Emergence of OXA-48-producing Enterobacter hormaechei in a Swiss companion animal clinic and their genetic relationship to clinical human isolates.

BACKGROUND: Enterobacter hormaechei producing the carbapenemase OXA-48 was identified repeatedly in infections in companion animals hospitalized at a Swiss veterinary clinic where OXA-48-producing Klebsiella pneumoniae was previously reported. OBJECTIVES: To determine the genetic relatedness of animal and human E. hormaechei strains collected in Switzerland during 2017-22 and their mobile genetic elements. METHODS: Hybrid assemblies for phylogenetic and comparative analysis of animal (n = 9) and human (n = 25) isolates were obtained by sequencing with Illumina, PacBio and Oxford Nanopore Technologies. Antimicrobial susceptibility was tested by broth microdilution. RESULTS: The animal strains were identified as E. hormaechei subsp. xiangfangensis ST114 (n = 6) and ST418 (n = 2), and E. hormaechei subsp. hoffmannii ST78 (n = 1). Human E. hormaechei belonged to subspecies steigerwaltii (n = 10), xiangfangensis (n = 13), hoffmannii (n = 1) and hormaechei (n = 1), with a heterogeneous ST distribution differing from the animal strains, except for two ST114. Core-gene SNP analysis confirmed the clonality of the animal ST114 and ST418 isolates (0 to 10 SNPs), and close relatedness of animal and human ST114 strains (80-120 SNPs). The strains harboured the blaOXA-48 gene on ca. 63 kb IncL-type plasmids (n = 27); on ca. 72 kb IncL plasmids co-harbouring blaCTX-M-14 (n = 2); and on ca. 150-180 kb IncFIB (n = 4) or hybrid IncFIB/IncL (n = 1) plasmids. The blaOXA-48-harbouring plasmids and the blaDHA-1-carrying ISCR1 element in one animal ST114 and both ST418 clones were likely acquired from previously spreading K. pneumoniae strains. CONCLUSIONS: Common ecological niches favour the spread of plasmid-borne carbapenemases among Enterobacterales and the emergence of MDR E. hormaechei clones.

The Novel Macrolide Resistance Genes mef(D), msr(F), and msr(H) Are Present on Resistance Islands in Macrococcus canis, Macrococcus caseolyticus, and Staphylococcus aureus.

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.

Mismatch Amplification Mutation Assay-Based Real-Time PCR for Rapid Detection of Neisseria gonorrhoeae and Antimicrobial Resistance 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.

Heterogeneous Genetic Location of mcr-1 in Colistin-Resistant Escherichia coli Isolates from Humans and Retail Chicken Meat in Switzerland: Emergence of mcr-1-Carrying IncK2 Plasmids.

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.

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

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 (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for 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 Susceptibility and Sequence Type Distribution of Ureaplasma Species Isolated from Genital Samples in Switzerland.

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.

Whole-Genome Sequences of a Lactobacillus melliventris Strain and Its Myovirus Temperate Phage, phIBH004, Isolated from the Digestive Tract of Apis mellifera in Switzerland.

The complete genome sequence of Lactobacillus melliventris strain IBH004, isolated from the gut of a honeybee worker (Apis mellifera) and containing two plasmids and a temperate phage, was determined using hybrid assembly of Oxford Nanopore and Illumina reads. Phage-sequence relationships were identified from the coding sequences, and a proteomic tree was constructed.

Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae.

Genomic insights into methicillin-resistant Staphylococcus pseudintermedius isolates from dogs and humans of the same sequence types reveals diversity in prophages and pathogenicity islands.

Methicillin-resistant Staphylococcus 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.

Surface expression of MPT64 as a fusion with the PE domain of PE_PGRS33 enhances Mycobacterium bovis BCG protective activity against Mycobacterium tuberculosis in mice.

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 ß chain variable region-ß 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.

Investigating the use of bacteriophages as a new decolonization strategy for intestinal carriage of CTX-M-15-producing ST131 Escherichia coli: An in vitro continuous culture system model.

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 48h. 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 10h 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 (≤101CFU/mL after 6h). In contrast, during study II, a phage-resistant mutant of 4901.28 persisted in the continuous culture (104CFU/mL at 48h). 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 108CFU/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.

Gradual in vitro Evolution of Cefepime Resistance in an ST131 Escherichia coli Strain Expressing a Plasmid-Encoded CMY-2 ß-Lactamase.

BACKGROUND: In a previous report, a clinical ST131 Escherichia coli isolate (Ec-1),producing a plasmid-encoded AmpC ß-lactamase CMY-2, evolved in vivo under cefepime (FEP) treatment to the FEP-resistant Ec-2 strain expressing an extended-spectrum ß-lactamase CMY-33. To compare factors responsible for in vitro and in vivo FEP resistance, we reproduced in vitro FEP resistance evolution in Ec-1. METHODS: FEP-resistant mutants were generated by subjecting Ec-1 (FEP MIC = 0.125 mg/L) to sub-inhibitory concentrations of FEP. MICs were obtained by broth microdilution or Etest. Strains were sequenced on an Illumina HiSeq platform. Transcriptional levels and plasmid copy numbers were determined by real-time PCR. Outer membrane proteins (OMPs) were extracted and separated by SDS-PAGE. Growth kinetics was evaluated by measuring OD450. RESULTS: The CMY-2 expressed by Ec-1 evolved to a CMY-69 (strain EC-4) by an Ala294Pro substitution after 24 passages. After 30 passages, the FEP MIC increased to 256 mg/L (strain EC-32). SDS PAGE did not reveal any lack of OMPs in the mutant strains. However, bla CMY transcription levels were up to 14-times higher than in Ec-1, which was partially explained by mutations in the upstream region of repA resulting in a higher copy number of the bla CMY-harboring IncI1 plasmid. All mutants showed a slight growth defect but no significant difference in relative growth rates compared to Ec-1. CONCLUSION: In vitro sub-inhibitory concentrations of FEP resulted in the selection of resistance mutations altering the H-10 helix of the CMY-2 and increasing the plasmid copy number. Appropriate dosing strategies may help preventing resistance evolution during treatments.

Evidence of complex transcriptional, translational, and posttranslational regulation of the extracytoplasmic function sigma factor sigmaE in Mycobacterium tuberculosis.

The extracytoplasmic factor (ECF) sigma factor sigma(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, sigma(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 sigma(H) dependent. In this paper, we further characterize the regulation of sigma(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 sigma(E) not only is subjected to complex transcriptional regulation but is also controlled at the translational and posttranslational levels.

SigF controls carotenoid pigment production and affects transformation efficiency and hydrogen peroxide sensitivity in Mycobacterium smegmatis.

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that sigma(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 sigma(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.

Comparative Genomics of the First and Complete Genome of "Actinobacillus porcitonsillarum" Supports the Novel Species Hypothesis.

"Actinobacillus porcitonsillarum" is considered a nonpathogenic member of the Pasteurellaceae family, which phenotypically resembles the pathogen Actinobacillus pleuropneumoniae. Previous studies suggested that "A. porcitonsillarum" may represent a new species closely related to Actinobacillus minor, yet no full genome has been sequenced so far. We implemented the Oxford Nanopore and Illumina sequencing technologies to obtain the highly accurate and complete genome sequence of the "A. porcitonsillarum" strain 9953L55. After validating our de novo assembly strategy by comparing the A. pleuropneumoniae S4074T genome sequence obtained by Oxford Nanopore Technology combined with Illumina reads with a PacBio-sequenced S4074T genome from the NCBI database, we performed comparative analyses of the 9953L55 genome with the A. minor type strain NM305T, A. minor strain 202, and A. pleuropneumoniae S4074T. The 2,263,191 bp circular genome of 9953L55 consisted of 2168 and 2033 predicted genes and proteins, respectively. The lipopolysaccharide cluster resembled the genetic organization of A. pleuropneumoniae serotypes 1, 9, and 11, possibly explaining the positive reactions observed previously in serotyping tests. In contrast to NM305T, we confirmed the presence of a complete apxIICABD operon in 9953L55 and 202 accounting for their hemolytic phenotype and Christie-Atkins-Munch-Petersen (CAMP) reaction positivity. Orthologous gene cluster analysis provided insight into the differential ability of strains of the A. minor/"porcitonsillarum" complex and A. pleuropneumoniae to ferment lactose, raffinose, trehalose, and mannitol. The four strains showed distinct and shared transposable elements, CRISPR/Cas systems, and integrated prophages. Genome comparisons based on average nucleotide identity and in silico DNA-DNA hybridization confirmed the close relationship among strains belonging to the A. minor/"porcitonsillarum" complex compared to other Actinobacillus spp., but also suggested that 9953L55 and 202 belong to the same novel species closely related to A. minor, namely, "A. porcitonsillarum." Recognition of the taxon as a separate species would improve diagnostics and control strategies of pig pleuropneumonia.

In Vitro Activity of 3 Commercial Bacteriophage Cocktails Against Salmonella and Shigella spp. Isolates of Human Origin.

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.

Antimicrobial resistance prediction and phylogenetic analysis of Neisseria gonorrhoeae isolates using the Oxford Nanopore MinION sequencer.

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.

Recent advances in the development and use of molecular tests to predict antimicrobial resistance in Neisseria gonorrhoeae.

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.

In vitro activity of three commercial bacteriophage cocktails against multidrug-resistant Escherichia coli and Proteus spp. strains of human and non-human origin.

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-ß-lactamase (ESBL)-producers (18 CTX-M-15-like, including ST131/ST648 E. coli); 27 plasmid-mediated AmpC ß-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.

First two cases of severe multifocal infections caused by Klebsiella pneumoniae in Switzerland: characterization of an atypical non-K1/K2-serotype strain causing liver abscess and endocarditis.

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 ß-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.