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Objectives Escherichia coli can cause infections in the urinary tract and in normally sterile body sites leading to invasive E. coli disease (IED), including bacteraemia and sepsis, with older populations at increased risk. We aimed to estimate the theoretical coverage rate by the ExPEC4V and 9V vaccine candidates. In addition, we aimed at better understanding the diversity of E. coli isolates, including their genetic and phenotypic antimicrobial resistance (AMR), sequence types (STs), O-serotypes and the bacterial population structure. Methods Blood and urine culture E. coli isolates (n  = 304) were collected from hospitalized patients ≥60 years (n = 238) with IED during a multicentric, observational study across three continents. All isolates were tested for antimicrobial susceptibility, O-serotyped, whole-genome sequenced and bioinformatically analysed. Results A large diversity of STs and of O-serotypes were identified across all centres, with O25b-ST131, O6-ST73 and O1-ST95 being the most prevalent types. A total of 45.4% and 64.7% of all isolates were found to have an O-serotype covered by the ExPEC4V and ExPEC9V vaccine candidates, respectively. The overall frequency of MDR was 37.4% and ST131 was predominant among MDR isolates. Low in-patient genetic variability was observed in cases where multiple isolates were collected from the same patient. Conclusions Our results highlight the predominance of MDR O25b-ST131 E. coli isolates across diverse geographic areas. These findings provide further baseline data on the theoretical coverage of novel vaccines targeting E. coli associated with IED in older adults and their associated AMR levels. [ABSTRACT FROM AUTHOR]

Objectives To investigate the global distribution of an optrA -harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage. Methods Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed. Results Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3–916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn 6674. Only three isolates displayed an optrA -carrying plasmid with complete or partial Tn 6674. The WT OptrA protein was most widespread in the ST476 lineage. Conclusions Linezolid-resistant optrA -carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage. [ABSTRACT FROM AUTHOR]

Background Streptococcus suis is an important pig pathogen and an emerging zoonotic agent. In a previous study, we described a high proportion of penicillin-resistant serotype 9 S. suis (SS9) isolates on pig farms in Italy. Objectives We hypothesized that resistance to penicillin emerged in some SS9 lineages characterized by substitutions at the PBPs, contributing to the successful spread of these lineages in the last 20 years. Methods Sixty-six SS9 isolates from cases of streptococcosis in pigs were investigated for susceptibility to penicillin, ceftiofur and ampicillin. The isolates were characterized for ST, virulence profile, and antimicrobial resistance genes through WGS. Multiple linear regression models were employed to investigate the associations between STs, year of isolation, substitutions at the PBPs and an increase in MIC values to β-lactams. Results MIC values to penicillin increased by 4% each year in the study period. Higher MIC values for penicillin were also positively associated with ST123, ST1540 and ST1953 compared with ST16. The PBP sequences presented a mosaic organization of blocks. Within the same ST, substitutions at the PBPs were generally more frequent in recent isolates. Resistance to penicillin was driven by substitutions at PBP2b, including K479T, D512E and K513E, and PBP2x, including T551S, while reduced susceptibility to ceftiofur and ampicillin were largely dependent on substitutions at PBP2x. Conclusions Here, we identify the STs and substitutions at the PBPs responsible for increased resistance of SS9 to penicillin on Italian pig farms. Our data highlight the need for monitoring the evolution of S. suis in the coming years. [ABSTRACT FROM AUTHOR]

Objectives: Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) represent a major public health concern due to limited treatment options. Among invasive isolates of VREfm, ST117, ST80 and ST78 represent the most frequently detected STs by MLST in Germany. In this study, we investigated the genetic diversity of isolates of VREfm recovered from different nosocomial outbreaks in Bavaria, Germany, by WGS.Methods: Between January 2018 and April 2019, 99 non-replicate isolates of VREfm originating from nosocomial outbreaks at eight different hospitals in Bavaria were investigated for genetic diversity by WGS. In detail, complex types (CTs) were identified by core-genome MLST. Furthermore, an SNP analysis was performed for all VREfm strains.Results: Most of the isolates of this study (76%) belonged to three major clonal groups, which occurred in at least three hospitals: ST80/CT1065 vanB (n = 45; six hospitals), ST117/CT71 vanB (n = 11; four hospitals) and ST78/CT894like vanA (n = 19; three hospitals). Moreover, isolates of the predominant lineage ST80/CT1065 vanB showed a maximum difference of 36 SNPs as revealed by SNP analysis.Conclusions: Whole-genome analysis of VREfm causing nosocomial outbreaks suggests the occurrence of few endemic clonal lineages in Bavarian hospital settings, namely ST80/CT1065 vanB, ST117/CT71 vanB and ST78/CT894like vanA. Further studies are needed for a better understanding of the factors affecting the successful spread of the above-mentioned lineages. [ABSTRACT FROM AUTHOR]

Background: Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic.Objectives: To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production.Methods: RNA expression was evaluated using real-time RT-PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting.Results: Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21-30 min for ST131 isolates compared with <2-23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with -4- to 70-fold for non-ST131 isolates).Conclusions: Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment. [ABSTRACT FROM AUTHOR]

Objectives: The continuous emergence of ceftriaxone-resistant Neisseria gonorrhoeae strains threatens the effectiveness of current treatment regimens for gonorrhoea. The objective of the present study was to characterize three ceftriaxone-resistant N. gonorrhoeae strains with a novel mosaic penA allele isolated in China.Methods: Three ceftriaxone-resistant Neisseria gonorrhoeae strains (GC150, GC161 and GC208) isolated in 2017 were characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST), MLST and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR). Recombination analyses were performed using the SimPlot software.Results: Three strains had the same antibiotic resistance profiles, with resistance to ceftriaxone (MIC 0.5 mg/L), ciprofloxacin (MIC 8.0 mg/L), penicillin (MIC 2.0 mg/L) and tetracycline (MIC 2.0-8.0 mg/L). STs were assigned as MLST7360, NG-MAST14292 and NG-STAR1611/NG-STAR1612. The penA gene of these three strains differed from previous ceftriaxone-resistant gonococcal strains and harboured a novel mosaic allele (penA-121.001). Like N. gonorrhoeae FC428, a widely disseminated ceftriaxone-resistant strain that was initially described in Japan in 2015, all strains also possessed substitutions A311V and T483S in PBP2, which are associated with resistance to ceftriaxone. Potential recombination events were detected in penA between N. gonorrhoeae strain FC428 and commensal Neisseria species. Our results provide further evidence that the commensal Neisseria species (Neisseria cinerea and Neisseria perflava) can serve as a reservoir of ceftriaxone resistance-mediating penA sequences in clinical gonococcal strains.Conclusions: The emergence of such strains may be the result of the interspecies recombination of penA genes between N. gonorrhoeae strain FC428 and commensal Neisseria species. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus pseudintermedius is a common pathogen in dogs and methicillin resistance has emerged over recent decades. According to the current guidelines, S. pseudintermedius displaying oxacillin resistance should be reported as resistant to all β-lactams.Objectives: To identify possible associations between β-lactam resistance levels and clonal complexes (CCs) and/or staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant S. pseudintermedius (MRSP).Methods: MICs of oxacillin, penicillin, ampicillin, amoxicillin/clavulanic acid and cefalotin were determined by broth microdilution for 86 clinical canine MRSP isolates from Denmark and the Netherlands. PCR and sequencing were used for SCCmec typing and MLST.Results: Isolates belonged to CC71 (n = 36), CC258 (n = 33), CC45 (n = 11), CC68 (n = 1) and five singleton STs. SCCmecII-III was exclusively found in CC71 and SCCmecIV was significantly associated with CC258. SCCmecV and non-typeable SCCmec types occurred in 4 and 14 isolates, respectively. SCCmecIV was associated with lower MICs of oxacillin (<2 mg/L), ampicillin (<8 mg/L) and amoxicillin/clavulanic acid (<4 mg/L) and with susceptibility to cefalotin (<4 mg/L). All isolates harbouring SCCmecV were susceptible to cefalotin as well.Conclusions: SCCmec types were associated with different CCs and with either high- or low-level resistance to different β-lactams. The finding of amoxicillin/clavulanic acid (20%) and cefalotin (70%) in vitro susceptibility across all CCs might have clinical implications, since amoxicillin/clavulanic acid and first-generation cephalosporins are first-choice antibiotics for treatment of S. pseudintermedius infections. Pharmacokinetic/pharmacodynamic and clinical outcome studies are warranted to evaluate the in vivo efficacy of these β-lactams for treatment of MRSP infections. [ABSTRACT FROM AUTHOR]

Objectives: To determine the association of Neisseria gonorrhoeae antimicrobial resistance and genotypes using N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR).Methods: We characterized 124 N. gonorrhoeae isolates for their antimicrobial susceptibility profiles and NG-STAR ST characteristics using the guidelines of CLSI and EUCAST. The NG-STAR STs of seven loci were analysed. N. gonorrhoeae multiantigen sequence typing (NG-MAST) and MLST analysis was conducted in isolates with specific NG-STAR STs.Results: NG-STAR differentiated 124 N. gonorrhoeae isolates into 84 STs, of which 66 STs were novel to the NG-STAR database. NG-STAR ST-199, ST-348, ST-428, ST-497 and ST-1138 were the predominant STs. Three N. gonorrhoeae isolates with ceftriaxone and cefixime MICs ≥1.0 mg/L were grouped as NG-STAR ST-233. NG-STAR ST-202 isolates (n=4) were associated with high azithromycin MICs and had an identical NG-MAST ST. The NG-STAR ST-348 group (n=5) comprised more isolates with reduced susceptibility to cefixime (n=4) than cefixime-susceptible isolates (n=1).Conclusions: NG-STAR analysis differentiated N. gonorrhoeae isolates in settings with a high prevalence of antimicrobial resistance. Specific NG-STAR STs are associated with reduced susceptibility to ceftriaxone or cefixime and resistance to azithromycin in N. gonorrhoeae. [ABSTRACT FROM AUTHOR]

Background: The population structure of extraintestinal pathogenic Escherichia coli evolves over time, notably due to the emergence of antibiotic-resistant clones such as ESBL-producing Enterobacteriaceae (ESBL-E).Objectives: To analyse by WGS the genetic diversity of a large number of ESBL-E isolated from urinary tract infections in children from paediatric centres across France between 2014 and 2017 and collected by the National Observatory of febrile urinary tract infection (FUTI) caused by ESBL-E.Methods: A total of 40 905 Enterobacteriaceae-positive urine cultures were identified. ESBL-E were found in 1983 samples (4.85%). WGS was performed on 251 ESBL-E causing FUTI. STs, core genome MLST (cgMLST), serotype, fimH allele, ESBL genes and presence of papGII key virulence factor were determined.Results: E. coli and Klebsiella pneumoniae were found in 86.9% (218/251) and 11.2% (28/251) of cases, respectively. Several STs predominate among E. coli such as ST131, ST38, ST69, ST73, ST95, ST405, ST12 and ST1193, while no ST emerged in K. pneumoniae. E. coli ST131, ST38 and ST1193 increased during the study period, with a heterogeneity in papGII prevalence (64.5%, 35% and 20% respectively). Most isolates harboured the CTX-M type (97%) with a predominance of blaCTX-M-15. blaCTX-M-27, an emerging variant in E. coli, is found in various STs. cgMLST enabled discrimination of clusters within the main STs.Conclusions: The predominance of ST131, and the emergence of other STs such as ST38 and ST1193 combined with ESBL genes deserves close epidemiological surveillance considering their high threat in infectious disease. cgMLST could be a discriminant complementary tool for the analyses. [ABSTRACT FROM AUTHOR]

Objectives: To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India.Methods: E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes.Results and Conclusions: Analysis of isolates collected without selection (n=33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15. [ABSTRACT FROM AUTHOR]

Objectives: Third-generation cephalosporin-resistant Escherichia coli from community-acquired urinary tract infections are increasingly reported worldwide. We sought to determine and characterize the mechanisms of cefotaxime resistance employed by urinary E. coli obtained from primary care, over 12 months, in Bristol and surrounding counties in South-West England.Methods: Cefalexin-resistant E. coli isolates were identified from GP-referred urine samples using disc susceptibility testing. Cefotaxime resistance was determined by subsequent plating onto MIC breakpoint plates. β-Lactamase genes were detected by PCR. WGS was performed on 225 isolates and analyses were performed using the Center for Genomic Epidemiology platform. Patient information provided by the referring general practices was reviewed.Results: Cefalexin-resistant E. coli (n=900) isolates were obtained from urines from 146 general practices. Following deduplication by patient approximately 69% (576/836) of isolates were cefotaxime resistant. WGS of 225 isolates identified that the most common cefotaxime-resistance mechanism was blaCTX-M carriage (185/225), followed by plasmid-mediated AmpCs (pAmpCs) (17/225), AmpC hyperproduction (13/225), ESBL blaSHV variants (6/225) or a combination of both blaCTX-M and pAmpC (4/225). Forty-four STs were identified, with ST131 representing 101/225 isolates, within which clade C2 was dominant (54/101). Ciprofloxacin resistance was observed in 128/225 (56.9%) of sequenced isolates, predominantly associated with fluoroquinolone-resistant clones ST131 and ST1193.Conclusions: Most cefalexin-resistant E. coli isolates were cefotaxime resistant, predominantly caused by blaCTX-M carriage. The correlation between cefotaxime resistance and ciprofloxacin resistance was largely attributable to the high-risk pandemic clones ST131 and ST1193. Localized epidemiological data provide greater resolution than regional data and can be valuable for informing treatment choices in the primary care setting. [ABSTRACT FROM AUTHOR]

Objectives: AmpC-β-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common β-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance.Methods: We tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs.Results: In total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79-0.94) on the training set, 0.79 (95% CI=0.64-0.89) on validation set 1 and 0.85 (95% CI=0.71-0.94) on validation set 2.Conclusions: This approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing. [ABSTRACT FROM AUTHOR]

Objectives: In 2015 and 2016 Poland recorded rapid proliferation of New Delhi MBL (NDM)-producing Enterobacterales, with at least 470 and 1780 cases, respectively. We addressed the roles of the Klebsiella pneumoniae ST11 NDM-1 outbreak genotype, already spreading in 2012-14, and of newly imported organisms in this increase.Methods: The study included 2136 NDM-positive isolates identified between April 2015 and December 2016, following transfer of patients with K. pneumoniae ST147 NDM-1 from Tunisia to Warsaw in March 2015. The isolates were screened by PCR mapping for variants of blaNDM-carrying Tn125-like elements. Selected isolates were typed by PFGE and MLST. NDM-encoding plasmids were analysed by nuclease S1/hybridization, transfer assays, PCR-based replicon typing and PCR mapping.Results: The organisms were mainly K. pneumoniae containing the Tn125A variant of the ST11 epidemic lineage (n = 2094; ∼98%). Their representatives were of the outbreak pulsotype and ST11, and produced NDM-1, encoded by specific IncFII (pKPX-1/pB-3002cz)-like plasmids. The isolates were recovered in 145 healthcare centres in 13/16 administrative regions, predominantly the Warsaw area. The 'Tunisian' genotype K. pneumoniae ST147 NDM-1 Tn125F comprised 18 isolates (0.8%) from eight institutions. The remaining 24 isolates, mostly K. pneumoniae and Escherichia coli of diverse STs, produced NDM-1 or NDM-5 specified by various Tn125 derivatives and plasmids.Conclusions: The K. pneumoniae ST11 NDM-1 outbreak has dramatically expanded in Poland since 2012, which may bring about a countrywide endemic situation in the near future. In addition, the so-far limited K. pneumoniae ST147 NDM-1 outbreak plus multiple NDM imports from different countries were observed in 2015-16. [ABSTRACT FROM AUTHOR]

Objectives: Most vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are derived from hospital-associated MRSA due to treatment failure; however, the prevalence of hVISA/VISA in community settings remains unclear.Methods: Four hundred and seventy-six community-associated isolates were collected between 2010 and 2011 during national surveillance for antimicrobial resistance in 31 county hospitals across China. Drug susceptibility evaluation and mecA detection were performed by using broth microdilution and PCR analysis, respectively. hVISA/VISA were identified by using macro-Etest and a modified population analysis profile (PAP)-AUC method. The genetic features of all hVISA/VISA isolates were genotyped.Results: Among 476 isolates, MRSA and MSSA accounted for 19.7% (n = 94) and 80.3% (n = 382), respectively. Two VISA and 36 hVISA isolates were identified by PAP-AUC testing. The VISA isolates and 29 of the hVISA isolates were MRSA. The proportion of hVISA/VISA was significantly higher in MRSA (30.9%) than in MSSA (1.8%). The hVISA/VISA isolates were assigned to 18 STs classified into seven clonal complexes (CCs). CC121 (n = 12) followed by ST239 (n = 11) was the most prevalent hVISA/VISA clone. All ST239-hVISA/VISA were MRSA, while 12 CC121-hVISA isolates included 6 MSSA and 6 MRSA isolates. SCCmec III was predominant among MRSA-hVISA/VISA isolates. agr I and agr IV were detected in ST239 and CC121, respectively. All except two strains were positive for Panton-Valentine leucocidin genes.Conclusions: To the best of our knowledge, this is the first report of CC121 as a prevalent hVISA clone in community settings, highlighting the necessity of surveillance and stricter infection control measures for this globally disseminated lineage. [ABSTRACT FROM AUTHOR]

Objectives: Fully sequenced IncI1 plasmids obtained from CTX-M-1-producing Escherichia coli of human and animal origin were compared.Methods: Twelve E. coli isolates sharing identical ESBL genes and plasmid multilocus STs sequenced on Illumina and MinION platforms were obtained from the Danish antimicrobial resistance surveillance programme, DANMAP. After de novo assembly, the sequences of plasmids harbouring blaCTX-M-1 were manually curated and ORFs annotated. Within-group comparisons were performed separately for the IncI1 ST3 plasmid type and the IncI1 ST7 plasmid type. The IncI1 ST3 plasmid group was obtained from 10 E. coli isolates (2 from patients with bloodstream infections, 6 from food and 2 from animals). The IncI1 ST7 plasmids originated from E. coli isolates obtained from a patient with bloodstream infection and from a pig. Sequences of IncI1 ST3 and IncI1 ST7 plasmids harbouring blaCTX-M-1 with determined origin were retrieved from GenBank and used for comparison within the respective group.Results: The 10 IncI1 ST3 blaCTX-M-1 plasmids were highly similar in structure and organization with only minor plasmid rearrangements and differences in the variable region. The IncI1 ST7 blaCTX-M-1 plasmids also showed high similarity in structure and organization. The high level of similarity was also observed when including plasmids from E. coli of animal origin from Australia, Switzerland, the Netherlands and France.Conclusions: This study shows broad spread of a very successful CTX-M-1-producing IncI1 type plasmid among E. coli of both human and animal origin. [ABSTRACT FROM AUTHOR]

Objectives: NDM-producing Enterobacteriaceae clinical isolates remain uncommon in the European region. We describe the emergence and broad dissemination of one successful NDM-1-producing Klebsiella pneumoniae clone in Greek hospitals.Methods: During a 4 year survey (January 2013-December 2016), 480 single-patient carbapenem non-susceptible K. pneumoniae isolates, phenotypically MBL positive, were consecutively recovered in eight Greek hospitals from different locations and subjected to further investigation. Antimicrobial susceptibility testing, combined-disc test, identification of resistance genes by PCR and sequencing, molecular fingerprinting by PFGE, plasmid profiling, replicon typing, conjugation experiments and MLST were performed.Results: Molecular analysis confirmed the presence of the blaNDM-1 gene in 341 (71%) K. pneumoniae isolates. A substantially increasing trend of NDM-1-producing K. pneumoniae was noticed during the survey (R2 = 0.9724). Most blaNDM-1-carrying isolates contained blaCTX-M-15, blaOXA-1, blaOXA-2 and blaTEM-1 genes. PFGE analysis clustered NDM-1 producers into five distinct clonal types, with five distinct STs related to each PFGE clone. The predominant ST11 PFGE clonal type was detected in all eight participating hospitals, despite adherence to the national infection control programme; it was identical to that observed in the original NDM-1 outbreak in Greece in 2011, as well as in a less-extensive NDM-1 outbreak in Bulgaria in 2015. The remaining four ST clonal types (ST15, ST70, ST258 and ST1883) were sporadically detected. blaNDM-1 was located in IncFII-type plasmids in all five clonal types.Conclusions: This study gives evidence of possibly the largest NDM-1-producing K. pneumoniae outbreak in Europe; it may also reinforce the hypothesis of an NDM-1 clone circulating in the Balkans. [ABSTRACT FROM AUTHOR]

Objectives: Our aim was to determine the antimicrobial susceptibilities of 2862 Listeria monocytogenes cultured from various foods in China and to use WGS to characterize the antimicrobial resistance and virulence genotypes of those expressing a resistance phenotype.Methods: The susceptibilities of 2862 L. monocytogenes were determined by broth microdilution. Twenty-eight L. monocytogenes were found to be resistant to one to four antibiotics. All 28 resistant isolates were subsequently sequenced using short-read high accuracy protocols. The corresponding genomes were assembled and further analysis was carried out using appropriate bioinformatics pipelines.Results: All 28 resistant L. monocytogenes were classified into five STs (ST3, ST8, ST9, ST155 and ST515). Both ST9 and ST155 were dominant and their genotypes correlated with their resistance phenotypes. All ST9 isolates were MDR and could be phylogenetically classified into two clusters. One was relatively close to clinical origins and one to food. Downstream analysis of the genetic contexts in which these resistance genotypes were found suggested that these may have been acquired from other bacteria by horizontal transfer or insertion into the chromosome. All isolates harboured Listeria pathogenicity island (LIPI)-1 and LIPI-2, and only two harboured LIPI-3.Conclusions: This study reported on the antimicrobial susceptibilities of 2862 foodborne L. monocytogenes along with the genomic characterization of 28 resistant isolates, 11 of which expressed an MDR phenotype. These data showed that this bacterium can acquire resistance by horizontal gene transfer in and between species. This study may necessitate a re-evaluation of risk to public health, associated with this bacterial species. [ABSTRACT FROM AUTHOR]

Objectives: The prevalence of infections caused by OXA-48-like carbapenemase-producing organisms in Ireland has increased dramatically since 2011 and is an urgent public health issue. Genome-based high-resolution genotyping was used to analyse clinical isolates submitted to the Irish Carbapenemase-Producing Enterobacteriaceae Reference Laboratory Service for a 13 month period (2016-17).Methods: A total of 109 OXA-48-producing non-duplicate clinical isolates from 16 submitting centres were sequenced. Using a gene-by-gene approach, isolate genomes were characterized by MLST and core genome MLST, and the presence of antimicrobial resistance determinants was determined. Reference mapping and a novel plasmid MLST-type approach was applied to determine plasmid background.Results: The OXA-48-like-producing isolates were Escherichia coli (n = 56), Klebsiella spp. (n = 46) and Enterobacter cloacae (n = 7). Amongst the E. coli isolates there were 37 different STs and amongst the Klebsiella spp. isolates there were 27 different STs. blaOXA-48 was present in 105/109 (96.3%) of isolates. Based on mapping analysis and detection of the pOXA-48 IncL-type plasmid replicon and backbone genes, a pOXA-48-like plasmid was identified in 93/109 isolates (85.3%). The remaining isolates (n = 16; 14.7%) harboured blaOXA-48-like genes in unknown environments. Using a gene-by-gene approach two pOXA-48-like plasmid groups with 2/71 pOXA-48-like locus differences between them were identified.Conclusions: In Ireland we found a diversity of genotypes associated with OXA-48-like-producing clinical isolates with the IncL pOXA-48 plasmid type predominating as the blaOXA-48 genetic environment. A plasmid MLST approach can rapidly identify plasmids associated with outbreaks and monitor spread of types temporally and geographically. [ABSTRACT FROM AUTHOR]

Background: Reliable phenotypic antimicrobial susceptibility testing can be a challenge in clinical settings in low- and middle-income countries. WGS is a promising approach to enhance current capabilities.Aim: To study diversity and resistance determinants and to predict and compare resistance patterns from WGS data of Acinetobacter baumannii with phenotypic results from classical microbiological testing at a tertiary care hospital in Tanzania.Methods and Results: MLST using Pasteur/Oxford schemes yielded eight different STs from each scheme. Of the eight, two STs were identified to be global clones 1 (n = 4) and 2 (n = 1) as per the Pasteur scheme. Resistance testing using classical microbiology determined between 50% and 92.9% resistance across all drugs. Percentage agreement between phenotypic and genotypic prediction of resistance ranged between 57.1% and 100%, with coefficient of agreement (κ) between 0.05 and 1. Seven isolates harboured mutations at significant loci (S81L in gyrA and S84L in parC). A number of novel plasmids were detected, including pKCRI-309C-1 (219000 bp) carrying 10 resistance genes, pKCRI-43-1 (34935 bp) carrying two resistance genes and pKCRI-49-1 (11681 bp) and pKCRI-28-1 (29606 bp), each carrying three resistance genes. New ampC alleles detected included ampC-69, ampC-70 and ampC-71. Global clone 1 and 2 isolates were found to harbour ISAba1 directly upstream of the ampC gene. Finally, SNP-based phylogenetic analysis of the A. baumannii isolates revealed closely related isolates in three clusters.Conclusions: The validity of the use of WGS in the prediction of phenotypic resistance can be appreciated, but at this stage is not sufficient for it to replace conventional antimicrobial susceptibility testing in our setting. [ABSTRACT FROM AUTHOR]

Objectives: To describe the epidemiological trend of linezolid-resistant enterococci (LRE) collected in France from 2006 to 2016 and to extensively characterize LRE isolates.Methods: The National Reference Center for Enterococci (NRC-Enc) received enterococcal isolates suspected to be VRE and/or LRE from all French hospitals between 2006 and 2016. LRE isolates were phenotypically characterized and their genomes were entirely sequenced by Miseq (Illumina). Transfer of linezolid resistance was attempted by filter mating experiments.Results: Out of 3974 clinical isolates of enterococci received at the NRC-Enc over the period, 9 (0.2%) were LRE (MICs 8 to >32 mg/L), including 6 Enterococcus faecium and 3 Enterococcus faecalis. This overall prevalence significantly increased over the study period, reaching 0.8% in 2016. The five LRE isolated before 2016 were vanA-positive E. faecium whereas strains isolated in 2016 (one E. faecium and three E. faecalis) were susceptible to vancomycin. None of these isolates was part of an outbreak, while E. faecium strains were assigned to four different STs [17 (1), 80 (3), 412 (1) and 650 (1)] and all three E. faecalis belonged to ST480. Except for the strain isolated in 2010, all LRE were positive for optrA, which was located on plasmids (5/8) or in the chromosome (3/8). Plasmid transfer of optrA was successful in three cases.Conclusions: There has been a significant increase in the prevalence of LRE in France over time; this is due to the spread of optrA among E. faecium and E. faecalis human clinical isolates (VRE or not). [ABSTRACT FROM AUTHOR]

Objectives: Our aim was to describe the molecular epidemiology and antimicrobial resistance determinants of isolates of Neisseria gonorrhoeae with decreased susceptibility and resistance to extended-spectrum cephalosporins (ESCs) in Argentina in 2011-16.Methods: Gonococcal isolates (n=158) with decreased susceptibility and resistance to ESCs collected in 2011-16 across Argentina were subjected to WGS and antimicrobial susceptibility testing for six antimicrobials.Results: In total, 50% of the isolates were resistant to cefixime, 1.9% were resistant to ceftriaxone, 37.3% were resistant to azithromycin and 63.9% of the isolates showed an MDR phenotype. Resistance and decreased susceptibility to ESCs was mainly associated with isolates possessing the mosaic penA-34.001, in combination with an mtrR promoter A deletion, and PorB1b amino acid substitutions G120K/A121N. Phylogenetic analysis revealed two main clades of circulating strains, which were associated with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 and closely related STs, and characterized by a high prevalence rate, wide geographical distribution and temporal persistence.Conclusions: N. gonorrhoeae isolates with decreased susceptibility and resistance to ESCs in Argentina have emerged and rapidly spread mainly due to two clonal expansions after importation of one or two strains, which are associated with the international MDR NG-MAST ST1407 clone. The identification of the geographical dissemination and characteristics of these predominant clones may help to focus action plans and public health policies to control the spread of ESC resistance in Argentina. Dual antimicrobial therapy (ceftriaxone plus azithromycin) for gonorrhoea needs to be considered in Argentina. [ABSTRACT FROM AUTHOR]

Objectives: To characterize the antimicrobial susceptibility, molecular epidemiology and carbapenem resistance mechanisms in Pseudomonas aeruginosa isolates recovered from respiratory tract samples from patients with ventilator-associated pneumonia enrolled in the MagicBullet clinical trial.Methods: Isolates were collected from 53 patients from 12 hospitals in Spain, Italy and Greece. Susceptibility was determined using broth microdilution and Etest. MALDI-TOF MS was used to detect carbapenemase activity and carbapenemases were identified by PCR and sequencing. Molecular epidemiology was investigated using PFGE and MLST.Results: Of the 53 isolates, 2 (3.8%) were considered pandrug resistant (PDR), 19 (35.8%) were XDR and 16 (30.2%) were MDR. Most (88.9%) of the isolates from Greece were MDR, XDR or PDR, whereas fewer of the isolates from Spain (33.3%) and Italy (43.5%) showed antibiotic resistance. Three Greek isolates were resistant to colistin. Overall, the rates of resistance of P. aeruginosa isolates to imipenem, ciprofloxacin, ceftolozane/tazobactam and ceftazidime/avibactam were 64.1%, 54.7%, 22.6% and 24.5%, respectively. All isolates resistant to ceftolozane/tazobactam and ceftazidime/avibactam (Greece, n = 10; and Italy, n = 2) carried blaVIM-2. Spanish isolates were susceptible to the new drug combinations. Forty-eight restriction patterns and 27 STs were documented. Sixty percent of isolates belonged to six STs, including the high-risk clones ST-111, ST-175 and ST-235.Conclusions: MDR/XDR isolates were highly prevalent, particularly in Greece. The most effective antibiotic against P. aeruginosa was colistin, followed by ceftolozane/tazobactam and ceftazidime/avibactam. blaVIM-2 is associated with resistance to ceftolozane/tazobactam and ceftazidime/avibactam, and related to highly resistant phenotypes. ST-111 was the most frequent and disseminated clone and the clonal diversity was lower in XDR and PDR strains. [ABSTRACT FROM AUTHOR]

Objectives: Rates of Clostridioides (Clostridium) difficile infection (CDI) are higher in North Wales than elsewhere in the UK. We used WGS to investigate if this is due to increased healthcare-associated transmission from other cases.Methods: Healthcare and community C. difficile isolates from patients across North Wales (February-July 2015) from glutamate dehydrogenase (GDH)-positive faecal samples underwent WGS. Data from patient records, hospital management systems and national antimicrobial use surveillance were used.Results: Of the 499 GDH-positive samples, 338 (68%) were sequenced and 299 distinct infections/colonizations were identified, 229/299 (77%) with toxin genes. Only 39/229 (17%) toxigenic isolates were related within ≤2 SNPs to ≥1 infections/colonizations from a previously sampled patient, i.e. demonstrated evidence of possible transmission. Independent predictors of possible transmission included healthcare exposure in the last 12 weeks (P = 0.002, with rates varying by hospital), infection with MLST types ST-1 (ribotype 027) and ST-11 (predominantly ribotype 078) compared with all other toxigenic STs (P < 0.001), and cephalosporin exposure in the potential transmission recipient (P = 0.02). Adjusting for all these factors, there was no additional effect of ward workload (P = 0.54) or failure to meet cleaning targets (P = 0.25). Use of antimicrobials is higher in North Wales compared with England and the rest of Wales.Conclusions: Levels of transmission detected by WGS were comparable to previously described rates in endemic settings; other explanations, such as variations in antimicrobial use, are required to explain the high levels of CDI. Cephalosporins are a risk factor for infection with C. difficile from another infected or colonized case. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the prevalence and molecular characteristics of ESBL-producing Escherichia coli (ESBL-EC) in faecal samples from dairy cows in China.Methods: In total, 651 faecal samples were collected from cows distributed among the 10 provinces of China. Potential ESBL-EC isolates were cultured on selective medium. The clonal relatedness of the ESBL-EC isolates was assessed using MLST. WGS was conducted on 3 mcr-positive isolates and 14 additional randomly selected ESBL-EC isolates. Southern blot, S1-PFGE and conjugation were performed for mcr-1-carrying isolates. The genetic environment of the pMCR-JLF4 plasmid was also analysed.Results: In total, 290 unique ESBL-EC isolates were detected from 284 cows (43.6%). Alleles of CTX-M were observed in 94.1% (273/290) of all isolates. The most prevalent genotypes observed in this study were blaCTX-M-14, blaCTX-M-15, blaCTX-M-17 and blaCTX-M-55. Differentiation of 79 STs with a polyclonal structure was accomplished using MLST. Clonal complex 10 was the most prevalent major complex detected here. Furthermore, the mcr-1 gene was detected in three isolates. The complete sequence of the mcr-1-containing pMCR-JLF4 was determined. The plasmid was 66.7 kb in length, with a genetic structure of nikA-nikB-mcr-1-pap2. Conjugation analysis confirmed that the mcr-1 gene in pMCR-JLF4 was transferable without the assistance of the ISApl1 gene.Conclusions: The data presented here suggest high prevalence of ESBL-EC in Chinese cow farms. Furthermore, it was clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M genes, potentially contributing to the dissemination and transfer of the mcr-1 gene to pathogenic bacteria among cows. [ABSTRACT FROM AUTHOR]

Background: Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm.Methods: A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid.Results: vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission.Conclusions: Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad. [ABSTRACT FROM AUTHOR]

Objectives: To determine the molecular characteristics of ESBL-producing Escherichia coli (ESBL-E) collected during a longitudinal study on an organic broiler farm in order to investigate clonal expansion and horizontal gene transfer.Methods: Isolates were obtained from a longitudinal study performed previously on an organic broiler fattening farm. Samples from individually followed-up broilers, the broiler house, the transport van and persons that took the samples, taken at several timepoints (days 1, 3, 4, 7, 10, 42 and 70) within a production round and during the consecutive one (days 1, 2, 3 and 70), had been investigated for the occurrence of ESBL-E. In the current study, ESBL genes and MLST STs of these ESBL-E were determined. Plasmids were characterized and subtyped.Results: On arrival in round_1, ESBL-E of ST88 predominated, while on days 3, 4, 7 and 10 ST10 was most often found and at slaughter age ST155 and ST1551 prevailed. A shift in STs was also observed in round_2. None of the 35 individually selected broilers followed up in round_1 was positive for the same ESBL-E ST at all sampling times. All isolates carried CTX-M-1 group genes, confirmed as blaCTX-M-1 in 158 isolates. Further analysis of 36 isolates of different STs showed blaCTX-M-1 on IncI1/ST3 plasmids.Conclusions: The rapid dissemination of ESBL-E on this broiler farm was not due to the spread of one specific E. coli clone, but most likely the result of horizontal transfer of an IncI1/ST3 plasmid carrying blaCTX-M-1 resulting in a shift in the predominant ESBL-E population in broilers. [ABSTRACT FROM AUTHOR]

Objectives: To ascertain whether the antimicrobial susceptibility of Neisseria gonorrhoeae isolates with differing susceptibilities to penicillin is associated with genogroups (GGs) and combined mutation patterns in PBP2 (penA), the multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB).Methods: The susceptibility of 146 clinical N. gonorrhoeae isolates to penicillin was determined using the agar dilution method and the interpretation criteria of CLSI. The DNA sequences of penA, mtrR and porB in isolates were compared with WT sequences and mutation patterns were determined. Isolates were typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and STs were grouped into specific GGs.Results: The isolates tested carried 9 mutation patterns in PBP2 and 12 mutation patterns in each of MtrR and PorB. Of the 146 isolates, 121 (82.9%) were grouped into 13 different GGs. Isolates with penicillin MICs of 0.03-0.06 mg/L were significantly associated with GG25 (P < 0.05) and PBP2/MtrR/PorB mutation pattern I/WT/WT (P < 0.01). Isolates with a penicillin MIC of 1.0 mg/L were associated (P < 0.05) with: (i) GG3655 and mutation pattern XXII/A-;G45D/G120K;A121N; (ii) GG921 and mutation pattern IX/G45D/G120D;A121N; and (iii) GG1109 and mutation pattern IX/G45D/WT. Sixty percent (9/15) of penicillin-resistant isolates (MIC ≥2 mg/L) were GG3654 (P < 0.0001) and carried mutation pattern IX/G45D/G120K;A121D or IX/G45D/G120D;A121D (P < 0.05).Conclusions: Specific mutation patterns in PBP2/MtrR/PorB were associated with specific GGs and penicillin susceptibility. This approach of typing strains and resistance patterns is ideal for predicting antimicrobial resistance and should be used in instances in which gonococcal culture is not available but DNA can be obtained from clinical specimens. [ABSTRACT FROM AUTHOR]

Objectives: To analyse VIM/IMP-type MBL-producing Enterobacteriaceae isolates identified in Poland during 2006-12.Methods: Isolates were typed by PFGE, followed by MLST. blaVIM/IMP genes were amplified and sequenced within class 1 integrons. Their plasmidic versus chromosomal location was assessed by nuclease S1 and I-CeuI plus hybridization experiments. Plasmids were characterized by transfer assays and PCR-based replicon typing.Results: One hundred and nineteen VIM/IMP-positive Enterobacteriaceae cases were reported in Poland from the first case in 2006 until 2012. The patients were in 54 hospitals and were infected or colonized by 121 organisms, including Enterobacter cloacae complex (n = 64), Klebsiella oxytoca (n = 23), Serratia marcescens (n = 20) and Klebsiella pneumoniae (n = 11). The isolates represented numerous pulsotypes and mainly original STs, and carried eight integrons with blaVIM-1-like genes (blaVIM-1/-4/-28/-37/-40; n = 101), three with blaVIM-2 variants (blaVIM-2/-20; n = 17) and one with blaIMP-19 (n = 3). Six integrons were new, and five and two formed prevalent families of In238-like (n = 96) and In1008-like (n = 16) elements, respectively. In238 (aacA4-blaVIM-4rpt) and In1008 (blaVIM-2-aacA4) had been originally observed in Polish Pseudomonas aeruginosa, suggestive of their transfer to enterobacteria, followed by spread and diversification. Four organisms have disseminated inter-regionally, i.e. Enterobacter hormaechei ST90 with plasmidic In238/In238a integrons (n = 36), K. oxytoca ST145 with a chromosomal In237-like element (n = 18) and two subclones of E. hormaechei ST89 with In1008- or In238-type variants (n = 8 and n = 7, respectively).Conclusions: The epidemiology of VIM/IMP-producing Enterobacteriaceae in Poland has revealed a remarkable number of specific or novel characteristics of the organisms, with some possible links to other mid-southern European countries. [ABSTRACT FROM AUTHOR]

Objectives: To discover the Escherichia coli STs and associated resistance mechanisms in the community in Islamabad, Pakistan by analysis of E. coli isolates in sewage.Methods: One hundred and ten E. coli were isolated from sewage across the city of Islamabad without antibiotic bias and confirmed as E. coli by MALDI-TOF MS. Isolates were characterized by fumC/fimH (CH) typing and core-genome MLST. Resistance mechanisms, virulence genes, phylotypes and plasmid incompatibility types were determined in a subset of isolates by in silico analysis. The genomic position of blaCTX-M-15 was determined using S1-PFGE, probing and Nanopore MinION sequencing.Results and conclusions: The most prevalent STs were ST394, ST10 and ST648, accounting for 39% of all isolates collected and were found at many sites across Islamabad. Carbapenemase genes were absent and only a single isolate of ST131 was found. The most prevalent resistance mechanisms were qnrS1 and blaCTX-M-15, with blaCTX-M-15 penetrating many STs and found in 31% of all collected isolates. However, the majority of the successful STs were blaCTX-M-15 negative indicating that resistance is not the main driver of prevalence. Twenty-three percent of blaCTX-M-15 genes were chromosomally encoded and large ISEcp1-mediated insertions included qnrS1 and several plasmid genes. In all chromosomally encoded isolates no plasmid copies of blaCTX-M-15 were found. The most prevalent ST (ST394) contained many enteroaggregative E. coli virulence genes and the fimH30 variant allele previously linked to the success of ST131. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the prevalence of clinical mcr-1-positive Escherichia coli and Klebsiella pneumoniae and characterize the antimicrobial resistance profiles of mcr-1-positive E. coli and mcr-1-negative E. coli in China.Methods: A total of 6264 clinical E. coli (n = 3854) and K. pneumoniae (n = 2410) were collected from hospitalized patients from 18 to 20 hospitals as part of the China Antimicrobial Resistance Surveillance Trial (CARST) between January 2007 and June 2016. PCR was used to screen for the mcr-1 gene among all isolates. Antibiotic susceptibility testing was performed using the broth microdilution method. mcr-1-positive pathogens were then characterized by MLST and minimum spanning tree analysis using the BURST algorithm for related STs.Results: We examined 39 (0.62%) clinical isolates of mcr-1-positive E. coli and K. pneumoniae over a 10 year period. Resistance to antimicrobial agents was significantly more severe in mcr-1-positive isolates than mcr-1-negative isolates, particularly piperacillin (P = 0.008), amikacin (P < 0.0001), nitrofurantoin (P < 0.004) and fosfomycin (P < 0.0001). Among mcr-1-carrying isolates, ESBL production was as high as 84.6% (33 of 39) and 92.3% (36 of 39) of them displayed an MDR phenotype. STs suggested ubiquitous dissemination of mcr-1-carrying pathogens.Conclusions: mcr-1-carrying E. coli and K. pneumoniae displayed a lower prevalence and abundant phylogenetic diversity in mainland China. mcr-1-positive E. coli showed significant differences in antimicrobial resistance profiles compared with mcr-1-negative E. coli strains, suggesting physicians may consider prescribing different antibiotics when faced with infections caused by mcr-1-positive pathogens. [ABSTRACT FROM AUTHOR]

Background: VRE are prevalent among patients in ICUs. Non-typeable vanA VRE, due to loss of one of the genes used for MLST (pstS), have increased in Australia, suggestive of a new, hospital-acquired lineage.Objectives: To understand the significance of this lineage and its transmission using WGS of strains isolated from patients in ICUs across New South Wales, Australia.Methods: A total of 240 Enterococcus faecium isolates collected between February and May 2016, and identified by conventional PCR as vanA positive, were sequenced. Isolates originated from 12 ICUs in New South Wales, grouped according to six local health districts, and represented both rectal screening swab (n = 229) and clinical (n = 11) isolates.Results: ST analysis revealed the absence of the pstS gene in 84.2% (202 of 240) of vanA isolates. Two different non-typeable STs were present based on different allelic backbone patterns. Loss of the pstS gene appeared to be the result of multiple recombination events across this region. Evidence for pstS-negative lineage spread across all six local health districts was observed suggestive of inter-hospital transmission. In addition, multiple outbreaks were detected, some of which were protracted and lasted for the duration of the study.Conclusions: These findings confirmed the evolution, emergence and dissemination of non-typeable vanA E. faecium. This study has highlighted the utility of WGS when attempting to describe accurately the hospital-based pathogen epidemiology, which in turn will continue to inform optimal infection control measures necessary to halt the spread of this important nosocomial organism. [ABSTRACT FROM AUTHOR]

Objectives: To determine the population structure and change in drug resistance of pneumococci colonizing children before and after the introduction of the 10-valent and 13-valent pneumococcal conjugate vaccines (PCV10/13) in Brazil. Methods: We used MLST to analyse 256 pneumococcal isolates obtained from children aged <6 years before (2009-10; n = 125) and after (2014; n = 131) the introduction of the PCV10 and PCV13. Antimicrobial susceptibility and capsular types were previously determined. Results: We identified 97 different STs. Ninety (35.2%) isolates were related to international clones. The most frequent lineages were serogroup 6-CC724 (where CC stands for clonal complex) and the MDR serotype 6C-CC386 in the pre- and post-PCV10/13 periods, respectively. Penicillin-non-susceptible pneumococci (PNSP) formed 24% and 38.9% of the pre- and post-PCV10/13 isolates, respectively (P = 0.01). In the pre-PCV10/13 period, serotype 14-ST156 was the predominant penicillin-non-susceptible lineage, but it was not detected in the post-PCV10/13 period. Serotype 14-ST156 and serotype 19A-ST320 complex isolates had the highest penicillin and ceftriaxone MICs in the pre- and post-PCV10/13 periods, respectively. In turn, serotype 6C-CC386 comprised almost 30% of the PNSP and over 40% of the erythromycin-resistant isolates (MIC >256 mg/L) in the post-PCV10/13 period. Conclusions: Although PNSP strains were polyclonal, most resistant isolates belonged to a single genotype from each period. Higher erythromycin resistance prevalence (42%) in the post-PCV10/13 period was mainly attributed to MDR serotype 6C-CC386. Ongoing surveillance of pneumococcal clonal composition is important to evaluate PCV use outcomes and to identify factors other than PCVs that drive pneumococcal drug resistance evolution. [ABSTRACT FROM AUTHOR]

Objectives: Information on clonal lineages and genetic platforms involved in the mobilization of carbapene-mases between Pseudomonas aeruginosa strains in Portugal is scarce. Here, we outline the genetic drivers contributing to the occurrence of blaVIM-2-producing P. aeruginosa over two decades. Methods: A collection of carbapenem-resistant P. aeruginosa clinical isolates (n = 263, 1995-2014) was screened for carbapenemase production by Blue-Carba and PCR. Antimicrobial susceptibility testing was performed according to EUCAST and clonal analysis by MLST. Nine isolates representing different integrons and STs were selected for WGS, followed by bioinformatics. Results: Twenty-seven blaVIM-2-producing P. aeruginosa belonging to 10 STs were identified, with ST179 and ST111 being the most prevalent and persistent clones. blaVIM-2 was associated with seven class I integrons frequently co-harbouring aminoglycoside resistance genes. In58 was commonly identified, followed by derivatives and In100. blaVIM-2-harbouring transposons of the Tn3 and Tn402 families were linked to different plasmids or integrative conjugative elements of the clc family. Conclusions: The dissemination of blaVIM-2 carrying integrons is associated with a complex interplay between different mobile genetic elements, including the overlooked integrative conjugative elements, and successful spread ofparticular clones. [ABSTRACT FROM AUTHOR]

Objectives: Since the first identification of the OXA-48 carbapenemase in 2001, Enterobacteriaceae harbouring OXA-48-like enzymes have been reported globally. Here, we applied WGS to characterize the molecular epidemiology of these bacterial isolates.Methods: Enterobacteriaceae non-susceptible to carbapenems isolated from patients between 2011 and 2014 were voluntarily submitted to the Canadian National Microbiology Laboratory where they were screened for carbapenemase genes. WGS was conducted on OXA-48-like producers using the Illumina MiSeq platform. WGS data were used for single nucleotide variant (SNV) analysis, MLST analysis, detection of resistance genes and partial plasmid characterization. Susceptibilities were determined using Vitek2 and Etest. Patient data provided from sites were reviewed.Results: Sixty-seven non-duplicated cases were identified among Escherichia coli (n = 21) and Klebsiella pneumoniae (n = 46). Recent international travel was observed in 40.4% of cases. OXA-181 (52.2%) and OXA-48 (31.3%) were the most common variants, one E. coli OXA-48 producer was found to harbour the acquired colistin resistance gene mcr-1. The dominant STs were ST38 and ST410 in E. coli and ST14 in K. pneumoniae. Three common plasmid types were observed among isolates: IncL/M associated with OXA-48 producers, and ColKP3 and IncX3 associated with OXA-181/232 producers.Conclusions: Enterobacteriaceae with OXA-48-like carbapenemases are emerging in Canada. This study highlights the complexity of OXA-48-types identified in Canada owing to travel and the successful clones and plasmids harbouring the OXA-48-like enzyme. [ABSTRACT FROM AUTHOR]

Objectives: Pseudomonas aeruginosa is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Ceftolozane/tazobactam is a new cephalosporin/β-lactamase inhibitor combination with potent activity against P. aeruginosa. This survey was carried out to evaluate the susceptibility of P. aeruginosa, circulating in Italy, to ceftolozane/tazobactam and comparators and to investigate the molecular epidemiology of carbapenemase-producing strains.Methods: Consecutive non-replicate P. aeruginosa clinical isolates (935) from bloodstream infections and lower respiratory tract infections were collected from 20 centres distributed across Italy from September 2013 to November 2014. Antimicrobial susceptibility testing was performed by broth microdilution and results were interpreted according to the EUCAST breakpoints. Isolates resistant to ceftolozane/tazobactam were investigated for carbapenemase genes by PCR, and for carbapenemase activity by spectrophotometric assay. WGS using an Illumina platform was performed on carbapenemase-producing isolates.Results: Ceftolozane/tazobactam was the most active molecule, retaining activity against 90.9% of P. aeruginosa isolates, followed by amikacin (88.0% susceptibility) and colistin (84.7% susceptibility). Overall, 48 isolates (5.1%) were positive for carbapenemase genes, including blaVIM (n = 32), blaIMP (n = 12) and blaGES-5 (n = 4), while the remaining ceftolozane/tazobactam-resistant isolates tested negative for carbapenemase production. Carbapenemase producers belonged to 10 different STs, with ST175 (n = 12) and ST621 (n = 11) being the most common lineages. Genome analysis revealed different trajectories of spread for the different carbapenemase genes.Conclusions: Ceftolozane/tazobactam exhibited potent in vitro activity against P. aeruginosa causing invasive infections in Italy. Carbapenemase production was the most common mechanism of resistance to ceftolozane/tazobactam. [ABSTRACT FROM AUTHOR]

Objectives: This study identified and characterized mcr-1-positive Enterobacteriaceae (MCRPE) and carbapenemase-producing Enterobacteriaceae (CPE) in hospital sewage water.Methods: Influent and effluent sewage samples were collected from five tertiary hospitals in Beijing in December 2016. Samples were screened for MCRPE and CPE using antibiotic selection media. Results were confirmed by PCR amplification of β-lactamase and colistin resistance (mcr-1 and mcr-2) genes and by sequencing. Antimicrobial susceptibility testing, MLST, conjugation and plasmid typing and S1-nuclease-PFGE/Southern blotting were performed for all MCRPE and CPE isolates.Results: Nine MCRPE and 12 CPE isolates were obtained. All mcr-1-positive isolates (n = 9) were Escherichia coli and belonged to eight different STs. The blaKPC-2-positive Enterobacteriaceae included Klebsiella pneumoniae (n = 4), Enterobacter cloacae (n = 4) and Citrobacter freundii (n = 1) isolates. Two C. freundii isolates and one E. cloacae isolate harboured the blaNDM-1 gene. MLST analysis revealed distinct genetic relatedness among all ST11 K. pneumoniae but not among any other carbapenemase-producing isolates. Conjugation and plasmid typing confirmed that three MCRPE isolates harboured mcr-1 on the self-transmissible IncX4 plasmid and the blaNDM-1 gene on the IncX3 plasmid. The sizes of the plasmids harbouring mcr-1, blaNDM-1 and blaKPC-2 were ∼33 to ∼240, ∼40 to ∼75 and ∼30 to ∼90 kb, respectively.Conclusions: To the best of our knowledge, this is the first report of mcr-1-positive E. coli and blaNDM-1-carrying E. cloacae and C. freundii in hospital sewage water. These findings, especially the diversity of MCRPE and K. pneumoniae ST11 that harbour the blaKPC-2 gene, suggest that monitoring and management of hospital sewage water should be enhanced. [ABSTRACT FROM AUTHOR]