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Chromosomal resistance islands containing the methicillin resistance gene mecD (McRI(mecD)) have been reported in Macrococcus caseolyticus. Here, we identified novel macrolide resistance genes in Macrococcus canis on similar elements, called McRI(msr). These elements were also integrated into the 3′ end of the 30S ribosomal protein S9 gene (rpsI), delimited by characteristic attachment (att) sites, and carried a related site-specific integrase gene (int) at the 5′ end. They carried novel macrolide resistance genes belonging to the msr family of ABC subfamily F (ABC-F)-type ribosomal protection protein [msr(F) and msr(H)] and the macrolide efflux mef family [mef(D)]. Highly related mef(D)-msr(F) fragments were found on diverse McRI(msr) elements in M. canis, M. caseolyticus, and Staphylococcus aureus. Another McRI(msr)-like element identified in an M. canis strain lacked the classical att site at the 3′ end and carried the msr(H) gene but no neighboring mef gene. The expression of the novel resistance genes in S. aureus resulted in a low-to-moderate increase in the MIC of erythromycin but not streptogramin B. In the mef(D)-msr(F) operon, the msr(F) gene was shown to be the crucial determinant for macrolide resistance. The detection of circular forms of McRI(msr) and the mef(D)-msr(F) fragment suggested mobility of both the island and the resistance gene subunit. The discovery of McRI(msr) in different Macrococcus species and S. aureus indicates that these islands have a potential for dissemination of antibiotic resistance within the Staphylococcaceae family.

Objectives We investigated the use of bacteriophages as a strategy to decolonize intestinal carriers of multidrug-resistant Escherichia coli. Methods A fermentor was used as a continuous culture system for 48 h. Two different pools of faeces (studies I and II) obtained from volunteers were spiked with a CTX-M-15-producing ST131 E. coli (strain 4901.28) susceptible to bacteriophages and challenged with three doses of INTESTI Bacteriophage cocktail administered at 2, 6 and 10 h after the inoculum. Bacterial typing was performed by implementing microdilution panels, spot test, rep-PCR and whole-genome sequencing (including cgMLST and single-nucleotide variant analysis) obtained using Nanopore and Illumina platforms. Results In study I, bacteriophages decreased the numbers of 4901.28 dramatically (≤101 CFU/mL after 6 h). In contrast, during study II, a phage-resistant mutant of 4901.28 persisted in the continuous culture (104 CFU/mL at 48 h). Whole-genome sequencing revealed the presence of two additional plasmids in the mutant as well as 11 single-nucleotide variants, including one chromosomal in a glycosyltransferase family 2 protein that is responsible for the transfer of sugars to polysaccharides and lipids. In both studies, the commensal E. coli population remained unchanged by the phage treatment maintaining itself at 108 CFU/mL. Conclusions Our data indicates that bacteriophage cocktails may be implemented to decolonize some intestinal carriers. However, the individual microbiota composition may have an impact on the development of phage resistance. Mechanisms underlying this phenomenon are likely to be various and complex. Further in vivo studies and protein expression experiments are needed to confirm our observations and hypotheses.

Methicillin-resistantStaphylococcus pseudintermedius(MRSP) is an important opportunistic pathogenic bacterium of dogs that also occasionally colonize and infect humans. However, whether MRSP can adapt to human hosts is not clear and whole genome sequences of MRSP from humans are still limited. Genomic comparative analyses of 3 couples of isolates from dogs (n = 3) and humans (n = 3) belonging to ST45, ST112, and ST181, the dominant clones in Thailand were conducted to determine the degree of similarities between human and animal MRSP of a same ST. Among eight prophages, three prophages associated with the leucocidins genes (lukF/S-I), φVB88-Pro1, φVB16-Pro1 and φAP20-Pro1, were distributed in the human MRSPs, while their remnants, φAH18-Pro1, were located in the dog MRSPs. A novel composite pathogenicity island, named SpPI-181, containing two integrase genes was identified in the ST181 isolates. The distribution of the integrase genes of the eight prophages and SpPI-181 was also analysed by PCR in 77 additional MRSP isolates belonging to different STs. The PCR screen revealed diversity in prophage carriage, especially in ST45 isolates. Prophage φAK9-Pro1 was only observed in ST112 isolates from dogs and SpPI-181 was found associated with ST181 clonal lineage. Among the 3 couple of isolates, ST45 strains showed the highest number of single nucleotide polymorphisms (SNP) in their core genomes (3,612 SNPs). The genomic diversity of ST45 isolates suggested a high level of adaptation that may lead to different host colonization of successful clones. This finding provided data on the genomic differences of MRSP associated with colonization and adaption to different hosts.

Objectives The aim of this study was to design a rapid and sensitive real-time PCR (rt-PCR) method for colistin resistance mcr-1 gene detection in human faecal samples. Methods Stools (n = 88) from 36 volunteers were analysed. To isolate mcr-1-producing Enterobacteriaceae, samples were enriched overnight in Luria–Bertani (LB) broth containing 2 mg/L colistin and were then plated on selective agar plates with 4 mg/L colistin. A SYBR® Green-based rt-PCR targeting mcr-1 was then designed. For method validation and to establish the limit of detection (LOD), total DNA was extracted from mcr-1-negative and mcr-1-positive Escherichia coli. rt-PCR was also performed with DNA extracted from 88 native stools and after enriching them in LB broth containing colistin. Results Based on the culture approach, three unique volunteers resulted colonised with mcr-1-harboring E. coli strains. For culture isolates, rt-PCR exhibited a LOD of 10 genomic copies/reaction, with both sensitivity and specificity of 100%. Nevertheless, when testing native stools, only two of the three mcr-1-positive specimens were detected. However, after enrichment in LB broth containing colistin, the rt-PCR was strongly positive for all culture-positive samples. The average cycle threshold was 22, granting rapid and confident detection of positive specimens within 30 cycles. No false positives were observed for the remaining 85 culture-negative specimens. Conclusions A rapid rt-PCR for detection of mcr-1 from stool specimens was developed. The detection rate was increased by testing selective broth enrichments. This approach also has the advantage of concomitant isolation of mcr-1-harboring strains for further antimicrobial susceptibility and genetic testing.

Background Neisseria gonorrhoeae cases are increasing in Europe, with decreasing susceptibility to first line antibiotics. Whole genome sequencing (WGS) provides detailed information about gonococcal molecular epidemiology and prediction of antimicrobial resistance (AMR), especially if linked to epidemiological data. The aim of this study was to examine molecular, clinical and social epidemiological aspects of gonorrhoea infections in Switzerland. Methods In 2015–2016, we cultured urethral, cervical, vaginal, rectal, and pharyngeal specimens from patients in three clinics predominantly attended by men who have sex with men (MSM) and female sex workers (FSW). MSM also completed a sexual behaviour questionnaire. Minimal inhibitory concentrations (MIC) were assessed by Etest, interpreted using EUCAST breakpoints except azithromycin (≥2 mg/L); WGS used an Illumina Miseq. Results We sequenced 140 isolates from 116 participants, MSM (107, 92%, mean age 35.8 years) and FSW (6, 5%, mean age 25.3 years). Amongst MSM, 48/105 respondents (45.7%) reported recent sex abroad. Three patients (two MSM and one FSW) carried different strains at different body sites. The isolates show large genomic diversity, with 69 NG-MAST types and 37 MLST sequence types, largely embedded within characterised European Union clusters. NG-MAST 1407 was identified in n=4 isolates from two patients (FSW, not travel-associated and MSM, sex elsewhere in Europe). Mosaic penAXXXIV was seen in these isolates, and also in an NG-MAST 13488 from an MSM, which was also not travel associated. One isolate (heterosexual male, not travel-associated) with elevated cefixime MIC (0.19 μg/ml) carried a mosaic penAX in an NG-MAST 10557 background. Ciprofloxacin resistance was seen in these six isolates, and overall in 59/140 (42%), all containing GyrA mutations S91F and D95A/G/N. Conclusion Switzerland has a high diversity of circulating gonorrhoea, generally related to European clusters. Multidrug resistant isolates were not identified in this study, but NG-MAST 1407 and penA mosaics, associated with elevated cephalosporin MICs, are circulating. Disclosure No significant relationships.

Enterovirulent Escherichia coli infections cause significant losses in the pig industry. However, information about the structures of the virulence and multidrug resistance (MDR) plasmids harboured by these strains is sparse. In this study, we used whole-genome sequencing with PacBio and Illumina platforms to analyse the molecular features of the multidrug-resistant enterotoxigenic E. coli (ETEC) strain 14OD0056 and the multidrug-resistant hybrid Shiga toxin-producing/enterotoxigenic E. coli (STEC/ETEC) strain 15OD0495 isolated from diarrheic pigs in Switzerland. Strain 14OD0056 possessed three virulence plasmids similar to others previously found in ETEC strains, while 15OD0495 harboured a 119-kb multivirulence IncFII/IncX1 hybrid STEC/ETEC plasmid (p15ODTXV) that co-carried virulence genes of both ETEC and STEC pathotypes, confirming the key role of plasmids in the emergence of hybrid pathotypes. All resistance genes of 14OD0056 that conferred resistance to ampicillin (blaTEM-1b), gentamicin (aac(3)-IIa), kanamycin (aph(3′)-Ia), sulfonamide (sul1 and sul2), streptomycin (aph(3″)-Ib, aph(6)-Id), tetracycline (tet(B)) and trimethoprim (dfrA1) were identified on a single 207-kb conjugative MDR plasmid of incompatibility group (Inc) IncHI1/IncFIA (p14ODMR). Strain 15OD0495 carried two antimicrobial resistance plasmids (p15ODAR and p15ODMR). The 99-kb IncI1 plasmid p15ODAR harboured only aminoglycoside resistance genes (aac(3)-IIa, aph(3″)-Ib, aph(6)-Id, aph(4)-Ia), whilst the 49-kb IncN MDR plasmid p15ODMR carried genes conferring resistance to ampicillin (blaTEM-1b), sulfonamide (sul2), streptomycin (aph(6)-Id), tetracycline (tet(A)) and trimethoprim (dfrA14). Filter mating assays showed that p14ODMR, p15ODMR and p15ODAR were conjugative at room temperature and 37 °C. The co-localization of multiple resistance genes on MDR conjugative plasmids such as p14ODMR and p15ODMR poses the risk of simultaneous selection of several resistance traits during empirical treatment. Thus, preventive strategies and targeted therapy following antibiotic susceptibility testing should be encouraged to avoid further dissemination of such plasmids.

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Molecular methods are often used for Neisseria gonorrhoeae detection, but complete definition of antimicrobial resistance (AMR) patterns still requires phenotypic tests. We developed an assay that both identifies N. gonorrhoeae and detects AMR determinants in clinical specimens., Molecular methods are often used for Neisseria gonorrhoeae detection, but complete definition of antimicrobial resistance (AMR) patterns still requires phenotypic tests. We developed an assay that both identifies N. gonorrhoeae and detects AMR determinants in clinical specimens. We designed a mismatch amplification mutation assay (MAMA)-based SYBR green real-time PCR targeting one N. gonorrhoeae-specific region (opa); mosaic penA alleles (Asp345 deletion [Asp345del], Gly545Ser) associated with decreased susceptibility to cephalosporins; and alterations conferring resistance to ciprofloxacin (GyrA Ser91Phe), azithromycin (23S rRNA A2059G and C2611T), and spectinomycin (16S rRNA C1192T). We applied the real-time PCR to 489 clinical specimens, of which 94 had paired culture isolates, and evaluated its performance by comparison with the performance of commercial diagnostic molecular and phenotypic tests. Our assay exhibited a sensitivity/specificity of 93%/100%, 96%/85%, 90%/91%, 100%/100%, and 100%/90% for the detection of N. gonorrhoeae directly from urethral, rectal, pharyngeal, cervical, and vaginal samples, respectively. The MAMA strategy allowed the detection of AMR mutations by comparing cycle threshold values with the results of the reference opa reaction. The method accurately predicted the phenotype of resistance to four antibiotic classes, as determined by comparison with the MIC values obtained from 94 paired cultures (sensitivity/specificity for cephalosporins, azithromycin, ciprofloxacin, and spectinomycin resistance, 100%/95%, 100%/100%, 100%/100%, and not applicable [NA]/100%, respectively, in genital specimens and NA/72%, NA/98%, 100%/97%, and NA/96%, respectively, in extragenital specimens). False-positive results, particularly for the penA Asp345del reaction, were observed predominantly in pharyngeal specimens. Our real-time PCR assay is a promising rapid method to identify N. gonorrhoeae and predict AMR directly in genital specimens, but further optimization for extragenital specimens is needed.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is common, compromising gonorrhoea treatment internationally. Rapid characterisation of AMR strains could ensure appropriate and personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of emergence and dissemination of AMR determinants, predicting AMR, in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that generates bacterial genomes within one day. However, accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench including de novo assembly and optimised BLAST algorithms, we show that 2D ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended antimicrobials in gonococcal isolates. We also show that the 2D ONT-derived sequences are useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes.

most colistin-resistant (Col-R) Klebsiella pneumoniae strains possess alterations of the two-component systems PhoP/Q and PmrA/B. These systems respond to environmental stimuli increasing the expression of the operon pmrHFIJKLM whose products are responsible for lipid A modifications leading to decreased affinity for polymyxins. This process is regulated by a negative feedback of the mgrB gene that encodes for a small protein repressing the PhoP/Q system. Thus, inactivation of mgrB leads to polymyxin resistance.

Background: Salmonella and Shigella spp. are 2 of the most frequent and deadly enteric bacterial pathogens recorded worldwide. In developing countries Salmonella infections are responsible for many deaths annually and these mortality rates are prone to increase due to the emergence of resistance to antibiotics. In this overall scenario new alternative therapeutic approaches are needed. Methods: For the first time, we investigated the activity of 3 commercial bacteriophage cocktails ( INTESTI , Septaphage , PYO ) against a collection of contemporary Salmonella spp. (n = 30) and Shigella spp. (n = 20) strains isolated in Switzerland. Phage susceptibility was determined by implementing the spot test. Results: The overall susceptibility of Salmonella spp. to INTESTI and Septaphage was 87% and 77%, respectively. With regard to Shigella spp., the overall susceptibility to INTESTI and Septaphage was 95% and 55%, respectively. PYO was observed to be active against only 10% of Salmonella spp. but against 95% of Shigella spp. Conclusions: Our results seem promising, especially for the INTESTI biopreparation against Salmonella enterica infections. Nevertheless, such speculation should be supported by further in vivo studies to confirm efficacy and safety of the cocktails. We also emphasize the importance of large in vitro screening analyses aimed to assess the activity of such biopreparations against contemporary multidrug-resistant strains that are emerging worldwide. Keywords: commercial; bacteriophages; Salmonella; Shigella cocktails

We characterized the genetic environment of mcr-1 in colistin-resistant Escherichia coli strains isolated in Switzerland during 2014 to 2016 from humans ( n = 3) and chicken meat ( n = 6). Whole-genome and plasmid sequencing identified the mcr-1 gene integrated in IncX4 (of which, one strain carried the mcr-1.2 variant), IncI2, IncHI2, and novel IncK2 plasmids (overall, n = 7), as well as in the bacterial chromosome ( n = 2) in single or duplicate copies. Our study supports the easy mobilization of mcr-1 across diverse genetic locations.

INTRODUCTION The number of genetic tests, mostly real-time PCRs, to detect antimicrobial resistance (AMR) determinants and predict AMR in Neisseria gonorrhoeae is increasing. Several of these assays are promising, but there are important shortcomings and few assays have been adequately validated and quality assured. Areas covered: Recent advances, focusing on publications since 2012, in the development and use of molecular tests to predict gonococcal AMR for surveillance and for clinical use, advantages and disadvantages of these tests and of molecular AMR prediction compared with phenotypic AMR testing, and future perspectives for effective use of molecular AMR tests for different purposes. Expert commentary: Several challenges for direct testing of clinical, especially extra-genital, specimens remain. The choice of molecular assay needs to consider the assay target, quality controls, sample types, limitations intrinsic to molecular technologies, and specific to the chosen methodology, and the intended use of the test. Improved molecular- and particularly genome-sequencing-based methods will supplement AMR testing for surveillance purposes, and translate into point-of-care tests that will lead to personalized treatments, while sparing the last available empiric treatment option (ceftriaxone). However, genetic AMR prediction will never completely replace phenotypic AMR testing, which detects also AMR due to unknown AMR determinants.

Introduction Strategies to identify antimicrobial resistance (AMR) and improve antibiotic stewardship to control the spread of AMR in Neisseria gonorrhoeae (NG) are urgently needed. As part of a project to develop a point-of-care (POC) test for AMR in NG, we investigated factors that could help identify infections due to antibiotic resistant NG. Methods We enrolled men who have sex with men (MSM) at sexual health centres in Zurich and Bern, Switzerland, from May 2015 to June 2016. All had samples taken for NG detection from urethra, rectum and pharynx. In culture positive specimens we obtained minimum inhibitory concentrations (MICs) using Etest for ciprofloxacin, ceftriaxone, cefixime and spectinomycin (EUCAST AMR breakpoints) and azithromycin (EuroGASP, >2 mg/L). We collected clinical data and patients completed an online questionnaire. We compared cases (positive NG culture and AMR) with controls (NG and no AMR) with odds ratios (OR) and 95% confidence intervals (CI). We used multivariable logistic regression in MSM with complete data for all included variables. Results Of 230 MSM enrolled, 117 had a positive NG culture. There were 46 (39%) cases with resistant NG (ciprofloxacin, n=45, azithromycin, n=1) and 71 controls. Clinical findings did not differ between cases and controls. Cases were more likely than controls to have had sex outside Switzerland in the previous 3 months (OR 2.2, 95% CI 1.0–4.7, p=0.05), to have received oral sex (OR 5.6, 95% CI 0.7–46.8, p=0.08) and to have concurrent partnerships (OR 2.2, 95% CI 0.8–6.5, p=0.11). In multivariable analysis (39 cases, 54 controls), the association with sex abroad remained (OR 2.0, 95% CI 0.9–4.8, p=0.10), controlling for concurrency. Conclusion In this population of MSM in Switzerland, AMR in NG might be more common in MSM who have sex abroad and who receive oral sex, possibly from asymptomatic pharyngeal NG. No clinical factors distinguished AMR from non-AMR NG infections in MSM. Strategies such as development of POC tests that detect AMR are needed to conserve last-line antibiotic treatment for NG.

We characterized the genetic environment of mcr-1 in colistin-resistant Escherichia coli strains isolated in Switzerland during 2014 to 2016 from humans (n = 3) and chicken meat (n = 6). Whole-genome and plasmid sequencing identified the mcr-1 gene integrated in IncX4 (of which, one strain carried the mcr-1.2 variant), IncI2, IncHI2, and novel IncK2 plasmids (overall, n = 7), as well as in the bacterial chromosome (n = 2) in single or duplicate copies. Our study supports the easy mobilization of mcr-1 across diverse genetic locations.

The aim of this study was to design a rapid and sensitive real-time PCR (rt-PCR) method for colistin resistance mcr-1 gene detection in human faecal samples.Stools (n=88) from 36 volunteers were analysed. To isolate mcr-1-producing Enterobacteriaceae, samples were enriched overnight in Luria-Bertani (LB) broth containing 2mg/L colistin and were then plated on selective agar plates with 4mg/L colistin. A SYBRBased on the culture approach, three unique volunteers resulted colonised with mcr-1-harboring E. coli strains. For culture isolates, rt-PCR exhibited a LOD of 10 genomic copies/reaction, with both sensitivity and specificity of 100%. Nevertheless, when testing native stools, only two of the three mcr-1-positive specimens were detected. However, after enrichment in LB broth containing colistin, the rt-PCR was strongly positive for all culture-positive samples. The average cycle threshold was 22, granting rapid and confident detection of positive specimens within 30 cycles. No false positives were observed for the remaining 85 culture-negative specimens.A rapid rt-PCR for detection of mcr-1 from stool specimens was developed. The detection rate was increased by testing selective broth enrichments. This approach also has the advantage of concomitant isolation of mcr-1-harboring strains for further antimicrobial susceptibility and genetic testing.

Background We describe the first two multifocal invasive infections due to Klebsiella pneumoniae recently observed in Switzerland. Methods Phenotypic (MIC assays and string test) and molecular analyses (PCR/Sequencing for bla, virulence factor genes and whole genome sequencing for one strain) were performed to characterize the causative K. pneumoniae isolates. Results Both K. pneumoniae isolates (Kp1 and Kp2) were pan-susceptible to antibiotics and produced narrow-spectrum SHV β-lactamases. However, only Kp1 was string test positive. Kp1 was of ST380 and caused liver abscess as well as pneumonia and orbital phlegmon in an Eritrean patient. It belonged to the hypervirulent capsular serotype K2 and harboured the classic virulence-associated rmpA and aerobactin genes, fulfilling both the clinical and microbiological definitions for an invasive K. pneumoniae syndrome. Kp2 was of ST1043 and caused both liver abscess and endocarditis in a Swiss patient. Moreover, it did not possess the classic virulence-associated genes. Whole genome sequencing identified less well-known virulence factors in Kp2 that might have contributed to its virulence. Among these there were genes important for intestinal colonization and/or invasion, such as genes involved in adhesion (e.g., fimABCD and mrkABCD), regulation of capsule polysaccharide biosynthesis (e.g., evgS-evgA), as well as iron uptake (iroN), energy conversion, and metabolism. Discussion This report confirms the continuous dissemination of hypervirulent K. pneumoniae strains among patients of non-Asian descent in Europe. Moreover, it highlights the genetic background of an atypical hypervirulent K. pneumoniae causing a severe invasive infection despite not possessing the classical virulence characteristics of hypermucoviscous strains.

Objectives Bacteriophages may represent a therapeutic alternative to treat infections caused by multidrug-resistant (MDR) pathogens. However, studies analysing their activity against MDR Enterobacteriaceae are limited. Methods The in vitro lytic activity of three commercial bacteriophage cocktails (PYO, INTESTI and Septaphage) was evaluated against 70 Escherichia coli and 31 Proteus spp. of human and non-human origin. Isolates were characterised by phenotypic and genotypic methods and included 82 MDR strains [44 extended-spectrum-β-lactamase (ESBL)-producers (18 CTX-M-15-like, including ST131/ST648 E. coli); 27 plasmid-mediated AmpC β-lactamase (pAmpC)-producers (23 CMY-2-like, including ST131 E. coli); 3 ESBL + pAmpC-producers; and 8 carbapenemase-producers]. Phage susceptibility was determined by the spot test. Results E. coli susceptibility to PYO, INTESTI and Septaphage was 61%, 67% and 9%, whereas that of Proteus spp. was 29%, 39% and 19%, respectively. For the subgroup of ESBL-producing E. coli/Proteus spp., the following susceptibility rates were recorded: PYO, 57%; INTESTI, 59%; and Septaphage, 11%. With regard to pAmpC-producers, 59%, 70% and 11% were susceptible to PYO, INTESTI and Septaphage, respectively. Five of eight carbapenemase-producers and three of four colistin-resistant E. coli were susceptible to PYO and INTESTI. Conclusions This is the first study analysing the activity of the above three cocktails against well-characterised MDR E. coli and Proteus spp. The overall narrow spectrum of activity observed could be related to the absence of specific bacteriophages targeting these contemporary MDR strains that are spreading in different settings. Therefore, bacteriophages targeting emerging MDR pathogens need to be isolated and integrated in such biopreparations.

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10 3 to 10 4 genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

Antibiotic resistance in Ureaplasma urealyticum / Ureaplasma parvum and Mycoplasma hominis is an issue of increasing importance. However, data regarding the susceptibility and, more importantly, the clonality of these organisms are limited. We analyzed 140 genital samples obtained in Bern, Switzerland, in 2014. Identification and antimicrobial susceptibility tests were performed by using the Mycoplasma IST 2 kit and sequencing of 16S rRNA genes. MICs for ciprofloxacin and azithromycin were obtained in broth microdilution assays. Clonality was analyzed with PCR-based subtyping and multilocus sequence typing (MLST), whereas quinolone resistance and macrolide resistance were studied by sequencing gyrA, gyrB , parC , and parE genes, as well as 23S rRNA genes and genes encoding L4/L22 ribosomal proteins. A total of 103 samples were confirmed as positive for U. urealyticum / U. parvum , whereas 21 were positive for both U. urealyticum / U. parvum and M. hominis . According to the IST 2 kit, the rates of nonsusceptibility were highest for ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.9%), erythromycin (1.9%), and azithromycin (1%). However, inconsistent results between microdilution and IST 2 kit assays were recorded. Various sequence types (STs) observed previously in China (ST1, ST2, ST4, ST9, ST22, and ST47), as well as eight novel lineages, were detected. Only some quinolone-resistant isolates had amino acid substitutions in ParC (Ser83Leu in U. parvum of serovar 6) and ParE (Val417Thr in U. parvum of serovar 1 and the novel Thr417Val substitution in U. urealyticum ). Isolates with mutations in 23S rRNA or substitutions in L4/L22 were not detected. This is the first study analyzing the susceptibility of U. urealyticum / U. parvum isolates in Switzerland and the clonality outside China. Resistance rates were low compared to those in other countries. We hypothesize that some hyperepidemic STs spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important issue.

To improve the current vaccine against tuberculosis, a recombinant strain of Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing a Mycobacterium tuberculosis vaccine candidate antigen (MPT64) in strong association with the mycobacterial cell wall was developed. To deliver the candidate antigen on the surface, we fused the mpt64 gene to the sequence encoding the PE domain of the PE_PGRS33 protein of M. tuberculosis (to create strain H PE -ΔMPT64-BCG), which we have previously shown to transport proteins to the bacterial surface. In a series of protection experiments in the mouse model of tuberculosis, we showed that (i) immunization of mice with H PE -ΔMPT64-BCG provides levels of protection significantly higher than those afforded by the parental BCG strain, as assessed by bacterial colonization in lungs and spleens and by lung involvement (at both 28 and 70 days postchallenge), (ii) rBCG strains expressing MPT64 provide better protection than the parental BCG strain only when this antigen is surface expressed, and (iii) the H PE -ΔMPT64-BCG-induced MPT64-specific T cell repertoire when characterized by β chain variable region-β chain joining region (BV-BJ) spectratyping indicates that protection is correlated with the ability to recruit gamma interferon (IFN-γ)-secreting T cells carrying the BV8.3-BJ1.5 (172 bp) shared rearrangement. These results demonstrate that H PE -ΔMPT64-BCG is one of the most effective new vaccines tested so far in the mouse model of tuberculosis and underscore the impact of antigen cellular localization on the induction of the specific immune response induced by rBCG.

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that σ F is required for carotenoid pigment production in Mycobacterium smegmatis . We further show that a sigF mutant exhibits a transformation efficiency 10 4 -fold higher than that of the parental strain, suggesting that σ F regulates the production of components affecting cell wall permeability. In addition, a sigF mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the M. smegmatis genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation.

The extracytoplasmic factor (ECF) sigma factor σ E is one of the most studied sigma factors of Mycobacterium tuberculosis . It has been shown to be involved in virulence as well as in survival under conditions of high temperature, alkaline pH, and exposure to detergents and oxidative stress. Unlike many ECF sigma factors, σ E does not directly regulate the transcription of its own gene. Two promoters have been identified upstream of the sigE gene; one is regulated by the two-component system MprAB, while the other has been shown to be σ H dependent. In this paper, we further characterize the regulation of σ E by identifying its anti-sigma factor and a previously unknown promoter. Finally, we show that sigE can be translated from three different translational start codons, depending on the promoter used. Taken together, our data demonstrate that σ E not only is subjected to complex transcriptional regulation but is also controlled at the translational and posttranslational levels.

Background Molecular tests to detect antimicrobial resistance in Neisseria gonorrhoeae (NG) are urgently needed. Genetic methods are the mainstay of NG detection in many settings, but resistance testing still requires conventional phenotypic tests. The objective of this study was to develop an assay to detect the most clinically important genetic markers conferring resistance to antibiotics used for the treatment of gonorrhoea. Methods We designed a fluorescent dye real-time PCR assay combined with high resolution melting (HRM) analysis. Several triplex and duplex reactions included target sequences for: two NG-specific genes ( porA and opa ); targets specific for the penA mosaic XXXIV (A501, G545S), associated with reduced cephalosporin susceptibility; single nucleotide polymorphisms conferring resistance to ciprofloxacin (GyrA S91F), azithromycin (23S rRNA A2059G and C2611T) and spectinomycin (16S rRNA C1192T and 5S rRNA T24P). We tested >50 characterised NG isolates, including: clinical isolates resistant to ciprofloxacin, azithromycin, spectinomycin, ceftriaxone (strain F89), strains with reduced cephalosporin susceptibility, the wild-type reference strain ATCC49226, and commensal Neisseria spp. Results HRM analysis correctly identified: all NG strains and all mutations, except for the A501P mutation in strain F89. However, cross-reactions with commensal Neisseria spp. occurred for: penA mosaic XXXIV (n = 3 species) and 23S rRNA (n = 3 species). Conclusion Our multiplex PCR assay accurately identified NG and detected the most frequent mutations associated with antimicrobial resistance in cultured isolates. The assay provides results significantly quicker than current culture-based methods. The analytical sensitivity and specificity of the assay for use with urethral, rectal, pharyngeal and vaginal specimens should be evaluated in the near future.

Background Antibiotic resistance in U. urealyticum (UUA), U. parvum (UPA) and M. hominis (MH) poses an increasing issue. However, data regarding antibiotic susceptibility is limited to several countries, whereas information about clonality is available only from China. Methods We analysed 140 genital samples collected in two laboratories from unique patients in Bern during 2014. Identification and antimicrobial susceptibility tests were obtained using the mycoplasma IST 2 kit (bioMerieux) and sequencing of 16S rDNA. Clonality was analysed with multilocus sequence typing (MLST) and expanded MLST (eMLST), whereas quinolone and macrolide resistance were studied by sequencing gyrA/B , parC/E , as well as genes encoding 23S rRNA and L4/22 ribosomal proteins. Results One-hundred-three samples (74%) were confirmed being positive for UUA/UPA, whereas 21 (15%) were positive for both UUA/UPA and MH. Non-susceptibility was highest to ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.8%), erythromycin (1.9%), azithromycin and tetracycline (both Conclusion This is the first study analysing susceptibility of Ureaplasma spp. isolates detected in Switzerland and the clonal distribution outside China. Resistance rates are low compared to other surrounding countries, but the empirical use of quinolones is compromised. We hypothesise that some hyperepidemic STs (e.g., ST4) spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important aspect.

PPE represent a peculiar family of mycobacterial proteins characterized by a 180 aminoacids conserved N-terminal domain. Several PPE genes are co-transcribed with a gene encoding for a protein belonging to another family of mycobacterial specific proteins named PE. Only one PE-PPE couple has been extensively characterized so far (PE25-PPE41) and it was shown that these two proteins form a heterodimer and that this interaction is essential for PPE41 stability and translocation through the mycobacterial cell wall. In this study we characterize the PE11-PPE17 couple. In contrast with what was found for PE25-PPE41, we show that PPE17 is not secreted but surface exposed. Moreover, we demonstrate that the presence of PE11 is not necessary for PPE17 stability or for its localization on the mycobacterial surface. Finally, we show that the PPE domain of PPE17 targets the mycobacterial cell wall and that this domain can be used as a fusion partner to expose heterologous proteins on the mycobacterial surface.

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PE(Rv1818c)), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PE(Rv1818c) domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation.