Development of Resistance of Gonococci to Penicillin: An Eight-Year Study

During the last eight years, 5700 strains of Neisseria gonorrhoeae have been isolated and tested for sensitivity to penicillin and sulfadiazine in the Public Health Laboratory (Toronto). At the beginning of the study 63% of the strains tested were sensitive to a concentration of 0.01 unit of penicillin per ml. of diluent. Since then the gonococcus has gradually developed resistance to this antibiotic until 27% of the strains isolated are now resistant to a concentration of 0.3 unit/ml., and 8% are resistant to 1.0 unit/ml. To overcome this degree of resistance it is necessary to give a soluble penicillin preparation intramuscularly in very high dosage (2-8 million units). At the present time there is an urgent need for a satisfactory substitute for penicillin in the treatment of gonorrhea. None has yet been found.     

Antibiotic sensitivities of Neisseria gonorrhoeae in the Toronto area

The antibiotic sensitivity pattern of 3872 isolates of N. gonorrhoeae tested in Toronto from 1969 to 1973 is reviewed. An increase in resistance to both penicillin and tetracycline was noted up to 1971, but no further increase has occurred since then. Ninety-seven percent of 135 patients with “sensitive” strains (inhibited by 0.3 U/ml of penicillin and/or 0.5 μg/ml of tetracycline) were cured by either 8 g of tetracycline or 5,000,000 U of penicillin, whereas only 59% of 58 patients with “resistant” strains (requiring 1.0 U/ml of penicillin and/or 2.0 μg/ml of tetracycline for inhibition) were cured by the same dosage. Spectinomycin appears to be an acceptable alternative therapy. Maximum doses of the chosen drug are recommended in the hope of retarding further spread of more resistant organisms.     

Prevalence of and risk factors for quinolone-resistant Neisseria gonorrhoeae infection in Ontario

Background

Quinolone-resistant Neisseria gonorrhoeae has swiftly emerged in Canada. We sought to determine its prevalence in the province of Ontario and to investigate risk factors for quinolone-resistant N. gonorrhoeae infection in a Canadian setting.

Methods

We used records from the Public Health Laboratory of the Ontario Agency for Health Protection and Promotion in Toronto, Ontario, and the National Microbiology Laboratory in Winnipeg, Manitoba, to generate epidemic curves for N. gonorrhoeae infection. We extracted limited demographic data from 2006 quinolone-resistant N. gonorrhoeae isolates and from a random sample of quinolone-susceptible isolates. We also extracted minimum inhibitory concentrations for commonly tested antibiotics.

Results

Between 2002 and 2006, the number of N. gonorrhoeae infections detected by culture decreased by 26% and the number of cases detected by nucleic acid amplification testing increased 6-fold. The proportion of N. gonorrhoeae isolates with resistance to quinolones increased from 4% to 28% over the same period. Analysis of 695 quinolone-resistant N. gonorrhoeae isolates and 688 quinolone-susceptible control isolates from 2006 showed a higher proportion of men (odds ratio [OR] 3.1, 95% confidence interval [CI] 2.3–4.1) and patients over 30 years of age (OR 3.1, 95% CI 2.4–3.8) in the quinolone-resistant group. The proportion of men who have sex with men appeared to be relatively similar in both groups (OR 1.4, 95% CI 1.1–1.8). Quinolone-resistant strains were more resistant to penicillin (p < 0.001), tetracycline (p < 0.001) and erythromycin (p < 0.001). All isolates were susceptible to cefixime, ceftriaxone, azithromycin and spectinomycin.

Interpretation

During 2006 in Ontario, 28% of N. gonorrhoeae isolates were resistant to quinolones. Infections in heterosexual men appear to have contributed significantly to the quinolone resistance rate. Medical practitioners should be aware of the widespread prevalence of quinolone-resistant N. gonorrhoeae and avoid quinolone use for empiric therapy.After declining for a number of years, Neisseria gonorrhoeae infections are once more on the rise in Canada. Between 1997 and 2007, reported incidence of the disease more than doubled, from 15 to 35 cases per 100 000.¹ To address the emergence of quinolone-resistant N. gonorrhoeae strains, the empiric treatment regimens for N. gonorrhoeae infection were recently revised in the 2006 Canadian Guidelines on Sexually Transmitted Infections.^2,3 Quinolones are no longer recommended for empiric therapy for N. gonorrhoeae infection.³In Canada, quinolone resistance in N. gonorrhoeae isolates increased from an estimated 2% in 2001 to 16% in 2005.⁴ Demographic risk factors for quinolone-resistant N. gonorrhoeae infection have not been studied. American studies have associated quinolone-resistant N. gonorrhoeae infection with men who have sex with men,^5,6 antibiotic use,^5,7 age above 35 years,⁵ HIV infection⁵ and travel to Asia.⁶ Public health data from the provinces of Quebec⁸ and Alberta² have also suggested an association between quinolone-resistant infection and men who have sex with men. In this study we generated epidemic curves for N. gonorrhoeae and quinolone-resistant N. gonorrhoeae infection in the province of Ontario. We also investigated demographic risk factors for quinolone-resistant N. gonorrhoeae infection.     

High-Throughput Screening for Novel Inhibitors of Neisseria gonorrhoeae Penicillin-Binding Protein 2

The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents.     

Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species

Summary The two pathogenic species of Neisseria, N. meningitidis and N. gonorrhoeae, have evolved resistance to penicillin by alterations in chromosomal genes encoding the high molecular weight penicillin-binding proteins, or PBPs. The PBP 2 gene (penA) has been sequenced from over 20 Neisseria isolates, including susceptible and resistant strains of the two pathogenic species, and five human commensal species. The genes from penicillin-susceptible strains of N. meningitidis and N. gonorrhoeae are very uniform, whereas those from penicillin-resistant strains consist of a mosaic of regions resembling those in susceptible strains of the same species, interspersed with regions resembling those in one, or in some cases, two of the commensal species. The mosaic structure is interpreted as having arisen from the horizontal transfer, by genetic transformation, of blocks of DNA, usually of a few hundred base pairs. The commensal species identified as donors in these interspecies recombinational events (N. flavescens and N. cinerea) are intrinsically more resistant to penicillin than typical isolates of the pathogenic species. Transformation has apparently provided N. meningitidis and N. gonorrhoeae with a mechanism by which they can obtain increased resistance to penicillin by replacing their penA genes (or the relevant parts of them) with the penA genes of related species that fortuitously produce forms of PBP 2 that are less susceptible to inhibition by the antibiotic. The ends of the diverged blocks of DNA in the penA genes of different penicillin-resistant strains are located at the same position more often than would be the case if they represent independent crossovers at random points along the gene. Some of these common crossover points may represent common ancestry, but reasons are given for thinking that some may represent independent events occurring at recombinational hotspots. Offprint requests to: B.G. Spratt     

Treatment of gonorrheal urethritis with spectinomycin hydrochloride

One hundred and eighty-seven males with uncomplicated gonorrheal urethritis were treated with spectinomycin hydrochloride in a dosage of 2 g. given intramuscularly. A failure rate of 3.2% was observed and no complications of therapy were encountered. In addition, 310 strains of N. gonorrhoeae were tested for susceptibility to penicillin G and spectinomycin. All strains were sensitive to 20 μg./ml. of spectinomycin and this susceptibility appeared to decrease as penicillin resistance increased. A greater incidence of relative resistance to penicillin G was observed than in similar studies from other Canadian areas.     

Phase Variation Leads to the Misidentification of a Neisseria Gonorrhoeae Virulence Gene

Neisseria gonorrhoeae is the causative agent of gonorrhea and an obligate pathogen of humans. The Opa proteins of these bacteria are known to mediate attachment and internalization by host cells, including neutrophils. The Opa protein repertoire of a typical N. gonorrhoeae isolate is encoded on ∼11 genes distributed throughout the chromosome and is subject to stochastic changes in expression through phase variation. Together, these characteristics make Opa proteins a critical yet unpredictable aspect of any experimental investigation into the interaction of N. gonorrhoeae with host cells. The goal of this study was to identify novel virulence factors of N. gonorrhoeae by assessing the contribution of a set of uncharacterized hydrogen peroxide-induced genes to bacterial survival against neutrophil-mediated killing. To this end, a strain harboring an engineered mutation in the NGO0322 gene was identified that exhibited increased sensitivity to neutrophil-mediated killing, enhanced internalization by neutrophils, and the ability to induce high levels of neutrophil-generated reactive oxygen species. Each of these phenotypes reverted to near wild-type levels following genetic complementation of the NGO0322 mutation. However, after immunoblot analysis of Opa proteins expressed by the isogenic parent, mutant, and genetically complemented strains, it was determined that phase variation had resulted in a disparity between the Opa profiles of these strains. To determine whether Opa phase variation, rather than NGO0322 mutation, was the cause of the observed neutrophil-related phenotypes, NGO0322 function was investigated in N. gonorrhoeae strains lacking all Opa proteins or constitutively expressing the OpaD variant. In both cases, mutation of NGO0322 did not alter survival of gonococci in the presence of neutrophils. These results demonstrate the importance of controlling for the frequent and random variation in Opa protein production by N. gonorrhoeae when investigating host cell interactions.     

Susceptibility of Neisseria gonorrhoeae to seven antibiotics in vitro

One hundred consecutive isolates of N. gonorrhoeae were tested for susceptibility to penicillin, ampicillin, tetracycline, erythromycin, kanamycin, cephaloridine and cephalexin by an agar dilution method. Relative resistance to penicillin was frequent. For 39% of isolates the minimum inhibitory concentration (MIC) of penicillin was 0.05 U./ml. or less; in 55% the MIC was 0.5 to 2.0 U./ml. Ampicillin was slightly more active than penicillin G: all isolates were inhibited by 0.5μg./ml. or less. Resistance to tetracycline and erythromycin was frequent with MIC of 1 μg./ml. or greater observed in 32 and 24% of isolates respectively. The MIC of kanamycin for all gonococci was 8 μg./ml. or greater. Cephalexin was slightly more active than cephaloridine, though each drug exhibited a wide range of MIC values. Gonococcus isolates resistant to penicillin (MIC of 1.0 U./ml. or greater) tended to be resistant to the other antibiotics tested.     

Emerging azithromycin-resistance among the <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> strains isolated in Hungary

Background

In the 1990s, azithromycin became the drug of choice for many infectious diseases but emerging resistance to the drug has only been reported in the last decade. In the last 5 years, the National Neisseria gonorrhoeae Reference Laboratory of Hungary (NNGRLH) has also observed an increased number of N. gonorrhoeae strains resistant to azithromycin. The aim of this study was to determine the most frequent sequence types (ST) of N. gonorrhoeae related to elevated levels of azithromycin MIC (minimal inhibitory concentration). Previously and currently isolated azithromycin-resistant strains have been investigated for the existence of molecular relationship.

Methods

Maldi-Tof technic was applied for the identification of the strains isolated from outpatients attending the reference laboratory. Testing antibiotic susceptibility of azithromycin, cefixime, ceftriaxone, tetracycline, spectinomycin and ciprofloxacin was carried out for all the identified strains, using MIC strip test Liofilchem^®. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed exclusively on azithromycin-resistant isolates. A phylogenetic tree was drawn using MEGA6 (Molecular Evolutionary Genetics Analysis Version 6.0) Neighbour-Joining method.

Results

Out of 192 N. gonorrhoeae isolates, 30.0 % (58/192) proved resistant to azithromycin (MIC > 0.5 mg/L). Of the azithromycin-resistant isolates, ST1407, ST4995 and ST11064 were the most prevalent. Based on the phylogenetic analysis, the latter two STs are closely related.

Conclusions

In contrast to West-European countries, in our region, resistance to azithromycin has increased up to 30 % in the last 5 years, so the recommendation of the European Guideline ?500 mg of ceftriaxone combined with 2 g of azithromycin as first choice therapy against N. gonorrhoeae- should be seriously considered in case of Hungary.

     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

Arterial Hypertension and Ischemic Heart Disease: Comparison in Epidemiological Studies

Forty cases of gonococcal urethritis were treated with oxytetracycline using various dosage schedules; there were 37 cures and three failures. The most convenient and most effective dosage was found to be 250 mg. oxytetracycline, given as a single intramuscular injection of 5 c.c.A series of 40 patients with non-gono-coccal urethritis was also collected. Two cases of urethritis due to Trichomonas vaginalis and two due to Candida albicans were removed from the series. Of the 36 cases which remained, cure was obtained with the use of oxytetracycline in different dosages in 30 cases; six cases were failures. The dosage which gave the best result in the therapy of non-gonococcal urethritis was 250 mg. oxytetracycline (5 c.c.), given as a single intramuscular injection, plus 250 mg. orally, four times a day for four days.The effectiveness of oxytetracycline in the treatment of urethritis has not decreased.     

Microarray genomotyping of key experimental strains of Neisseria gonorrhoeae reveals gene complement diversity and five new neisserial genes associated with Minimal Mobile Elements.

Background  

There are four widely used experimental strains of N. gonorrhoeae, one of which has been sequenced and used as the basis for the construction of a multi-strain, mutli-species pan-neisserial microarray. Although the N. gonorrhoeae population structure is thought to be less diverse than N. meningitidis, there are some recognized gene-complement differences between strains, including the 59 genes of the Gonococcal Genetic Island. In this study we have investigated the three experimental strains that have not been sequenced to determine the extent and nature of their similarities and differences.     

Characterization of DNA restriction and modification activities in Neisseria species

Type II restriction endonuclease activities detected in various Neisseria species were characterized for sequence specificity and precise site of cleavage. NsiCI isolated from N. sicca C351 cleaves the sequence 5′-GAT↓ATC-3′ (EcoRV isoschizomer); NmeCI from N. meningitidis C114 and NphI from N. pharyngis C245 cleave 5′-N↓GATCN-3′ (MboI isoschizomers); NgoPII and NgoPIII from N. gonorrhoeae P9-2 cleave at 5′-CC↓GCGG-3′ (SacII isoschizomer) and 5′-GG↓CC-3′ (HaeIII isoschizomer), respectively. Chromosomal DNA isolated from these strains and two other N. meningitidis strains (which lacked detectable endonuclease activities), was found to be refractive to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases.     

Trends in antibiotic resistance in Prevotella species from patients of the University Hospital of Maxillofacial Surgery,Sofia, Bulgaria,in 2003–2009

Head-and-neck infections often involve anaerobes such as Prevotella species. Aim of the present study was to assess the evolution and the factors associated with resistance in Prevotella species to penicillin, clindamycin, metronidazole, tetracycline and β-lactams/β-lactamase inhibitors (BL/BLIs). In total, 192 Prevotella strains, isolated from patients with oral and head-and-neck infections, were evaluated. Common isolates were Prevotella intermedia and Prevotella melaninogenica within the pigmented species as well as Prevotella oris and Prevotella oralis group within the non-pigmented species. Overall resistance was 43.2% for penicillin, 10.9% for clindamycin, 0% for metronidazole. Nonsusceptibility to tetracycline was 29.1% without significant differences in resistance rates between pigmented and other species. Penicillin resistant strains were β-lactamase positive. From 2003–2004 to 2007–2009, penicillin resistance rates increased about four-fold (from 15.4% to 60.6%). Clindamycin resistance did not show evolution, whereas tetracycline nonsusceptibility decreased from 43.3% in 2003–2004 to 20.7% in 2007–2009. Except for one (0.5%) P. oralis strain with intermediate susceptibility to BL/BLIs, the other strains were susceptible to the agents. In conclusion, in Prevotella strains from patients with head-and-neck infections, the resistance rate to penicillin increased, that to clindamycin remained stable and the nonsusceptibility rate to tetracycline decreased during the period. Activity against >99% of Prevotella strains was observed with metronidazole and BL/BLIs. The penicillin resistance and tetracycline nonsusceptibility were associated with the year of study, national antibiotic consumption and possibly with previous treatment (for tetracycline). The evolution of penicillin resistance in Prevotella strains was highly dynamic.     

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae

Resistance to penicillin in non-β-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42–581) into an ATG expression vector. When the recombinant PBP 2 molecules were over-expressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2′ (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2′ for [³H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other β-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.     

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.     

Genetic Studies of Sulfadiazine-resistant and Methionine-requiring Neisseria Isolated From Clinical Material

Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met⁺) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met⁺). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met⁻). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B₁₂, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met⁺ and Sul-r/Met⁻ loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met⁻ properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met⁻ clones tested against recipients having nonidentical Sul-r/Met⁻ mutant sites yielded Sul-s Met⁺ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species.