Genomic Analysis Reveals Antibiotic-Susceptible Clones and Emerging Resistance in Neisseria gonorrhoeae in Saskatchewan, Canada.

Whole-genome sequencing was used to identify mutations in antibiotic resistance-conferring genes to compare susceptibility predictions with MICs and to ascertain strain types in 99 isolates of Neisseria gonorrhoeae Genotypes associated with susceptibility, as well as MIC creep or emerging resistance, were noted. Phylogenomic analysis revealed three distinctive clades and putative gonococcal transmission linkages involving a tetracycline-resistant N. gonorrhoeae outbreak and the clonal spread of susceptible isolates in men.

A ß-lactamase-producing plasmid from Neisseria gonorrhoeae carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDa TEM-1 penicillinase that hydrolyses ampicillin slowly.

BACKGROUND: Seven structurally related ß-lactamase-producing plasmids have been characterized in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates. We characterized a variant (i.e. pJRD20, Canada type) of the Africa-type (pJD5) plasmid isolated from N. gonorrhoeae strain 8903. OBJECTIVES: To compare the DNA sequence of pJRD20 with that of pJD5 and pJD4 (Asia-type) and their TEM-1 ß-lactamases. METHODS: N. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. ß-Lactamase production was assessed using nitrocefin. MICs were determined by agar dilution and Etest methods (CLSI). The DNA sequences of pJRD20, pJD5 and pJD4 were assembled and annotated. The structure of TEM-1 and its penicillin-binding properties were determined by in silico molecular modelling and docking. TEM-1 proteins were characterized by western blot, mass spectrometry and ampicillin hydrolysis assays. RESULTS: N. gonorrhoeae 8903 exhibited intermediate susceptibility to penicillin with slow ß-lactamase activity (i.e. 35 min to hydrolyse nitrocefin). Except for a novel 6 bp deletion starting at the G of the ATG start codon of blaTEM-1, the DNA sequence of pJRD20 was identical to that of pJD5. The TEM-1 ß-lactamase produced by pJRD20 is 24 kDa and hydrolyses ampicillin only after several hours. CONCLUSIONS: This unusual PPNG isolate might have been characterized as a non-PPNG owing to its low MIC of penicillin and its very slow hydrolysis of nitrocefin. Given the unusual nature of its TEM-1 ß-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays.

Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status.

A real-time PCR (RT-PCR) assay was designed for the simultaneous identification of Neisseria gonorrhoeae and its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) in gyrA of N. gonorrhoeae The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254 N. gonorrhoeae isolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeae species isolates. The performance of the primers was validated using genomic DNA from 100 different N. gonorrhoeae isolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeae isolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of the N. gonorrhoeae isolates and 23 non-N. gonorrhoeae isolates. These primers detected N. gonorrhoeae and its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status.

Purification of the Bacterial Amyloid "Curli" from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues.

Microbiologists are learning to appreciate the importance of "functional amyloids" that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic Salmonella enterica and Escherichia coli strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from S. enterica, acknowledging that the same method can be applied to E. coli. In addition, we describe methods for detection of curli within animal tissues (i.e., GI tract) and discuss purifying curli intermediates in a S. enterica msbB mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.

A GMMA-CPS-Based Vaccine for Non-Typhoidal Salmonella.

Non-typhoidal Salmonella are a major cause of gastroenteritis worldwide, as well as causing bloodstream infections in sub-Saharan Africa with a high fatality rate. No vaccine is currently available for human use. Current vaccine development strategies are focused on capsular polysaccharides (CPS) present on the surface of non-typhoidal Salmonella. This study aimed to boost the amount of CPS purified from S. Typhimurium for immunization trials. Random mutagenesis with Tn10 transposon increased the production of CPS colanic acid, by 10-fold compared to wildtype. Immunization with colanic acid or colanic acid conjugated to truncated glycoprotein D or inactivated diphtheria toxin did not induce a protective immune response in mice. However, immunization with Generalized Modules for Membrane Antigens (GMMAs) isolated from colanic acid overproducing isolates reduced Salmonella colonization in mice. Our results support the development of a GMMA-CPS-based vaccine against non-typhoidal Salmonella.

Characterization of antimicrobial resistance genes from Neisseria gonorrhoeae positive remnant Aptima urine specimens.

Aim: To ascertain the antimicrobial resistance and strain types (STs) of Neisseria gonorrhoeae from 50 remnant Aptima urine specimens using molecular methods. Methods: Mutations predictive of resistance to six antibiotics were identified in eight genes. STs were determined using NG-MAST and NG-STAR. Results: All eight antimicrobial resistance genes could be characterized in 36 specimens. A total of 17 specimens were predicted to be susceptible to all antibiotics, including ceftriaxone. Decreased susceptibility to cefixime and ciprofloxacin resistance was predicted in 11 specimens (PBP2 type 34.001). Overall, 38/50 specimens were predicted to be ciprofloxacin susceptible; three were azithromycin resistant. Nineteen NG-MAST and 21 NG-STAR STs were noted. Conclusion: Molecular analysis of remnant Aptima specimens enabled the prediction of emerging gonococcal cefixime and azithromycin resistance which would otherwise have been undetected.

Evaluation of a Hydrogel-Based Diagnostic Approach for the Point-of-Care Based Detection of Neisseria gonorrhoeae.

Eleven primer pairs were developed for the identification of Neisseria gonorrhoeae. The sensitivity and specificity of these primers were evaluated by Real Time (RT)-PCR melt curve analyses with DNA from 145 N. gonorrhoeae isolates and 40 other Neisseria or non-Neisseria species. Three primer pairs were further evaluated in a hydrogel-based RT-PCR detection platform, using DNA extracted from 50 N. gonorrhoeae cultures. We observed 100% sensitivity and specificity in the hydrogel assay, confirming its potential as a point-of-care test (POCT) for N. gonorrhoeae diagnosis.