Sensitive and specific assay for the simultaneous detection of <Emphasis Type="Italic">Mycoplasma genitalium</Emphasis> and macrolide resistance-associated mutations

Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg Macrolide^R qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg Macrolide^R qPCR. The Mg Macrolide^R qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg Macrolide^R qPCR, indicating specificity. The Mg Macrolide^R qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.     

Fluoroquinolone and Macrolide Resistance-Associated Mutations in Mycoplasma genitalium

Mycoplasma genitalium is a significant sexually transmitted pathogen, causing up to 25% of cases of nongonococcal urethritis in men, and it is strongly associated with cervicitis and pelvic inflammatory disease in women. Currently, the usual first-line treatment is the macrolide antibiotic azithromycin, but an increasing incidence of treatment failure over the last 5 years suggests the emergence of antibiotic resistance. The mutations responsible for macrolide resistance have been found in the 23S rRNA gene in numerous M. genitalium populations. A second-line antibiotic, the fluoroquinolone moxifloxacin, was thought to be a reliable alternative when azithromycin began to fail, but recent studies have identified mutations that may confer fluoroquinolone resistance in the genes parC and gyrA. The aim of this study was to determine the prevalence of antibiotic resistance in M. genitalium in Sydney, Australia, by detecting relevant mutations in the 23S rRNA gene, parC, and gyrA. M. genitalium-positive DNA extracts of specimens, collected from patients attending sexual health clinics in Sydney, were tested by PCR amplification and DNA sequence alignment. The 186 specimens tested included 143 initial patient specimens and 43 second, or subsequent, specimens from 24 patients. We identified known macrolide resistance-associated mutations in the 23S rRNA gene in 43% of the initial patient samples and mutations potentially associated with fluoroquinolone resistance in parC or gyrA sequences in 15% of the initial patient samples. These findings support anecdotal clinical reports of azithromycin and moxifloxacin treatment failures in Sydney. Our results indicate that further surveillance is needed, and testing and treatment protocols for M. genitalium infections may need to be reviewed.     

<Emphasis Type="Italic">Mycoplasma hominis</Emphasis> and <Emphasis Type="Italic">Gardnerella vaginalis</Emphasis> display a significant synergistic relationship in bacterial vaginosis

Gardnerella vaginalis plays an important role in bacterial vaginosis (BV,) while the role of genital Mollicutes is less obvious. The diagnosis of BV by use of the current Gram stain Nugent score is also suboptimal for defining the role of Mollicutes that lack a cell wall. Since bacterial load and diversity is an important prerequisite for BV, real-time quantitative polymerase chain reaction (qPCR) assays enable these to be assessed. The purpose of this study was to define the role of genital Mollicutes and potential patterns of synergy with G. vaginalis in women with BV. Vaginal swabs from 130 women categorised by Nugent score as BV (n?=?28), intermediate (n?=?22) and non-BV (n?=?80) were tested against four qPCR TaqMan assays targeting G. vaginalis, Mycoplasma hominis, M. genitalium, Ureaplasma urealyticum and U. parvum. Statistical analyses were used to compare bacterial prevalence and load between the three groups of women. Mycoplasma hominis and G. vaginalis co-infection was significantly more common in BV (60.7 %) compared to intermediate (36.4 %) and non-BV (8.8 %) Nugent scores (p?<?0.001). Significantly higher loads of M. hominis (p?=?0.001) and G. vaginalis (p?<?0.001) were detected in women with BV and the respective loads in M. hominis and G. vaginalis co-infections displayed a significant positive correlation (p?<?0.001; r?=?0.60). No significant associations were seen with the other Mollicutes. The findings strengthen the evidence of a role for M. hominis in BV and a potential synergy with G. vaginalis. This synergy could be an important trigger of the condition and sexual contact the conduit for the transmission of an otherwise commensal bacterium that could initiate it.     

The molecular characteristic of multidrug-resistant strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolated in Northwestern Russia

The goal of this work was to obtain genotypic characteristics of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (i.e., strains that are resistant at least to rifampicin and isoniazid) isolated from consumptives in Northwestern Russia in 2011–2012. Spoligotyping of 195 strains of Mycobacterium tuberculosis revealed 14 spolingotypes belonging to genetic families Beijing (n = 162), LAM (n = 15), H3/URAL (n = 14), T, Harleem, and X. Spolingotypes SIT1 (Beijing), SIT42 (LAM), and SIT62 (H3/URAL) were predominant. Regardless of genotype, all studied strains were resistant to streptomycin. Multidrug-resistant strains were resistant to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The fractions of the strains resistant to ethambutol were 71 (n = 115) and 42% (n = 14) among the Beijing and nonBeijing strains, respectively (p < 0.05). Representatives of the Beijing genetic family remain predominant in Northwestern Russia in the population of multidrug-resistant strains of Mycobacterium tuberculosis (83%).     

Characterisation of <Emphasis Type="Italic">Clostridium difficile</Emphasis> strains isolated from Groote Schuur Hospital,Cape Town,South Africa

The C. difficile infection rate in South Africa is concerning. Many strains previously isolated from diarrhetic patients at Groote Schuur Hospital were ribotype 017. This study further characterised these strains with respect to their clonal relationships, antibiotic susceptibility, toxin production and various attributes impacting on pathogen colonisation. Multilocus variable-number tandem-repeat analysis (MLVA) was used to characterise all C. difficile isolates. Antibiotic susceptibility was determined by E-test and PCR-based analysis of the ermB, gyrA and gyrB genes. Auto-aggregation of cells was measured in broth, and biofilm formation observed in 24-well plates. Toxins were measured using the Wampole C DIFF TOX A/B II kit. Most isolates belonged to the ribotype 017 group. Identical MLVA types occurred in different wards over time, and several patients were infected with identical strains. All isolates were susceptible to vancomycin and metronidazole, but some ribotype 017 isolates showed reduced metronidazole susceptibility (≥2 mg l^?1). Sixty-nine percent of ribotype 017 isolates were resistant to moxifloxacin, and 94 % to erythromycin, compared to 0 % and 17 % resistance, respectively, in non-ribotype 017 isolates. The ermB gene and mutations in the gyrA and/or gyrB genes were linked to erythromycin and moxifloxacin resistance, respectively. Ribotype 017 isolates auto-aggregated more strongly than other isolates and produced lower levels of the TcdB toxin than a reference strain. Certain strains produced strong biofilms. Patient-to-patient transfer and unique infection events could cause the predominance of ribotype 017 strains in the cohort. Multi-drug resistant strains are a potential reservoir for future infections.     

New Mutations in Ciprofloxacin Resistant Strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Guilan Province,Northern Iran

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative bacterium that continues to be a major cause of opportunistic nosocomial infections. In P. aeruginosa, several fluoroquinolones resistance mechanisms have been proposed such as mutations in the target enzymes GyrA and ParC and upregulation of efflux pump MexAB-OprM. Fluoroquinolones inhibit the target enzymes DNA gyrase and topoisomerase IV, each comprised of two subunits: GyrA and GyrB, and ParC and ParE, respectively. In addition, fluoroquinolones were blocked by overexpression of MexAB-OprM induced through mutations in the regulatory genes mexR and nalC.

Materials and methods

In this study, 44 P. aeruginosa strains were isolated from different clinical samples of burn and infection from patients in some hospitals and laboratories in Guilan, which were identified by biochemical tests. The antibiotic resistance and susceptibility of strains was determined by Kirby Bauer method and microdilution method, and then PCR-sequencing was carried out to assess mutation in several genes involved in ciprofloxacin resistance.

Results

Of 44 isolates, 14 isolates were fluoroquinolone resistant. All 14 strains were nalidixic acid resistant with MIC = 1024 μg/mL. While ciprofloxacin resistance was showed in resistant isolates with MIC to be 32–1024 μg/mL. PCR-sequencing analysis showed that all 14 fluoroquinolone resistant isolates had one or two mutations in gyrA mutation. Mutations in parC, mexR and nalC were shown in some ciprofloxacin resistant isolates. Moreover, three strains had new mutation in mexR gene (111InsC and 262-263delAG).

Conclusions

It seems that high resistance to ciprofloxacin can simultaneously be the result of mutation in several genes such as topoisomerase enzymes and negative regulatory genes implicated in the expression of efflux pump systems in Guilan Province.

     

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.     

Comparative study of all <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis strains isolated from food and food animals in Greece from 2008 to 2010 with clinical isolates

The aim of the present work was to study the epidemiology of Salmonella enterica serovar Enteritidis (S. Enteritidis) in Greece, comparing all the food and food animal isolates during a 3-year period with clinical isolates. Submission of the generated data to the PulseNet Europe database was carried out in order to study the population structure of this particular serovar and indicate possible connections with European strains. One hundred and sixty-eight (168) S. Enteritidis strains of human, animal, and food origin, isolated during the period 2008–2010 in Greece, were studied. Strains were characterized by phenotypic (antibiotic resistance) and molecular [pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)] methods. PFGE revealed 39 XbaI, 48 BlnI, and 80 XbaI–BlnI distinct pulsotypes, suggesting several clones circulating through the food chain and multiple sources of transmission. Submission to the PulseNet Europe database indicated that PFGE profile SENTXB.0001, the most common PFGE profile in Europe, was also predominant in Greece (33.3 %). MLST showed that all the strains studied shared the same sequence type (ST11), representing the most common ST in Europe. High rates of resistance to nalidixic acid were observed among human and poultry isolates (~25 %), indicating the potential fluoroquinolone treatment failure. Our data suggest that strains originating from multiple reservoirs circulated in Greece through the food chain during the study period. Predominant profiles in Greece were common to PulseNet Europe profiles, indicating similarities between the S. Enteritidis populations in Greece and Europe.     

Dioxidine induces bacterial resistance to antibiotics

The capability of the dioxidine drug to cause bacterial resistance to antibiotics was studied. The study was performed with the use of a bioluminescent test and the recombinant E. coli strains MG 1655 (pSoxS-lux), MG1655 (pKatG-lux), MG1655 (pRecA-lux), and MG1655 (pColD-lux). The strains harbored plasmids with the operon luxCDABE from the photobacteria Photorhabdus luminescens under control of the corresponding E. coli stress-inducible promoters. The mutation frequency of the stable mutants was found by conventional methods for nonpathogenic and opportunistic strains of E. coli, Bacillus amyloliquefaciens, and Klebsiella pneumoniae. Dioxidine was shown to induce the promoters PrecA and Pcda in E. coli MG1655, which suggests the induction of the SOS-response in bacterial cells. The Pcda induction was higher than with PrecA. The value of the induction coefficient was highest if the dioxidine concentration was 2.25 × 10^–5 M. Moreover, this drug enhanced the induction of the SoxS and KatG promoters responding to the superoxide anion radical and hydrogen peroxide, which suggests possible participation of oxidative mechanisms in dioxidine DNA damage. The maximal value of the induction coefficient was also observed under conditions of 2.25 × 10^–5 M. Dioxidine can induce mutations leading to antibiotic resistance in all bacterial strains studied. An increase in mutation frequency was observed for rifampicin (twofold), ciprofloxacin (sixfold), and azithromycin (fourfold). These data show the necessity for an antibiogram for every patient who is taking or has taken dioxidine.     

Clinical characteristics of infections caused by <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> P1 genotypes in children

Mycoplasma pneumoniae (M. pneumoniae) isolates can be classified into two major genetic groups, P1 type 1 (MP1) and P1 type 2 (MP2), based on the DNA sequence of the P1 adhesion protein gene. The aim of our study was to determine if M. pneumoniae P1 genotype is associated with disease manifestation and severity of acute M. pneumoniae infection. We compared epidemiological and clinical data of children infected with either MP1 or MP2. In addition, we separately analysed data of patients presenting with individual manifestations of M. pneumoniae infection. Data of 356 patients infected with MP1 were compared with those of 126 patients infected with MP2. MP2-infected children presented with higher median baseline C-reactive protein levels and were admitted to the hospital more often. The distribution of P1 genotype varied among groups of patients with different manifestations of M. pneumoniae infection. MP2 was more common than MP1 among patients with neurological and cardiovascular manifestations, whereas MP1 was more prevalent in other manifestations. The results from our large cohort indicate that the two P1 subtypes may have different pathogenic potential and that infections with MP2 strains could be more virulent than those with MP1 strains.     

The improvement of M1 polarization in macrophages by glycopeptide derived from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

Ganoderma lucidum (Fr.) Karst (Ganodermataceae) is a medicinal mushroom that has been extensively used in China for centuries to promote longevity and improve vigor without significant adverse effects. There is continuous interest in the bioactive properties of G. lucidum in view of its newly developed popularity in other regions besides Asia, such as Europe. Glycopeptide derived from G. lucidum (Gl-PS) is one of the main effective components isolated from this mushroom. The Gl-PS has been demonstrated pleiotropic with many bioactivities including immunomodulatory and antitumor effects. Macrophages are important cells involved in innate and adaptive immunity. Classically activated macrophages (M1) and alternatively activated macrophages (M2), with their different roles, display distinct cytokine profiles: M1 preferentially produces TNF-α, IL-6, and IL-12; conversely, M2 generates more IL-10 and arginase. Gl-PS might have the potential to promote macrophage M1 polarization by lipopolysaccharide (LPS). In this study, LPS was used to induce the M1 polarization. It was shown that the level of the TNF-α, IL-6, and IL-12 were increased and the IL-10 and arginase I were decreased in the polarized M1 macrophages after application of Gl-PS compared to the control. The results indicated the potential of Gl-PS to promote M1 polarization vs M2, with the health beneficial understanding of the bioactivities of Gl-PS.     

Comparative analysis of glucose metabolism in strains of <Emphasis Type="Italic">Vibrio cholera</Emphasis> biovar El Tor

Comparative analysis of glucose fermentation in typical strains of V. cholerae biovar El Tor isolated in the Russian Federation in 1970–1990 and highly virulent strains of genovariants imported in 1993–2012 was carried out. It was demonstrated that glucose metabolism of V. cholerae biovar El Tor genovariants changed as a result of acquisition of classical CTX prophage (or only its ctxB gene) and a corresponding increase in virulence. A phenotypical manifestation of these changes is a lack of ability to grow on a minimal nutrient medium supplemented with 1% carbohydrate, as well as compromised capacity for acetoin fermentation in the course of the Voges–Proskauer test. Possible causes of the degraded glucose metabolism are associated with the presence of SNP in gene alsD that encodes acetolactate decarboxylase enzyme and is incorporated into als operon, which, in turn, is involved in acetoin generation, as well as with hyperexpression of regulatory protein AphA that controls acetoin biosynthesis.     

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.     

Preliminary antimycobacterial study on selected Turkish plants (Lamiaceae) against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and search for some phenolic constituents

Background

The global resurgence of tuberculosis is a significant threat. Lamiaceae members have been used in folk remedies for centuries. This study was designed to assess the in-vitro antimycobacterial activity of eighteen crude extracts from six plants (Lamiaceae) and to characterize their phenolic and flavonoid compounds.

Methods

Six Turkish medicinal plants of the family Lamiaceae (Stachys tmolea Boiss., Stachys thirkei C. Koch, Ballota acetabulosa (L.) Benth., Thymus sipthorpii Benth., Satureja aintabensis P.H. Davis, and Micromeria juliana (L.) Benth. ex Reich.) were collected in 2009 – 2010. Dried and crushed plant samples were subjected to sequential extraction with petroleum ether, ethyl acetate, and methanol in order of increasing polarity. A broth microdilution method was employed to screen extracts against four mycobacterial strains of Mycobacterium tuberculosis. Phenolic and flavonoid compounds were characterized by liquid chromatography–mass spectrometry.

Results

S. aintabensis, T. sibthorpii, and M. juliana were found to develop considerable activity against the four strains of M. tuberculosis with the minimal inhibitory concentrations value of 12.5-100 μg/ml. S. aintabensis and T. sibthorpii extracts killed M. tuberculosis with the minimum bactericidal concentration value of 50–800 μg/ml. On the basis of these prominent antimycobacterial activity, we suggest that they could be a source of natural anti-tuberculosis agents.

Conclusion

S. aintabensis and T. sibthorpii showed activity by killing Mycobacteria strains. The major phenolic compound was rosmarinic for T. sibthorpii and S. aintabensis. Flavonoids might be “a modal” for the drug design.

     

<Emphasis Type="Italic">Helicobacter bilis</Emphasis>-Associated Suppurative Cholangitis in a Patient with X-Linked Agammaglobulinemia

?

Helicobacter bilis is a commensal bacterium causing chronic hepatitis and colitis in mice. In humans, enterohepatic Helicobacter spp. are associated with chronic hepatobiliary diseases.

Purpose

We aimed at understanding the microbial etiology in a patient with X-linked agammaglobulinemia presenting with suppurative cholangitis.

Methods

16S rDNA PCR directly performed on a liver biopsy retrieved DNA of H. bilis.

Results

Clinical outcome resulted in the normalization of clinical and biological parameters under antibiotic treatment by a combination of ceftriaxone, metronidazole, and doxycyclin followed by a 2-week treatment with moxifloxacin and a 2-month treatment with azithromycin.

Conclusion

In conclusion, these data suggest a specific clinical and microbiological approach in patients with humoral deficiency in order to detect H. bilis hepatobiliary diseases.

     

The Phenomenon of the Formation of Biofilms by <Emphasis Type="Italic">Brevibacillus</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. in the Presence of Clinical Strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The formation of biofilms by M. tuberculosis on Shkolnikova’s medium (synthetic medium, analogue of Sauton’s medium) has been researched. We studied 150 clinical and 20 laboratory strains of M. tuberculosis. None of the 150 strains isolated from human beings produced biofilms (pellicle), but all yielded abundant planktonic growth. Twenty reference strains of M. tuberculosis produced both biofilms (pellicle) and planktonic growth. The phenomenon of biofilm formation by mixed cultures was observed when inoculating sputum treated with NALC-NaOH from patients with tuberculosis. We obtained 63 mixed biofilms. In 30.2% (19/63) of cases, biofilms contained the DNA of the causative agent of tuberculosis. The RV-PCR method was used to select six samples with the highest concentration of mycobacterial DNA. Molecular cloning and sequencing of a fragment of the 16S rRNA gene from one of the biofilms was carried out. The nucleotide sequence had 99% homology with the Bacillus thermoamylovorans species. From the mixed biofilms obtained, three strains of spore-forming bacilli were isolated. Strains are identified by Sanger’s sequencing of the 16S rRNA gene, one as Bacillus licheniformis, and the other two as Brevibacillus spp. A study of the resistance of isolated strains of spore bacilli against 12 antituberculosis drugs of the first and second series was carried out. All three strains were resistant to maximum concentrations of isoniazid, streptomycin, ethionamide, and ethambutol. Strains of Brevibacillus spp. were additionally resistant to para-aminosalicylic acid (PAS) and kanamycin. In a model experiment, the possibility of cogrowth of clinical strains of M. tuberculosis and B. licheniformis was demonstrated with prolonged co-incubation in Shkolnikova’s medium. In the first few days of growth, B. licheniformis produced a biofilm that remained stable for the entire observation period of 45 days. The hypothesis suggesting the possibility of a short-term persistence of some “saprophytic” species of bacilli in the caseous contents of necrosis foci in the late stages of pulmonary tuberculosis has been postulated.     

RD7 genotyping of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains isolated from patients with lung tuberculosis in various areas of Kazakhstan

A three-primer PCR assay has been designed for detecting possible deletions in the RD7 chromosomal region of the Mycobacterium tuberculosis complex. The assay gives rise to amplicons of different sizes depending on the presence or absence of deletions. The PCR assay was applied to 176 isolates from lung tuberculosis cases collected in various areas of Kazakhstan in the summer of 2004. Prior to assay, the isolates were characterized by culture and biochemical tests. The RD7 genotyping showed neither polymorphism nor deletions in the RD7 genome region. Some strains were additionally characterized by a PCR-RFLP analysis of the gyrB and hsp64 genes. The RFLP patterns corresponded to M. tuberculosis. The results of the study were consistent with certain previous studies, which indicates the population stability of RD7 in M. tuberculosis strains. Species identification of the isolates showed that M. tuberculosis sensu stricto was the principal causative agent of human lung tuberculosis in Kazakhstan.     

Hypervirulent <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> induced ventilator-associated pneumonia in mechanically ventilated patients in China

The purpose of this study was to investigate the clinical characteristics of hypervirulent K. pneumoniae (hvKP) induced ventilator-associated pneumonia (VAP) and the microbiological characteristics and epidemiology of the hvKP strains. A retrospective study of 49 mechanically ventilated patients with K. pneumoniae induced VAP was conducted at a university hospital in China from January 2014 to December 2014. Clinical characteristics and K. pneumoniae antimicrobial susceptibility and biofilm formation were analyzed. Genes of capsular serotypes K1, K2, K5, K20, K54 and K57 and virulence factors plasmid rmpA(p-rmpA), iroB, iucA, mrkD, entB, iutA, ybtS, kfu and allS were also evaluated. Multilocus sequence typing (MLST) and random amplified polymorphic DNA (RAPD) analyses were used to study the clonal relationship of the K. pneumoniae strains. Strains possessed p-rmpA and iroB and iucA were defined as hvKP. Of 49 patients, 14 patients (28.6 %) were infected by hvKP. Antimicrobial resistant rate was significantly higher in cKP than that in hvKP. One ST29 K54 extended-spectrum-beta-lactamase (ESBL) producing hvKP strain was detected. The prevalence of K1 and K2 in hvKP was 42.9 % and 21.4 %, respectively. The incidences of K1, K2, K20, p-rmpA, iroB, iucA, iutA, Kfu and alls were significantly higher in hvKP than those in cKP. ST23 was dominant among hvKP strains, and all the ST23 strains had identical RAPD pattern. hvKP has become a common pathogen of VAP in mechanically ventilated patients in China. Clinicians should increase awareness of hvKP induced VAP and enhance epidemiologic surveillance.     

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.