Increasing azithromycin resistance in Neisseria gonorrhoeae due to NG-MAST 12302 clonal spread in Canada, 2015-2018

Objectives: Azithromycin resistant (AZIR) gonorrhea has been steadily increasing in Canada over the past decade which is cause for alarm as azithromycin (AZI) has been part of the combination therapy recommended by the Canadian Guidelines on Sexually Transmitted Infections (CGSTI) since 2012. Method: Neisseria gonorrhoeae (NG) with AZI MICs ≥ 1 mg/L collected between 2015 and 2018 as part of the Gonococcal Antimicrobial Surveillance Program-Canada underwent antimicrobial susceptibility testing, molecular typing and whole genome sequencing. Regional, demographic and clinical isolation site comparisons were made to aid in our understanding of AZI susceptibility trending. Results: 3,447 NG with AZI MICs ≥ 1 mg/L were identified in Canada, increasing from 6.3% in 2015 to 26.5% of isolates in 2018. Central Canada had the highest proportion rising from 9.2% in 2015 to 31.2% in 2018. 273 different NG-MAST sequence types were identified among these isolates with ST-12302 the most prevalent (50.9%). Whole genome sequencing identified the Neisseria lactamica -like mosaic mtr locus as the mechanism of AZIR in isolates of ST-12302 and isolates genetically similar (differ by ≤ 5 base pairs) designated as the ST-12302 genogroup, accounting for 65.2% of study isolateswhich were originally identified in central Canada but spread to other regions by 2018. Conclusion: Genomic analysis indicated that AZIR in Canadian NG expanded rapidly due to clonal spread of the ST-12302 genogroup. The rapid expansion of this AZIR clonal group in all regions of Canada is of concern. CGSTI are currently under review to address the increase in AZIR in Canada.

Unsuspected clonal spread of Methicillin-resistant Staphylococcus aureus causing bloodstream infections in hospitalized adults detected using whole genome sequencing

Background: Though detection of transmission clusters of methicillin-resistant Staphylococcus aureus (MRSA) infections is a priority for infection control personnel in hospitals, the transmission dynamics of MRSA among hospitalized patients with bloodstream infections (BSIs) has not been thoroughly studied. Whole genome sequencing (WGS) of MRSA isolates for surveillance is valuable for detecting outbreaks in hospitals, but the bioinformatic approaches used are diverse and difficult to compare. Methods: We combined short-read WGS with genotypic, phenotypic, and epidemiological characteristics of 106 MRSA BSI isolates collected for routine microbiological diagnosis from inpatients in two hospitals over 12 months. Clinical data and hospitalization history were abstracted from electronic medical records. We compared three genome sequence alignment strategies to assess similarity in cluster ascertainment. We conducted logistic regression to measure the probability of predicting prior hospital overlap between clustered patient isolates by the genetic distance of their isolates. Results: While the three alignment approaches detected similar results, they showed some variation. A pangenome-based alignment method was most consistent across MRSA clonal complexes. We identified nine unique clusters of closely-related BSI isolates. Most BSI were healthcare-associated and community-onset. Our logistic model showed that with 13 single nucleotide polymorphisms the likelihood that any two patients in a cluster overlapped in a hospital was 50 percent. Conclusions: Multiple clusters of closely related MRSA isolates can be identified using WGS among strains cultured from BSI in two hospitals. Genomic clustering of these infections suggest that transmission resulted from a mix of community spread and healthcare exposures long before BSI diagnosis.

Prevalence and Molecular Characterization of Fluoroquinolone-Resistant Escherichia coli in Healthy Children

Faecal E. coli can act as reservoirs for resistance genes. Here, we analyzed prevalence of drug resistance in faecal E. coli isolated from healthy children at a single kindergarten in Beijing, China, then used whole genome sequencing to characterize fluoroquinolone-non-susceptible strains. Our results revealed high resistance to ampicillin (54.0%), trimethoprim/sulphurmethoxazole (47.5%) and tetracycline (58.9%) among 576 faecal E. coli isolates, 49.2% of which exhibited multidrug resistance. A total of 113 E. coli isolates were not susceptible to ciprofloxacin, with four sequence types, namely ST1193 (25.7%), ST773 (13.3%), ST648 (8.8%) and ST131 (7.1%) found to be the most prevalent (54.9%). With regards to resistance to quinolones, we detected chromosomal mutations in gyrA, parC, and parE in 111 (98.2%), 105 (92.9%), and 67 (61.1%) isolates, respectively. blaCTX-M (37.2%) was the major ESBL gene, whereas blaCTX-M-14 (12.4%) and blaCTX-M-27 (11.5%) were the most frequent subtypes. A total of 90 (79.6%) ExPEC and 65 (57.5%) UPEC isolates were classified. Overall, these findings revealed clonal spread of certain prevalent STs, namely ST1193, ST773, ST648 and ST131 E. coli isolates in healthy children within a single kindergarten in Beijing, China, affirming the seriousness of the multidrug resistance problem and potential pathogenicity of E. coli isolates in healthy children. Therefore, there is an urgent need for increased surveillance to enhance control of this problem.

First Report of Enteroaggregative E. Coli Harbouring NDM-4 and NDM-7 From Asymptomatic Children in India

For the first time in the world, we report Enteroaggregative E.coli harbouring blaNDM−4 and blaNDM−7 that were isolated from healthy children. NDM-4 and NDM-7 are associated with increased carbapenemase activity. Clonal spread, inter-strain and interspecies horizontal transfer of such resistant determinants in a healthy population will be a cause of concern.

Capsular Genotype and Lipooligosaccharide Class Associated Genomic Characterizations of Campylobacter jejuni Isolates From Food Animals in China

Campylobacter jejuni (C. jejuni) is the leading causative agent of gastroenteritis and Guillain–Barré syndrome (GBS). Capsular polysaccharide (CPS) and lipooligosaccharide (LOS) contribute to the susceptibility of campylobacteriosis, which have been concern the major evaluation indicators of C. jejuni isolates from clinical patients. As a foodborne disease, food animal plays a primary role in the infection of campylobacteriosis. To assess the pathogenic characterizations of C. jejuni isolates from various ecological origins, 1609 isolates sampled from 2005 to 2019 in China were analyzed using capsular genotyping. Strains from cattle and poultry were further characterized by LOS classification and multilocus sequence typing (MLST), compared with the isolates from human patients worldwide with enteritis and GBS. Results showed that the disease associated capsular genotypes and LOS classes over-represented in human isolates were also dominant in animal isolates, especially cattle isolates. Based on the same disease associated capsular genotype, more LOS class types were represented by food animal isolates than human disease isolates. Importantly, high-risk lineages CC-22, CC-464, and CC-21 were found dominated in human isolates with GBS worldwide, which were also represented in the food animal isolates with disease associated capsular types, suggesting a possibility of clonal spread of isolates across different regions and hosts. This is the first study providing genetic evidence for food animal isolates of particular capsular genotypes harbor similar pathogenic characteristics to human clinical isolates. Collective efforts for campylobacteriosis hazard control need to be focused on the zoonotic pathogenicity of animal isolates, along the food chain “from farm to table.”

Characterization of Haemophilus influenzae Strains with Non-Enzymatic Resistance to β-Lactam Antibiotics Caused by Mutations in the PBP3 Gene in the Czech Republic in 2010–2018

The surveillance data on antibiotic resistance of Haemophilus influenzae have shown that strains with non-enzymatic resistance to β-lactam antibiotics have been on the rise in the Czech Republic over the last decade. This type of resistance is more difficult to detect than β-lactamase production. Analysis of 228 H. influenzae strains revealed that isolates with non-enzymatic resistance to β-lactams due to mutations in the ftsI gene could be reliably demonstrated by single run testing of susceptibility to amoxicillin/clavulanic acid (sensitivity of detection is 84.6%), cefuroxime (92.6%), ampicillin and penicillin (both 95.7%). Thirty-seven different amino acid substitution combinations were detected in the PBP3 protein at 23 positions (V329I, D350N, S357N, A368T, M377I, S385T, A388V, L389F, P393L, A437S, I449V, G490E, I491V, R501L, A502S, A502T, A502V, V511A, R517H, I519L, N526K, A530S, and T532S). The most common combination (35%) of amino acid substitutions was the combination D350N, M377I, A502V, N526K. Epidemiological typing does not indicate a clonal spread of a particular MLST type. Altogether there has been detected 74 STs. The most prevalent ST 1034 was associated mainly with a combination D350N, M377I, A502V, N526K. Clonal analysis revealed six clonal complexes (CCs) with the founder found, eight CCs without founder and 33 singletons.

Clonal Spread and Intra- and Inter-Species Plasmid Dissemination Associated With Klebsiella pneumoniae Carbapenemase-Producing Enterobacterales During a Hospital Outbreak in Barcelona, Spain

Objectives: The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in Catalonia, Spain.Methods: Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids.Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 and additional isolates of K. pneumoniae, Escherichia coli and Enterobacter sp. from different STs. All isolates but one carried the blaKPC–2 allelic variant. The blaKPC–2 gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn4401 element designated NTEKPC-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC-pMC-2-1 module was highly conserved.Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPC-producing ST15 K. pneumoniae but also the intra- and inter-species transmission of the blaKPC–2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high-risk clones.

Deciphering the Epidemiological Characteristics and Molecular Features of blaKPC–2- or blaNDM–1-Positive Klebsiella pneumoniae Isolates in a Newly Established Hospital

The emergence of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) was regarded as an emerging threat in clinical settings. Here, we investigated the prevalence of CRKP strains among inpatients in a new hospital over 1 year since its inception with various techniques, and carried out a WGS-based phylogenetic study to dissect the genomic background of these isolates. The genomes of three representative blaNDM–1-positive strains and the plasmids of four blaKPC–2-positive strains were selected for Nanopore long-read sequencing to resolve the complicated MDR structures. Thirty-five CRKP strains were identified from 193 K. pneumoniae isolates, among which 30 strains (85.7%) harbored blaKPC–2, whereas the remaining five strains (14.3%) were positive for blaNDM–1. The antimicrobial resistance profiles of blaNDM–1-positive isolates were narrower than that of blaKPC–2-positive isolates. Five isolates including two blaNDM–1-positive isolates and three blaKPC–2-positive strains could successfully transfer the carbapenem resistance phenotype by conjugation. All CRKP strains were categorized into six known multilocus sequence types, with ST11 being the most prevalent type. Phylogenetic analysis demonstrated that the clonal spread of ST11 blaKPC–2-positive isolates and local polyclonal spread of blaNDM–1-positive isolates have existed in the hospital. The blaNDM–1 gene was located on IncX3, IncFIB/IncHI1B, and IncHI5-like plasmids, of which IncFIB/IncHI1B plasmid has a novel structure. By contrast, all ST11 isolates shared the similar blaKPC–2-bearing plasmid backbone, and 11 of them possessed pLVPK-like plasmids. In addition, in silico virulome analysis, Galleria mellonella larvae infection assay, and siderophore secretion revealed the hypervirulence potential of most blaKPC–2-positive strains. Given that these isolates also had remarkable environmental adaptability, targeted measures should be implemented to prevent the grave consequences caused by hv-CRKP strains in nosocomial settings.

Spread of clonal genovar E Chlamydia trachomatis among men who have sex with men

In a previous study, we developed a Multi-Locus VNTRs Analysis (MLVA) typing system, called MLVA-5, for the discrimination of Chlamydia trachomatis genovar E strain. The results suggested the clonal spread of a MLVA-5 type 21 strain among men who have sex with men (MSM). We applied the MLVA-5 typing method on 157 French anorectal genovar E specimens and 19 Swedish specimens collected between 2010 and 2015. A total of 29 MLVA-5 types was obtained, with three predominant types among French samples: 78 specimens belonged to MLVA-5 type 21, two other types, 11 and 13, included 9 and 14 specimens, respectively. In 15 cases, one unique MLVA-5 type was observed for a single patient, 7 of which were new types not previously described. The distribution of MLVA-5 types according to sexual orientation showed that the 7 anorectal specimens from heterosexual patients belonged to 6 genotypes, and the 12 anorectal specimens from bisexual patients comprised eight types. The 95 anorectal specimens from MSM were distributed into 22 types, but 55 (57.9%) of them belonged to MLVA-5 type 21. Among the Swedish specimens from MSM, eight were from MLVA-type 21 (4 urines and 4 anorectal specimens). The results support the hypothesis of the spread of clonal genovar E strain among MSM.

Clonal Spread of Carbapenemase-Producing Enterobacteriaceae in a Region, China

Background The increasing number of carbapenemase-producing Enterobacteriaceae (CPE) has become a serious problem globally. This study aimed to elucidate their geographically epidemiological characteristics. Results A total of 930 phenotypically confirmed carbapenem-resistant Enterobacteriaceae (CRE) isolates collected from 19 hospitals were genotypically characterised. K.pneumoniae (KP) and E.coli isolates were 785 (85.14%) and 96 (10.41%) among 922 CPE isolates. Two major carbapenemase genes KPC-2 and NDM in CPE isolates accounted for 84.6% (n = 780) and 13.77% (n = 127). ST11 comprised 86.83% (633/729) of KPC-2 KP isolates. The wzi typing method could further subdivide ST11 KP group into at least five subgroups, among which wzi209 (69.83%, 442/633) was the most frequently isolated, followed by wzi141 (25.28%, 160/633). Conjugation assays showed that high conjugation rates were observed in CPE (15.24%, 32/210) for NDM plasmids, but relatively low (8.1%, 17/210) for KPC-2 plasmids. No associations between the rates of antibiotics consumption and CPE prevalence were observed. The number of intra-hospital and inter-hospital transfers of CPE patients increased gradually from 18 (17.82%, 101) and 12 (11.88%, 101) in 2015 to 63 (30.73%, 205) and 51 (24.88%, 205) in 2018 (p = 0.016 and p = 0.008), respectively. Evidence-based measures could effectively reduce the prevalence of ST11-wzi209 clone but failed to control the dissemination of ST11-wzi141 KP clone. Conclusions Clonal spread of CPE, especially KPC-2 ST11 KP was the key factor contributing to the CPE increase in the region. Continued vigilance for the importations should be maintained. Coordinated regional interventions are urgently needed to reduce CPE threat.