌细胞生长,2周后疤痕组织不明显;而对照组心梗坏死区有大量以中性粒细胞为主的炎症细胞浸润,无CD34+细胞浸润及新生心肌细胞生长,2周后出现较大量的疤痕组织。结论:G-CSF动员CD34⁺细胞具有向心肌细胞分化的潜能,用G-CSF 动员造血干细胞的“干细胞自身移植”,可治疗急性心肌梗死。     

Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping

Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.     

Evaluation of molecular typing methods for identification of outbreak‐associated Neisseria meningitidis isolates

It is essential in an outbreak investigation that strain characterization of Neisseria meningitidis is performed in a rapid and accurate manner. This study evaluated two new molecular typing methods, multiple‐locus variable number tandem repeat analysis (MLVA) and repetitive sequence‐based PCR (rep‐PCR) (DiversiLab; bioMérieux) and compared them with current recommended methodologies. This retrospective study included 36 invasive N. meningitidis serogroup C isolates collected in Sweden 2001 through 2009 and previously subjected to outbreak investigation. All strains were typed with highly variable‐MLVA (HV‐MLVA) and rep‐PCR. The isolates were further characterized by multilocus sequence typing (MLST) and sequencing of the fetA, fHbp, penA, porA and porB genes. The results showed that HV‐MLVA had the highest index of diversity (0.99) and rep‐PCR had the highest congruence (40%) with the currently recommended typing methods. The HV‐MLVA correlated best to the spatiotemporal connections and had the overall highest Adjusted Wallace coefficients, suggesting that HV‐MLVA can predict the results of the other typing methods in the study. We therefore suggest that after initial confirmation of species, serogroup and genosubtype, HV‐MLVA should be used as the most discriminatory method for first hand investigation of N. meningitidis serogroup C isolates.     

Bacterial and epidemiologic study of the resistance to oxyiminocephalosporins in Escherichia coli in a French hospital

Objective: To understand the mechanisms and epidemiology of resistance to oxyiminocephalosporins in Escherichia coli over a 2-year period in a French hospital.
Methods: Forty-four strains, resistant or intermediately resistant to one of the oxyiminocephalosporins or aztreonam, were collected from 35 patients. MIC determinations were carried out for the 44 isolates using a panel of β-lactam antibiotics, and characterization of the β-lactamases they produced by isoelectric focusing and catalytic activity measurement. Extended-spectrum β-lactamase production was studied by use of the double disk diffusion test. Conjugation experiments were used to search for plasmidic cephalosporinase. An epidemiologic study was then performed, by use of molecular typing of the strains with an ERIC-PCR method and a case-control analysis.
Results: Less than 1% of all the E. coli isolates at our hospital showed decreased susceptibility to oxyiminocephalosporins. Only three of the 44 isolates showed synergy between clavulanate and a third-generation cephalosporin and produced an extended-spectrum β-lactamase. For the other strains, a β-lactamase with a highly basic isoelectric point was detected. Spectrophotometric measures confirmed that most of these isolates were AmpC hyper-producers. No plasmidic cephalosporinase could be detected by conjugation experiments. Molecular typing showed all isolates to be different, except for two strains isolated in two patients of the same hospital unit, and for the repeated isolates of some patients. When 20 case patients were compared to 40 randomly selected control patients, prior receipt of an antimicrobial and more specifically of a β-lactam agent was significantly associated with case patients.
Conclusions: Although it appears to be very rare, the resistance to broad-spectrum cephalosporins needs our attention, because of the high frequency of E. coli infections and β-lactam use in their treatment.