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Seven countries in Latin America and the Caribbean report on (2010 and 2011) the susceptibility of 2235 isolates of Neisseria gonorrhoeae to 6 antibiotics. Thirteen isolates had ceftriaxone minimum inhibitory concentrations (MICs) of 0.125 to ≥ 0.25 mg/L. The percentage of resistant isolates to the following antibiotics was: azithromycin, 1.0% to 1.7%; ciprofloxacin, 42.1% to 36.2%; penicillin, 31% to 35%; tetracycline, 21.8% to 22.6%.

A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible ( https://ngstar.canada.ca ). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes ( penA , mtrR , porB , ponA , gyrA , parC , and 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA , porB , gyrA , and parC ; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) ( n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs ( n = 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 ( n = 100; 13.0%), ST-42 and ST-91 ( n = 45; 5.9%), ST-64 ( n = 44; 5.72%), and ST-139 ( n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 ( n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 ( n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 ( n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 ( n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains.

Chlamydia trachomatis infections are the most prominent bacterial sexually-transmitted disease world-wide and a lot of effort is put into the development of an effective vaccine. Pigs have been shown to be a valuable animal model for C. trachomatis vaccine development. The aim of this study was to decipher the T-cell-mediated immune response to chlamydial infections including C. trachomatis and C. suis , the chlamydia species naturally infecting pigs with a demonstrated zoonotic potential. Vaginal infection of pigs with C. suis and C. trachomatis lasted from 3 to 21 days and intra-uterine infection was still present after 21 days in 3 out of 5 C. suis - and 4 out of 5 C. trachomatis -inoculated animals and caused severe pathological changes. Humoral immune responses including neutralizing antibodies were found predominantly in response to C. suis starting at 14 days post inoculation. The T-cell-mediated immune responses to C. trachomatis and C. suis -infections started at 7 days post inoculation and consisted mainly of CD4 + T cells which were either IFN-γ single cytokine-producing or IFN-γ/TNF-α double cytokine-producing T-helper 1 cells. IL-17-producing CD4 + T cells were rare or completely absent. The T-cell-mediated immune responses were triggered by both homologous or heterologous re-stimulation indicating that cross-protection between the two chlamydia species is possible. Thus, having access to a working genital C. suis and C. trachomatis infection model, efficient monitoring of the host-pathogen interactions, and being able to accurately assess the responses to infection makes the pig an excellent animal model for vaccine development which also could bridge the gap to the clinical phase for C. trachomatis vaccine research.

Neisseria gonorrhoeae isolates were grown on chocolate agar slants comprising GC Agar Medium Base supplemented with 1% modified Kellogg's supplement and 1% beef blood hemoglobin. The bacterial growth was covered with sterilized paraffin oil. Cultures were used for transportation and storage of Neisseria gonorrhoeae for 3months at room temperature.

A longitudinal study combining multilocus sequence typing with molecular evolutionary analysis determined the distribution, population structure, and evolution of antibiotic resistance in Neisseria gonorrhoeae isolates in Saskatchewan that were collected between 2005 and 2008. Of 195 gonococcal isolates examined, 29 sequence types (STs) were identified with 3 major circulating strains (ST-1 through ST-3) comprising 52% of all gonococcal isolates studied. The prevalences, persistence, distribution patterns, and clonalities of these isolates strongly suggest that gonorrhea endemicity within this broad geographic region was driven by these 3 circulating strains. ST-1 exhibited a significantly ( P = 0.001) higher prevalence throughout the study than did the others, accounting for ∼25% of the tested isolates each year. The spatial distributions of the gonococcal strains indicated that ST-1 in 2007 entered a linear component of the sexual network, reaching the remote north and resulting in the further spread and maintenance of infection. Ciprofloxacin and azithromycin resistances were observed in distantly related gonococcal lineages, clearly indicating the convergent acquisition of these antibiotic-resistant phenotypes. In addition, all ciprofloxacin- and azithromycin-resistant lineages were found at the edges of the minimum spanning tree, far from the major lineages, suggesting that these antibiotic phenotypes were most likely introduced into the province. In contrast, resistance to penicillin was found mostly in the endemic gonococcal lineages, suggesting that penicillin resistance was probably acquired in Saskatchewan as a result of spontaneous mutations in already-established lineages. Tetracycline resistance was present in all STs except one, indicating its ubiquitous nature in the gonococcal population studied.

The majority of Staphylococcus aureus infections from Isfahan, Iran, were caused by epidemic methicillin-susceptible S. aureus (MSSA) lineages, sequence type 8 (ST8), ST22, ST30, and ST6. The predominant methicillin-resistant S. aureus strain was ST239. We observed a high prevalence of Panton-Valentine leukocidin-positive MSSA strains (19.7%), which is a matter of considerable concern, since these strains have the ability to cause severe infections.

SUMMARY Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae , together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a serious public health problem, compromising the management and control of gonorrhoea globally. Resistance in N. gonorrhoeae to ceftriaxone, the last option for first-line empirical monotherapy of gonorrhoea, has been reported from many countries globally, and sporadic failures to cure especially pharyngeal gonorrhoea with ceftriaxone monotherapy and dual antimicrobial therapies (ceftriaxone plus azithromycin or doxycycline) have been confirmed in several countries. In 2018, the first gonococcal isolates with ceftriaxone resistance plus high-level azithromycin resistance were identified in England and Australia. The World Health Organization (WHO) Global Gonococcal Antimicrobial Surveillance Program (GASP) is essential to monitor AMR trends, identify emerging AMR and provide evidence for refinements of treatment guidelines and public health policy globally. Herein we describe the WHO GASP data from 67 countries in 2015–16, confirmed gonorrhoea treatment failures with ceftriaxone with or without azithromycin or doxycycline, and international collaborative actions and research efforts essential for the effective management and control of gonorrhoea. In most countries, resistance to ciprofloxacin is exceedingly high, azithromycin resistance is present and decreased susceptibility or resistance to ceftriaxone has emerged. Enhanced global collaborative actions are crucial for the control of gonorrhoea, including improved prevention, early diagnosis, treatment of index patient and partner (including test-of-cure), improved and expanded AMR surveillance (including surveillance of antimicrobial use and treatment failures), increased knowledge of correct antimicrobial use and the pharmacokinetics and pharmacodynamics of antimicrobials and effective drug regulations and prescription policies (including antimicrobial stewardship). Ultimately, rapid, accurate and affordable point-of-care diagnostic tests (ideally also predicting AMR and/or susceptibility), new therapeutic antimicrobials and, the only sustainable solution, gonococcal vaccine(s) are imperative.

Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10-12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVA(Ef) self-interacts and forms a 10-12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVA(Ef) self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVA(Ef) did not affect DivIVA(Ef) oligomerization. The introduction of DivIVA(Ef) mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVA(Ef) in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVA(Ef) are indispensable for proper biological function. Overexpression of wild-type DivIVA(Ef) in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVA(Ef) mutations failed to induce such alterations.

While bacterial cell division has been widely studied in rod-shaped bacteria, the mechanism of cell division in round (coccal) bacteria remains largely enigmatic. In the present study, interaction between the cell division inhibitor MinC from Neisseria gonorrhoeae (MinC(Ng)) and the gonococcal cell division proteins MinD(Ng) and FtsZ(Ng) are demonstrated. Protein truncation and site-directed mutagenic approaches determined which N-terminal residues were essential for cell division inhibition by MinC(Ng) using cell morphology as an indicator of protein functionality. Truncation from or mutation at the 13th amino acid of the N terminus of MinC(Ng) resulted in loss of protein function. Bioinformatic analyses predicted that point mutations of L35P and L68P would affect the alpha-helical conformation of the protein and we experimentally showed that these mutations alter the functionality of MinC(Ng). The bacterial two-hybrid system showed that interaction of MinC(Ng) with FtsZ(Ng) is abrogated upon truncation of 13 N-terminal residues while MinC(Ng)-MinD(Ng) interaction or MinC(Ng) homodimerization is unaffected. These data confirm interactions among gonococcal cell division proteins and determine the necessity of the 13th amino acid for MinC(Ng) function.

Data from the Canadian National Gonococcal Antimicrobial Susceptibility Comparison Program, including results from 25 proficiency panels distributed between 2003 and 2012, were analyzed. The average MIC agreement between the participating laboratories ranged from 85.6% to 98.8% over the 10-year period, with the interpretation agreement ranging from 85.7% to 98.1%.

Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum, namely, MinC, MinD, and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation; however, little is actually known about the structure of this functionally critical region. To understand how MinE can perform its anti-MinCD function, we have therefore investigated the conformation of the full-length MinE from Neisseria gonorrhoeae by solution NMR. Although solubility considerations required the use of sample conditions that limit the observation of amide resonances to regions that are protected from solvent exchange, backbone chemical shifts from both N- and C-terminal domains could be assigned. In contrast to previous models, secondary chemical shift analysis of these solvent-protected regions shows that parts of the N-terminal anti-MinCD domain are stably folded with many functionally important residues localizing to a beta-structure. In addition, this N-terminal domain may be interacting with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The nonfunctional MinE mutant L22D showed even larger evidence of structural perturbations in both domains, with significant destabilization of the entire MinE structure. Overall, these results suggest that there is an intimate structural association between the anti-MinCD and topological specificity domains, allowing the functional properties of the two domains to be modulated through this interaction.

Enterococcus faecalis divIVA (divIVA Ef) is an essential gene implicated in cell division and chromosome segregation. This gene was disrupted by insertional inactivation creating E. faecalis JHSR1, which was viable only when a wild-type copy of divIVA Ef was expressed in trans, confirming the essentiality of the gene. The absence of DivIVAEf in E. faecalis JHSR1 inhibited proper cell division, which resulted in abnormal cell clusters possessing enlarged cells of altered shape instead of the characteristic diplococcal morphology of enterococci. The lower viability of the divIVA Ef mutant is caused by improper nucleoid segregation and impaired septation within the numerous cells generated in each cluster. Overexpression of DivIVAEf in Escherichia coli KJB24 resulted in enlarged cells with disrupted cell division, suggesting that this round E. coli mutant strain could be used as an indicator for functionality of DivIVAEf. A Bacillus subtilis divIVA mutant was not complemented by DivIVAEf, indicating that this protein does not recognize DivIVA-specific target sites in B. subtilis, or that it does not interact with other proteins of the cell division machinery of this micro-organism. DivIVAEf also failed to complement a Streptococcus pneumoniae divIVA mutant, supporting the phylogenetic distance between Enterococcus and Streptococcus. Our results indicate that DivIVA is a species-specific multifunctional protein implicated in cell division and chromosome segregation in E. faecalis.

A region in the cell division site determinant MinD required for stimulation by MinE and which determines MinD topological specificity along coil-like structures has been identified. Structural modeling of dimeric MinD and sequence alignment of 24 MinD proteins revealed a conserved polar region in Gram-negative bacterial MinD proteins, corresponding to residues 92-94 of Neisseria gonorrhoeae MinD (MinD(Ng)). Using MinD(Ng) as a paradigm for MinD functionality in Gram-negative organisms, mutation of these conserved residues did not abrogate MinD(Ng) self-association, nor its interaction with MinE(Ng) and the cell division inhibitor MinC. Although the MinD(Ng) mutant dimerized in the presence of ATP, its ATPase activity was not stimulated by MinE(Ng), unlike wild-type MinD(Ng). GFP fusions to either MinD(Ng) or to Escherichia coli MinD bearing simultaneous or individual mutations to residues 92-94 localized within coiled arrays along the E. coli inner cell periphery, similar to wild-type GFP-MinD. However, unlike wild-type GFP-fusions, the mutant proteins were distributed uniformly throughout the array, despite the presence of MinE, which normally imparts topological specificity to MinD by inducing the latter to oscillate from pole-to-pole and away from midcell. Hence, despite localizing along the inner cell periphery as a polymeric structure, the mutant MinD proteins in this study have lost the ability to be efficiently stimulated by MinE(Ng), resulting in a loss of distinct pole-to-pole oscillation.

The composition of 19 garlic natural health products (NHPs) and fresh garlic extracts were determined, as was their antibacterial activity. The 19 NHPs and 5 fresh garlic extract standards were analysed for their principal active constituents. They were also extracted for 5, 10 or 15 min in water to fresh garlic equivalents of 200 mg/mL. The extract's minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against three indicator microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis) were determined by the broth microdilution method. While 47% of the aqueous garlic NHP extracts exhibited activity against N. gonorrhoeae, only 16% of the aqueous extracts inhibited S. aureus or E. faecalis at all three timepoints. Generally, products with high antimicrobial activity contained higher levels of garlic constituents with comparable activity to fresh garlic extracts, while products with marginal antibacterial activity often contained lower concentrations of constituents than their product labels indicated. Different extraction times affected antibacterial activity only against N. gonorrhoeae and tended to be correlated with levels of allicin. Thus, many extracts showed discrepancies in both composition, allicin:alliin ratio and antimicrobial activity, raising concerns as to standards of preparation and quality control for these products.

Background: In many regions the susceptibility of Neisseria gonorrhoeae isolates to antimicrobial agents is rarely tested. The Gonococcal Antimicrobial Surveillance Program (GASP) in Cuba was established as part of a larger regional GASP program to facilitate the collection and reporting of antimicrobial susceptibility data for N gonorrhoeae isolates. Goal: The goal was to retrospectively determine the antimicrobial susceptibility and molecular epidemiology of 91 isolates of N gonorrhoeae isolated from 11 centers in Cuba. Study Design: Isolates of N gonorrhoeae were collected and presumptively identified from 11 Cuban provincial health centers. They were then forwarded to the National Laboratory of Pathogenic Neisseria Havana for confirmatory identification and were subsequently analyzed at the Center for GASP in Ottawa. Isolates were tested for susceptibility to penicillin tetracycline spectinomycin ceftriaxone ciprofloxacin and azithromycin by the agar dilution method. To establish baseline data for molecular epidemiologic profiles the auxotype (A) serovar (S) plasmid content (P) and TetM type of the isolates were determined. Certain A/S/P classes were further analyzed by pulsed field gel electrophoresis (PFGE). Results: High percentages of the 91 N gonorrhoeae isolates were resistant to penicillin (68%) and tetracycline (83.5%) with 56% being penicillinase-producing (PPNG) and 64% carrying plasmid-mediated tetracycline resistance (TRNG; 50% were PP/TRNG). An additional 14% of the isolates carried chromosomal resistance (CMRNG) to either tetracycline or penicillin or both antibiotics. All isolates were susceptible to spectinomycin ceftriaxone and ciprofloxacin. However nine isolates were resistant to azithromycin (MIC >/=1.0 µg/ml) and 43 other isolates displayed reduced susceptibility to this antibiotic (MIC 0.25–0.5 µg/ml). Although a total of 21 different A/S classes were identified most of the isolates (61) belonged to three A/S classes: NR/IA-6 (35 isolates) NR/IB-1 (15 isolates) and P/IA-6 (11 isolates). Thirty-two of 45 PP/TRNG were A/S class NR/IA-6 and nine of the P/IA-6 isolates were TRNG. By contrast most of A/S class NR/IB-1 (8) were CMRNG. PFGE analysis following digestion with NheI or SpeI further clustered the isolates into separate groups. Conclusions: This study demonstrates high percentages of N gonorrhoeae isolates with penicillin and tetracycline resistance in Cuba. As has been noted in other studies in the Caribbean region and Latin America resistance and reduced susceptibility to azithromycin are developing as emerging problems. Since penicillin and tetracycline continue to be widely used for the treatment of gonococcal infections in Cuba this study indicates the importance of antimicrobial susceptibility surveillance so that effective antibiotics may be recommended for treatment of gonococcal infections. (authors)

The successful treatment of gonorrhea, presently the second most prevalent bacterial sexually transmitted infection worldwide, has historically taken full advantage of the introduction of successive new categories of antimicrobial agents to successfully eliminate the microorganism causing this disease, Neisseria gonorrhoeae. New antibiotics have replaced older ones for treatment because N. gonorrhoeae has progressively developed resistance to each class introduced; first to sulphonamides, then to penicillins, tetracyclines, and then quinolones and macrolides [1, 2]. While the last class of antimicrobial effective for the treatment of gonorrhea is the third-generation cephalosporins, more and more reports are emerging of in vitro decreased susceptibility and treatment failures to the oral cephalosporins, such as cefixime, coupled with recent reports of strains with highlevel resistance to ceftriaxone [3–5]. The strains with high-level resistance to cephalosporins also exhibited resistance or reduced susceptibility to several other antibiotics. Thus, there is a great concern that gonorrhea has become an untreatable superbug in certain instances, and the search for effective alternative treatment strategies and therapies, possibly a combination of antibiotics, is urgent [5, 6]. The emergence of resistance to some classes of antimicrobial has been so rapid and sustained that a basic understanding of the factors underscoring the ability of N. gonorrhoeae isolates to survive in potentially lethal antimicrobial environments, a measure of fitness, is lacking. The report by Kunz et al in this issue of the Journal of Infectious Diseases describes fluoroquinolone resistance in N. gonorrhoeae as it relates to microbial fitness. Fluoroquinolones were introduced for the treatment of gonorrhea infections in the late 1980s following the rise and worldwide dissemination of both plasmid-mediated and chromosomal resistance to penicillins and tetracyclines [1, 2]. However, the introduction of fluoroquinolones, such as ciprofloxacin, for treatment was quickly followed by reports of resistance, first in Asia and subsequently worldwide [7, 8]. Once resistance to fluoroquinolones reached 5% of the N. gonorrhoeae isolates tested in any region, the accepted cutoff where it is recommended that treatment be changed, the third-generation cephalosporins were then recommended as the primary antibiotics to treat gonorrhea infections [9, 10]. Interestingly, some countries reported almost 100% resistance of N. gonorrhoeae isolates to fluoroquinolones [11], and this high percentage of resistance has not diminished appreciably. In other countries, despite some diminution of total percentages of resistant isolates, fluoroquinolone resistance persists at levels higher than 5% despite the withdrawal of these antimicrobials for the treatment of gonorrhea [11, 12]. The withdrawal of antibiotics can result in some modest diminution of resistance levels, possibly because antibiotic resistant isolates may have less survival advantage, or be less fit, than susceptible isolates [13]. However, given the maintenance of high percentages of ciprofloxacin resistant N. gonorrhoeae, it seems that, in some circumstances, antibioticresistant N. gonorrhoeae isolates may have a fitness advantage and not a fitness deficit. Bacterial fitness has been broadly described as the ability of a particular strain to survive and reproduce [13]. Fitness, often measured by growth rate and generation time, is frequently adversely affected by the development of antibiotic resistance. Most models used for bacterial fitness analysis involve in vitro and in vivo competition assays [13], and a mathematical model with high predictive value that associates fitness costs with drug-resistant bacteria has been reported [14]. Overall, the challenge in measuring bacterial fitness in antimicrobial environments is to choose a set of assays in the context of different bacterial environmental niches, genetic Received 5 September 2011; accepted 16 December 2011; electronically published 5 April 2012. Correspondence: Jo-Anne R Dillon, PhD, Professor, Dept of Biology, and Research Scientist, Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Rd, Saskatoon, Saskatchewan, Canada S7N-5E3 ( j.dillon@usask.ca). The Journal of Infectious Diseases 2012;205:1775–7 © The Author 2012. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@ oup.com. DOI: 10.1093/infdis/jis281

OBJECTIVES: We aimed to identify demographic and behavioural determinants associated with risk of repeat STI infection and coinfection with gonorrhea and chlamydia in the Regina Qu’Appelle Health Region, Saskatchewan. METHODS: We extracted data from a cross-sectional sample of laboratory confirmed gonorrhea cases between 2003 and 2012 from the notifiable disease files of the Regina Qu’Appelle Health Region. Risk factors for repeater status were examined using logistic regression and for coinfection with gonorrhea and chlamydia using mixed-effects logistic regression to account for multiple diagnoses for individual clients. RESULTS: Data from 1,143 cases (representing 1,027 unique individuals) and 1,524 reported contacts (representing 1,383 unique individuals) were extracted from the 10-year period. Factors associated with repeat infection entries in the database included younger age at first visit ( p = 0.01), coinfection ( p = 0.01), and sex trade involvement ( p < 0.01). Factors associated with coinfection at the time of diagnosis included younger age at diagnosis ( p < 0.001) and reported alcohol or drug abuse ( p = 0.04). CONCLUSION: In one of the first epidemiologic studies on gonorrhea in Saskatchewan, we have identified age, engagement in the sex trade, and drug and alcohol abuse as potential markers to identify clients with a high risk of reinfection and coinfection in the Regina Qu’Appelle Health Region. This information can help health care professionals in Saskatchewan’s urban centres personalize their approach to counselling and treatment to optimize patient outcomes and disease control efforts, including potentially using expedited partner therapy and/or dual therapy where indicated.

Neisseria gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted infection (STI) of major importance. As a result of antibiotic resistance, there are now limited options for treating patients. We collected draft genome sequence data and associated metadata data on 76 N. gonorrhoeae strains from around the globe and searched for known determinants of antibiotics resistance within the strains. The population structure and evolutionary forces within the pathogen population were analyzed. Our results indicated a cosmopolitan gonoccocal population mainly made up of five subgroups. The estimated ratio of recombination to mutation (r/m = 2.2) from our data set indicates an appreciable level of recombination occurring in the population. Strains with resistance phenotypes to more recent antibiotics (azithromycin and cefixime) were mostly found in two of the five population subgroups.

Min proteins are involved in the correct placement of division septa in many bacterial species. In Escherichia coli (Ec) cells, these proteins oscillate from pole to pole, ostensibly to prevent unwanted polar septation. Here, we show that Min proteins from the coccus Neisseria gonorrhoeae (Ng) also oscillate in E. coli. Green fluorescent protein (GFP) fusions to gonococcal MinD and MinE localized dynamically in different E. coli backgrounds. GFP-MinD N g moved from pole to pole in rod-shaped E. coil cells with a 70 ′ 25 s localization cycle when MinE N g was expressed in cis. The oscillation time of GFP-MinD N g was reduced when wild-type MinE N g was replaced with MinE N g carrying a R30D mutation, but lengthened by 15 s when activated by MinE E c . Several mutations in the N-terminal domain of MinD N g , including K16Q and 4-and 19-amino acid truncations, prevented oscillation; these MinD N g mutants showed decreased or lost interaction with themselves and MinE N g . Like MinE E c -GFP, MinE N g -GFP formed MinE rings and oscillated in E. coli cells when MinD E c was expressed in cis. Finally, in round E. coli cells, GFP-MinD N g appeared to move in a plane parallel to completed septa. This pattern of movement is predicted to be similar in gonococcal cells, which also divide in alternating perpendicular planes.

Neisseria gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted infection (STI) of major importance. As a result of antibiotic resistance, there are now limited options for treating patients. We collected whole genome sequences and associated metadata data on 76 N. gonorrhoeae strains from around the globe and searched for known determinants of antibiotics resistance within the strains. The population structure and evolutionary forces within the pathogen population were analyzed. Our results indicated a cosmopolitan gonoccocal population mainly made up of five subgroups. The estimated ratio of recombination to mutation (r/m=2.2) from our data set indicates an appreciable level of recombination occurring in the population. Strains with resistance phenotypes to more recent antibiotics (azithromycin and cefixime) were mostly found in two of the five population subgroups.

Objectives To determine the effects of using discrete versus continuous quantities of people in a compartmental model examining the contribution of antimicrobial resistance (AMR) to rebound in the prevalence of gonorrhoea. Methods A previously published transmission model was reconfigured to represent the occurrence of gonorrhoea in discrete persons, rather than allowing fractions of infected individuals during simulations. Results In the revised model, prevalence only rebounded under scenarios reproduced from the original paper when AMR occurrence was increased by 10 5 times. In such situations, treatment of high-risk individuals yielded outcomes very similar to those resulting from treatment of low-risk and intermediate-risk individuals. Otherwise, in contrast with the original model, prevalence was the lowest when the high-risk group was treated, supporting the current policy of targeting treatment to high-risk groups. Conclusions Simulation models can be highly sensitive to structural features. Small differences in structure and parameters can substantially influence predicted outcomes and policy prescriptions, and must be carefully considered.

Using crude whole-genome assemblies, we analyzed 25 isolates of Neisseria gonorrhoeae by using a high-resolution single nucleotide polymorphism (SNP) approach for nine housekeeping genes, characterizing penA alleles, and antimicrobial susceptibility phenotypes coupled with population structure analysis. Two clonal complexes, characterized by their spatial and geographical persistence, were identified. In addition, the clonal spread of penicillin-resistant/intermediate phenotypes and a novel introduction of the azithromycin resistance phenotype in Saskatchewan, Canada, were ascertained using this method.

Mitigating the Emergence and Spread of Multidrug- and Extensively Drug-Resistant Gonorrhea : Is There Sufficient Support in Resource-Poor Settings in Africa?

The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae (MinD Ng ) in this mutant. Hence, MinD Ng is required for maintaining proper cell division and growth in N. gonorrhoeae . Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinD Ng is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinC Ng and MinD Ng from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinC Ng or MinD Ng alone did not display noticeable morphological changes. These studies suggest that MinD Ng is involved in inhibiting gonococcal cell division, likely in conjunction with MinC Ng . The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinD Ng in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinD Ng revealed a novel MinD Ng self-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinD Ng . These results indicate that MinD Ng is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.

The activity of the promoter regions of the cell division genes ftsZ, ftsE, minC, minD and minE from Neisseria gonorrhoeae (Ng) was studied under different environmental conditions using lacZ translational fusions. The promoters of the min Ng genes have not been previously determined and we identified promoter regions upstream of each gene ( minC p, minD p and minE p). We determined that minD p had the strongest activity. Expression of the promoter regions of ftsZ Ng and ftsE Ng , which we had previously identified, as well as minD Ng , were then studied under conditions reflecting the environment of the genitourinary tract. These conditions included anaerobiosis, presence of isoleucine or urea (3 mM and 400 mM, respectively) and acidity of pH 6. Both β -galactosidase expression and northern blot analysis indicated that all three genes were upregulated under anaerobiosis. The addition of isoleucine as well as media at pH 6 did not have any significant effects on the promoter activity of these genes while the presence of urea significantly decreased ftsZ Ng promoter activity. The expression of the minD Ng promoter region was analyzed during different growth phases and shown to follow the growth behavior of the culture. By contrast, the ftsZ Ng promoter activity continued to rise after the onset of the stationary phase. When gonococcal ftsZ promoter 1 (Pz1) was altered by site-directed mutagenesis, a significant decrease in the expression of ftsZ Ng was observed under both aerobic and anaerobic conditions. These data infer that gonococci regulate their cell division in response to different environments.

An origin of replication (ori) was obtained from a naturally occurring β-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.

The exact nature of the sequence differences between the medically important family of gonococcal penicillinase-producing plasmids has been ascertained. The entire DNA sequence of the Asia-type plasmid, pJD4, demonstrated that it is 7426 bp and contains two direct repeats (DR30) that are implicated in the formation of deletion variant plasmids, such as the Africa-type plasmid. We have identified putative DnaA and IHF binding sites, various open reading frames that are thought to specify functional proteins, and some important DNA sequences involved with conjugative transfer of gonococcal beta-lactamase plasmids. The deletion in the Africa-type plasmid is 1827 bp and one of the DR30 repeats is also missing. The deletion in the Rio-type plasmid and several Toronto-type plasmids was determined to be 2273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. TheNcirc;imes-type plasmid is an Africa-type plasmid and also contains an IS5 insertion sequence. Since IS5 has not been identified in gonococcal isolates, we suggest that this sequence may have been inserted after the original gonococcal plasmid was transformed into Escherichia coli. The New Zealand plasmid is an Asia-type plasmid that contains an endogenous tandem duplication of 1883 bp and the direct DR2 is implicated in this duplication. The nature of the defined truncation of Tn2 present in the various plasmids is also discussed.

One hundred Peptostreptococcus isolates from five species were assessed for their ability to hydrolyze gelatin. Most Peptostreptococcus magnus (95.8%) and Peptostreptococcus micros isolates (79.0%) hydrolyzed gelatin in contrast to Peptostreptococcus asaccharolyticus (8.0%), Peptostreptococcus anaerobius (10.0%), and Peptostreptococcus prevotii isolates (16.7%). Gelatin hydrolysis in Peptostreptococcus magnus and Peptostreptococcus micros isolates correlated (r = 0.80; P = 0.0019) with more aminopeptidases produced than Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, or Peptostreptococcus prevotii. The five species were further classified into three groups using the extended Tukey test (P < 0.0001) based on the mean percentage of aminopeptidases produced by each species with Peptostreptococcus magnus and Peptostreptococcus micros belonging to group I, Peptostreptococcus asaccharolyticus and Peptostreptococcus prevotii belonging to group II, and Peptostreptococcus anaerobius forming group III. An analysis of possible proteolytic activity of four selected Peptostreptococcus magnus isolates indicated that only 5 of 11 substrates were hydrolyzed as compared to a control isolate of Porphyromonas gingivalis W83, which had a strong proteolytic profile. Therefore, gelatin hydrolysis by Peptostreptococcus spp., in particular Peptostreptococcus magnus and Peptostreptococcus micros, is probably due to a variety of aminopeptidases rather than proteinases.Key words: Peptostreptococcus, aminopeptidases, proteolytic activity.

Globally, incidence of Neisseria gonorrhoeae infection is once again the highest of the bacterial sexually transmitted infections. The bacterium can produce serious complications in those infected, and emerging resistance to third generation cephalosporins could usher in an era of potentially untreatable gonorrhea. This research aimed to identify risk factors for antibiotic resistant gonorrhea infection among clients at a Shanghai sexually transmitted infection clinic over two time periods, 2004–2005 and 2008–2011. Demographic and risk factor behavior data, and biological samples for antimicrobial resistance analysis, were collected. Statistical models were built to identify risk factors associated with probable resistance to ceftriaxone and resistance to penicillin and tetracycline. High levels of ciprofloxacin resistance (98%) in our sample precluded examining its risk factors; all isolates were susceptible to spectinomycin. Overall (P

Protein L (L) and albumin-binding (A) proteins may be virulence factors ofPeptostreptococcus magnusisolates. When 32P. magnusisolates from different countries were screened for protein L gene sequences by the polymerase chain reaction (PCR), the only positive isolates were three which had been previously identified. Protein L-containing (L+A−) or albumin-binding isolates (L−A+) and unrelated isolates (L−A−) were analysed using three genotyping methods not previously used to distinguish strains with these putative virulence factors — PCR-restriction endonuclease analysis (REA) of 16 SrRNA, ribotyping, and macrorestriction endonuclease analysis of DNA by pulsed-field gel electrophoresis (PFGE). PFGE produced the greatest discrimination (ten banding patterns), followed by ribotyping (nine banding patterns) and PCR-REA of 16S rRNA amplicons (two banding patterns). Based on patterns from PFGE and ribotyping, cluster analysis showed that the three L+A−isolates were more closely related to each other than with other isolates. Six of eight L−A+isolates formed two indistinguishable groups of three isolates each, but these two groups were genetically distant from the other two unrelated L−A+isolates. L−A−isolates were not related, either among themselves, or to any other isolates. Thus, L+and A+phenotypes and genotyping methods (PFGE and ribotyping) are useful for differentiatingP. magnusisolates and identifying specific strain types.

SUMMARY The carbamoyl-phosphate synthase (CPS) enzyme in prokaryotes is a heterodimer, encoded by genes commonly called carA and carB. In most prokaryotes examined, these genes are separated by up to 24 bp and are cotranscribed. In Pseudomonas aeruginosa, carA and carB are also co-transcribed, but are separated by 682 bp. We have determined the complete DNA sequence of the carA and carB genes of Neisseria gonorrhoeae strain CH811. carA (1125 bp) and carB (3237 bp) are similar in size and sequence to other prokaryotic CPS genes, however they are separated by an intervening sequence of 3290 bp which has no similarity to the intervening sequence between other CPS genes; furthermore, putative transcription terminators are found downstream of both carA and carB. Several neisserial repetitive sequences were identified within the 9 kb sequenced, as well as novel 120 and 150 bp repeats (designated RS6 and RS7, respectively) which were found within the intervening sequence between carA and carB. To determine whether the intervening sequence observed in N. gonorrhoeae CH811 was not unusual, the sequence between carA and carB was amplified by PCR from 30 isolates of N. gonorrhoeae. The intervening sequence was found to vary in size, from approximately 2·2 to 3·7 kb, although the carA and carB genes themselves did not vary in size in isolates with functional CPS enzyme. A similar large, variably sized intervening sequence was also found between the carA and carB genes of 12 isolates of N. meningitidis and 18 commensal Neisseria isolates comprising nine species. This unexpected organization of the CPS genes in N. gonorrhoeae is therefore widespread throughout the genus Neisseria.

The emergence of resistance and treatment failures to third generation cephalosporins prompted the revitalization of the global Gonococcal Antimicrobial Surveillance Program (GASP) to ensure that information regarding trends of the antimicrobial susceptibility of Neisseria gonorrhoeae isolates is up-to-date. Accordingly, former and potential GASP participants in Latin America and the Caribbean were contacted to reinitiate the GASP network in the region and to undertake a retrospective analysis of the antimicrobial susceptibility of N. gonorrhoeae isolates between 2000 and 2009.Eleven countries participated in this retrospective analysis reporting on the susceptibility of N. gonorrhoeae isolates to up to 6 antibiotics as well as national treatment guidelines over the period. Antimicrobial susceptibility determination was carried out using combination of agar dilution and disk diffusion (Clinical Laboratory and Standards Institute) or Etest. Antimicrobial susceptibility data from each country were aggregated and analyzed for antimicrobial resistance trends in the region.More than 11,400 N. gonorrhoeae isolates were tested for antimicrobial susceptibility: 6 countries tested N. gonorrhoeae over the entire period and 5 countries tested sporadically. Decreased susceptibility to ceftriaxone was reported from 1 country (7 isolates, MICs0.25 μg/ml) in 2007. No resistance to spectinomycin was reported. From 2000 to 2009, aggregated ciprofloxacin resistance increased from 2% (19/784) to 31% (311/1015) in 9 countries and azithromycin resistance increased from 6% (39/646) to 23% (225/962) in 4/6 reporting countries. Overall, resistance to penicillin and tetracycline decreased from 35% (441/1241) to 26% (258/975) and from 60% (476/792) to 35% (323/931), respectively.In 2009, resistance to gentamicin (3%, 4/122), chloramphenicol (5%, 6/120), and ofloxacin (2%, 6/120) was reported from 1 country.The report of ceftriaxone-resistant isolates coupled with the emergence and spread of resistance to ciprofloxacin and azithromycin in Latin America and the Caribbean in the 2000s indicates the importance of active surveillance of N. gonorrhoeae antimicrobial susceptibility to determine antimicrobial resistance emerging trends so as to promptly inform and guide the development of effective treatment options for gonococcal infections.

In Neisseria gonorrhoeae, cytokinesis involves Escherichia coli homologues of minC, minD and minE which are encoded as part of a min operon. MinD, a 30 kD protein component of the MinCeMinD septum inhibitory complex, together with MinE, mediates cell division site selection. Gonococci mutated in minD display aberrant cytokinesis, abnormal morphology, defective microcolony formation and virulence. minD is 274 bp upstream of oxyR, another min operon gene in N. gonorrhoeae, which encodes a redox-responsive transcriptional regulator implicated in responses to oxidative stress. In this study, we aimed to examine the oxyR-mediated regulation of minD. We observed the cotranscription of oxyR with the minCDE gene cluster. The mutation of oxyR resulted in non-midline formation of the division septum, anomalous DNA segregation, and increased aggregation of bacterial cells. qRT-PCR and Western Blot analysis revealed upregulation of minD in an oxyR mutant as compared to its isogenic wild-type N. gonorrhoeae strain in stationary phase. Furthermore, the exposure to oxidative stress in the form of H2O2 increased MinD expression levels in wild-type N. gonorrhoeae. Using b-galactosidase activity-based promoter assays, we found that oxyR negatively regulates the promoter region (PminD) upstream of minD. Our results demonstrate the involvement of oxyR in cell division and minD expression in N. gonorrhoeae. Crown Copyright 2013 Published by Elsevier Masson SAS on behalf of Institut Pasteur. All rights reserved.

HE INCIDENCE OF GONORRHEA HAS BEEN DECLINING IN Canada and other industrialized countries: in 1981. 56.330 cases of gonorrhea were reported in Canada compared with 13,822 cases in 1990 (l). The reasons for this decline are unknown: however. educa­ tional programs for U1e primary prevention of acquired immune deficiency syndrome as well as effective therapy may have played a role. Persons aged 15 to 29 years accounted for more than 75% of a ll reported cases of gonon·hea with twice as many female cases in the ·under 20' age group. Females with gonorrhea are at increased risk for serious compli cations. such as pelvic inflammatory disease (P!D) which. in turn. may lead to sequelae such as infertility and ectopic pregnancy. Un ­ treated or ineffectively treated gonococcal infections are a significant cause of PID . Despite U1e decrease in the number of cases of gonorrhea. the prevalence of isolates resistant to anti ­ microbial agents used for treatment has steadily in ­ creased in Canada. This resistance is caused by eiU1 er chromosomal mutations or plasmids conferring anti­ biotic resistance. A number of chromosomal loci which produce phenotypes wiU1 reduced susceptibili ty to antimicrobia l agents have been described (2). Some chromosomal mutations are characterized by s imul­ taneous resistance to other unrelated antimicrobia l agents and some act in concert to produce step-wise. additive increases in resistance. Decreased antimicro­ bial susceptibility in gonococci caused by chromosomal

Objectives: To elucidate loci in Neisseria gonorrhoeae implicated in reduced susceptibility to ceftriaxone. Methods: N. gonorrhoeae isolates were collected in Shanghai, China, in 2005 and 2008. Twenty-eight isolates with reduced susceptibility to ceftriaxone (CRO Red ; MIC=0.12 5 -0.25 mg/L) were studied for mutations in PorB (porB), MtrR (mtrR), PBP2 (penA) and PBP1 (ponA). The mutation profiles of the 28 CRO Red isolates were compared with those of 32 ceftriaxone-susceptible isolates (CRO S ; MIC= 0 .0 04 - 0 . 01 6 mg/L). porB-based DNA sequence typing and N. gonorrhoeae multi-antigen sequence typing (NG-MAST) analyses were performed. Results: Significantly more CRO Red isolates (89.3%) exhibited a PIB phenotype as compared with the CRO S isolates (59.4%) (P=0.02). Double mutations (G45D/H105Y or A39T/H105Y) in MtrR were associated with CRO Red phenotypes. A 'wild-type' MtrR protein characterized CRO Red isolates (50.0%, 14/28), while a single H105Y mutation was observed only in CRO S isolates (43.8%, 14/32). Both CRO Red and CRO S isolates carried an '―A' deletion in the mtrR promoter. Six of 15 mutation patterns observed in PBP2 were new. Mutation patterns XIII (17.9% of CRO Red isolates) and XVII or XVIII (25.0% of CRO Red isolates) of PBP2 comprised A501V/G542S or A501V/P551S double mutations and were associated with a CRO Red phenotype. The mosaic PBP2 (pattern X) was not observed. The L421P mutation in PBP1 was observed in all CRO Red and in 97.0% of CRO S isolates. CRO Red isolates were non-clonal. Conclusions: Reduced susceptibility to ceftriaxone in N. gonorrhoeae is mediated by porB1b alleles and is associated with specific mutations in PBP2 and in the DNA binding and dimerization domains of MtrR.

Neisseria gonorrhoeae, a sexually-transmitted gram-negative bacterium, causes gonorrhoea in humans. The min genes of N. gonorrhoeae are involved in cell division site selection with oxyR co-transcribed with these genes. The mutation in min genes and oxy R cause aberrant cell morphology and aggregation patterns, respectively. Our objective was to assess the contribution of neisserial min operon cell division genes i.e. minC, minD and oxyR in virulence. Compared to the N. gonorrhoeae parental strain (Ng CH811Str(R)), its isogenic mutants with insertionally inactivated minC (Ng CSRC1), minD (Ng CJSD1) or oxyR (Ng KB1) showed reduced adherence to and invasion of urethral epithelial cells. This may be explained by defective microcolony formation in the mutant strains, possibly owing to abnormal morphology and aggregation. The expression levels of surface virulence factors like Opa, pilin and lipooligosaccharide in the mutants were unchanged relative to Ng CH811Str(R). Furthermore, in urethral epithelial cells, the min and oxyR mutants induced the release of proinflammatory cytokines like IL6 and IL8 to levels similar to that induced by the parental strain. Taken together, our studies indicate that inactivation of minC, minD or oxyR in N. gonorrhoeae attenuates its ability to bind to and invade urethral epithelial cells without altering its potential to induce IL6 and IL8 release.

MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first similar to 30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae, with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for full-length and N-terminal peptides from MinE demonstrated that residues that are buried in the dimeric interface nonetheless participate in direct interactions with MinD. According to results from NMR spin relaxation experiments, access to these buried residues may be facilitated by the presence of conformational exchange. We suggest that this concealment of MinD-binding residues by the MinE dimeric interface provides a mechanism for prevention of nonspecific interactions, particularly with the lipid membrane, to allow the free diffusion of MinE that is critical for Min protein oscillation. Original Publication:Houman Ghasriani, Thierry Ducat, Chris T Hart, Fatima Hafizi, Nina Chang, Ali Al-Baldawi, Saud H Ayed, Patrik Lundström, Jo-Anne R Dillon and Natalie K Goto, Appropriation of the MinD protein-interaction motif by the dimeric interface of the bacterial cell division regulator MinE, 2010, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (107), 43, 18416-18421.http://dx.doi.org/10.1073/pnas.1007141107Copyright: National Academy of Sciences; 1999http://www.nas.edu/

Background: The successful treatment of Neisseria gonorrhoeae (NG) infections is increasingly problematic because of the resistance of this pathogen to multiple antimicrobial agents. This development underscores the need for new antimicrobial sources. In the current study, 21 crude methanol extracts, from 19 plants used in Colombian traditional medicine for cutaneous infections, were screened for antimicrobial activity against NG. Methods: Extracts were screened by disc susceptibility assay. In addition, the minimum inhibitory concentrations of active compounds from P. lanceaefolium were assayed using a panel of 26 NG strains comprising 12 antibiotic-resistant phenotypes. Results: In all, 71% of the crude extracts exhibited antibacterial activity against the antibiotic susceptible NG strain WHO V, whereas 10% of the extracts inhibited penicillinase-producing NG strain GC1– 182. The crude extract of Piper lanceaefolium was the only extract to show significant activity without ultraviolet (UV) light activation. Preliminary screening identified 3 compounds in this plant possessing antimicrobial activity: the flavonoids 5,7-dihydroxyflavanone (pinocembrin), 2,4,6-trihydroxychalcone (pinocembrin chalcone), and the prenylated benzoic acid derivative cyclolanceaefolic acid methyl ester. Pinocembrin and pinocembrin chalcone inhibited 100% of the NG panel at 64 g/mL and 128 g/mL, respectively, whereas cyclolanceaefolic acid methyl ester inhibited 44% of the strains at 128 g/mL. Conclusions: This is the first report of the antibacterial activity of Columbian plants against NG. The activity of the 2 flavonoids, pinocembrin, and pinocembrin chalcone, toward both susceptible and resistant NG strains makes them promising candidates for further research.

Adherence to epithelial cells is a prerequisite for intestinal colonization by the bacterial pathogen, enterohemorrhagic Escherichia coli (EHEC). The deletion of minD, a cell division gene, in EHEC caused reduced adherence to human epithelioid cervical carcinoma (HeLa) and human colonic adenocarcinoma (Caco-2) cells as compared to wild-type. The minD mutant formed minicells and filaments owing to aberrant cytokinesis. Moreover, its ability to form microcolonies as typically seen in the co-cultures of wild-type with Caco-2 cells, was abolished. In conclusion, the present study highlights the importance of minD in regards to EHEC adherence to human epithelial cells.

porB DNA sequence analysis and Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) methods were compared for their abilities to discriminate strains and to identify epidemiologically congruent pairs of N. gonorrhoeae . Both methods provided high-level discrimination of strains. NG-MAST further differentiated large porB -based clusters. However, considerations of cost suggest that porB DNA sequence analysis is a useful tool for preliminary molecular analysis of the epidemiology of N. gonorrhoeae .

(i) To distinguish Neisseria gonorrhoeae isolates in Shanghai by porB typing; (ii) to ascertain the congruence of porB DNA sequence typing with cases linked epidemiologically; (iii) to determine the association of specific PorB mutations with antimicrobial resistance to penicillin or tetracycline.porB DNA sequences of 174 N. gonorrhoeae isolates, collected from 143 male patients and 31 female sexual partners in Shanghai were determined. Phylogenetic analysis was used to determine sequence associations and concordance with epidemiologically linked cases. PorB protein sequences were compared with the wild-type sequence to identify mutations associated with antimicrobial resistance to penicillin and tetracycline.porB1a genotypes comprised 27.0% of the isolates and included 15 distinct DNA sequences, while 73.0% of the isolates carried porB1b genotypes with 63 distinct DNA sequences. porB DNA sequence typing was congruent with patient-reported sexual contacts. In addition, porB DNA sequence analysis revealed a number of strains with identical DNA sequences not identified through traditional epidemiological methods. The porB1b isolates had a significantly higher percentage of chromosomally mediated resistance to tetracycline and higher MIC50s to penicillin and ciprofloxacin. G120K/A121D mutations were observed in 71.1% of PIB isolates and were associated with resistance to penicillin and/or tetracycline. The majority of the PIA isolates (82.1%) also carried G120D/A121G double mutations. The index of discrimination for porB DNA sequence analysis was 95%.The porB1b genotype was found to be predominant in Shanghai. porB DNA sequence typing was sufficiently discriminatory for differentiating N. gonorrhoeae isolates and was congruent with epidemiological linkages. Novel porB sequences of N. gonorrhoeae and novel mutations of PorB proteins were identified.

The threat of antimicrobial resistance (AMR) in bacteria has been escalated to a rightful seat on the global health agenda. In September 2016, for only the fourth time in United Nations (UN) history, the UN General Assembly in New York will meet to focus on a health threat – antimicrobial resistance. Other diseases afforded this level of consultation at the UN were human immunodeficiency virus (HIV), non-communicable diseases and Ebola virus. There are grim predictions for the future in terms of AMR and health security that span income settings. These predictions challenge the premise that minor bacterial infections of childhood are innocuous, and threaten to halt the medical advancements dependant on antibiotic therapy. Those with compromised immune systems, whether endogenous or induced, will be at highest risk. The development and spread of AMR has been, and will continue to be, fanned by the relentless selection pressure of exposure to antibiotics whether used appropriately, unnecessarily or suboptimally, in human health, animal health and agriculture. The distribution of antibiotic resistant bacteria is facilitated by travel and transport. Antimicrobial resistance will affect those in the community and the hospital.

Introduction Every year, Neisseria gonorrhoeae (Ng) causes 106 million new gonorrhoea infections worldwide and has recently joined the fast-expanding group of multi-drug resistant superbugs. Treatment is often empirical/delayed as culture-based antibiotic susceptibility tests in current use can take several days. A Point-of-Care Test (POCT) for antimicrobial susceptibility would change this; for example, no longer recommended antibiotics such as ciprofloxacin could be used in certain regions where high percentages of isolates remain susceptible. The objective of the present study was to develop and validate a portfolio of diagnostic primer pairs for application in a Nucleic Acid Amplification-based POCT for the concurrent diagnosis of Ng and ciprofloxacin susceptibility. Methods A bioinformatics analysis of >30,000 bacterial genomes identified 9 unique signature sequences specific to Ng FA1090 that were used as targets in a SYBR green-based qPCR assay. 9 diagnostic primer pairs were evaluated for specificity and sensitivity on 271 Ng and non-gonococcal isolates. Two primer pairs targeting ciprofloxacin resistance-conferring single nucleotide polymorphisms in gyrA were tested on 200 resistant and susceptible Ng isolates. qPCR was performed by an Applied Biosystems StepOnePLus TM qPCR system. Results Out of the 9 diagnostic primer pairs tested for Ng identification, one probe failed to detect 28 positive samples of Ng out of a total of 234 isolates. Another primer pair, which amplifies the DR9 repeat region used in the COBAS4800 was not able to detect one Ng isolate collected from Hong Kong. The remaining 7 primer pairs showed 100% specificity in terms of Ng detection and were highly sensitive in detecting Ng DNA in concentrations as low as 0.00001 ng/ul. A multiplexed assay using ciprofloxacin susceptibility-determining primer pairs distinctly differentiated between resistant and susceptible isolates based on melt curve analysis. Conclusion A POCT-adaptable assay has been developed for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status. Disclosure of interest statement The present work was supported by Grand Challenges Canada ((#S5 398). No grants were received from any company in the development of this study.

Neisseria gonorrhoeae isolates continue to develop an impressive arsenal of resistance mechanisms to antimicrobial agents, including resistance to some of the antibiotics presently recommended for the treatment of gonococcal infections.

Objectives: To determine the antimicrobial susceptibility of Neisseria gonorrhoeae from Shanghai and to type the quinolone resistance-determining regions (QRDRs) of ciprofloxacin-resistant isolates. Methods: N. gonorrhoeae isolates (n = 159) were consecutively collected from male patients in Shanghai and examined for their antimicrobial susceptibilities to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone. The mutation profiles of the QRDRs of gyrA and parC were determined for 103 isolates including one susceptible isolate and one isolate with intermediate levels of susceptibility to ciprofloxacin. Results: High percentages of the 159 isolates were resistant to ciprofloxacin (98.7%), penicillin (93.1%) and tetracycline (56.5%). Penicillinase-producing N. gonorrhoeae (PPNG, 37.8%) or penicillinase-producing/ tetracycline-resistant N. gonorrhoeae (PP/TRNG, 13.8%) accounted for 51.6% of the isolates. Chromosomal resistance to penicillin was observed in 41.5% of the isolates. Tetracycline resistance was noted in 56.5% of the isolates with 20.1% carrying plasmid-mediated resistance and 36.4% being chromosomally resistant. All isolates were susceptible to ceftriaxone and spectinomycin, although a trend to decreased susceptibility was noted. QRDR mutations were observed in the 101 ciprofloxacin-resistant isolates and the one ciprofloxacin-intermediate isolate, in contrast to the ciprofloxacin-susceptible isolate tested. Mutations in the QRDRs comprised four predominant (65.0% of the 103 isolates) patterns of a total of 19 patterns. Mutations in parC were significantly associated with higher MICs of ciprofloxacin. Conclusions: Spectinomycin and ceftriaxone are currently recommended for the treatment of gonorrhoea in Shanghai. Although the present study indicates that these antimicrobials should remain effective, the identification of isolates with decreased susceptibility underscores the importance of ongoing antimicrobial susceptibility surveillance to monitor and respond to the emergence of resistant isolates.