Defining Fluoroquinolone Resistance-Mediating Mutations from Non-Resistance Polymorphisms in Mycoplasma hominis Topoisomerases

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24–48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics’ surveillance.

Contribution of Different Mechanisms to Ciprofloxacin Resistance in Salmonella spp.

Development of fluoroquinolone resistance can involve several mechanisms that include chromosomal mutations in genes (gyrAB and parCE) encoding the target bacterial topoisomerase enzymes, increased expression of the AcrAB-TolC efflux system, and acquisition of transmissible quinolone-resistance genes. In this study, 176 Salmonella isolates from animals with a broad range of ciprofloxacin MICs were collected to analyze the contribution of these different mechanisms to different phenotypes. All isolates were classified according to their ciprofloxacin susceptibility pattern into five groups as follows: highly resistant (HR), resistant (R), intermediate (I), reduced susceptibility (RS), and susceptible (S). We found that the ParC T57S substitution was common in strains exhibiting lowest MICs of ciprofloxacin while increased MICs depended on the type of GyrA mutation. The ParC T57S substitution appeared to incur little cost to bacterial fitness on its own. The presence of PMQR genes represented an route for resistance development in the absence of target-site mutations. Switching of the plasmid-mediated quinolone resistance (PMQR) gene location from a plasmid to the chromosome was observed and resulted in decreased ciprofloxacin susceptibility; this also correlated with increased fitness and a stable resistance phenotype. The overexpression of AcrAB-TolC played an important role in isolates with small decreases in susceptibility and expression was upregulated by MarA more often than by RamA. This study increases our understanding of the relative importance of several resistance mechanisms in the development of fluoroquinolone resistance in Salmonella from the food chain.

Characterisation of Campylobacter spp. Isolated from Poultry in KwaZulu-Natal, South Africa

This study investigated the antibiotic resistance, virulence profiles, and clonality of Campylobacter jejuni and Campylobacter coli isolated from an intensive poultry farming system in KwaZulu-Natal, South Africa. Following ethical approval, samples were collected over six weeks using the farm-to-fork approach. Campylobacter spp. were identified using culture, confirmed and differentiated to species level by PCR, and subjected to antibiotic susceptibility testing. Selected antibiotic resistance (and mutations) and virulence genes were screened by PCR and confirmed by DNA sequencing. Genetic relatedness amongst the isolates was ascertained using pulsed-field gel electrophoresis. In all, 105 isolates were confirmed as belonging to both Campylobacter coli (60; 57%) and C. jejuni (45; 43%). The highest resistance was recorded against erythromycin and clindamycin. The gyrA mutation, A20175C/A2074G point mutation, tet(O), and cmeB, all associated with antibiotic resistance, were detected. All the virulence genes (pldA, ciaB, cdtA, cdtB, cdtC, dnaJ, except for cadF) were also detected. Isolates were grouped into five pulsotypes displaying 85% similarity, irrespective of their resistance profiles. The numerous permutations of clonality, antibiotic resistance, and virulence profiles evident in Campylobacter spp. pose a challenge to food safety and necessitate a comprehensive understanding of the molecular epidemiology of this organism to decrease its spread in the food chain.

The parC mutation G248T (S83I), and concurrent gyrA mutations, are associated with moxifloxacin and sitafloxacin treatment failure for Mycoplasma genitalium

Background The basis of fluoroquinolone treatment failure for Mycoplasma genitalium is poorly understood. Methods To identify mutations associated with failure we sequenced key regions of the M. genitalium parC and gyrA genes for patients undergoing sequential therapy with doxycycline-moxifloxacin (201 patients, including 21 failures) or doxycycline-sitafloxacin (126 patients, including 13 failures). Results The parC G248T/S83I mutation was more common among patients who failed sequential doxycycline-moxifloxacin (present in 76.2% of failures vs 7.8% cures, p<0.001) and doxycycline-sitafloxacin (50% failures vs 16.8% cures, p = 0.014). Doxycycline-sitafloxacin was more efficacious than doxycycline-moxifloxacin against infections carrying the ParC S83I mutation. Infections with a ParC S83I were more likely to fail treatment with a concurrent gyrA mutation (M95I or D99N) (for doxycycline- moxifloxacin group p = 0.067, for doxycycline-sitafloxacin group p = 0.0093), suggesting an additive effect. Conclusions This study indicates that parC G248T/S83I mutations contribute to failure of moxifloxacin and sitafloxacin, and will inform the development of quinolone resistance assays needed to ensure optimal selection of antimicrobials for M. genitalium.

The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis

Fluoroquinolones (FQ) form the backbone in experimental treatment regimens against drug-susceptible tuberculosis. However, little is known on whether the genetic variation present in natural populations of Mycobacterium tuberculosis (Mtb) affects the evolution of FQ-resistance (FQ-R). To investigate this question, we used nine genetically distinct drug-susceptible clinical isolates of Mtb and measured their frequency of resistance to the FQ ofloxacin (OFX) in vitro. We found that the Mtb genetic background led to differences in the frequency of OFX-resistance (OFX-R) that spanned two orders of magnitude and substantially modulated the observed mutational profiles for OFX-R. Further, in vitro assays showed that the genetic background also influenced the minimum inhibitory concentration and the fitness effect conferred by a given OFX-R mutation. To test the clinical relevance of our in vitro work, we surveyed the mutational profile for FQ-R in publicly available genomic sequences from clinical Mtb isolates, and found substantial Mtb lineage-dependent variability. Comparison of the clinical and the in vitro mutational profiles for FQ-R showed that 51% and 39% of the variability in the clinical frequency of FQ-R gyrA mutation events in Lineage 2 and Lineage 4 strains, respectively, can be attributed to how Mtb evolves FQ-R in vitro. As the Mtb genetic background strongly influenced the evolution of FQ-R in vitro, we conclude that the genetic background of Mtb also impacts the evolution of FQ-R in the clinic.

Genetic Diversity of Salmonella Derby from the Poultry Sector in Europe

Salmonella Derby (S. Derby) is emerging in Europe as a predominant serovar in fattening turkey flocks. This serovar was recorded as being predominant in the turkey sector in 2014 in the United Kingdom (UK). Only two years later, in 2016, it was also recorded in the turkey and broiler sectors in Ireland and Spain. These S. Derby isolates were characterised as members of the multilocus sequence type (MLST) profile 71 (ST71). For the first time, we characterise by whole genome sequencing (WGS) analysis a panel of 90 S. Derby ST71 genomes to understand the routes of transmission of this emerging pathogen within the poultry/turkey food trade. Selected panel included strains isolated as early as 2010 in five leading European g countries for turkey meat production. Twenty-one of the 90 genomes were extracted from a public database—Enterobase. Five of these originated from the United States (n = 3), China (n = 1) and Taiwan (n = 1) isolated between 1986 and 2016. A phylogenomic analysis at the core-genome level revealed the presence of three groups. The largest group contained 97.5% of the European strains and included both, turkey and human isolates that were genetically related by an average of 35 ± 15 single nucleotide polymorphism substitutions (SNPs). To illustrate the diversity, the presence of antimicrobial resistance genes and phages were characteised in 30, S. Derby ST71 genomes, including 11 belonging to this study This study revealed an emergent turkey-related S. Derby ST71 clone circulating in at least five European countries (the UK, Germany, Poland, Italy, and France) since 2010 that causes human gastroenteritis. A matter of concern is the identification of a gyrA mutation involved in resistance to quinolone, present in the Italian genomes. Interestingly, the diversity of phages seems to be related to the geographic origins. These results constitute a baseline for following the spread of this emerging pathogen and identifying appropriate monitoring and prevention measures.