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author:("nemo, Magnus")
1.  Treatment efficacy, treatment failures and selection of macrolide resistance in patients with high load of Mycoplasma genitalium during treatment of male urethritis with josamycin 
Background
Azithromycin has been widely used for Mycoplasma genitalium treatment internationally. However, the eradication efficacy has substantially declined recent decade. In Russia, josamycin (another macrolide) is the recommended first-line treatment for M. genitalium infections, however, no data regarding treatment efficacy with josamycin and resistance in M. genitalium infections have been internationally published. We examined the M. genitalium prevalence in males attending an STI clinic in Moscow, Russia from December 2006 to January 2008, investigated treatment efficacy with josamycin in male urethritis, and monitored the M. genitalium DNA eradication dynamics and selection of macrolide resistance in M. genitalium during this treatment.
Methods
Microscopy and real-time PCRs were used to diagnose urethritis and non-viral STIs, respectively, in males (n = 320). M. genitalium positive patients were treated with recommended josamycin regimen and treatment efficacy was monitored using quantitative real-time PCR. Macrolide resistance mutations were identified using sequencing of the 23S rRNA gene.
Results
Forty-seven (14.7%) males were positive for M. genitalium only and most (85.1%) of these had symptoms and signs of urethritis. Forty-six (97.9%) males agreed to participate in the treatment efficacy monitoring. All the pre-treatment M. genitalium specimens had wild-type 23S rRNA. The elimination of M. genitalium DNA was substantially faster in patients with lower pre-treatment M. genitalium load, and the total eradication rate was 43/46 (93.5%). Of the six patients with high pre-treatment M. genitalium load, three (50%) remained positive post-treatment and these positive specimens contained macrolide resistance mutations in the 23S rRNA gene, i.e., A2059G (n = 2) and A2062G (n = 1).
Conclusions
M. genitalium was a frequent cause of male urethritis in Moscow, Russia. The pre-treatment M. genitalium load might be an effective predictor of eradication efficacy with macrolides (and possibly additional antimicrobials) and selection of macrolide resistance. Additional in vivo and in vitro data are crucial to support the recommendation of using josamycin as first-line treatment for M. genitalium infections in Russia. It would be valuable to develop international M. genitalium management guidelines, and quantitative diagnostic PCRs determining also M. genitalium load and resistance mutations (for macrolides and ideally also moxifloxacin) should ideally be recommended.
doi:10.1186/s12879-015-0781-7
PMCID: PMC4318211  PMID: 25645440
Mycoplasma genitalium; Treatment; Treatment efficacy; Treatment failure; Antimicrobial resistance; 23S rRNA; Josamycin; Macrolides; Russia; Male urethritis
2.  Antimicrobial susceptibility/resistance and NG-MAST characterisation of Neisseria gonorrhoeae in Belarus, Eastern Europe, 2010–2013 
Background
Gonorrhoea and widely spread antimicrobial resistance (AMR) in its etiological agent Neisseria gonorrhoeae are major public health concerns worldwide. Gonococcal AMR surveillance nationally and internationally, to identify emerging resistance and inform treatment guidelines, is imperative for public health purposes. In 2009, AMR surveillance was initiated in Belarus, Eastern Europe because no gonococcal AMR data had been available for at least two decades. Herein, the prevalence and trends of gonococcal AMR and molecular epidemiological characteristics of N. gonorrhoeae strains from 2010 to 2013 in Belarus, are described.
Methods
N. gonorrhoeae isolates (n=193) obtained in the Mogilev (n=142), Minsk (n=36) and Vitebsk (n=15) regions of Belarus in 2010 (n=72), 2011 (n=6), 2012 (n=75) and 2013 (n=40) were analyzed in regards to AMR using the Etest method and for molecular epidemiology with N. gonorrhoeae multi-antigen sequence typing (NG-MAST).
Results
During 2010–2013, the proportions of resistant N. gonorrhoeae isolates were as follows: tetracycline 36%, ciprofloxacin 28%, penicillin G 9%, azithromycin 5%, and cefixime 0.5%. Only one (0.5%) β-lactamase producing isolate was detected. No isolates resistant to ceftriaxone and spectinomycin were identified. Overall, the resistance levels to tetracycline, ciprofloxacin and penicillin G were relatively stable. Interestingly, the level of resistance to azithromycin declined from 12% in 2010 to 0% in 2013 (P < 0.05). In total, 70 NG-MAST STs were identified. The predominant STs were ST1993 (n=53), ST807 (n=13), ST285 (n=8) and ST9735 (n=8). Many novel STs (n=43, 61%), representing 41% of all isolates, were found.
Conclusions
During 2010–2013, the N. gonorrhoeae population in Belarus displayed high and relatively stable resistance levels to tetracycline, ciprofloxacin, and penicillin G, while the resistance to azithromycin declined. One isolate was resistant to cefixime, but no resistance to ceftriaxone or spectinomycin was found. The results of the present surveillance initiated in 2009 were also used to replace penicillin G with ceftriaxone (1 g single dose intramuscularly) as the first-line drug for empiric treatment of gonorrhoea in the national treatment guidelines in Belarus in late 2009. It is essential to further strengthen the surveillance of gonococcal AMR and ideally survey also treatment failures and molecular epidemiological genotypes in Belarus.
doi:10.1186/s12879-015-0755-9
PMCID: PMC4316755  PMID: 25637258
Neisseria gonorrhoeae; Gonorrhoea; Antimicrobial resistance; Surveillance; Extended-spectrum cephalosporins (ESCs); Ceftriaxone; Cefixime; Treatment; N. gonorrhoeae multiantigen sequence typing (NG-MAST); Belarus
3.  In Vitro Activity of the New Fluoroketolide Solithromycin (CEM-101) against Macrolide-Resistant and -Susceptible Mycoplasma genitalium Strains 
Mycoplasma genitalium has become well established as an etiological agent of sexually transmitted infections, but due to its fastidious growth requirements, only a few M. genitalium strains are available to determine the MICs of currently used and new antimicrobial agents. Recent clinical trials have suggested that treatment with azithromycin has decreasing efficacy due to an increasing prevalence of macrolide resistance, and alternative treatment with moxifloxacin is similarly under pressure from emerging resistance. Thus, there is an urgent need for new antimicrobials. The in vitro activity of the newly developed fluoroketolide solithromycin (CEM-101) was evaluated against a collection of 40 M. genitalium strains, including 15 with high-level macrolide resistance and 5 multidrug-resistant strains with resistance to both macrolides and quinolones. Furthermore, the MIC of solithromycin was correlated with mutations in the 23S rRNA gene and in the genes encoding ribosomal proteins L4 and L22. The in vitro results showed that solithromycin has activity against M. genitalium superior to that of other macrolides, doxycycline, and fluoroquinolones. Accordingly, this new fluoroketolide might be an effective option for treatment of M. genitalium infections. However, the efficacy of solithromycin in clinical trials with follow-up for test of cure and detection of genotypic and phenotypic resistance needs to be evaluated prior to widespread use. In a phase 2 clinical trial, solithromycin was highly effective as a single oral dose against C. trachomatis and Neisseria gonorrhoeae, suggesting that solithromycin could be a treatment option for several sexually transmitted infections, including in syndromic treatment of urethral and vaginal discharge.
doi:10.1128/AAC.02411-14
PMCID: PMC4068426  PMID: 24637681
4.  Importance of Multidrug Efflux Pumps in the Antimicrobial Resistance Property of Clinical Multidrug-Resistant Isolates of Neisseria gonorrhoeae 
The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae that express extensively drug-resistant or multidrug-resistant phenotypes has heretofore not been examined. Accordingly, we assessed the effect on antimicrobial resistance of loss of the three gonococcal efflux pumps associated with a known capacity to export antimicrobials (MtrC-MtrD-MtrE, MacA-MacB, and NorM) in such clinical isolates. We report that the MIC of several antimicrobials, including seven previously and currently recommended for treatment was significantly impacted.
doi:10.1128/AAC.00038-14
PMCID: PMC4068427  PMID: 24733458
5.  Characterisation of blaTEM genes and types of β-lactamase plasmids in Neisseria gonorrhoeae – the prevalent and conserved blaTEM-135 has not recently evolved and existed in the Toronto plasmid from the origin 
BMC Infectious Diseases  2014;14(1):454.
Background
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern worldwide. It has been recently feared that the blaTEM-1 gene is, via blaTEM-135, evolving into an extended-spectrum β-lactamase (ESBL), which could degrade all cephalosporins including ceftriaxone. The aims of the present study were to characterize the blaTEM genes, types of β-lactamase plasmids, the degradation of ampicillin by TEM-135 compared to TEM-1, and to perform molecular epidemiological typing of β-lactamase-producing N. gonorrhoeae strains internationally.
Methods
β-lactamase producing N. gonorrhoeae isolates (n = 139) cultured from 2000 to 2011 in 15 countries were examined using antibiograms, blaTEM gene sequencing, β-lactamase plasmid typing, and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, the blaTEM gene was sequenced in the first described Toronto plasmid (pJD7), one of the first Asian plasmids (pJD4) and African plasmids (pJD5) isolated in Canada. The degradation of ampicillin by TEM-135 compared to TEM-1 was examined using a MALDI-TOF MS hydrolysis assay.
Results
Six different blaTEM sequences were identified (among isolates with 125 different NG-MAST STs), i.e. blaTEM-1 (in 104 isolates), blaTEM-135 (in 30 isolates), and four novel blaTEM sequences (in 5 isolates). The blaTEM-1 allele was only found in the African and Asian plasmids, while all Rio/Toronto plasmids possessed the blaTEM-135 allele. Most interesting, the first described gonococcal Toronto plasmid (pJD7), identified in 1984, also possessed the highly conserved blaTEM-135 allele. The degradation of ampicillin by TEM-135 compared to TEM-1 was indistinguishable in the MALDI-TOF MS hydrolysis assay.
Conclusions
blaTEM-135, encoding TEM-135, is predominantly and originally associated with the Rio/Toronto plasmid and prevalent among the β-lactamase producing gonococcal strains circulating globally. blaTEM-135 does not appear, as previously hypothesized, to have recently evolved due to some evolutionary selective pressure, for example, by the extensive use of extended-spectrum cephalosporins worldwide. On the contrary, the present study shows that blaTEM-135 existed in the Toronto plasmid from its discovery and that blaTEM-135 is highly conserved (not further evolved in the past >30 years). Nevertheless, international studies for monitoring the presence of different blaTEM alleles, the possible evolution of the blaTEM-135 allele, and the types of β-lactamase producing plasmids, remain imperative.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2334-14-454) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2334-14-454
PMCID: PMC4152594  PMID: 25149062
Gonorrhoea; Antimicrobial resistance; blaTEM-1; blaTEM-135; TEM-1; TEM-135; Rio/Toronto plasmid, Extended-spectrum β-lactamase (ESBL)
7.  Russian gonococcal antimicrobial susceptibility programme (RU-GASP) – resistance in Neisseria gonorrhoeae during 2009–2012 and NG-MAST genotypes in 2011 and 2012 
BMC Infectious Diseases  2014;14:342.
Background
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern worldwide and gonococcal AMR surveillance globally is imperative for public health purposes. In Eastern Europe, gonococcal AMR surveillance is exceedingly rare. However, in 2004 the Russian gonococcal antimicrobial susceptibility programme (RU-GASP) was initiated. The aims of this study were to describe the prevalence and trends of gonococcal AMR from 2009 to 2012, and molecular epidemiological genotypes in 2011 and 2012 in Russia.
Methods
Gonococcal isolates from 12–46 surveillance sites distributed across Russia, obtained in 2009 (n = 1200), 2010 (n = 407), 2011 (n = 423), and 2012 (n = 106), were examined for antimicrobial susceptibility using agar dilution method. Gonococcal isolates from 2011 and 2012 were investigated with N. gonorrhoeae multi-antigen sequence typing (NG-MAST).
Results
During 2009–2012, the proportions of gonococcal isolates resistant to ciprofloxacin, penicillin G, azithromycin and spectinomycin ranged from 25.5% to 44.4%, 9.6% to 13.2%, 2.3% to 17.0% and 0.9% to 11.6%, respectively. Overall, the resistance level to penicillin G was stable, the resistance level to ciprofloxacin was decreasing, however, the level of resistance to azithromycin increased. All isolates were susceptible to ceftriaxone using the US CLSI breakpoints. However, using the European breakpoints 58 (2.7%) of the isolates were resistant to ceftriaxone. Interestingly, this proportion was decreasing, i.e. from 4.8% in 2009 to 0% in 2012.
Conclusions
In Russia, the diversified gonococcal population showed a high resistance to ciprofloxacin, penicillin G and azithromycin. In general, the MICs of ceftriaxone were relatively high, however, they were decreasing from 2009 to 2012. Ceftriaxone should be the first-line for empiric antimicrobial monotherapy of gonorrhoea in Russia. It is essential to further strengthen the surveillance of gonococcal AMR (ideally also gonorrhoea treatment failures) in Russia.
doi:10.1186/1471-2334-14-342
PMCID: PMC4075497  PMID: 24947981
Neisseria gonorrhoeae; Gonorrhoea; Antimicrobial resistance; National surveillance; Russian gonococcal antimicrobial susceptibility programme (RU-GASP); Extended-spectrum cephalosporins (ESCs); Ceftriaxone; Treatment; N. gonorrhoeae multiantigen sequence typing (NG-MAST); Russia
8.  Antimicrobial Resistance and Molecular Typing of Neisseria gonorrhoeae Isolates in Kyoto and Osaka, Japan, 2010 to 2012: Intensified Surveillance after Identification of the First Strain (H041) with High-Level Ceftriaxone Resistance 
Antimicrobial Agents and Chemotherapy  2013;57(11):5225-5232.
In 2009, the first high-level ceftriaxone-resistant Neisseria gonorrhoeae strain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) of Neisseria gonorrhoeae isolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193 N. gonorrhoeae isolates were collected and the MICs (μg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, <0.5 μg/ml), and spectinomycin. The rates of resistance (intermediate susceptibility) to azithromycin, penicillin G, and ciprofloxacin were 3.6% (19.7%), 24.4% (71.0%), and 78.2% (0.5%), respectively. Multilocus sequence typing (MLST) showed that 40.9%, 19.2%, and 17.1% of isolates belonged to ST1901, ST7359, and ST7363, respectively. Furthermore, N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 μg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic type penA alleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of the penA mosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistant N. gonorrhoeae strain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.
doi:10.1128/AAC.01295-13
PMCID: PMC3811299  PMID: 23939890
9.  Antimicrobial susceptibility/resistance and genetic characteristics of Neisseria gonorrhoeae isolates from Poland, 2010-2012 
Background
In Poland, gonorrhoea has been a mandatorily reported infection since 1948, however, the reported incidences are likely underestimated. No antimicrobial resistance (AMR) data for Neisseria gonorrhoeae has been internationally reported in nearly four decades, and data concerning genetic characteristics of N. gonorrhoeae are totally lacking. The aims of this study were to investigate the AMR to previously and currently recommended gonorrhoea treatment options, the main genetic resistance determinant (penA) for extended-spectrum cephalosporins (ESCs), and genotypic distribution of N. gonorrhoeae isolates in Poland in 2010-2012.
Methods
N. gonorrhoeae isolates cultured in 2010 (n = 28), 2011 (n = 92) and 2012 (n = 108) in Warsaw and Bialystok, Poland, were examined using antimicrobial susceptibility testing (Etest), pyrosequencing of penA and N. gonorrhoeae multi-antigen sequence typing (NG-MAST).
Results
The proportions of N. gonorrhoeae isolates showing resistance were as follows: ciprofloxacin 61%, tetracycline 43%, penicillin G 22%, and azithromycin 8.8%. No isolates resistant to ceftriaxone, cefixime or spectinomycin were found. However, the proportion of isolates with an ESC MIC = 0.125 mg/L, i.e. at the resistance breakpoint, increased significantly from none in 2010 to 9.3% and 19% in 2012 for ceftriaxone and cefixime, respectively. Furthermore, 3.1% of the isolates showed multidrug resistance, i.e., resistance to ciprofloxacin, penicillin G, azithromycin, and decreased susceptibility to cefixime (MIC = 0.125 mg/L). Seventy-six isolates (33%) possessed a penA mosaic allele and 14 isolates (6.1%) contained an A501V/T alteration in penicillin-binding protein 2. NG-MAST ST1407 (n = 58, 25% of isolates) was the most prevalent ST, which significantly increased from 2010 (n = 0) to 2012 (n = 46; 43%).
Conclusions
In Poland, the diversified gonococcal population displayed a high resistance to most antimicrobials internationally previously recommended for gonorrhoea treatment and decreasing susceptibility to the currently recommended ESCs. The decreasing susceptibility to ESCs was mostly due to the introduction of the internationally spread multidrug-resistant NG-MAST ST1407 in 2011. It is essential to promptly revise the gonorrhoea treatment guidelines, improve the gonorrhoea laboratory diagnostics, and implement quality assured surveillance of gonococcal AMR (ideally also treatment failures) in Poland.
doi:10.1186/1471-2334-14-65
PMCID: PMC3922028  PMID: 24502606
Neisseria gonorrhoeae; Gonorrhoea; Poland; Antimicrobial resistance (AMR); Extended-spectrum cephalosporins (ESCs); Ceftriaxone; Cefixime; penA; NG-MAST
10.  Molecular Diagnostics for Gonorrhoea: Implications for Antimicrobial Resistance and the Threat of Untreatable Gonorrhoea 
PLoS Medicine  2014;11(2):e1001598.
This Essay from Nicola Low and colleagues discusses the importance of the nucleic acid amplification tests for rapid detection of N. gonorrhoeae and its resistance determinants, as well as the importance of ensuring their rational use, as priorities for controlling both gonorrhoea and antimicrobial resistance.
Please see later in the article for the Editors' Summary
doi:10.1371/journal.pmed.1001598
PMCID: PMC3913554  PMID: 24503544
11.  Identification of Amino Acids Conferring High-Level Resistance to Expanded-Spectrum Cephalosporins in the penA Gene from Neisseria gonorrhoeae Strain H041 
The recent identification of a high-level-ceftriaxone-resistant (MIC = 2 to 4 μg/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empirical antimicrobial monotherapy. The penA gene from H041 (penA41) is a mosaic penA allele similar to mosaic alleles conferring intermediate-level cephalosporin resistance (Cephi) worldwide but has 13 additional mutations compared to the mosaic penA gene from the previously studied Cephi strain 35/02 (penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MICs for ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction fragment exchange and site-directed mutagenesis, we identified three mutations, A311V, T316P, and T483S, that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. A311V and T316P are close to the active-site nucleophile Ser310 that forms the acyl-enzyme complex, while Thr483 is predicted to interact with the carboxylate of the β-lactam antibiotic. These three mutations have thus far been described only for penA41, but dissemination of these mutations in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.
doi:10.1128/AAC.00093-13
PMCID: PMC3697319  PMID: 23587946
12.  Innate Recognition by Neutrophil Granulocytes Differs between Neisseria gonorrhoeae Strains Causing Local or Disseminating Infections 
Infection and Immunity  2013;81(7):2358-2370.
Members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as cellular receptors for Neisseria gonorrhoeae. More specifically, neisserial colony opacity (OpaCEA) proteins bind to epithelial CEACAMs (CEACAM1, CEA, CEACAM6) to promote bacterial colonization of the mucosa. In contrast, recognition by CEACAM3, expressed by human granulocytes, results in uptake and destruction of OpaCEA-expressing bacteria. Therefore, CEACAM3-mediated uptake might limit the spread of gonococci. However, some strains can cause disseminating gonococcal infections (DGIs), and it is currently unknown how these strains escape detection by granulocyte CEACAM3. Therefore, the opa gene loci from N. gonorrhoeae strain VP1, which was derived from a patient with disseminated gonococcal disease, were cloned and constitutively expressed in Escherichia coli. Similar to Opa proteins of the nondisseminating strain MS11, the majority of Opa proteins from strain VP1 bound epithelial CEACAMs and promoted CEACAM-initiated responses by epithelial cells. In sharp contrast to the Opa proteins of strain MS11, the Opa proteins of strain VP1 failed to interact with the human granulocyte receptor CEACAM3. Accordingly, bacteria expressing VP1 Opa proteins were not taken up by primary human granulocytes and did not trigger a strong oxidative burst. Analysis of Opa variants from four additional clinical DGI isolates again demonstrated a lack of CEACAM3 binding. In summary, our results reveal that particular N. gonorrhoeae strains express an Opa protein repertoire allowing engagement of epithelial CEACAMs for successful mucosal colonization, while avoiding recognition and elimination via CEACAM3-mediated phagocytosis. A failure of CEACAM3-mediated innate immune detection might be linked to the ability of gonococci to cause disseminated infections.
doi:10.1128/IAI.00128-13
PMCID: PMC3697628  PMID: 23630956
13.  Analytical Evaluation of GeneXpert CT/NG, the First Genetic Point-of-Care Assay for Simultaneous Detection of Neisseria gonorrhoeae and Chlamydia trachomatis 
Journal of Clinical Microbiology  2013;51(6):1945-1947.
GeneXpert CT/NG was evaluated with 372 characterized bacterial strains. Sensitivity of 10 genome copies/reaction was obtained for both agents. Four Neisseria mucosa and two Neisseria subflava isolates were positive for one of two gonococcal targets; however, the assay flagged all as negative. The assay was analytically highly sensitive and specific.
doi:10.1128/JCM.00806-13
PMCID: PMC3716077  PMID: 23554203
14.  The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array 
PLoS ONE  2013;8(10):e76696.
Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e.g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.
doi:10.1371/journal.pone.0076696
PMCID: PMC3792038  PMID: 24116138
15.  Target Gene Sequencing To Define the Susceptibility of Neisseria meningitidis to Ciprofloxacin 
Meningococcal gyrA gene sequence data, MICs, and mouse infection were used to define the ciprofloxacin breakpoint for Neisseria meningitidis. Residue T91 or D95 of GyrA was altered in all meningococcal isolates with MICs of ≥0.064 μg/ml but not among isolates with MICs of ≤0.032 μg/ml. Experimental infection of ciprofloxacin-treated mice showed slower bacterial clearance when GyrA was altered. These data suggest a MIC of ≥0.064 μg/ml as the ciprofloxacin breakpoint for meningococci and argue for the molecular detection of ciprofloxacin resistance.
doi:10.1128/AAC.02184-12
PMCID: PMC3623314  PMID: 23357770
16.  Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea 
Future microbiology  2012;7(12):1401-1422.
The new superbug Neisseria gonorrhoeae has retained resistance to antimicrobials previously recommended for first-line treatment and has now demonstrated its capacity to develop resistance to the extended-spectrum cephalosporin, ceftriaxone, the last remaining option for first-line empiric treatment of gonorrhea. An era of untreatable gonorrhea may be approaching, which represents an exceedingly serious public health problem. Herein, we review the evolution, origin and spread of antimicrobial resistance and resistance determinants (with a focus on extended-spectrum cephalosporins) in N. gonorrhoeae, detail the current situation regarding verified treatment failures with extended-spectrum cephalosporins and future treatment options, and highlight essential actions to meet the large public health challenge that arises with the possible emergence of untreatable gonorrhea. Essential actions include: implementing action/response plans globally and nationally; enhancing surveillance of gonococcal antimicrobial resistance, treatment failures and antimicrobial use/misuse; and improving prevention, early diagnosis and treatment of gonorrhea. Novel treatment strategies, antimicrobials (or other compounds) and, ideally, a vaccine must be developed.
doi:10.2217/fmb.12.117
PMCID: PMC3629839  PMID: 23231489
action plan; antimicrobial resistance; cefixime; ceftriaxone; cephalosporins; Neisseria gonorrhoeae; penA; surveillance; treatment failure
17.  Neisseria gonorrhoeae Strain with High-Level Resistance to Spectinomycin Due to a Novel Resistance Mechanism (Mutated Ribosomal Protein S5) Verified in Norway 
Gonorrhea may become untreatable, and new treatment options are essential. Verified resistance to spectinomycin is exceedingly rare. However, we describe a high-level spectinomycin-resistant (MIC, >1,024 μg/ml) Neisseria gonorrhoeae strain from Norway with a novel resistance mechanism. The resistance determinant was a deletion of codon 27 (valine) and a K28E alteration in the ribosomal protein 5S. The traditional spectinomycin resistance gene (16S rRNA) was wild type. Despite this exceedingly rare finding, spectinomycin available for treatment of ceftriaxone-resistant urogenital gonorrhea would be very valuable.
doi:10.1128/AAC.01775-12
PMCID: PMC3553714  PMID: 23183436
18.  The evolution of infectious agents in relation to sex in animals and humans: brief discussions of some individual organisms 
The following series of concise summaries addresses the evolution of infectious agents in relation to sex in animals and humans from the perspective of three specific questions: (1) what have we learned about the likely origin and phylogeny, up to the establishment of the infectious agent in the genital econiche, including the relative frequency of its sexual transmission; (2) what further research is needed to provide additional knowledge on some of these evolutionary aspects; and (3) what evolutionary considerations might aid in providing novel approaches to the more practical clinical and public health issues facing us currently and in the future?
doi:10.1111/j.1749-6632.2011.06133.x
PMCID: PMC3694397  PMID: 21824167
evolution; infectious agents; sexual transmission; econiche
19.  Genome Sequencing of a Neisseria gonorrhoeae Isolate of a Successful International Clone with Decreased Susceptibility and Resistance to Extended-Spectrum Cephalosporins 
Antimicrobial Agents and Chemotherapy  2012;56(11):5633-5641.
The recent emergence of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins is a major concern globally. We sequenced the genome of an N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 isolate (SM-3) with decreased susceptibility and resistance to oral extended-spectrum cephalosporins. The isolate was cultured in 2008 in San Francisco, CA, and possessed mosaic penA allele XXXIV, which is associated with an international clone that possesses decreased susceptibility as well as resistance to oral extended-spectrum cephalosporins globally. The genome sequence of strain NCCP11945 was used as a scaffold, and our assembly resulted in 91 contigs covering 2,029,064 bp (91%; >150× coverage) of the genome. Numerous instances of suspected horizontal genetic transfer events with other Neisseria species were identified, and two genes, opa and txf, acquired from nongonococcal Neisseria species, were identified. Strains possessing mosaic penA alleles (n = 108) and nonmosaic penA alleles (n = 169) from the United States and Europe (15 countries), cultured in 2002 to 2009, were screened for the presence of these genes. The opa gene was detected in most (82%) penA mosaic-containing isolates (mainly from 2007 to 2009) but not in any penA nonmosaic isolates. The txf gene was found in all strains containing opa but also in several (18%) penA nonmosaic strains. Using opa and txf as genetic markers, we identified a strain that possesses mosaic penA allele XXXIV, but the majority of its genome is not genetically related to strain SM-3. This implies that penA mosaic allele XXXIV was transferred horizontally. Such isolates also possessed decreased susceptibility and resistance to oral extended-spectrum cephalosporins. These findings support that genetic screening for particular penA mosaic alleles can be a valuable method for tracking strains with decreased susceptibility as well as resistance to oral extended-spectrum cephalosporins worldwide and that screening using only NG-MAST may not be sufficient.
doi:10.1128/AAC.00636-12
PMCID: PMC3486552  PMID: 22908152
20.  Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible? 
PLoS ONE  2013;8(4):e60670.
Background and Objective
Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.
Materials and Methods
Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.
Results
Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.
Conclusions
Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.
doi:10.1371/journal.pone.0060670
PMCID: PMC3621988  PMID: 23585843
21.  Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae isolates from Vietnam, 2011 
Background
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health concern worldwide. In Vietnam, knowledge regarding N. gonorrhoeae prevalence and AMR is limited, and data concerning genetic characteristics of N. gonorrhoeae is totally lacking. Herein, we investigated the phenotypic AMR (previous, current and possible future treatment options), genetic resistance determinants for extended-spectrum cephalosporins (ESCs), and genotypic distribution of N. gonorrhoeae isolated in 2011 in Hanoi, Vietnam.
Methods
N. gonorrhoeae isolates from Hanoi, Vietnam isolated in 2011 (n = 108) were examined using antibiograms (Etest for 10 antimicrobials), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and sequencing of ESC resistance determinants (penA, mtrR and penB).
Results
The levels of in vitro resistance were as follows: ciprofloxacin 98%, tetracycline 82%, penicillin G 48%, azithromycin 11%, ceftriaxone 5%, cefixime 1%, and spectinomycin 0%. The MICs of gentamicin (0.023-6 mg/L), ertapenem (0.002-0.125 mg/L) and solithromycin (<0.016-0.25 mg/L) were relatively low. No penA mosaic alleles were found, however, 78% of the isolates contained an alteration of amino acid A501 (A501V (44%) and A501T (34%)) in the encoded penicillin-binding protein 2. A single nucleotide (A) deletion in the inverted repeat of the promoter region of the mtrR gene and amino acid alterations in MtrR was observed in 91% and 94% of the isolates, respectively. penB resistance determinants were detected in 87% of the isolates. Seventy-five different NG-MAST STs were identified, of which 59 STs have not been previously described.
Conclusions
In Vietnam, the highly diversified gonococcal population displayed high in vitro resistance to antimicrobials previously recommended for gonorrhoea treatment (with exception of spectinomycin), but resistance also to the currently recommended ESCs were found. Nevertheless, the MICs of three potential future treatment options were low. It is essential to strengthen the diagnostics, case reporting, and epidemiologic surveillance of gonorrhoea in Vietnam. Furthermore, the surveillance of gonococcal AMR and gonorrhoea treatment failures is imperative to reinforce. Research regarding novel antimicrobial treatment strategies (e.g., combination therapy) and new antimicrobials is crucial for future treatment of gonorrhoea.
doi:10.1186/1471-2334-13-40
PMCID: PMC3574855  PMID: 23351067
Neisseria gonorrhoeae; Gonorrhoea; Vietnam; Antimicrobial resistance; Extended-spectrum cephalosporins (ESCs); Ceftriaxone; Cefixime; Resistance determinants; penA; NG-MAST
22.  Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae isolates from India, Pakistan and Bhutan in 2007–2011 
Background
Knowledge on antimicrobial drug resistance and genetic characteristics of Neisseria gonorrhoeae isolates circulating in India, Pakistan, and Bhutan is sorely lacking. In this paper, we describe the prevalence of antimicrobial resistance and molecular characteristics of N. gonorrhoeae isolates from India, Pakistan, and Bhutan in 2007–2011.
Methods
Antimicrobial susceptibility and β-lactamase production were tested for 65 N. gonorrhoeae isolates from India (n=40), Pakistan (n=18) and Bhutan (n=7) using Etest methodology (eight antimicrobials) and nitrocefin solution, respectively. Resistance determinants, i.e. penA, mtrR, porB1b, gyrA, and parC, were sequenced. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed for molecular epidemiology.
Results
The highest resistance level was observed for ciprofloxacin (94%), followed by penicillin G (68%), erythromycin (62%), tetracycline (55%), and azithromycin (7.7%). All the isolates were susceptible to ceftriaxone, cefixime, and spectinomycin. Thirty-four (52%) of the isolates were producing β-lactamase. No penA mosaic alleles or A501-altered alleles of penicillin-binding protein 2 were identified. Forty-nine NG-MAST STs were identified, of which 42 STs have not been previously described worldwide.
Conclusions
Based on this study, ceftriaxone, cefixime, and spectinomycin can be used as an empirical first-line therapy for gonorrhoea in India, Pakistan, and Bhutan, whereas ciprofloxacin, penicillin G, tetracycline, erythromycin, and azithromycin should not be. It is imperative to strengthen the laboratory infrastructure in this region, as well as to expand the phenotypic and genetic surveillance of antimicrobial resistance, emergence of new resistance, particularly, to extended-spectrum cephalosporins, and molecular epidemiology.
doi:10.1186/1471-2334-13-35
PMCID: PMC3565906  PMID: 23347339
Gonorrhoea; Antimicrobial resistance; penA; Ceftriaxone; Southeast Asia; India; Bhutan; Pakistan
23.  In Vitro Activity of Ertapenem versus Ceftriaxone against Neisseria gonorrhoeae Isolates with Highly Diverse Ceftriaxone MIC Values and Effects of Ceftriaxone Resistance Determinants: Ertapenem for Treatment of Gonorrhea? 
Clinical resistance to the currently recommended extended-spectrum cephalosporins (ESCs), the last remaining treatment options for gonorrhea, is being reported. Gonorrhea may become untreatable, and new treatment options are crucial. We investigated the in vitro activity of ertapenem, relative to ceftriaxone, against N. gonorrhoeae isolates and the effects of ESC resistance determinants on ertapenem. MICs were determined using agar dilution technique or Etest for international reference strains (n = 17) and clinical N. gonorrhoeae isolates (n = 257), which included the two extensively drug-resistant (XDR) strains H041 and F89 and additional isolates with high ESC MICs, clinical ESC resistance, and other types of clinical high-level and multidrug resistance (MDR). Genetic resistance determinants for ESCs (penA, mtrR, and penB) were sequenced. In general, the MICs of ertapenem (MIC50 = 0.032 μg/ml; MIC90 = 0.064 μg/ml) paralleled those of ceftriaxone (MIC50 = 0.032 μg/ml; MIC90 = 0.125 μg/ml). The ESC resistance determinants mainly increased the ertapenem MIC and ceftriaxone MIC at similar levels. However, the MIC ranges for ertapenem (0.002 to 0.125 μg/ml) and ceftriaxone (<0.002 to 4 μg/ml) differed, and the four (1.5%) ceftriaxone-resistant isolates (MIC = 0.5 to 4 μg/ml) had ertapenem MICs of 0.016 to 0.064 μg/ml. Accordingly, ertapenem had in vitro advantages over ceftriaxone for isolates with ceftriaxone resistance. These in vitro results suggest that ertapenem might be an effective treatment option for gonorrhea, particularly for the currently identified ESC-resistant cases and possibly in a dual antimicrobial therapy regimen. However, further knowledge regarding the genetic determinants (and their evolution) conferring resistance to both antimicrobials, and clear correlates between genetic and phenotypic laboratory parameters and clinical treatment outcomes, is essential.
doi:10.1128/AAC.00326-12
PMCID: PMC3393402  PMID: 22547617
25.  In Vitro Activity of the New Fluoroketolide Solithromycin (CEM-101) against a Large Collection of Clinical Neisseria gonorrhoeae Isolates and International Reference Strains, Including Those with High-Level Antimicrobial Resistance: Potential Treatment Option for Gonorrhea? 
Gonorrhea may become untreatable, and new treatment options are essential. We investigated the in vitro activity of the first fluoroketolide, solithromycin. Clinical Neisseria gonorrhoeae isolates and reference strains (n = 246), including the two extensively drug-resistant strains H041 and F89 and additional isolates with clinical cephalosporin resistance and multidrug resistance, were examined. The activity of solithromycin was mainly superior to that of other antimicrobials (n = 10) currently or previously recommended for gonorrhea treatment. Solithromycin might be an effective treatment option for gonorrhea.
doi:10.1128/AAC.00036-12
PMCID: PMC3346660  PMID: 22354296

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