Multilocus Sequence Typing System Research Articles

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Analysis of antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery.

To analyze antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery with the focus on coagulase-negative staphylococci (CNS) during cataract surgery and discuss the implications of this colonization as a potential risk of acquiring endophthalmitis. After approval of the institutional review board and informed consent from patients had been obtained, conjunctival swabs for culture from 59 patients undergoing cataract surgery were taken of the fellow eye at baseline (C0) and from the eye to be operated before (T0) and after (T1) irrigation with povine-iodine 5%, and at the end of surgery (T2). Genes responsible for virulence (mecA, ica and atlE) and antibiotic profile were determined; strain clonality of persistent colonizing Staphylococcus epidermidis strains was established by the Multi-locus sequence typing (MLST). The frequency of CNS was significantly reduced in T1 (13.6%) from 81.4% in T0 and 86.4% in C0. The frequency of mecA, ica and atlE genes was 34.4%, 37.5% and 61.4%, respectively; and methicillin phenotypic resistance was 35.4%. S. epidermidis was the most frequent species isolated in every time point. MLST revealed in 7 patients 100% coincidence of the seven alleles of the S. epidermidis isolated previous to povine-iodine 5% disinfection and at the end of the surgery. CNS isolates from T1 or T2 corresponded to the same species, antibiotic and virulence profile as those isolates from C0 or T0. Povidone-iodine 5% prophylaxis before surgery significantly reduced conjunctival contamination; in those that persisted, the source of contamination was mostly the patient's microbiota confirmed by the MLST system.

Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains.

Tenacibaculosis is a bacterial ulcerative disease affecting marine fish and represents a major threat to aquaculture worldwide. Its aetiological agents, bacteria belonging to the genus Tenacibaculum, have been present in Norway since at least the late 1980's and lead to regular ulcerative outbreaks and high mortalities in production of farmed salmonids. Studies have shown the presence of several Tenacibaculum species in Norway and a lack of clonality in outbreak-related strains, thus preventing the development of an effective vaccine. Hence, a thorough examination of the bacterial diversity in farmed fish presenting ulcers and the geographical distribution of the pathogens should provide important insights needed to strengthen preventive actions. In this study, we investigated the diversity of Tenacibaculum strains isolated in 28 outbreaks that occurred in Norwegian fish farms in the period 2017-2020. We found that 95% of the 66 strains isolated and characterized, using an existing MultiLocus Sequence Typing system, have not previously been identified, confirming the high diversity of this genus of bacteria in Norway. Several of these Tenacibaculum species seem to be present within restricted areas (e.g., Tenacibaculum dicentrarchi in western Norway), but phylogenetic analysis reveals that several of the strains responsible of ulcerative outbreaks were isolated from different localities (e.g., ST- 172 isolated from northern to southern parts of Norway) and/or from different hosts. Understanding their reservoirs and transmission pathways could help to address major challenges in connection with prophylactic measures and development of vaccines.

Development of a multi-locus sequence typing system helps reveal the evolution of Cardinium hertigii, a reproductive manipulator symbiont of insects

BackgroundCardinium is an intracellular bacterial symbiont in the phylum Bacteroidetes that is found in many different species of arthropods and some nematodes. This symbiont is known to be able to induce three reproductive manipulation phenotypes, including cytoplasmic incompatibility. Placing individual strains of Cardinium within a larger evolutionary context has been challenging because only two, relatively slowly evolving genes, 16S rRNA gene and Gyrase B, have been used to generate phylogenetic trees, and consequently, the relationship of different strains has been elucidated in only its roughest form.ResultsWe developed a Multi Locus Sequence Typing (MLST) system that provides researchers with three new genes in addition to Gyrase B for inferring phylogenies and delineating Cardinium strains. From our Cardinium phylogeny, we confirmed the presence of a new group D, a Cardinium clade that resides in the arachnid order harvestmen (Opiliones). Many Cardinium clades appear to display a high degree of host affinity, while some show evidence of host shifts to phylogenetically distant hosts, likely associated with ecological opportunity. Like the unrelated reproductive manipulator Wolbachia, the Cardinium phylogeny also shows no clear phylogenetic signal associated with particular reproductive manipulations.ConclusionsThe Cardinium phylogeny shows evidence of diversification within particular host lineages, and also of host shifts among trophic levels within parasitoid-host communities. Like Wolbachia, the relatedness of Cardinium strains does not necessarily predict their reproductive phenotypes. Lastly, the genetic tools proposed in this study may help future authors to characterize new strains and add to our understanding of Cardinium evolution.

Strain diversity of Treponema pallidum subsp. pertenue suggests rare interspecies transmission in African nonhuman primates

In our most recent study, we found that in Tanzania infection with Treponema pallidum (TP) subsp. pertenue (TPE) is present in four different monkey species. In order to gain information on the diversity and epidemiological spread of the infection in Tanzanian nonhuman primates (NHP), we identified two suitable candidate genes for multi-locus sequence typing (MLST). We demonstrate the functionality of the MLST system in invasively and non-invasively collected samples. While we were not able to demonstrate frequent interspecies transmission of TPE in Tanzanian monkeys, our results show a clustering of TPE strains according to geography and not host species, which is suggestive for rare transmission events between different NHP species. In addition to the geographic stability, we describe the relative temporal stability of the strains infecting NHPs and identified multi-strain infection. Differences between TPE strains of NHP and human origin are highlighted. Our results show that antibiotic resistance does not occur in Tanzanian TPE strains of NHP origin.

High Wolbachia Strain Diversity in a Clade of Dung Beetles Endemic to Madagascar

Determining the drivers of diversity is a major topic in biology. Due to its high level of micro-endemism in many taxa, Madagascar has been described as one of Earth's biodiversity hotspot. The exceptional Malagasy biodiversity has been shown to be the result of various eco-evolutionary mechanisms that have taken place on this large island since its isolation from other landmasses. Extensive phylogenetic analyses have, for example, revealed that most of the dung beetle radiation events have arisen due to allopatric speciation, and adaptation to altitudinal and/or longitudinal gradients. But other biotic factors, that have yet to be identified, might also be at play. Wolbachia is a maternally transmitted endosymbiotic bacterium widespread in insects. The bacterium is well-known for its ability to modify its host reproductive system in ways that may lead to either discordance patterns between the host mitochondrial and nuclear phylogenies, and in some cases to speciation. Here, we used the MultiLocus Sequence Typing system, to identify and characterize five Wolbachia strains infecting several species within the Nanos clypeatus dung beetle clade. We discuss the implications of these Wolbachia strains for the evolution and diversification of their dung beetle hosts in Madagascar. (Less)

Specific clones of Trichomonas tenax are associated with periodontitis.

Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflammatory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 periodontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from periodontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%°) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p<0.05). More frequently detected in periodontal cases, T. tenax is likely to be related to the onset or/and evolution of periodontal diseases.

Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles.

Treponema pallidum subsp. pallidum, the causative agent of sexually transmitted syphilis, detected in clinical samples from France, was subjected to molecular typing using the recently developed Multilocus Sequence Typing system. The samples (n = 133) used in this study were collected from 2010–2016 from patients with diagnosed primary or secondary syphilis attending outpatient centers or hospitals in several locations in France. Altogether, 18 different allelic profiles were found among the fully typed samples (n = 112). There were five allelic variants identified for TP0136, 12 for TP0548, and eight for TP0705. Out of the identified alleles, one, seven, and three novel alleles were identified in TP0136, TP0548, and TP0705, respectively. Partial allelic profiles were obtained from 6 samples. The majority of samples (n = 110) belonged to the SS14-like cluster of TPA isolates while 7 clustered with Nichols-like isolates. Patients infected with Nichols-like samples were more often older (p = 0.041) and more often diagnosed with secondary syphilis (p = 0.033) compared to patients infected with SS14-like samples. In addition, macrolide resistance caused by the A2058G mutation was found to be associated with allelic profile 1.3.1 or with strains belonging to the 1.3.1 lineage (p<0.001). The genetic diversity among TPA strains infecting the European population was surprisingly high, which suggests that additional studies are needed to reveal the full genetic diversity of TPA pathogens infecting humans.

Genotyping Mycoplasma hyopneumoniae isolates based on multi-locus sequence typing, multiple-locus variable-number tandem repeat analysis and analysing gene p146

Mycoplasma hyopneumoniae is a swine pathogen bacterium, causing significant economic losses worldwide. Epidemiological investigations based on molecular typing methods support the prevention and eradication strategies for the control of M. hyopneumoniae, through tracing the spreading of the pathogen. The present study describes the genotyping of 44 M. hyopneumoniae strains isolated from Hungarian, Czech and Slovakian porcine lung samples by multi-locus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA) and analysing gene p146, and the evaluation of the used methods. The resolution of the three-gene (adk, rpoB, tpiA) and the seven-gene (efp, metG, pgiB, recA, adk, rpoB, tpiA) based MLST systems was identical with 27 sequence types. MLVA utilising loci P97-RR1 and Locus1 extended with the serine repeat numbers of gene p146 showed the highest resolution power among the studied methods differentiating 40 genotypes. The independent analysis of gene p146 revealed 31 different types among the isolates.High variability of M. hyopneumoniae strains was detected by the used typing methods. The results confirmed that utilization of the minimal MLST is suitable for phylogenetic analyses of M. hyopneumoniae strains. The MLVA method extended with the evaluation of serine repeat numbers of gene p146 is adequate for the resolution of genetic relationships within MLST groups. Examination of the p146 gene is suitable to complement both MLST and MLVA methods in order to refine closer genetic relationships.

Current state of knowledge on Wolbachia infection among Coleoptera: a systematic review.

BackgroundDespite great progress in studies on Wolbachia infection in insects, the knowledge about its relations with beetle species, populations and individuals, and the effects of bacteria on these hosts, is still unsatisfactory. In this review we summarize the current state of knowledge about Wolbachia occurrence and interactions with Coleopteran hosts.MethodsAn intensive search of the available literature resulted in the selection of 86 publications that describe the relevant details about Wolbachia presence among beetles. These publications were then examined with respect to the distribution and taxonomy of infected hosts and diversity of Wolbachia found in beetles. Sequences of Wolbachia genes (16S rDNA, ftsZ) were used for the phylogenetic analyses.ResultsThe collected publications revealed that Wolbachia has been confirmed in 204 beetle species and that the estimated average prevalence of this bacteria across beetle species is 38.3% and varies greatly across families and genera (0–88% infected members) and is much lower (c. 13%) in geographic studies. The majority of the examined and infected beetles were from Europe and East Asia. The most intensively studied have been two groups of herbivorous beetles: Curculionidae and Chrysomelidae. Coleoptera harbor Wolbachia belonging to three supergroups: F found in only three species, and A and B found in similar numbers of beetles (including some doubly infected); however the latter two were most prevalent in different families. A total of 59% of species with precise data were found to be totally infected. Single infections were found in 69% of species and others were doubly- or multiply-infected. Wolbachia caused numerous effects on its beetle hosts, including selective sweep with host mtDNA (found in 3% of species), cytoplasmic incompatibility (detected in c. 6% of beetles) and other effects related to reproduction or development (like male-killing, possible parthenogenesis or haplodiploidy induction, and egg development). Phylogenetic reconstructions for Wolbachia genes rejected cospeciation between these bacteria and Coleoptera, with minor exceptions found in some Hydraenidae, Curculionidae and Chrysomelidae. In contrast, horizontal transmission of bacteria has been suspected or proven in numerous cases (e.g., among beetles sharing habitats and/or host plants).DiscussionThe present knowledge about Wolbachia infection across beetle species and populations is very uneven. Even the basic data about infection status in species and frequency of infected species across genera and families is very superficial, as only c. 0.15% of all beetle species have been tested so far. Future studies on Wolbachia diversity in Coleoptera should still be based on the Multi-locus Sequence Typing system, and next-generation sequencing technologies will be important for uncovering Wolbachia relations with host evolution and ecology, as well as with other, co-occurring endosymbiotic bacteria.

Revisiting the phylogeny of Wolbachia in Collembola.

The endosymbiont Wolbachia has been detected in a few parthenogenetic collembolans sampled in Europe and America, including three species of Poduromorpha, two species of Entomobryomorpha, and two species of Neelipleona. Based on 16S rRNA and ftsZ gene sequences, most of the Wolbachia infecting parthenogenetic collembolans were characterized as members of supergroup E and showed concordant phylogeny with their hosts. However, the two neelipleonan symbionts form another unique group, indicating that Wolbachia has infected parthenogenetic collembolans multiple times. In this study, five parthenogenetic collembolan species were identified as hosts of Wolbachia, and four new Wolbachia strains were reported for four collembolan species sampled in China, respectively, including a neelipleonan strain from Megalothorax incertus (wMinc). Our results demonstrated that the Wolbachia multilocus sequence typing (MLST) system is superior to the 16S rRNA + ftsZ approach for phylogenetic analyses of collembolan Wolbachia. The MLST system assigned these Wolbachia of parthenogenetic collembolans to supergroup E as a unique clade, which included wMinc, supporting the monophyletic origin of Wolbachia in parthenogenetic collembolan species. Moreover, our data suggested supergroup E as one of the most divergent lineages in Wolbachia and revealed the discrepancy between the phylogenies of Wolbachia from parthenogenetic collembolans and their hosts, which may result from the high level of genetic divergence between collembolan Wolbachia, in association with the geographic differentiation of their hosts, or the possible horizontal transmission of Wolbachia between different collembolan species.

Selective sweep of Wolbachia and parthenogenetic host genomes - the example of the weevil Eusomus ovulum.

Most parthenogenetic weevil species are postulated to have originated via hybridization, but Wolbachia has also been speculated to play a role via the induction of parthenogenesis. Here, we examine the molecular diversity of Wolbachia and parthenogenetic host genomes. The host species studied here, Eusomus ovulum, is known to be exclusively parthenogenetic and triploid. The E. ovulum populations that we examined had a low genetic diversity of mitochondrial (cytochrome oxidase I gene) and nuclear markers (internal transcribed spacer 2 and elongation factor 1-α gene), and they all were infected by only single bacteria strains (genotyped for five genes according to the multilocus sequence typing system). We found significant signs of linkage disequilibrium and a lack of recombination amongst all of the examined genomes (bacteria and host), which strongly indicates a selective sweep. The lack of heterozygosity in host nuclear genes, missing bisexual populations and selective sweep between the parthenogenetic host and bacteria genomes suggest that parthenogenesis in this species could have originated as a result of infection rather than hybridization. However, the finding that highly similar Wolbachia strains are also present in other parthenogenetic weevils from the same habitat suggests the opposite scenario: bacteria may have infected the already parthenogenetic lineage and taken advantage of the host's unisexual reproduction.

Wolbachia Multilocus Sequence Typing of Singly Infected and Multiply Infected Populations of Northern Corn Rootworm (Coleoptera: Chrysomelidae)

Abstract The northern corn rootworm (Diabrotica barberi Smith &amp; Lawrence) in eastern and central North America exhibits at least three distinct populations with respect to Wolbachia infection: uninfected; singly infected; multiply infected. The infected states are associated with different mtDNA haplotypes and reduced mtDNA variability. Multilocus sequence typing (MLST) was used to define strain types and examine the diversity of Wolbachia infecting northern corn rootworm. Four of the five MLST genes (coxA, hcpA, fbpA, and ftsZ) were represented by three alleles each. The remaining MLST gene (gatB) had four to six alleles. The uncertainty arose from whether to call two small clusters new alleles or artifacts of the amplification process. Singly infected individuals had a common set of alleles that defined one strain (wBarB). This strain was also a component of the multiple infections. A second strain (wBarA), restricted to the multiple infections, was defined by alleles that appeared with substantially greater frequency for each of the five loci. By default a third strain would comprise the less frequent third allele of the four loci, but it is not possible to determine to which gatB allele they are linked. Therefore, the strain is not fully defined. The diversity of gatB indicates that four to six Wolbachia strains are present in northern corn rootworm, a number that is similar to the five variants reported previously for the wsp gene. These results also highlight the very real difficulty of using the MLST system to define specific strains in a multiply infected host.

Rapid and Specific Detection, Molecular Epidemiology, and Experimental Virulence of the O16 Subgroup within Escherichia coli Sequence Type 131

Escherichia coli sequence type 131 (ST131), a widely disseminated multidrug-resistant extraintestinal pathogen, typically exhibits serotype O25b:H4. However, certain ST131 isolates exhibit serotype O16:H5 and derive from a phylogenetic clade that is distinct from the classic O25b:H4 ST131 clade. Both clades are assigned to ST131 by the Achtman multilocus sequence typing (MLST) system and a screening PCR assay that targets ST131-specific sequence polymorphisms in the mdh and gyrB genes. However, they are classified as separate STs by the Pasteur Institute MLST system, and an ST131 PCR method that targets the O25b rfb region and an ST131-specific polymorphism in pabB detects only the O25b-associated clade. Here, we describe a novel PCR-based method that allows for rapid and specific detection of the O16-associated ST131 clade. The clade members uniformly contained allele 41 of fimH (type 1 fimbrial adhesin) and a narrow range of alleles of gyrA and parC (fluoroquinolone target genes). The virulence genotypes of the clade members resembled those of classic O25b:H4 ST131 isolates; representative isolates were variably lethal in a mouse subcutaneous sepsis model. Several pulsotypes spanned multiple sources (adults, children, pets, and human fecal samples) and locales. An analysis of recent clinical E. coli collections showed that the O16 ST131 clade is globally distributed, accounts for 1 to 5% of E. coli isolates overall, and, when compared with other ST131 isolates, it is associated with resistance to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole and with susceptibility to fluoroquinolones and extended-spectrum cephalosporins. Attention to this O16-associated ST131 clade, which is facilitated by our novel PCR-based assay, is warranted in future epidemiological studies of ST131 and, conceivably, in clinical applications.

Clonality, recombination, and hybridization in the plumbing-inhabiting human pathogen Fusarium keratoplasticum inferred from multilocus sequence typing.

BackgroundRecent work has shown that Fusarium species and genotypes most commonly associated with human infections, particularly of the cornea (mycotic keratitis), are the same as those most commonly isolated from plumbing systems. The species most dominant in plumbing biofilms is Fusarium keratoplasticum, a cosmopolitan fungus known almost exclusively from animal infections and biofilms. To better understand its diversity and population dynamics, we developed and utilized a nine-locus sequence-based typing system to make inferences about clonality, recombination, population structure, species boundaries and hybridization.ResultsHigh levels of genetic diversity and evidence for recombination and clonality were detected among 75 clinical and 156 environmental isolates of Fusarium keratoplasticum. The multilocus sequence typing system (MLST) resolved 111 unique nine-locus sequence types (STs). The single locus bifactorial determinants of mating compatibility (mating types MAT1-1 and MAT1-2), were found in a ratio of 70:30. All but one of the 49 isolates of the most common ST (FSSC 2d-2) came from human infections, mostly of the cornea, and from biofilms associated with contact lenses and plumbing surfaces. Significant levels of phylogenetic incongruence were found among loci. Putative clonal relationships among genotypes were estimated, showing a mixture of large clonal complexes and unrelated singletons. Discordance between the nuclear ribosomal rRNA and other gene genealogies is consistent with introgression of ribosomal RNA alleles of phylogenetic species FSSC 9 into F. keratoplasticum. No significant population subdivision based on clinical versus non-clinical sources was found.ConclusionsIncongruent phylogenetic trees and the presence of both mating types within otherwise identical STs were observed, providing evidence for sexuality in F. keratoplasticum. Cryptic speciation suggested in a published three-locus MLST system was not supported with the addition of new loci, but evidence of introgression of ribosomal RNA genes from another strongly supported phylogenetic species (FSSC 9), also known from plumbing systems and human infections, was detected in two isolates. Overall, F. keratoplasticum is a diverse and geographically unstructured species with a mixed clonal and recombinant life history.

Multilocus sequence typing system (MLST) reveals a significant association of Entamoeba histolytica genetic patterns with disease outcome

The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needed to be explored. Proper identification and genetic characterization of Entamoeba histolytica clinical isolates is an effective tool for exploring this relation. Along with conventional polymorphic marker (Chitinase), tRNA linked short tandem repeat (STR) loci has been employed as multilocus genotyping tool due to its better resolution and evolutionary significance. Some common as well as exclusive repeat patterns showing significant relation with disease outcome have been identified. Phylogenetic analysis revealed that repeat patterns exclusively found in asymptomatic and amoebic liver abscess derived isolates are placed in a common lineage and has similar association pattern with the disease outcome. Assumption can be made that isolates of E. histolytica remaining asymptomatic is genetically closer siblings of those causing liver abscess rather than the diarrheal isolates.

Identification and Biological Role of the EndosymbiontsWolbachiain Rice Water Weevil (Coleoptera: Curculionidae)

Wolbachia spp. are obligate intracellular bacteria present in reproductive tissues of many arthropod species. Wolbachia infection status and roles in host reproduction were studied in the rice water weevil, Lissorhoptrus oryzophilus Kuschel (Coleoptera, Curculionidae), an introduced species in China. We examined Wolbachia infection status in five populations in China where it reproduces parthenogenetically, and one native population in Southeast Texas, where it reproduces bisexually. All populations were infected by Wolbachia, and all specimens in each population were infected by Wolbachia of a single strain. Phylogenetic analyses based on multilocus sequence typing system indicated that Wolbachia in non-native L. oryzophilus weevils diverges evidently from those in native weevils. After treatments with tetracycline, parthenogenetic weevils reduced the fecundity significantly and eggs were not viable. Our results suggest that Wolbachia are necessary for oocyte production in L oryzophilus.

Multilocus Sequence Typing Scheme for the Characterization of 936-Like Phages Infecting Lactococcus lactis

Lactococcus lactis phage infections are costly for the dairy industry because they can slow down the fermentation process and adversely impact product safety and quality. Although many strategies have been developed to better control phage populations, new virulent phages continue to emerge. Thus, it is beneficial to develop an efficient method for the routine identification of new phages within a dairy plant to rapidly adapt antiphage tactics. Here, we present a multilocus sequence typing (MLST) scheme for the characterization of the 936-like phages, the most prevalent phage group infecting L. lactis strains worldwide. The proposed MLST system targets the internal portion of five highly conserved genomic sequences belonging to the packaging, morphogenesis, and lysis modules. Our MLST scheme was used to analyze 100 phages with different restriction fragment length polymorphism (RFLP) patterns isolated from 11 different countries between 1971 and 2010. PCR products were obtained for all the phages analyzed, and sequence analysis highlighted the high discriminatory power of the MLST system, detecting 93 different sequence types. A conserved locus within the lys gene (coding for endolysin) was the most discriminative, with 65 distinct alleles. The locus within the mcp gene (major capsid protein) was the most conserved (54 distinct alleles). Phylogenetic analyses of the concatenated sequences exhibited a strong concordance of the clusters with the phage host range, indicating the clonal evolution of these phages. A public database has been set up for the proposed MLST system, and it can be accessed at http://pubmlst.org/bacteriophages/.

Multilocus Sequence Typing of Genital Chlamydia trachomatis in Norway Reveals Multiple New Sequence Types and a Large Genetic Diversity

BackgroundThe Chlamydia trachomatis incidence rate in Finnmark, the most northern and sparsely populated county in Norway, has been twice the national average. This population based cross-sectional study among Finnmark high school students had the following aims: i) to examine distribution of multilocus sequence types (STs) of C. trachomatis in a previously unmapped area, ii) to compare chlamydia genetic diversity in Finnmark with that of two urban regions, and iii) to compare discriminatory capacity of multilocus sequence typing (MLST) with conventional ompA sequencing in a large number of chlamydia specimens.Methodology ompA sequencing and a high-resolution MLST system based on PCR amplification and DNA sequencing of five highly variable genetic regions were used. Eighty chlamydia specimens from adolescents aged 15–20 years in Finnmark were collected in five high schools (n = 60) and from routine clinical samples in the laboratory (n = 20). These were compared to routine clinical samples from adolescents in Tromsø (n = 80) and Trondheim (n = 88), capitals of North and Central Norway, respectively.Principal Findings ompA sequencing detected 11 genotypes in 248 specimens from all three areas. MLST displayed 50 STs providing a five-fold higher resolution. Two-thirds of all STs were novel. The common ompA E/Bour genotype comprised 46% and resolved into 24 different STs. MLST identified the Swedish new variant of C. trachomatis not discriminated by ompA sequencing. Simpson's discriminatory index (D) was 0.93 for MLST, while a corrected Dc was 0.97. There were no statistically significant differences in ST genetic diversity between geographic areas. Finnmark had an atypical genovar distribution with G being predominant. This was mainly due to expansion of specific STs of which the novel ST161 was unique for Finnmark.Conclusions/SignificanceMLST revealed multiple new STs and a larger genetic diversity in comparison to ompA sequencing and proved to be a useful tool in molecular epidemiology of chlamydia infections.

Comment on: AbaR4 replaces AbaR3 in a carbapenem-resistant Acinetobacter baumannii isolate belonging to global clone 1 from an Australian hospital

Sir, I read with great interest the valuable article by Hamidian and Hall regarding the AbaR4 genomic island in D36, an Acinetobacter baumannii isolate belonging to global clone (GC) 1, and proposing a new family of transposons. However, I would like to make some remarks on the authors’ statement that D36 was of a rare sequence type (ST), ST247, and represented a new GC 1 lineage. In their study, the ST of A. baumannii strain D36 was determined by the Oxford multilocus sequence typing (MLST) scheme, which employs gltA, gyrB, gdhB, recA, cpn6, gpi and rpoD as housekeeping genes. As a result, strain D36 showed the allelic profile 10-12-4-11-4-58-5 and was assigned to ST247, slightly different from ST109 (10-12-4-11-4-9-5), which includes the GC 1 reference strain RUH875 (A297). Therefore, the difference between ST247 and ST109 was in their gpi alleles. However, in a recent study it was demonstrated that the gene gpi resided in a part of the A. baumannii genome prone to recombination and showed significant variations compared with other housekeeping genes, and a similar observation was also described in another study. Thus it appears to be problematic to include gpi in the MLST system and therefore the conclusion that ST109 and ST247 are two distinct lineages is also doubtful. Besides AbaR4-associated features, such as lack of Tn6018 and the resistance genes catA1, blaTEM, tet(A) and aphA1, strain D36 also showed other characteristics (aadB gene cassette and sul2 gene) different from other GC 1 strains included in the study by Hamidian and Hall. It has been suggested that the accessory genome (genes present in some isolates, but not in others) can be used as a tool for epidemiological investigation, and that the distinct features of strain D36 might support its role as the representative of a new lineage. On the one hand, it is difficult to select epidemiological markers from the hundreds of genes in the accessory genome, which varies between isolates. On the other hand, comparative genomic analysis has demonstrated that there are frequent homologous recombination events across up to 20% of GC 2 genomes, and the recombination also took place across a large phylogenetic distance, including GC 1. The horizontal transfer of aminoglycoside resistance genes and gene cassettes representing the variable region of integrons between GC 1 and 2 isolates from the Czech Republic has also been described. Therefore, the usefulness of the accessory genome in epidemiological research remains to be determined. As a result, the taxonomic position of strain D36 should be determined by a new scheme, for example, the MStree analysis-based MLST system, and further evidence is required to support the conclusion that strain D36 belongs to a new GC 1 lineage.