Antimicrobial susceptibilities and genomic epidemiology of Neisseria gonorrhoeae in Stockholm, Sweden.

The aim of this study was to investigate the genomic epidemiology and antimicrobial susceptibilities of N. gonorrhoeae isolates in Stockholm, Sweden. In total, 6723 isolates detected in Stockholm, Sweden, from January 2016 to September 2022, were examined for antimicrobial susceptibilities by using E-test. Whole-genome sequencing (WGS) was applied to isolates in sentinel surveillance and isolates resistant to extended-spectrum cephalosporins (ESCs) or high-level azithromycin (HLAzi-R, MIC ≥ 256 mg/L). As sentinel surveillance, consecutive clinical isolates (n = 396) detected every 4th week from January 2021 to September 2022 were enrolled in the study. Of the 6723 isolates investigated, 33 isolates (< 1%) were found to be resistant to cefixime, one of which was co-resistant to ceftriaxone and ciprofloxacin and was detected in September 2022. Ten isolates presented a high level of azithromycin resistance. Resistant rates to ciprofloxacin varied from 32 in 2017 to 68-69% in 2021-2022. Elevated MIC50 and MIC90 of azithromycin were observed over the years. No resistance to spectinomycin was identified. The most frequently occurring MLST in the sentinel surveillance was ST9362 (23%), followed by ST11706 (9%), ST7359 (8%), ST10314 (7%), and ST11422 (6%). The ceftriaxone-resistant isolate belonged to ST8130 and the novel NG-STAR ST4859. Genomic resistance traits found in this strain included mutations in genes mtrR (A39T), parC (S87N), and gyrA (S91F and D95A), as well as the presence of blaTEM-135 and tetM genes. A predominance of ST9362 was observed in Stockholm. The high number of azithromycin and ciprofloxacin-resistant isolates and the emergence of a strain with a novel NG-STAR are of great concern.

Azithromycin Resistance in Shiga Toxin-Producing Escherichia coli in France between 2004 and 2020 and Detection of mef(C)-mph(G) Genes.

We described and characterized Shiga-toxin-producing Escherichia coli (STEC) strains with high levels of resistance to azithromycin isolated in France between 2004 and 2020. Nine of 1,715 (0.52%) STEC strains were resistant to azithromycin, with an increase since 2017. One isolate carried a plasmid-borne mef(C)-mph(G) gene combination, described here for the first time for E. coli. Azithromycin resistance, although rare, needs consideration, as this treatment may be useful in cases of STEC infection.

Extensively drug-resistant (XDR) Neisseria gonorrhoeae causing possible gonorrhoea treatment failure with ceftriaxone plus azithromycin in Austria, April 2022.

We describe a gonorrhoea case with ceftriaxone plus high-level azithromycin resistance. In April 2022, an Austrian heterosexual male was diagnosed with gonorrhoea after sexual intercourse with a female sex worker in Cambodia. Recommended treatment with ceftriaxone (1 g) plus azithromycin (1.5 g) possibly failed. Worryingly, this is the second strain in an Asian Neisseria gonorrhoeae genomic sublineage including high-level azithromycin-resistant strains that developed ceftriaxone resistance by acquisition of mosaic penA-60.001. Enhanced resistance surveillance and actions are imperative to prevent spread.

Spontaneous Emergence of Azithromycin Resistance in Independent Lineages of Salmonella Typhi in Northern India.

BACKGROUND: The emergence and spread of antimicrobial resistance (AMR) pose a major threat to the effective treatment and control of typhoid fever. The ongoing outbreak of extensively drug-resistant Salmonella Typhi (S. Typhi) in Pakistan has left azithromycin as the only remaining broadly efficacious oral antimicrobial for typhoid in South Asia. Ominously, azithromycin-resistant S. Typhi organisms have been subsequently reported in Bangladesh, Pakistan, and Nepal. METHODS: Here, we aimed to understand the molecular basis of AMR in 66 S. Typhi organisms isolated in a cross-sectional study performed in a suburb of Chandigarh in Northern India using whole-genome sequencing and phylogenetic analysis. RESULTS: We identified 7 S. Typhi organisms with the R717Q mutation in the acrB gene that was recently found to confer resistance to azithromycin in Bangladesh. Six out of the seven azithromycin-resistant S. Typhi isolates also exhibited triple mutations in gyrA (S83F and D87N) and parC (S80I) genes and were resistant to ciprofloxacin. These contemporary ciprofloxacin/azithromycin-resistant isolates were phylogenetically distinct from each other and from those reported from Bangladesh, Pakistan, and Nepal. CONCLUSIONS: The independent emergence of azithromycin-resistant typhoid in Northern India reflects an emerging broader problem across South Asia and illustrates the urgent need for the introduction of typhoid conjugate vaccines in the region.

Multi-year prevalence and macrolide resistance of Mycoplasma genitalium in clinical samples from a southern Italian hospital.

The use of azithromycin for the treatment of Mycoplasma genitalium infections has led to resistance to macrolides. From July 2014 to July 2020, 7150 samples were analysed for the detection of sexually transmitted infections at the Policlinico of Bari. A total of 67/7150 samples (0.93%) were positive for MG DNA and 47 samples were analysed for the evaluation of six azithromycin resistance-associated mutations. In 5/47 samples, the A2058G mutation was detected (10.63%). Although the cases of positive MG samples and mutations are low in our reality, diagnostic tests to detect azithromycin resistant-associated genes may provide a convenient way to monitor resistance rate.

Drug-resistant enteric fever worldwide, 1990 to 2018: a systematic review and meta-analysis.

BACKGROUND: Antimicrobial resistance (AMR) is an increasing threat to global health. There are > 14 million cases of enteric fever every year and > 135,000 deaths. The disease is primarily controlled by antimicrobial treatment, but this is becoming increasingly difficult due to AMR. Our objectives were to assess the prevalence and geographic distribution of AMR in Salmonella enterica serovars Typhi and Paratyphi A infections globally, to evaluate the extent of the problem, and to facilitate the creation of geospatial maps of AMR prevalence to help targeted public health intervention. METHODS: We performed a systematic review of the literature by searching seven databases for studies published between 1990 and 2018. We recategorised isolates to allow the analysis of fluoroquinolone resistance trends over the study period. The prevalence of multidrug resistance (MDR) and fluoroquinolone non-susceptibility (FQNS) in individual studies was illustrated by forest plots, and a random effects meta-analysis was performed, stratified by Global Burden of Disease (GBD) region and 5-year time period. Heterogeneity was assessed using the I2 statistics. We present a descriptive analysis of ceftriaxone and azithromycin resistance. FINDINGS: We identified 4557 articles, of which 384, comprising 124,347 isolates (94,616 S. Typhi and 29,731 S. Paratyphi A) met the pre-specified inclusion criteria. The majority (276/384; 72%) of studies were from South Asia; 40 (10%) articles were identified from Sub-Saharan Africa. With the exception of MDR S. Typhi in South Asia, which declined between 1990 and 2018, and MDR S. Paratyphi A, which remained at low levels, resistance trends worsened for all antimicrobials in all regions. We identified several data gaps in Africa and the Middle East. Incomplete reporting of antimicrobial susceptibility testing (AST) and lack of quality assurance were identified. INTERPRETATION: Drug-resistant enteric fever is widespread in low- and middle-income countries, and the situation is worsening. It is essential that public health and clinical measures, which include improvements in water quality and sanitation, the deployment of S. Typhi vaccination, and an informed choice of treatment are implemented. However, there is no licenced vaccine for S. Paratyphi A. The standardised reporting of AST data and rollout of external quality control assessment are urgently needed to facilitate evidence-based policy and practice. TRIAL REGISTRATION: PROSPERO CRD42018029432.

Emergence and Spread of Neisseria gonorrhoeae Strains with High-Level Resistance to Azithromycin in Taiwan from 2001 to 2018.

A total of 598 Neisseria gonorrhoeae isolates obtained from patients in Taiwan from 2001 to 2018 were evaluated. The MICs of ceftriaxone (CRO) and azithromycin (AZM) against the isolates were determined by the agar dilution method. N. gonorrhoeae isolates with AZM MICs of ≥1 µg/ml were identified and characterized by the presence of AZM resistance determinants. For high-level AZM-resistant (AZM-HLR) isolates (MIC ≥ 256 µg/ml), genotyping was performed using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Among the N. gonorrhoeae isolates studied, 8.7% (52/598) exhibited AZM MICs of ≥1 µg/ml. Thirteen of the 52 isolates contained A2059G (23S rRNA NG-STAR type 1) or C2611T (23S rRNA NG-STAR type 2) mutations. The prevalence of the A2059G mutation was higher in AZM-HLR isolates (P < 0.001). The -35A deletion in the promoter region of the mtrR gene did not differ between AZM-HLR isolates (100%, 10/10) and the isolates with AZM MICs of 1 µg/ml to 64 µg/ml (95.2%, 40/42) (P = 1.000). The presence of mutations in the mtrR coding region was significantly different between these two groups at 90% (9/10) and 26.2% (11/42), respectively (P < 0.001). The AZM-HLR isolates, all carrying four mutated A2059G alleles, a -35A deletion, and G45D, were classified as MLST 12039/10899 and NG-MAST 1866/16497. In conclusion, Taiwan is among the countries reporting gonococci with high-level resistance to AZM so that a single dose of 1 g ceftriaxone intramuscularly as the first choice for management of N. gonorrhoeae infection should be evaluated.

Emergence of Neisseria gonorrhoeae Strains Harboring a Novel Combination of Azithromycin-Attenuating Mutations.

The nimbleness of Neisseria gonorrhoeae to evade the effect of antibiotics has perpetuated the fight against antibiotic-resistant gonorrhea for more than 80 years. The ability to develop resistance to antibiotics is attributable to its indiscriminate nature in accepting and integrating exogenous DNA into its genome. Here, we provide data demonstrating a novel combination of the 23S rRNA A2059G mutation with a mosaic-multiple transferable resistance (mosaic-mtr) locus haplotype in 14 N. gonorrhoeae isolates with high-level azithromycin MICs (≥256 µg/ml), a combination that may confer more fitness than in previously identified isolates with high-level azithromycin resistance. To our knowledge, this is the first description of N. gonorrhoeae strains harboring this novel combination of resistance determinants. These strains were isolated at two independent jurisdictions participating in the Gonococcal Isolate Surveillance Project (GISP) and in the Strengthening the U.S. Response to Resistant Gonorrhea (SURRG) project. The data suggest that the genome of N. gonorrhoeae continues to shuffle its genetic material. These findings further illuminate the genomic plasticity of N. gonorrhoeae, which allows this pathogen to develop mutations to escape the inhibitory effects of antibiotics.

Four-color multiplex real-time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium.

BACKGROUND: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. METHODS: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. RESULTS: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. CONCLUSIONS: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.

Sensitive and specific assay for the simultaneous detection of Mycoplasma genitalium and macrolide resistance-associated mutations.

Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg MacrolideR qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg MacrolideR qPCR. The Mg MacrolideR qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg MacrolideR qPCR, indicating specificity. The Mg MacrolideR qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.

Mycoplasma genitalium: Accurate Diagnosis Is Necessary for Adequate Treatment.

Background: Mycoplasma genitalium is very difficult to grow in culture but has been more able to be studied for disease associations since the advent of research molecular amplification assays. Polymerase chain reaction (PCR) and other molecular assays have demonstrated an association with adverse disease outcomes, such as urethritis or nongonococcal urethritis in men and adverse reproductive sequelae in women-for example, cervicitis, endometritis, and pelvic inflammatory disease (PID), including an association with risk for human immunodeficiency virus. The lack of commercially available diagnostic assays has limited widespread routine testing. Increasing reports of high rates of resistance to azithromycin detected in research studies have heightened the need available commercial diagnostic assays as well as standardized methods for detecting resistance markers. This review covers available molecular methods for the diagnosis of M. genitalium and assays to predict the antibiotic susceptibility to azithromycin. Methods: A PubMed (US National Library of Medicine and National Institutes of Health) search was conducted for literature published between 2000 and 2016, using the search terms Mycoplasma genitalium, M. genitalium, diagnosis, and detection. Results: Early PCR diagnostic tests focused on the MPa adhesion gene and the 16S ribosomal RNA gene. Subsequently, a transcription-mediated amplification assay targeting ribosomes was developed and widely used to study the epidemiology of M. genitalium. Newer methods have proliferated and include quantitative PCR for organism load, AmpliSens PCR, PCR for the pdhD gene, a PCR-based microarray for multiple sexually transmitted infections, and multiplex PCRs. None yet are cleared by the Food and Drug Administration in the United States, although several assays are CE marked in Europe. As well, many research assays, including PCR, gene sequencing, and melt curve analysis, have been developed to detect the 23S ribosomal RNA gene mutations that confer resistance to azithromycin. One recently developed assay can test for both M. genitalium and azithromycin resistance mutations at the same time. Conclusions: It is recommended that more commercial assays to both diagnose this organism and guide treatment choices should be developed and made available through regulatory approval. Research is needed to establish the cost-effectiveness of routine M. genitalium testing in symptomatic patients and screening in all individuals at high risk of acquiring and transmitting sexually transmitted infections.

Mycoplasma genitalium: whole genome sequence analysis, recombination and population structure.

BACKGROUND: Although Mycoplasma genitalium is a common sexually transmitted pathogen causing clinically distinct diseases both in male and females, few genomes have been sequenced up to now, due mainly to its fastidious nature and slow growth. Hence, we lack a robust phylogenetic framework to provide insights into the population structure of the species. Currently our understanding of the nature and diversity of M. genitalium relies on molecular tests targeting specific genes or regions of the genome and knowledge is limited by a general under-testing internationally. This is set against a background of drug resistance whereby M. genitalium has developed resistance to mainly all therapeutic antimicrobials. RESULTS: We sequenced 28 genomes of Mycoplasma genitalium from temporally (1980-2010) and geographically (Europe, Japan, Australia) diverse sources. All the strain showed essentially the same genomic content without any accessory regions found. However, we identified extensive recombination across their genomes with a total of 25 regions showing heightened levels of SNP density. These regions include the MgPar loci, associated with host interactions, as well as other genes that could also be involved in this role. Using these data, we generated a robust phylogeny which shows that there are two main clades with differing degrees of genomic variability. SNPs found in region V of 23S rRNA and parC were consistent with azithromycin/erythromycin and fluoroquinolone resistances, respectively, and with their phenotypic MIC data. CONCLUSIONS: The sequence data here generated is essential for designing rational approaches to type and track Mycoplasma genitalium as antibiotic resistance increases. It represents a first approach to its population genetics to better appreciate the role of this organism as a sexually transmitted pathogen.

Emerging azithromycin-resistance among the Neisseria gonorrhoeae 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.

Transmission of azithromycin-resistant gene, erm(T), of Gram-positive bacteria origin to Klebsiella pneumoniae.

The erm(T) gene encodes the 23 s rRNA methyltransferase and confers erythromycin resistance in Gram-positive bacteria, while has rarely been identified in Gram-negative bacteria. In this study, we identified a small IncQ1 plasmid of 6135 bp harboring the erm(T) gene in a clinical K. pneumoniae strain and confirmed the role of the erm(T) gene in mediating azithromycin resistance. This plasmid was found to be generated by incorporating the erm(T) gene from mobile elements into an IncQ1 plasmid. Our data indicated the spread of the erm(T) gene from Gram-positive bacteria to Gram-negative bacteria and the clonal spread of the ST11-KL47 type K. pneumoniae strains carrying this plasmid. Generation of this kind of multi-host plasmid will promote the dissemination of the erm(T) gene among Gram-negative bacteria and result in failures of azithromycin in treating bacterial infections.

Retrospective Analysis of Azithromycin-Resistant Ureaplasma urealyticum and Mycoplasma hominis Cervical Infection Among Pregnant Women.

Purpose: Ureaplasma urealyticum and Mycoplasma hominis began to show resistance to azithromycin, a macrolide antibiotic commonly used in pregnancy. Unfortunately, there are few effective and safe drugs in the clinic for genital mycoplasmas in pregnant women. In the present study, we investigated the prevalence of azithromycin-resistant U. urealyticum and M. hominis infections in pregnant women. The secondary research objects were possible influencing factors and consequences of insensitive Mycoplasma infection. Patients and methods: A retrospective analysis was carried out in pregnant women who underwent cervical Mycoplasma culture between October 2020 and October 2021 at a large general hospital in eastern China. The sociological characteristics and clinical information of these women were collected and analyzed. Results: A total of 375 pregnant women were enrolled, and 402 cultured mycoplasma specimens were collected. Overall, 186 (49.60%) patients tested positive cervical Mycoplasma infection, and 37 (9.87%) had infections caused by azithromycin-resistant Mycoplasma. In total, 39 mycoplasma samples were insensitive to azithromycin in vitro, also showing extremely high resistance to erythromycin, roxithromycin, and clarithromycin. Azithromycin was the only antibiotic used in women with Mycoplasma cervical infection, regardless of azithromycin resistance in vitro. Statistical results showed that azithromycin-resistant cervical Mycoplasma infection in pregnant women was unrelated to age, body mass index (BMI), gestational age, number of embryos, and assisted reproductive technology (ART) use, but led to a significantly increased incidence of adverse pregnancy outcomes (spontaneous abortion (SA), preterm birth (PTB), preterm prelabor rupture of membranes (PPROM), and stillbirth). Conclusion: Azithromycin-resistant U. urealyticum and M. hominis cervical infections are relatively common during pregnancy, and can increase the risk of adverse pregnancy outcomes; however, there is currently a lack of safe and effective drug treatments. Herein, we show that azithromycin-resistant mycoplasma infection requires timely intervention.

Azithromycin Susceptibility Testing and Molecular Investigation of Neisseria gonorrhoeae Isolates Collected in Russia, 2020-2021.

The aim of this work was to study the resistance to macrolides (azithromycin) in the modern Russian population of N. gonorrhoeae with the analysis of genetic resistance determinants. Azithromycin is not used to treat gonococcal infection in Russia. However, among 162 isolates collected in 2020-2021, 22 isolates (13.6%) were phenotypically resistant to azithromycin. Mutations in 23S rRNA genes were found only in two isolates; erm and mefA genes were absent. Azithromycin resistance was shown to be predominantly associated with mutations in the mtrR and mtrD genes of the MtrCDE efflux pump and their mosaic alleles which may have formed due to a horizontal transfer from N. meningitidis. A total of 30 types of mtrR alleles and 10 types of mtrD alleles were identified including mosaic variants. Matching between the mtrR and mtrD alleles was revealed to indicate the cooperative molecular evolution of these genes. A link between the mtrR and mtrD alleles and NG-MAST types was found only for NG-MAST 228 and 807, typical of N. gonorrhoeae in Russia. The high level of resistance to azithromycin in Russia may be related to the spread of multiple transferable resistance to antimicrobials regardless of their use in the treatment of gonococcal infection.

Antimicrobial Resistance and Mechanisms of Azithromycin Resistance in Nontyphoidal Salmonella Isolates in Taiwan, 2017 to 2018.

Antimicrobial resistance was investigated in 2,341 nontyphoidal Salmonella (NTS) isolates recovered from humans in Taiwan from 2017 to 2018 using antimicrobial susceptibility testing. Azithromycin resistance determinants were detected in 175 selected isolates using PCR and confirmed in 81 selected isolates using whole-genome sequencing. Multidrug resistance was found in 47.3% of total isolates and 96.2% of Salmonella enterica serovar Anatum and 81.7% of S. enterica serovar Typhimurium isolates. Resistance to the conventional first-line drugs (ampicillin, chloramphenicol, and cotrimoxazole), cefotaxime and ceftazidime, and ciprofloxacin was found in 32.5 to 49.0%, 20.3 to 20.4%, and 3.2% of isolates, respectively. A total of 76 (3.1%) isolates were resistant to azithromycin, which was associated with mph(A), erm(42), erm(B), and possibly the enhanced expression of efflux pump(s) due to ramAp or defective ramR. mph(A) was found in 53% of the 76 azithromycin-resistant isolates from 11 serovars and located in an IS26-mph(A)-mrx(A)-mphR(A)-IS6100 unit in various incompatibility plasmids and the chromosomes. erm(42) in S. enterica serovar Albany was carried by an integrative and conjugative element, ICE_erm42, and in S. enterica serovar Enteritidis and S. Typhimurium was located in IS26 composite transposons in the chromosomes. erm(B) was carried by IncI1-I(α) plasmids in S. Enteritidis and S. Typhimurium. ramAp was a plasmid-borne ramA, a regulatory activator of efflux pump(s), found in only S. enterica serovar Goldcoast. Since the azithromycin resistance determinants are primarily carried on mobile genetic elements, they could easily be disseminated among human bacterial pathogens. The ramAp-carrying S. Goldcoast isolates displayed azithromycin MICs of 16 to 32 mg/L. Thus, the epidemiological cutoff value of ≤16 mg/L of azithromycin proposed for wild-type NTS should be reconsidered. IMPORTANCE Antimicrobial resistance in NTS isolates is a major public health concern in Taiwan, and the mechanisms of azithromycin resistance are rarely investigated. Azithromycin and carbapenems are the last resort for the treatment of invasive salmonellosis caused by multidrug-resistant (MDR) and extensively drug-resistant Salmonella strains. Our study reports the epidemiological trend of resistance in NTS in Taiwan and the genetic determinants involved in azithromycin resistance. We point out that nearly half of NTS isolates from 2017 to 2018 are MDR, and 20% are resistant to third-generation cephalosporins. The azithromycin resistance rate (3.1%) for the NTS isolates from Taiwan is much higher than those for the NTS isolates from the United States and Europe. Our study also indicates that azithromycin resistance is primarily mediated by mph(A), erm(42), erm(B), and ramAp, which are frequently carried on mobile genetic elements. Thus, the azithromycin resistance determinants could be expected to be disseminated among diverse bacterial pathogens.

Emergence of Azithromycin-Resistant Neisseria gonorrhoeae Isolates Belonging to the NG-MAST Genogroup 12302 in Russia.

The goal of this work was to determine the factors affecting the emergence of azithromycin-resistant Neisseria gonorrhoeae isolates in Russia, where azithromycin was never recommended for the treatment of gonococcal infections. Clinical N. gonorrhoeae isolates collected in 2018-2021 (428 isolates) were analyzed. No azithromycin-resistant isolates were found in 2018-2019, but in 2020-2021, a significant increase in the ratio of azithromycin-resistant isolates was observed: 16.8% and 9.3%, respectively. A hydrogel DNA microarray was developed for the analysis of resistance determinants: mutations in the genes encoding the mtrCDE efflux system and in all four copies of the 23S rRNA gene (position 2611). A majority of the azithromycin-resistant Russian isolates belonged to the NG-MAST G12302 genogroup, and the resistance was associated with the presence of a mosaic structure of the mtrR gene promoter region with the -35 delA deletion, an Ala86Thr mutation in the mtrR gene, and a mosaic structure of the mtrD gene. A comparative phylogenetic study of modern Russian and European N. gonorrhoeae populations allowed us to conclude that the emergence of azithromycin resistance in Russia in 2020 was the result of the appearance and spread of European N. gonorrhoeae strains belonging to the G12302 genogroup due to possible cross-border transfer.

Identification and genetic characterization of two conjugative plasmids that confer azithromycin resistance in Salmonella.

With the development of multidrug resistance in Salmonella spp. in recent years, ciprofloxacin, ceftriaxone, and azithromycin have become the principal antimicrobial agents used for the treatment of Salmonella infections. The underlying mechanisms of plasmid-mediated ciprofloxacin and ceftriaxone resistance have attracted extensive research interest, but not much is focused on azithromycin resistance in Salmonella. In this study, we investigated the genetic features of two conjugative plasmids and a non-transferable virulence plasmid that encode azithromycin resistance in food-borne Salmonella strains. We showed that the azithromycin resistance phenotype of these strains was conferred by erm(B) gene and/or the complete genetic structure IS26-mph(A)-mrx-mphR-IS6100. Comparative genetic analysis showed that these conjugative plasmids might originate from Escherichia coli and play a role in the rapid dissemination of azithromycin resistance in Salmonella. These conjugative plasmids may also serve as a reservoir of antimicrobial resistance (AMR) genes in Salmonella in which these AMR genes may be acquired by the virulence plasmids of Salmonella via genetic transposition events. Importantly, the formation of a novel macrolide-resistance and virulence-encoding plasmid, namely pS1380-118 kb, was observed in this study. This plasmid was found to exhibit transmission potential and pose a serious health threat as the extensive transmission of azithromycin resistant and virulent Salmonella strains would further compromise the effectiveness of treatment for salmonellosis. Further surveillance and research on the dissemination and evolution routes of pS1380-118kb-like plasmids in potential human pathogens of the family of Enterobacteriaceae are necessary.

An IncB/O/K/Z conjugative plasmid encodes resistance to azithromycin and mediates transmission of virulence plasmid in Klebsiella pneumoniae.

INTRODUCTION: Azithromycin resistance in bacterial pathogens has increased worldwide, and Klebsiella pneumoniae (K. pneumoniae) carries a variety of azithromycin resistance encoding genes. METHODS: Genomic DNA of K. pneumoniae strain 16HN-12 was subjected to whole-plasmid sequencing using both the 150-bp paired-end Illumina NextSeq 500 platform and the long-read Oxford Nanopore Technologies MinION platform. Transferability of the azithromycin-resistance plasmid and the virulence plasmid was assessed by performing the conjugation assay. RESULTS: This study identified an IncB/O/K/Z conjugative plasmid that harboured erm(B) and mph(A) genes from a clinical K. pneumoniae strain. The plasmid was readily able to conjugate to Escherichia coli (E. coli) strain J53 and Salmonella enterica subsp. enterica serovar Typhimurium strain PY1 and promoted phenotypic resistance to azithromycin. Furthermore, the virulence plasmid harboured by this K. pneumoniae strain could be conjugated to E. coli strain EC600 and K. pneumoniae strain WZ1-2 via the help of this resistance plasmid through formation of a fusion plasmid. The fusion process was generated by homologous recombination through a homologous region located in both the virulence plasmid and resistance plasmid. CONCLUSIONS: Generation of this kind of conjugative plasmid simultaneously carrying virulence and resistance determinants could accelerate dissemination of these determinants and generate bacterial pathogens encoding these phenotypes. These data provide more information about transmission of azithromycin resistance and virulence determinants and call for action to further investigate and prevent such an evolutionary trend.