Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate of Neisseria gonorrhoeae Possessing a Mosaic-Like mtr Efflux Pump Locus.

Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae, likely originating from commensal Neisseria sp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018, https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.

Expansion of a urethritis-associated Neisseria meningitidis clade in the United States with concurrent acquisition of N. gonorrhoeae alleles.

BACKGROUND: Increased reports of Neisseria meningitidis urethritis in multiple U.S. cities during 2015 have been attributed to the emergence of a novel clade of nongroupable N. meningitidis within the ST-11 clonal complex, the "U.S. NmNG urethritis clade". Genetic recombination with N. gonorrhoeae has been proposed to enable efficient sexual transmission by this clade. To understand the evolutionary origin and diversification of the U.S. NmNG urethritis clade, whole-genome phylogenetic analysis was performed to identify its members among the N. meningitidis strain collection from the Centers for Disease Control and Prevention, including 209 urogenital and rectal N. meningitidis isolates submitted by U.S. public health departments in eleven states starting in 2015. RESULTS: The earliest representatives of the U.S. NmNG urethritis clade were identified from cases of invasive disease that occurred in 2013. Among 209 urogenital and rectal isolates submitted from January 2015 to September 2016, the clade accounted for 189/198 male urogenital isolates, 3/4 female urogenital isolates, and 1/7 rectal isolates. In total, members of the clade were isolated in thirteen states between 2013 and 2016, which evolved from a common ancestor that likely existed during 2011. The ancestor contained N. gonorrhoeae-like alleles in three regions of its genome, two of which may facilitate nitrite-dependent anaerobic growth during colonization of urogenital sites. Additional gonococcal-like alleles were acquired as the clade diversified. Notably, one isolate contained a sequence associated with azithromycin resistance in N. gonorrhoeae, but no other gonococcal antimicrobial resistance determinants were detected. CONCLUSIONS: Interspecies genetic recombination contributed to the early evolution and subsequent diversification of the U.S. NmNG urethritis clade. Ongoing acquisition of N. gonorrhoeae alleles by the U.S. NmNG urethritis clade may facilitate the expansion of its ecological niche while also increasing the frequency with which it causes urethritis.

A Case of Decreased Susceptibility to Ceftriaxone in Neisseria gonorrhoeae in the Absence of a Mosaic Penicillin-Binding Protein 2 (penA) Allele.

We report a case of Neisseria gonorrhoeae with a non-mosaic penA allele that exhibited decreased susceptibility to extended-spectrum cephalosporins, including a ceftriaxone minimum inhibitory concentration of 0.5 µg/mL. An analysis of resistance determinants suggested that the observed phenotype might have resulted from the combined effects of mutations in multiple genes.

Large Cluster of Neisseria meningitidis Urethritis in Columbus, Ohio, 2015.

Background: Neisseria meningitidis (Nm) is a Gram-negative diplococcus that normally colonizes the nasopharynx and rarely infects the urogenital tract. On Gram stain of urethral exudates, Nm can be misidentified as the more common sexually transmitted pathogen Neisseria gonorrhoeae. Methods: In response to a large increase in cases of Nm urethritis identified among men presenting for screening at a sexually transmitted disease clinic in Columbus, Ohio, we investigated the epidemiologic characteristics of men with Nm urethritis and the molecular and phylogenetic characteristics of their Nm isolates. The study was conducted between 1 January and 18 November 2015. Results: Seventy-five Nm urethritis cases were confirmed by biochemical and polymerase chain reaction testing. Men with Nm urethritis were a median age of 31 years (interquartile range [IQR] = 24-38) and had a median of 2 sex partners in the last 3 months (IQR = 1-3). Nm cases were predominantly black (81%) and heterosexual (99%). Most had urethral discharge (91%), reported oral sex with a female in the last 12 months (96%), and were treated with a ceftriaxone-based regimen (95%). A minority (15%) also had urethral chlamydia coinfection. All urethral Nm isolates were nongroupable, ST-11 clonal complex (cc11), ET-15, and clustered together phylogenetically. Urethral Nm isolates were similar by fine typing (PorA P1.5-1,10-8, PorB 2-2, FetA F3-6), except 2, which had different PorB types (2-78 and 2-52). Conclusions: Between January and November 2015, 75 urethritis cases due to a distinct Nm clade occurred among primarily black, heterosexual men in Columbus, Ohio. Future urogenital Nm infection studies should focus on pathogenesis and modes of sexual transmission.

Genomic sequencing of Neisseria gonorrhoeae to respond to the urgent threat of antimicrobial-resistant gonorrhea.

The development of resistance of Neisseria gonorrhoeae to available first-line antibiotics, including penicillins, tetracyclines, fluoroquinolones and cephalosporins, has led to the circulation of multidrug-resistant gonorrhea at a global scale. Advancements in high-throughput whole-genome sequencing (WGS) provide useful tools that can be used to enhance gonococcal detection, treatment and management capabilities, which will ultimately aid in the control of antimicrobial resistant gonorrhea worldwide. In this minireview, we discuss the application of WGS of N. gonorrhoeae to strain typing, phylogenomic, molecular surveillance and transmission studies. We also examine the application of WGS analyses to the public health sector as well as the potential usage of WGS-based transcriptomic and epigenetic methods to identify novel gonococcal resistance mechanisms.

Azithromycin Resistance and Decreased Ceftriaxone Susceptibility in Neisseria gonorrhoeae, Hawaii, USA.

During 2016, eight Neisseria gonorrhoeae isolates from 7 patients in Hawaii were resistant to azithromycin; 5 had decreased in vitro susceptibility to ceftriaxone. Genomic analysis demonstrated a distinct phylogenetic clade when compared with local contemporary strains. Continued evolution and widespread transmission of these strains might challenge the effectiveness of current therapeutic options.

Use of whole-genome sequencing data to analyze 23S rRNA-mediated azithromycin resistance.

The whole-genome sequences of 24 isolates of Neisseria gonorrhoeae with elevated minimum inhibitory concentrations (MICs) to azithromycin (≥2.0 µg/mL) were analyzed against a modified sequence derived from the whole-genome sequence of N. gonorrhoeae FA1090 to determine, by signal ratio, the number of mutant copies of the 23S rRNA gene and the copy number effect on 50S ribosome-mediated azithromycin resistance. Isolates that were predicted to contain four mutated copies were accurately identified compared with the results of direct sequencing. Fewer than four mutated copies gave less accurate results but were consistent with elevated MICs.

Complete Genome Sequences of 11 Haemophilus ducreyi Isolates from Children with Cutaneous Lesions in Vanuatu and Ghana.

Haemophilus ducreyi causes chancroid and has recently been shown to be a significant cause of cutaneous lesions in tropical or subtropical regions where yaws is endemic. Here, we report the draft genome assemblies for 11 cutaneous strains of Haemophilus ducreyi, isolated from children in Vanuatu and Ghana.

Notes from the Field: Increase in Neisseria meningitidis-Associated Urethritis Among Men at Two Sentinel Clinics - Columbus, Ohio, and Oakland County, Michigan, 2015.

Neisseria meningitidis (Nm) urogenital infections, although less common than infections caused by Neisseria gonorrhoeae (Ng), have been associated with urethritis, cervicitis, proctitis, and pelvic inflammatory disease. Nm can appear similar to Ng on Gram stain analysis (gram-negative intracellular diplococci) (1-5). Because Nm colonizes the nasopharynx, men who receive oral sex (fellatio) can acquire urethral Nm infections (1,3,5). This report describes an increase in Nm-associated urethritis in men attending sexual health clinics in Columbus, Ohio, and Oakland County, Michigan.

Complete Genome Sequences of Three Neisseria gonorrhoeae Laboratory Reference Strains, Determined Using PacBio Single-Molecule Real-Time Technology.

Neisseria gonorrhoeae, the etiological agent that causes the sexually transmitted infection gonorrhea, is a significant public health concern due to the emergence of antimicrobial resistance. We report the complete genome sequences of three reference isolates with varied antimicrobial susceptibility that will aid in elucidating the genetic mechanisms that confer resistance.

Strain typing and antimicrobial resistance of fluoroquinolone-resistant Neisseria gonorrhoeae causing a California infection outbreak.

Antimicrobial-resistant Neisseria gonorrhoeae is an emerging public health problem as a result of the alarming limitation in treatment options. We examined an outbreak in California of fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) by evaluation of a combination of routine isolates from the Gonococcal Isolate Surveillance Project and isolates collected by expanded surveillance performed between April 2000 and June 2002. QRNG isolates were characterized by two methods: (i) determination of a combination of antibiogram, auxotype, serovar, Lip type, and patterns of amino acid alteration in the quinolone resistance-determining region of GyrA and ParC (ASLGP) and (ii) pulsed-field gel electrophoresis (PFGE). Strain typing was used to describe the QRNG outbreak strains and the associated antimicrobial resistance profiles. Among 79 isolates that were completely characterized, we identified 20 different ASLGP strain types, and 2 of the types were considered to belong to outbreak strains that comprised 65% (51/79) of the isolates. By PFGE typing, there were 24 different strain types, and 4 of these were considered outbreak types and comprised 66% (52/79) of the isolates. The overall agreement between the typing methods in distinguishing outbreak strains and non-outbreak strains was 84% (66/79). The most common QRNG ASLGP strain type had chromosomally mediated resistance to penicillin and tetracycline and an azithromycin MIC of 0.5 microg/ml. The occurrence of an outbreak caused by QRNG strains that could fail to be eradicated by most antibiotic classes reinforces the serious problem with antimicrobial resistance in Neisseria gonorrhoeae that the public health system faces. Adherence to a regimen with the recommended antibiotics at the appropriate dose is critical, and monitoring for antimicrobial susceptibility needs to be actively maintained to adapt treatment guidelines appropriately.

The use of combination subtyping in the forensic evaluation of a three-year-old girl with gonorrhea.

The use of pulsed field gel electrophoresis and neisserial lipoprotein gene sequencing for subtyping Neisseria gonorrhoeae has not been reported in the evaluation of sexually abused children. We report the application and implications of combining pulsed field gel electrophoresis and lipoprotein subtyping in the evaluation of a 3-year-old girl with N. gonorrhoeae infection.

Fluoroquinolone resistance among Neisseria gonorrhoeae isolates in Hawaii, 1990-2000: role of foreign importation and increasing endemic spread.

OBJECTIVES: In 1999, an increase in ciprofloxacin-resistant Neisseria gonorrhoeae isolates was identified in Hawaii, prompting initiation of investigative studies. GOALS: The goal of this study was epidemiologic evaluation of this increase. STUDY: The authors conducted a review of laboratory data; case-series and case-control studies based on medical record review; and a prospective case-control study based on patient interviews. RESULTS: A total of 10.4% (21 of 201) of gonococcal isolates from Hawaii in 2000 were ciprofloxacin-resistant compared with <1.5% per year from 1990 to 1997. From medical record review for patients diagnosed with ciprofloxacin-resistant N. gonorrhoeae infection from 1990 to 1999, 59% were Asian/Pacific Islanders and 91% were heterosexual. From review of 1998 and 1999 sexually transmitted disease (STD) clinic medical records, patients with ciprofloxacin-resistant N. gonorrhoeae were more likely to report recent foreign travel or a sex partner with recent foreign travel than patients with ciprofloxacin-susceptible N. gonorrhoeae (6 of 12 vs. 10 of 117, P <0.001), but 50% (6 of 12) acquired a ciprofloxacin-resistant strain locally from a partner with no recent travel. In 2000, 70% (7 of 10) of STD clinic patients with ciprofloxacin-resistant N. gonorrhoeae acquired their infection locally from partners with no reported recent travel. CONCLUSIONS: Infections with ciprofloxacin-resistant N. gonorrhoeae are increasing and evolving in Hawaii.

The emergence of Neisseria gonorrhoeae with decreased susceptibility to Azithromycin in Kansas City, Missouri, 1999 to 2000.

BACKGROUND AND OBJECTIVES: We describe the first cluster of persons with Neisseria gonorrhoeae with decreased susceptibility to azithromycin (AziDS; minimum inhibitory concentration >/=1.0 microg/mL) in the United States. GOAL The goal of this study was to identify risk factors for AziDS N. gonorrhoeae and to describe isolate microbiology. STUDY DESIGN: Persons with AziDS N. gonorrhoeae (cases) were identified in Kansas City, Missouri, through the Gonococcal Isolate Surveillance Project (GISP) in 1999 and expanded surveillance, January 2000 to June 2001. A case-control study using 1999 GISP participants was conducted; control subjects had azithromycin-susceptible N. gonorrhoeae. RESULTS: Thirty-three persons with AziDS N. gonorrhoeae were identified. Case patients were older than control patients (median age, 33 years vs. 23 years; P <0.001). Fifty percent of cases and 13% of control subjects had a history of sex with a female commercial sex worker (odds ratio, 7.0; 95% confidence interval, 1.3-36.0); 50% of cases and 4% of control subjects met sex partners on street A (P <0.01). AziDS N. gonorrhoeae isolates were phenotypically and genotypically similar and contained an mtrR gene mutation. CONCLUSIONS: With few treatment options remaining, surveillance for antimicrobial-resistant N. gonorrhoeae is increasingly important, especially among persons at high risk.

Multidrug-resistant Neisseria gonorrhoeae with decreased susceptibility to cefixime-Hawaii, 2001.

We report 4 urogenital Neisseria gonorrhoeae isolates recovered from 3 patients that demonstrated resistance to penicillin, tetracycline, and ciprofloxacin and reduced susceptibility to cefixime. This report of the first 3 patients in the United States identified with this multidrug-resistant strain may portend an emerging problem for clinicians and public health officials.

Mutations causing in vitro resistance to azithromycin in Neisseria gonorrhoeae.

In 1999, a cluster of gonococcal isolates exhibiting high Minimal Inhibitory Concentrations (MICs), to azithromycin (2.0-4.0 mg/l) were identified in Kansas City, MO. Isolates were characterized by auxotype/serovar class, lipoprotein (Lip) subtyping and sequencing of the mtrR gene, which has been implicated in decreased azithromycin susceptibility in the gonococcus. Isolates were Pro/IB-3 and contained the 17c Lip subtype. Molecular characterization of the mtrR gene revealed a 153 base pair insertion sequence located between the mtrR/mtrC promoter and the mtrC gene. Some isolates also contained a frame shift within the mtrR gene. Transformation of these mutations into an azithromycin-sensitive recipient strain resulted in transformants with MICs as high as 2.0 mg/l and inactivation of the mtrD gene reduced azithromycin MICs 270-fold. These results demonstrated that the mtr mutations were responsible for the increased MICs in these isolates.

Multiclonal increase in ciprofloxacin-resistant Neisseria gonorrhoeae, Thailand, 1998-1999.

BACKGROUND: isolates exhibiting clinically significant resistance to fluoroquinolones have been isolated most frequently in Asian and western Pacific countries, including Thailand. In Bangkok, Thailand, ciprofloxacin has been used to treat gonorrhea since 1987. GOAL: Our goal was to determine the prevalence of isolates of that exhibit resistance to ciprofloxacin in Bangkok and to characterize these strains with regard to ciprofloxacin MICs, auxotype/serovar (A/S) classification, A and C mutations responsible for ciprofloxacin resistance, and outer membrane lipoprotein (Lip) subtype analysis. STUDY DESIGN: MICs of gonococcal isolates from consecutive patients attending the Bangrak Hospital STD Clinic in Bangkok were determined by agar dilution. A/S class was determined by established procedures. Mutations within A and C were determined by DNA sequencing. Lip subtypes were determined by PCR and DNA sequencing. RESULTS: In 1998 and 1999, 115 of 168 isolated strains of exhibited decreased susceptibility or resistance to ciprofloxacin, and three cases of possible ciprofloxacin treatment failure were identified. Ciprofloxacin-resistant (CipR) strains increased from 13.8% (8/58) in 1998 to 25.4% (28/110) in 1999 ( = 0.08). Ciprofloxacin MICs of CipR isolates ranged from 1.0 microg/ml to 32.0 microg/ml. CipR strains belonged to a number of A/S classes and Lip subtypes. Different CipR strains contained alterations at both amino acid 91 and amino acid 95 of A and also contained an amino acid alteration in C. These alterations are known to be involved in gonococcal resistance to ciprofloxacin. CONCLUSIONS: The prevalence of CipR strains of isolated in Bangkok increased substantially in the 1990s. Characterization of the CipR isolates revealed a number of different strain subtypes, indicating that CipR isolates in Bangkok are not from a single clonal source and therefore result from multiple cases of importation or local emergence. Because of the high level of CipR isolates at Bangrak Hospital, in 2000 the Thai Ministry of Public Health issued recommendations against the use of fluoroquinolones for the treatment of gonococcal infection in Thailand.