Structural organization of intact and damaged by <Emphasis Type="Italic">IS</Emphasis>100 element porin genes adjacent to the PGM region in <Emphasis Type="Italic">Yersinia pestis</Emphasis> and <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> strains

The porin gene, which is adjacent to the pigmentation region (pgm), is usually damaged by IS100 element in highly virulent Yersinia pestis strains. In addition, the pgm region, which carries the genes responsible for virulence (high pathogenicity island) and biofilm generation (hms-operon), is flanked by direct IS100 copies (causing its destabilization). The study of distribution of intact and truncated porin genes was conducted among 240 Y. pestis strains from 39 natural foci of Russia and countries of the near abroad and 68 Yersinia pseudotuberculosis strains from different geographical regions. Most highly virulent Y. pestis strains and some phylogenetic Y. pseudotuberculosis lines of O:1 serotype contain truncated porin genes. At the same time, deletion of the pgm region by flanked IS100 in Y. pseudotuberculosis is impossible, since IS100 is integrated in the porin gene in an orientation opposite to that of Y. pestis. The intact porin gene is carried by all Y. pestis strains with low epidemic significance and certain phylogenetic lines of highly virulent Y. pestis strains from desert foci and Caspian sandy focus, as well as most Y. pseudotuberculosis strains of O:1 serotype. A continuous deletion, which includes the porin gene and a part of the astE gene, was detected in less virulent Y. pseudotuberculosis strains of O:3 serotype. The nucleotide sequence of porin genes is identical in Y. pestis and Y. pseudotuberculosis strains from different geographical regions. Three porin gene allele only differ by IS100 integration site and orientation or absence of its integration. The nucleotide sequence of IS100 introduced in the porin gene of Yersinia has small differences only for two Y. pestis strains isolated in America. The correlation of low frequency of Hms-mutants with the intact porin gene state in Y. pestis and the absence of such a correlation in Y. pseudotuberculosis were established.     

The first molecular genetic identification of the tularemia pathogen in <Emphasis Type="Italic">Ixodes trianguliceps</Emphasis> Bir. ticks in Russia

A real-time PCR (RT PCR) assay for Francisella tularensis DNA in ixodes ticks Ixodes trianguliceps (140 mature individuals and 211 nymph pools) found on small mammals from the Middle Ural forests (Chusovskoi district, Perm Region) has been performed for the first time. Francisella DNA was detected in 12 mature ticks and 4 nymph pools when the 16SrRNA gene (amplicon size 1165–1170 bp) was used as the target. Amplification of a shorter fragment of the same gene (221–222 bp) resulted in identification of additional positive samples among mature ticks (17 of 128) and nymph pools (16 of 89). All 49 RT-PCR positive samples were identified as F. tularensis DNA using primers and probes complementary to a fragment of the lpnA (tul4) gene and the ISFtu2 element. The data obtained are indicative of the possible involvement of I. trianguliceps ticks in tularemia pathogen circulation in natural foci of forest type.     

Mutational pressure in genomes of human α-herpesviruses

Genomes of herpes simplex viruses (HSV1 and HSV2) possess highly GC-rich DNA (their G + C content is 0.68 and 0.70, respectively). This suggests a strong mutational GC pressure. The genome of varicella zoster virus (VZV) has a GC content of 0.46, which suggests a weak AT pressure. We have calculated the frequencies of nucleotide substitutions directed by mutational pressure for the genes encoding a major capsid protein (MCP) of human α-herpes viruses capable of infecting humans. For HSV1 the substitution frequencies were calculated from the MCP gene of HSV1 (G + C = 0.68, 3 GC = 0.89) to MCP gene of HSV2 (G + C = 0.70, 3 GC = 0.91) and to the homologous gene (G + C = 0.73, 3 GC = 0.99), which is phylogenetically related to HSV, i.e., primate herpes virus 16. In these two pairs of genes, transversions C → G and G → C were the most frequently observed. Less frequent were transitions in the direction of mutation GC pressure (T → C and A → G). Even less frequently, the transversions A → C and T → G were seen. For VZV, the substitution frequencies were calculated from an MCP gene of VZV (G + C = 0.47, 3GC = 0.41) to an MCP gene (G + C = 0.41, 3GC = 0.28) of the primate herpes virus 9 related to VZV. In this pair of genes, the most frequent transitions were seen in the direction of mutation AT pressure (C → T and G → A). The transversion frequencies of A → T and T → A were fairly lower; however, they exceeded the transversion frequencies of C → A and G → T. For the MCP gene of VZV, the probability of transition frequency induced by mutational pressure in the third codon positions (where the majority of transitions are synonymous) was 2.36-fold higher than in the MCP gene of HSV1 and 3-fold higher than in HSV2. These results tend to explain the rarity of recurrent zoster compared to herpes infection.     

Identification of Francisella tularensis by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry: Fast,Reliable, Robust,and Cost-Effective Differentiation on Species and Subspecies Levels

Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.Francisella tularensis is the causative agent of the zoonotic disease tularemia. Beside its medical and veterinary impact, it possesses a high potential to be used for bioterrorist attacks (7). The World Health Organization (WHO) has modeled a bioterrorist attack with Francisella tularensis leading to very high costs for society ($5.4 billion if 100,000 individuals are affected) due to the extreme infectivity of the pathogen. Tularemia appears in various clinical forms depending on the virulence of the involved F. tularensis strain as well as the route and dose of inoculation.The genus Francisella was recently rearranged (12, 23) and is currently divided into four species, including F. tularensis and F. philomiragia. The species Francisella tularensis is further subdivided into four subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida, all four showing significant differences in virulence regarding animal and human infections (28). F. tularensis subsp. tularensis (type A), which is almost exclusively found in North America, and F. tularensis subsp. holarctica (type B), which is endemic in areas all over the northern hemisphere, represent the two most virulent and clinically relevant subspecies. Several strains of F. tularensis subsp. mediasiatica have been isolated in Central Asia, but little is known about their virulence in humans. F. tularensis subsp. novicida is rarely isolated from human specimens but can cause a tularemia-like disease in immunocompromised individuals (5).Differentiation of the highly virulent F. tularensis subsp. tularensis from the less virulent F. tularensis subsp. holarctica is of substantial clinical interest (30) but may be even more important regarding the potential use of F. tularensis as a biological warfare agent.Identification and differentiation of bacteria based on the polymorphism of the 16S and 23S rRNA genes have become widely accepted (18). Although 16S rRNA gene sequencing is accepted as the reference method for species identification (3, 6, 8), 23S rRNA gene sequencing seems to be more suitable for the identification of F. tularensis subspecies (W. Splettstoesser, E. Seibold, E. Zeman, K. H. Trebesius, and A. Podbielski, submitted for publication).Cultivation of F. tularensis is fastidious. It has high requirements for the growth media used for its cultivation (e.g., Thayer-Martin and cysteine heart agar [CHA]), and cultivation may take up to 10 days (29). Identification and differentiation of Francisella strains by traditional phenotyping are difficult, often leading to ambiguous results. Additionally these methods include prolonged cultivation, thereby increasing the risk for laboratory-acquired tularemia (29).Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify bacterial species (2, 10, 13, 15, 20, 22, 24, 25) by measuring the mass of peptides and small proteins from whole cells. The resulting protein spectra are postulated to be characteristic for each bacterial species. The available literature regarding the identification of and discrimination between subspecies or even single strains by MALDI-TOF MS is limited (1, 24). Francisella tularensis subspecies were successfully discriminated with surface-enhanced laser desorption ionization-time of flight MS (SELDI-TOF MS) (19, 26), a modified MALDI-TOF MS method which allows selective absorption of proteins on a chromatographic array surface prior to MS (31, 32). Compared to SELDI-TOF MS, MALDI-TOF MS is less time-consuming and more cost-effective. Furthermore, there are several databases available for MALDI-TOF MS, which allow for matching spectra of unknown isolates with reference spectra.Although there are still concerns regarding the reproducibility of MALDI-TOF MS under different cultivation conditions, recent publications showed good reproducibility for various cultivation conditions in several bacterial groups (22, 24). A recent international study achieved 98.75% interlaboratory reproducibility regarding the identification of 60 blind-coded nonfermenting bacterial samples (21).The aim of our study was to identify and differentiate strains of the genus Francisella by the use of MALDI-TOF MS at the subspecies level by combining MALDI-TOF MS with dedicated bioinformatics and statistical methods, i.e., database search, pattern-matching algorithm, cluster analysis, and principal component analysis (PCA). Initial spectra from representative strains of each Francisella species and subspecies were used to set up database entries for reidentification of F. tularensis strains. This database was evaluated with 45 blind-coded Francisella strains that were also analyzed by 23S rRNA gene sequencing. Reproducibility of the method was tested with various growth media, cultivation times, numbers of strain passages, protein extraction protocols, and mass spectrometers.     

Multidrug-resistant <Emphasis Type="Italic">Mycoplasma genitalium</Emphasis> infections in Europe

In Japan and Australia, multidrug-resistant Mycoplasma genitalium infections are reported with increasing frequency. Although macrolide-resistant M. genitalium strains are common in Europe and North America, fluoroquinolone-resistant strains are still exceptional. However, an increase of multidrug-resistant M. genitalium in Europe and America is to be expected. The aim of this paper is to increase awareness on the rising number of multidrug-resistant M. genitalium strains. Here, one of the first cases of infection with a genetically proven multidrug-resistant M. genitalium strain in Europe is described. The patient was a native Dutch 47-year-old male patient with urethritis. Mycoplasma genitalium was detected, but treatment failed with azithromycin, doxycycline and moxifloxacin. A urogenital sample was used to determine the sequence of the 23S rRNA, gyrA, gyrB and parC genes. The sample contained an A2059G single nucleotide polymorphism (SNP) in the 23S rRNA gene and an SNP in the parC gene, resulting in an amino acid change of Ser83 → Ile, explaining both azithromycin and moxifloxacin treatment failure. The SNPs associated with resistance were probably generated de novo, as a link with high-prevalence areas was not established. It is, thus, predictable that there is going to be an increase of multidrug-resistant M. genitalium strains in Europe. As treatment options for multidrug-resistant M. genitalium are limited, the treatment of M. genitalium infections needs to be carefully considered in order to limit the rapid increase of resistance to macrolides and fluoroquinolones.     

Point mutations sites in <Emphasis Type="Italic">tox</Emphasis> promoter/operator and diphtheria toxin repressor (DtxR) gene associated with the level of toxin production by <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> strains isolated in Belarus

DNA fragments 129 bp in length containing the promoter region of the tox gene from 81 toxigenic Corynebacterium diphtheriae strains were analyzed using single strand conformational polymorphism (SSCP). We found that only two strains carried mutations; these strains also showed the highest levels of the toxin production (over 5120 VERO CD50/ml). Other strains were characterized either as high (640–5120 VERO CD50/ml, 41 strains) or low (40–320 VERO CD50/ml, 38 strains) toxin producers. The nucleotide sequence analysis revealed single T to C mutations at positions ?54 and ?184 within the ?232 to +85 region of the tox operon. The first mutation at position ?184 was mapped outside of the tox promoter/operator, whereas the second substitution at position ?54 modified the 9-bp interrupted palindromic sequence of the tox promoter/operator from ATAATTAGG in the wild-type bacteriophage β to ACAATTAGG in strains with an enhanced level of the toxin production. The nucleotide sequence analysis of region from ?76 to +681 of the diphtheria toxin repressor (dtxR) gene from 15 strains of C. diphtheriae revealed two missense mutations resulting in amino acid substitutions A147V and L214I in the C-terminal (the third) region of the DtxR protein. Seven of these strains were identified as high toxin producers; four strains, as low toxin producers. In addition, one low-level toxin-producing strain was shown to contain a missense mutation leading to the amino acid substitution I221T. Three strains—two strains exhibiting the highest level of the toxin production and the nontoxigenic C7(-) strain—contained no nucleotide substitutions. The ten strains belonging to the Sankt-Peterburg and Rossija epidemic ribotypes, as well as the NCTC 13129 strain (the etiologic agent of the diphtheria epidemic outbreak in the Eastern Europe), contained two mutations, A147V and L214I, in the C-terminal region of the diphtheria toxin repressor (DtxR).     

Three Genetically Different Lineages of <Emphasis Type="Italic">Yersinia pestis</Emphasis> subsp. <Emphasis Type="Italic">Microtus</Emphasis> bv. Caucasica (0.PE2) Strains Circulate among Common Voles in Natural Plague Foci in the Caucasus

Gram-negative bacteria Y. pestis subsp. pestis of 0.ANT-4.ANT, 1.ORI, and 2.MED SNP types are the cause of numerous epidemic outbreaks, epidemics, and three plague pandemics, claiming hundreds of millions of human lives. At the same time, strains of the microtus subspecies that belong to the 0.PE SNP type circulating in populations of different species of voles (Microtus spp.) are able to cause only extremely rare human infections that are not transmitted from person to person. It is suggested that the clinical form of the infection can develop only in individuals with impaired immune status. Strains of Y. pestis bv. caucasica (0.PE2), one of the most ancient phylogenetic groups of subsp. microtus, are isolated on the territory of the following natural foci: the Transcaucasian highland (including the Leninakan (4), Pre-Sevan (5), and Zanzegur- Karabakh (6)) mesofoci and the Dagestan-highland (39). In addition to the enumerated areas, similar strains of Y. pestis are isolated in the territories of the Pre-Araks focus (7) bordering the Transcaucasian highland one. Previously, we showed that passport data on the phenotypic differences of strains of the biovar caucasica, isolated from different foci, corresponded to their MLVA25, CRISPR, and DFR genotypes. In the present work, a comparative analysis of the clustering ability of MLVA25- and CRISPR-typing methods with the “gold standard” of phylogenetic studies—SNP typing—has been conducted on 21 strains of Y. pestis subsp. microtus bv. caucasica (0.PE2). The analysis of the obtained results confirms the existence of three clonal clusters of strains corresponding to the natural plague foci—39, 4, and the group of foci 5–7. Only the SNP-typing method made it possible to separate branch of focus 5 isolates from a group of strains isolated in foci 5–7. In addition, this method made it possible to reveal a greater genetic heterogeneity of strains from focus 39, in contrast to the strains of foci 4–7. When analyzing the genomes of Y. pestis strains isolated in the territory of focus 39, a deletion (~20 kb) was detected in the CRISPR region of the Ypb locus. The absence of this locus can serve as a marker for the determination of a given population of Y. pestis strains.     

Biofilm Forming Ability and <Emphasis Type="Italic">Spa</Emphasis> Gene Polymorphism in Methicillin Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Clinical Isolates in North of Iran

Spa typing has been shown to function as a genetic marker for Staphylococcus aureus outbreak investigations and epidemiological studies. This study was aimed to investigate biofilm formation capacity and spa gene polymorphism in methicillin resistant S. aureus (MRSA) strains isolated from clinical samples. A total of 102 S. aureus isolated during 2016, were analyzed for methicillin resistance and biofilm formation using phenotypic assays and PCR-based detection of associated genes. The polymorphic region of the spa gene was amplified by PCR using specific primers and subsequently in MRSA strains the amplified products were sequenced and spa types determined by using the spa database website. Out of 102 S. aureus, 41 isolates (40.2%) recognized as MRSA in phenotypic and genotypic investigations. In phenotypic assay, biofilm forming ability was detected in 71 isolates. The frequency of icaA and fnbA in test isolate were 53.9 and 65.7% respectively. Amplification of polymorphic region of the spa gene in all 102 tested isolates resulted in eight size fragments ranged between 168–336 bp. In MRSA strains thirteen distinct spa types with 5–12 repeats were observed. The most frequent types of spa were t030, t037, t325, t421, t937, t1814 and t084. spa types t2421, t1814, t359 and t2617 identified for the first time in Iran. The present results showed high biofilm formation capacity and great diversity of variable region of spa gene in MRSA strains and confirmed that, spa typing provides valuable information on the epidemiologic features and discrimination of this bacterium.     

Gene <Emphasis Type="Italic">sak0192</Emphasis> of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> contains direct repeats and spacers serving as genetic markers characterizing the strains

A new method of strain differentiation in Streptococcus agalactiae is proposed in this work. It is based on revealing the polymorphism of structure of gene sak0192 encoding a hypothetical protein, which in different strains of Str. agalactiae contains a different number of regularly alternating repeats and spacer sites having sizes of 16 and 44 bp, respectively. The sequences of forward repeats are mostly identical, while spacers are characterized with expressed heterogeneity. In general, the structure of gene sak0192 is similar to one of sites with direct repeats and spacers in genomes of Mycobacterium tuberculosis and Str. pyogenes. The possibility of using polymorphism of sak0192 in both identification of species affiliation and intraspecific differentiation of Str. agalactiae strains is demonstrated.     

Cytotoxic and antimicrobial effects of biosynthesized ZnO nanoparticles using of <Emphasis Type="Italic">Chelidonium majus</Emphasis> extract

The basic goal of this study was to synthesize zinc oxide nanoparticles using the Chelidonium majus extract and asses their cytotoxic and antimicrobial properties. The synthesized ZnO NPs were characterized by UV-Vis, Scanning Electron Microscopy (SEM) with EDS profile, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). The aforementioned methods confirmed that the size of synthesized ZnO nanoparticles was at the range of 10 nm. The antimicrobial activity of ZnO nanoparticles synthesized using the Ch. majus extract was tested against standard strains of bacteria (Staphylococcus aureus NCTC 4163, Pseudomonas aeruginosa NCTC 6749, Escherichia coli ATCC 25922), yeast (Candida albicans ATCC 10231), filamentous fungi (molds: Aspergillus niger ATCC 16404, dermatophytes: Trichophyton rubrum ATCC 28188), clinical strains of bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) and yeast (Candida albicans). The study showed that zinc oxide nanoparticles were excellent antimicrobial agents. What is more, biologically synthesized ZnO nanoparticles demonstrate high efficiency in treatment of human non-small cell lung cancer A549.     

Acid Phosphatases Do Not Contribute to the Pathogenesis of Type A Francisella tularensis

The intracellular pathogen Francisella tularensis is the causative agent of tularemia, a zoonosis that can affect humans with potentially lethal consequences. Essential to Francisella virulence is its ability to survive and proliferate within phagocytes through phagosomal escape and cytosolic replication. Francisella spp. encode a variety of acid phosphatases, whose roles in phagosomal escape and virulence have been documented yet remain controversial. Here we have examined in the highly virulent (type A) F. tularensis strain Schu S4 the pathogenic roles of three distinct acid phosphatases, AcpA, AcpB, and AcpC, that are most conserved between Francisella subspecies. Neither the deletion of acpA nor the combination of acpA, acpB, and acpC deletions affected the phagosomal escape or cytosolic growth of Schu S4 in murine and human macrophages, despite decreases in acid phosphatase activities by as much as 95%. Furthermore, none of these mutants were affected in their ability to cause lethality in mice upon intranasal inoculation. Hence, the acid phosphatases AcpA, AcpB, and AcpC do not contribute to intracellular pathogenesis and do not play a major role in the virulence of type A Francisella strains.The Gram-negative bacterium Francisella tularensis is a highly infectious, facultative intracellular pathogen that causes tularemia, a widespread zoonosis affecting humans. Human tularemia is a fulminant disease that can be contracted by exposure to as few as 10 bacteria, the pneumonic form of which can lead to mortality rates as high as 25% if untreated (35). Three subspecies of F. tularensis, Francisella tularensis subsp. tularensis (type A), Francisella tularensis subsp. holarctica (type B), and Francisella tularensis subsp. mediasiatica, are recognized, among which strains of the first two subspecies can cause tularemia in humans (15). While type B strains are geographically distributed all over the northern hemisphere, the highly virulent type A strains are restricted to North America and account for the most-severe cases of the disease. Francisella novicida, a species of low virulence in humans but high virulence in rodents, has been used extensively as a surrogate model of F. tularensis pathogenesis, based on the assumption that it uses conserved virulence mechanisms (4, 7, 8, 19, 23, 25-29, 31, 41-45, 47). As a facultative intracellular pathogen, F. tularensis is capable of infecting and proliferating in a variety of host cell types, including hepatocytes, epithelial cells, and mononuclear phagocytes (15). Macrophages constitute an important target for infection in vivo (21), and the pathogenesis of F. tularensis depends on the bacterium''s ability to survive and replicate within these host cells (15). Upon phagocytosis, Francisella ensures its effective survival and proliferation via rapid phagosomal escape followed by extensive replication in the cytosol (11, 14, 20, 42), thereby segregating itself from the degradative endosomal system and its associated bactericidal activities. Phagosomal escape is a tightly regulated process whose efficiency depends on conditions encountered within the early phagosome (12, 41), such as vacuolar acidification, although some controversy remains as to whether Francisella-containing phagosomes are significantly acidified prior to membrane disruption (13). Regardless of such discrepancies, phagosomal escape is an essential step in Francisella intracellular pathogenesis, since it is a prerequisite for cytosolic replication. Indeed, Francisella mutants that are defective in phagosomal escape do not grow intracellularly and are attenuated in vivo (6, 24, 43-45), and a belated phagosomal escape delays intracellular proliferation of the highly virulent type A strain Schu S4 (12).Much effort has focused on identifying bacterial factors that contribute to phagosomal escape. Several genes located within a 30-kb chromosomal locus known as the Francisella pathogenicity island (FPI) (31) are required for proper phagosomal escape of F. novicida (43, 44) and the attenuated F. tularensis subsp. holarctica live vaccine strain (LVS) (6, 24), since transposon insertions or targeted deletions in iglC, iglD, and pdpA affect the translocation of the mutants to the cytosol. Based on the homology of some FPI proteins with components of type VI secretion systems in other pathogens (30, 36), the FPI likely encodes a secretion apparatus that is required for phagosomal disruption. Yet a true understanding of FPI functions and the characterization of actual Francisella effectors of phagosomal escape are lacking. In addition to the FPI, Mohapatra et al. have recently reported for F. novicida that the acid phosphatases AcpA, AcpB, AcpC, and Hap are required for phagosomal escape and virulence in mice (27, 29). Acid phosphatases, which are ubiquitous in nature and hydrolyze phosphomonoesters at acidic pHs, have been associated with the survival of intracellular parasites within phagocytes through inhibition of the respiratory burst (1, 3, 9, 22, 37-40), suggesting that they act as virulence factors. In Francisella, a prominent role was established for AcpA, an unusual, respiratory-burst-inhibiting enzyme exemplifying a novel family of acid phosphatases (18, 37). AcpA accounts for most of the acid phosphatase and phospholipase activities in the outer membrane fraction of F. novicida (29). These reports assigned acid phosphatases a role in phagosomal escape yet contradicted a previous study by Baron et al., who concluded that AcpA was not required for the intracellular growth or virulence of F. novicida (4). While the acpA mutants were constructed differently in these studies, the acid phosphatase activity associated with AcpA was abolished in both situations. A proposed explanation for these conflicting results was that the truncated AcpA generated by Baron et al. remained functional as a phospholipase C (37), an activity that would be required for phagosomal escape and virulence (27). Yet this hypothesis has not been tested, leaving the role of AcpA in Francisella virulence a controversial matter.All studies of Francisella acid phosphatases have been carried out with F. novicida (4, 27, 29, 37), raising the question of significance with regard to the virulent F. tularensis subspecies. In particular, recent whole-genome comparisons between F. novicida and the different Francisella tularensis subspecies have highlighted important intervening sequence (IS)-mediated genome rearrangements in F. tularensis subsp. holarctica and F. tularensis subsp. tularensis strains relative to F. novicida (10). Such rearrangements have disrupted large numbers of open reading frames (ORFs), thereby creating pseudogenes (10) and likely inactivating many functions in virulent F. tularensis strains. For example, Mohapatra et al. (29) have reported that the virulent type A strain Schu S4 is missing a homolog of one of the two hap genes (FTN_0022) present in F. novicida, raising the question of conservation of acid phosphatase-encoding genes in virulent strains. Because phagosomal escape is an essential stage of the Francisella intracellular cycle that is common to F. novicida and F. tularensis, we have postulated that factors required to promote this process must be conserved between these organisms. Here we have compared acid phosphatase-encoding genes in F. novicida and virulent F. tularensis subspecies, and we have generated deletion mutants of the most conserved genes in Schu S4 in order to test their role in the phagosomal escape and pathogenesis of the highly virulent F. tularensis subspecies. We demonstrate that most acid-phosphatase-encoding genes are disrupted in virulent strains and that the most conserved loci are not required for phagosomal escape and virulence.     

Uncommonly isolated clinical <Emphasis Type="Italic">Pseudomonas</Emphasis>: identification and phylogenetic assignation

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.     

Genomic insights into the TTSS island of enteropathogenic <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">Salmonella</Emphasis> and its conjugational transfer

Whole genome sequences of the non-pathogenic K12 and pathogenic O127:H6 strains of Escherichia coli were analyzed using Mauve software. The genomes showed 80% similarity with few desert regions including a 35.99 kb region in pathogenic strain. This region contained Locus of Enterocyte Effacement (LEE) and Type III Secretion System (TTSS) genes. Whole genome alignment of this E. coli pathogenic strain and Salmonella enterica subsp. enterica serovar Newport str. SK254 showed 40% homology. Intimin protein coding escU gene of E. coli and ssaU gene of S. enterica were analyzed by BLASTn and ClustalW and cross referred with Pathogenicity Island Database (PDI DB) to check similarity with other foodborne bacterial species. The ssaU gene of Salmonella was found to be designated as escU in E. coli database. Comparison of these genes at nucleotide and amino acid sequence level revealed possible codon redundancies. Six clinical isolates of E. coli (designated as SBANU 1 to 6) were screened for salt aggregation and hemolysin production abilities followed by PCR analysis of escU gene. The E. coli isolate SBANU 6 was further used in induced conjugation assay with S. enterica as donor. PCR analysis and sequencing of the amplified DNA in E. coli transconjugant cells revealed the possible acquiring of ssaU gene from S. enterica.     

Association study of <Emphasis Type="Italic">Demodex</Emphasis> bacteria and facial dermatoses based on DGGE technique

The role of bacteria is unclear in the facial skin lesions caused by Demodex. To shed some light on this issue, we conducted a case-control study comparing cases with facial dermatoses with controls with healthy skin using denaturing gradient gel electrophoresis (DGGE) technique. The bacterial diversity, composition, and principal component were analyzed for Demodex bacteria and the matched facial skin bacteria. The result of mite examination showed that all 33 cases were infected with Demodex folliculorum (D. f), whereas 16 out of the 30 controls were infected with D. f, and the remaining 14 controls were infected with Demodex brevis (D. b). The diversity analysis showed that only evenness index presented statistical difference between mite bacteria and matched skin bacteria in the cases. The composition analysis showed that the DGGE bands of cases and controls were assigned to 12 taxa of 4 phyla, including Proteobacteria (39.37–52.78%), Firmicutes (2.7–26.77%), Actinobacteria (0–5.71%), and Bacteroidetes (0–2.08%). In cases, the proportion of Staphylococcus in Firmicutes was significantly higher than that in D. f controls and D. b controls, while the proportion of Sphingomonas in Proteobacteria was significantly lower than that in D. f controls. The between-group analysis (BGA) showed that all the banding patterns clustered into three groups, namely, D. f cases, D. f controls, and D. b controls. Our study suggests that the bacteria in Demodex should come from the matched facial skin bacteria. Proteobacteria and Firmicutes are the two main taxa. The increase of Staphylococcus and decrease of Sphingomonas might be associated with the development of facial dermatoses.     

Genomic analysis reveals the presence of a class D beta-lactamase with broad substrate specificity in animal bite associated <Emphasis Type="Italic">Capnocytophaga</Emphasis> species

Capnocytophga canimorsus and Capnocytophga cynodegmi can be transmitted from cats and dogs to humans, and can cause a wide range of infections including wound infections, sepsis, or endocarditis. We and others recently discovered two new Capnocytophaga species, C. canis and C. stomatis, mainly associated with wound infections. The first-line treatment of animal bite related infections is penicillin, and in case of allergy, doxycycline and trimethoprim/sulfamethoxazole. However, there is a lack of antibiotic susceptibility patterns for animal bite associated Capnocytophaga species. Thus, we ?set out to study the antibiotic profiles against animal bite associated Capnocytophaga species isolated from wound and blood cultures after cat and dog bites and coupled the findings to whole genome sequencing data. A total of 24 strains were included in the study. Phenotypic analysis of antibiotic resistance was performed with E-tests. The web-based tool ‘Resfinder’ was used to identify resistance genes in the whole genome dataset. Two strains of C. cynodegmi and two strains of the recently discovered C. stomatis were resistant to penicillin (MIC?> 24 mg?/L) and cephalosporins (MIC?>?24 mg/?L), and three out of these strains also exhibited resistance to imipenem (MIC?=?32 mg/?L). Genomic analysis revealed that these strains carried a class D beta-lactamase gene, which has not previously been found in Capnocytophaga spp. A class D beta lactamase with broad substrate specificity was found in animal bite associated Capnocytophaga species, which could have important implications when treating wound infections after cat and dog bites. It also suggests that pet animal bacteria can harbour resistance genes with relevance for human infections.     

Characterisation of VIM-2-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolates from lower tract respiratory infections in a Spanish hospital

To analyse the antimicrobial phenotype, carbapenem mechanisms, integrons, virulence factors and molecular typing of 164 Pseudomonas aeruginosa isolates recovered from lower tract respiratory samples in a Spanish hospital (1 year) as well as the patients’ clinical data. Susceptibility testing to 12 antipseudomonal agents was determined by microdilution and metallo-beta-lactamase (MBL) phenotype by double disc method. The oprD gene was studied by PCR, sequencing and comparison with P. aeruginosa PAO1 sequence. Detection and characterisation of MBLs, class 1, 2 and 3 integrons, and virulence genes were studied by PCR and sequencing. The prevalence of carbapenem-resistant P. aeruginosa (CRPA) was 26.8%. MBL phenotype was detected in 52.3% CRPA, and all of them were disseminated throughout the intensive care unit. Most of the MBL-carrying patients presented respiratory disease, mechanical ventilation, tracheostomy, bacteraemia, ≥?30 hospitalisation days and previous treatment with carbapenems and/or ≥?3 different antimicrobial families. The bla_VIM-2 gene was the unique MBL encoding gene and was detected inside class 1 integrons. The class 1 integrons detected in 39 strains (23.8%) were associated with aminoglycosides (aadB, aadA1, aadA6, aacA4, aac(3)-I) and carbapenems resistance genes (bla_VIM-2). The aac(3)-I?+?aadA1 and bla_VIM-2 arrangements were the most prevalent ones. Thirty-one different PFGE patterns and 4 STs (ST175, ST235, ST253, ST973) were detected among the 39 intI1-positive isolates, being ST235 the most frequent. CRPA showed a great variety of alterations in oprD gene. The exoU⁺/exoS^? genotype was detected in 82.6% of bla_VIM-2-producing strains (ST235) and the exoU^?/exoS⁺ in the remaining 17.4% (ST973).     

Molecular evidence of <Emphasis Type="Italic">Chlamydiales</Emphasis> in ticks from wild and domestic hosts in Sardinia,Italy

Ticks are well known to be important vectors for a wide range of bacteria, viruses and protozoa affecting human and animal health. Ixodid ticks are widely distributed in Sardinia, and an increasing number of tick-borne bacteria have been documented in the island. A growing number of evidence are supporting the hypothesis of alternative transmission routes for chlamydial bacteria such as the involvement of vectors. This study was conducted to provide possible molecular detection of members belonging to the Chlamydiales order in Sardinian ticks and to update information concerning the presence of new ectoparasite-borne bacteria in ticks collected from domestic and wild hosts in a typical Mediterranean environment. A total of 378 ticks were individually screened with a pan-Chlamydiales specific primers targeting the 16S rRNA gene. Chlamydiales DNA was detected in 28% of the total ticks analyzed. The analyses of sequences highlighted that Rhipicephalus sanguineus sensu lato, Rhipicephalus bursa, Rhipicephalus annulatus, Haemaphysalis sulcata, Haemaphysalis punctata and Dermacentor marginatus ticks exhibited DNA of Chlamydiaceae and Parachlamydiaceae members. Our results revealed that DNA of zoonotic microorganisms such as C. psittaci, C. abortus and the emerging pathogen Parachlamydia acanthamoebae are present in Sardinian ticks. Since routes of Chlamydia transmission are yet to be fully defined, the role of ticks as possible vectors for Chlamydiales remains the most challenging and interesting question to be addressed in future research. Continued monitoring of these pathogens in tick vectors is needed to provide strategies for controlling of possible chlamydial infections and disease outbreaks in the island.     

Strain variation and antigenic divergence among <Emphasis Type="Italic">Bordetella pertussis</Emphasis> circulating strains isolated from patients in Iran

Despite global efforts and widespread vaccination to control whooping cough (pertussis) caused by B. pertussis, the re-emergence of pertussis still is being reported all over the world. Antigenic divergence in B. pertussis virulence factors is one of the reasons of pertussis resurgence, resulting in dissimilarity of local and vaccine strains. In this study, clonal spread and variation of B. pertussis virulence factor in isolated strains from Iranian patients have been analyzed. A total of 100 B. pertussis isolates were obtained from Pertussis Reference Laboratory of Pasteur Institute of Iran. Real-time PCR were performed to confirm the B. pertussis strains. The genomic patterns of B. pertussis strains were analyzed by pulsed-field gel electrophoresis (PFGE). Predominant alleles of local strains were ptxP3, ptxA1, prn2, fim 2-1, fim3-2, and cya2. PFGE results showed 25 patterns clustered into 18 PFGE groups. A few similarities between the circulating isolates, vaccine, and standard strains were obtained. Significantly, 48% of the isolates showed dominant pattern with different allelic profiles from vaccine strains. According to the genomic profiles, the clonal spread was observed among the circulating strains. Predominant virulence factor profile was also comparable with other countries. It may be suggested that strain variation between vaccine and local strains may have an effect on pertussis resurgence in Iran like other parts of the world.     

Characterization of a <Emphasis Type="Italic">Pestivirus H</Emphasis> isolate originating from goats

Natural Pestivirus H infections in cattle have been reported worldwide; however, only a few cases of Pestivirus H have been described in non-bovine ruminants such as goats. A new Pestivirus H HN1507 strain was isolated from an infected goat in 2015 and the genome sequence was determined. The full-length genome sequence was 12,556 nucleotides. Phylogenetic analysis, based on the complete genome and Npro fragments, revealed that the isolate belonged to Pestivirus H and was closely related to strains from Italy. Two unique amino acid substitutions were found in the C-terminal of the E2 protein. To the best of our knowledge, this is first report determining the complete genome of a Pestivirus H strain from goat.     

Shaperone-Dependent Optimization of Expression in <Emphasis Type="Italic">E. coli</Emphasis> and Purification of Soluble Recombinant Lipid A Phosphatase LpxE from <Emphasis Type="Italic">Francisella tularensis</Emphasis>

Lipid-1a-phosphatase LpxE cleaves the phosphate group in the first position of lipid A of lipopolysaccharide in Gram-negative bacteria, which reduces toxicity and reactogenicity of bacterial endotoxin while maintaining its immunogenicity. Treatment with these enzymes in preparations of recombinant proteins obtained in bacterial producers, for example, in E. coli, may find application in the purification techniques of various pharmaceutical substances, primarily various protein components of vaccines. This work is devoted to the creation of an efficient method for obtaining an F. tularensis LpxE soluble protein in a heterologous expression system based on E. coli. Optimization of the bacterial production of LpxE enzyme is achieved by creating a SUMO-LpxE chimeric protein in which the SUMO polypeptide acts as a folding catalyst and an affinity target carrier for purifying the expression product. An additional increase in the level of synthesis of LpxE and reduction of its toxicity for a bacterial strain is achieved against the background of superproduction of bacterial periplasmic chaperones encoded by an auxiliary low copy plasmid. Subsequent chromatographic purification of the target recombinant protein involves the use of affinity metal chelate chromatography and a SUMO protease cleavage step of the SUMO peptide carrying the His6 N-terminal peptide with the reduction of natural primary structure of the enzyme. The developed method makes it possible to obtain a highly purified active enzyme LpxE F. tularensis with a yield of 0.26 g/L of bacterial culture without fermentation.