Invasive Isolates Research Articles

Parallel evolution leading to impaired biofilm formation in invasive Salmonella strains.

Pathogenic Salmonella strains that cause gastroenteritis are able to colonize and replicate within the intestines of multiple host species. In general, these strains have retained an ability to form the rdar morphotype, a resistant biofilm physiology hypothesized to be important for Salmonella transmission. In contrast, Salmonella strains that are host-adapted or even host-restricted like Salmonella enterica serovar Typhi, tend to cause systemic infections and have lost the ability to form the rdar morphotype. Here, we investigated the rdar morphotype and CsgD-regulated biofilm formation in two non-typhoidal Salmonella (NTS) strains that caused invasive disease in Malawian children, S. Typhimurium D23580 and S. Enteritidis D7795, and compared them to a panel of NTS strains associated with gastroenteritis, as well as S. Typhi strains. Sequence comparisons combined with luciferase reporter technology identified key SNPs in the promoter region of csgD that either shut off biofilm formation completely (D7795) or reduced transcription of this key biofilm regulator (D23580). Phylogenetic analysis showed that these SNPs are conserved throughout the African clades of invasive isolates, dating as far back as 80 years ago. S. Typhi isolates were negative for the rdar morphotype due to truncation of eight amino acids from the C-terminus of CsgD. We present new evidence in support of parallel evolution between lineages of nontyphoidal Salmonella associated with invasive disease in Africa and the archetypal host-restricted invasive serovar; S. Typhi. We hypothesize that the African invasive isolates are becoming human-adapted and ‘niche specialized’ with less reliance on environmental survival, as compared to gastroenteritis-causing isolates.

Emerging serotype III sequence type 17 group B streptococcus invasive infection in infants: the clinical characteristics and impacts on outcomes

BackgroundGroup B Streptococcus (GBS) is an important pathogen that causes high mortality and morbidity in young infants. However, data on clinical manifestations between different GBS serotypes and correlation with molecular epidemiology are largely incomplete. The aim of this study was to determine the serotype distribution, antimicrobial resistance, clinical features and molecular characteristics of invasive GBS isolates recovered from Taiwanese infants.MethodsFrom 2003 to 2017, 182 non-duplicate GBS isolates that caused invasive disease in infants less than one year of age underwent serotyping, multilocus sequence typing (MLST) and antibiotic susceptibility testing. The clinical features of these infants with GBS disease were also reviewed.ResultsOf the 182 patients with invasive GBS disease, 41 (22.5%) were early-onset disease, 121 (66.5%) were late-onset disease and 20 (11.0%) were late late-onset disease (> 90 days of age). All these patients were treated with effective antibiotics on time. Among them, 51 (28.0%) had meningitis, 29 (16.0%) had neurological complications, 12 (6.6%) died during hospitalization, and 15 (8.8%) out of 170 patients who survived had long-term neurological sequelae at discharge. Serotype III GBS strains accounted for 64.8%, followed by serotype Ia (18.1%) and Ib (8.2%). MLST analysis revealed 11 different sequence types among the 182 isolates and ST-17 was the most dominant sequence type (56.6%). The correlation between serotype III and ST17 was evident, as ST17 accounted for 87.3% of all serotype III isolates. There was an obvious increasing trend of type III/ST-17 GBS that caused invasive disease in infants. All isolates were susceptible to penicillin, cefotaxime, and vancomycin, while 68.1 and 65.9% were resistant to erythromycin and clindamycin, respectively.ConclusionsDespite timely and appropriate antibiotic treatment, a significant proportion of invasive GBS disease still inevitably causes adverse outcomes. Further study to explore preventive strategies and development of serotype-based vaccines will be necessary in the future.

Decoding the molecular epidemiology of group A streptococcus - an Indian perspective.

Unlike western countries the knowledge of group A streptococcus (GAS) epidemiology in India remains patchy and incomplete. Typing is crucial for surveillance as well as in predicting the efficacy of multivalent M protein vaccine. The present study aimed to explore the emm types of 206 invasive and non-invasive GAS isolates from South India as well as reviewing all the published literature on GAS molecular epidemiology from India thereby generating a pan-Indian data to predict the conjectural coverage of the 30-valent M-protein vaccine in this population. emm typing and superantigen (SAg) profiling of GAS along with reviewing literatures on GAS molecular epidemiology from India. This study revealed a high diversity of emm types with emm 63, 82, 183, 85, 92, 169, 42, 44, 106, 74, 12 being frequently encountered, belonging to twenty emm clusters. The pan-Indian data on prevalent emm types further supports our study findings with 135 emm different types. Six clusters dominated accounting for 80 % of the GAS isolates: E3(26 %), E6(20 %), E2(11 %), E4(10 %), D4(7 %), E1(6 %). No significant association was noted between emm types and the nature of infection (P≥0.05) while a few SAg profiles were significantly associated with certain emm types. Pan Indian data revealed that only 16 % of the emm types encountered were included in proposed 30-valent M protein based vaccine. The coverage among the South Indian GAS isolates was 28.2 % which increased to only 46.6 % with the cross-opsonic effect, thus highlighting the importance of developing a specific multivalent vaccine including the prevalent emm types in India or considering the use of conserved C-repeat vaccines and non-M protein based vaccines.

Pneumococcal lineages associated with serotype replacement and antibiotic resistance in childhood invasive pneumococcal disease in the post-PCV13 era: an international whole-genome sequencing study

SummaryBackgroundInvasive pneumococcal disease remains an important health priority owing to increasing disease incidence caused by pneumococci expressing non-vaccine serotypes. We previously defined 621 Global Pneumococcal Sequence Clusters (GPSCs) by analysing 20 027 pneumococcal isolates collected worldwide and from previously published genomic data. In this study, we aimed to investigate the pneumococcal lineages behind the predominant serotypes, the mechanism of serotype replacement in disease, as well as the major pneumococcal lineages contributing to invasive pneumococcal disease in the post-vaccine era and their antibiotic resistant traits.MethodsWe whole-genome sequenced 3233 invasive pneumococcal disease isolates from laboratory-based surveillance programmes in Hong Kong (n=78), Israel (n=701), Malawi (n=226), South Africa (n=1351), The Gambia (n=203), and the USA (n=674). The genomes represented pneumococci from before and after pneumococcal conjugate vaccine (PCV) introductions and were from children younger than 3 years. We identified predominant serotypes by prevalence and their major contributing lineages in each country, and assessed any serotype replacement by comparing the incidence rate between the pre-PCV and PCV periods for Israel, South Africa, and the USA. We defined the status of a lineage as vaccine-type GPSC (≥50% 13-valent PCV [PCV13] serotypes) or non-vaccine-type GPSC (>50% non-PCV13 serotypes) on the basis of its initial serotype composition detected in the earliest vaccine period to measure their individual contribution toward serotype replacement in each country. Major pneumococcal lineages in the PCV period were identified by pooled incidence rate using a random effects model.FindingsThe five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. The five most prevalent serotypes in the PCV13 period varied between countries, with only serotypes 5, 12F, 15B/C, 19A, 33F, and 35B/D common to two or more countries. These serotypes were associated with more than one lineage, except for serotype 5 (GPSC8). Serotype replacement was mainly mediated by expansion of non-vaccine serotypes within vaccine-type GPSCs and, to a lesser extent, by increases in non-vaccine-type GPSCs. A globally spreading lineage, GPSC3, expressing invasive serotypes 8 in South Africa and 33F in the USA and Israel, was the most common lineage causing non-vaccine serotype invasive pneumococcal disease in the PCV13 period. We observed that same prevalent non-vaccine serotypes could be associated with distinctive lineages in different countries, which exhibited dissimilar antibiotic resistance profiles. In non-vaccine serotype isolates, we detected significant increases in the prevalence of resistance to penicillin (52 [21%] of 249 vs 169 [29%] of 575, p=0·0016) and erythromycin (three [1%] of 249 vs 65 [11%] of 575, p=0·0031) in the PCV13 period compared with the pre-PCV period.InterpretationGlobally spreading lineages expressing invasive serotypes have an important role in serotype replacement, and emerging non-vaccine serotypes associated with different pneumococcal lineages in different countries might be explained by local antibiotic-selective pressures. Continued genomic surveillance of the dynamics of the pneumococcal population with increased geographical representation in the post-vaccine period will generate further knowledge for optimising future vaccine design.FundingBill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control.

Whole Genome Sequencing Based Surveillance of L. monocytogenes for Early Detection and Investigations of Listeriosis Outbreaks.

In Austria, all laboratories are legally obligated to forward human and food/environmental L. monocytogenes isolates to the National Reference Laboratory/Center (NRL) for Listeria. Two invasive human isolates of L. monocytogenes serotype 1/2a of the same pulsed-field gel electrophoresis (PFGE) pattern, previously unknown in Austria, were cultured for the first time in January 2016. Five further human isolates, obtained from patients with invasive listeriosis between April 2016 and September 2017, showed this PFGE pattern. In Austria the NRL started to use whole-genome sequencing (WGS) based typing in 2016, using a core genome MLST (cgMLST) scheme developed by Ruppitsch et al. 2015, which contains 1701 target genes. Sequence data are submitted to a publicly available nomenclature server (Ridom GmbH, Münster, Germany) for allocation of the core genome complex type (CT). The seven invasive human isolates differed from each other with zero to two alleles and were allocated to CT1234 (declared as outbreak strain). Among the Austrian strain collection of about 6,000 cgMLST-characterized non-human isolates (i.e., food/environmental isolates) 90 isolates shared CT1234. Out of these, 83 isolates were traced back to one meat processing-company. They differed from the outbreak strain by up to seven alleles; one isolate originated from the company's industrial slicer. The remaining seven CT1234-isolates were obtained from food products of four other companies (five fish-products, one ready-to-eat dumpling and one deer-meat) and differed from the outbreak strain by six to eleven alleles. The outbreak described shows the considerable potential of WGS to identify the source of a listeriosis outbreak. Compared to PFGE analysis, WGS-based typing has higher discriminatory power, yields better data accuracy, and allows higher laboratory through-put at lower cost. Utilization of WGS-based typing results of human and food/ environmental L. monocytogenes isolates by appropriate public health analysts and epidemiologists is indispensable to support a successful outbreak investigation.

Antibiotic resistance profiles and multidrug resistance patterns of Streptococcus pneumoniae in pediatrics: A multicenter retrospective study in mainland China.

Emergent resistance to antibiotics among Streptococcus pneumoniae isolates is a severe problem worldwide. Antibiotic resistance profiles for S pneumoniae isolates identified from pediatric patients in mainland China remains to be established.The clinical features, antimicrobial resistance, and multidrug resistance patterns of S pneumoniae were retrospectively analyzed at 10 children's hospitals in mainland China in 2016.Among the collected 6132 S pneumoniae isolates, pneumococcal diseases mainly occurred in children younger than 5 years old (85.1%). The resistance rate of S pneumoniae to clindamycin, erythromycin, tetracycline, and trimethoprim/sulfamethoxazole was 95.8%, 95.2%, 93.6%, and 66.7%, respectively. The resistance rates of S pneumoniae to penicillin were 86.9% and 1.4% in non-meningitis and meningitis isolates, while the proportions of ceftriaxone resistance were 8.2% and 18.1%, respectively. Pneumococcal conjugate vaccine was administered to only 4.1% of patients. Penicillin and ceftriaxone resistance, underling diseases, antibiotic resistant risk factors, and poor prognosis appeared more frequently in invasive pneumococcal diseases. The incidence of multidrug resistance (MDR) was 46.1% in patients with invasive pneumococcal disease which was more than in patients with non-invasive pneumococcal disease (18.3%). Patients with invasive pneumococcal disease usually have several MDR coexistence.S pneumoniae isolates showed high resistance to common antibiotics in mainland China. Penicillin and ceftriaxone resistance rate of invasive streptococcal pneumonia patients were significantly higher than that of non-invasive S pneumoniae patients. Alarmingly, 46.1% of invasive clinical isolates were multidrug resistant, so it is important to continued monitor the resistance of S pneumoniae when protein conjugate vaccine (PCV13) is coming in mainland China.

Source attribution of Campylobacter jejuni shows variable importance of chicken and ruminants reservoirs in non-invasive and invasive French clinical isolates

Campylobacter jejuni is the most common cause of bacterial gastroenteritis worldwide. Mainly isolated from stool samples, C. jejuni can also become invasive. C. jejuni belongs to the commensal microbiota of a number of hosts, and infection by this bacterium can sometimes be traced back to exposure to a specific source. Here we genome sequenced 200 clinical isolates (2010–2016) and analyzed them with 701 isolate genomes from human infection, chicken, ruminants and the environment to examine the relative contribution of different reservoirs to non-invasive and invasive infection in France. Host-segregating genetic markers that can discriminate C. jejuni source were used with STRUCTURE software to probabilistically attribute the source of clinical strains. A self-attribution correction step, based upon the accuracy of source apportionment within each potential reservoir, improved attribution accuracy of clinical strains and suggested an important role for ruminant reservoirs in non-invasive infection and a potentially increased contribution of chicken as a source of invasive isolates. Structured sampling of Campylobacter in the clinic and from potential reservoirs provided evidence for variation in the contribution of different infection sources over time and an important role for non-poultry reservoirs in France. This provides a basis for ongoing genomic epidemiology surveillance and targeted interventions.

Serotype distribution and antibiotic resistance among invasive Streptococcus pneumoniae from Oman post 13-valent vaccine introduction

ObjectiveThis study was undertaken to determine the serotype distribution and drug susceptibility patterns in pneumococcal isolates recovered from patients with invasive pneumococcal disease (IPD). MethodsAll invasive pneumococcal isolates received between June 2014 and June 2016 were included in the study as part of a national laboratory-based IPD surveillance program. Isolates recovered from clinical specimens of normally sterile body sites were included. ResultsA total of 41 different serotypes were identified among the 132 pneumococcal isolates included in this study. The most prevalent serotypes/serogroups were 12 (8.3%), 15 (8.3%), 19F (7.6%), 3 (6.1%), and 19A (6.1%);. It was observed that overall vaccine coverage rates for pneumococcal conjugate vaccines (PCV), PCV7, PCV10 and PCV13 were 15.9%, 24.2% and 37.1% respectively. 56.8% (n=75) of the isolates were non-susceptible to at least one antibiotic and 40.9% (n=54) of the isolates were resistant to PEN (M). 18.9% (n=25) of the isolates were multi-drug resistant (MDR).The case fatality rate was 15.9%. ConclusionOur study results call for broader vaccine coverage, emphasizes the need to introduce the conjugate pneumococcal vaccine for the high risk adult population and stress the importance of continuous surveillance of serotypes and antimicrobial resistance to guide vaccine development and antimicrobial stewardship activities.

Potentiation of Phase Variation in Multiple Outer-Membrane Proteins During Spread of the Hyperinvasive Neisseria meningitidis Serogroup W ST-11 Lineage.

BackgroundSince 2009, increases in the incidence of invasive meningococcal disease have occurred in the United Kingdom due to a sublineage of the Neisseria meningitidis serogroup W ST-11 clonal complex (hereafter, the “original UK strain”). In 2013, a descendent substrain (hereafter, the “2013 strain”) became the dominant disease-causing variant. Multiple outer-membrane proteins of meningococci are subject to phase-variable switches in expression due to hypermutable simple-sequence repeats. We investigated whether alterations in phase-variable genes may have influenced the relative prevalence of the original UK and 2013 substrains, using multiple disease and carriage isolates.MethodsRepeat numbers were determined by either bioinformatics analysis of whole-genome sequencing data or polymerase chain reaction amplification and sizing of fragments from genomic DNA extracts. Immunoblotting and sequence-translation analysis was performed to identify expression states.ResultsSignificant increases in repeat numbers were detected between the original UK and 2013 strains in genes encoding PorA, NadA, and 2 Opa variants. Invasive and carriage isolates exhibited similar repeat numbers, but the absence of pilC gene expression was frequently associated with disease.ConclusionsElevated repeat numbers in outer-membrane protein genes of the 2013 strain are indicative of higher phase-variation rates, suggesting that rapid expansion of this strain was due to a heightened ability to evade host immune responses during transmission and asymptomatic carriage.

Performance comparison of the fast track diagnostics multiplex real-time PCR assays on two different automated molecular platforms

Minim typing is derived from the multi-locus sequence typing (MLST). It targets the same genes, but sequencing is replaced by high resolution melt analysis. Typing can be performed by analysing six loci (6MelT), four loci (4MelT) or using data from four loci plus sequencing the tonB gene (HybridMelT). The aim of this study was to evaluate Minim typing to discriminate extended-spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KLPN) isolates at our hospital. In total, 380 isolates were analyzed. The obtained alleles were assigned according to both the 6MelT and 4MelT typing scheme. In 97 isolates, the tonB gene was sequenced to enable HybridMelT typing. We found that the presented method is suitable to quickly monitor isolates of ESBL-KLPN; results are obtained in less than 2 hours and at a lower cost than MLST. We identified a local ESBL-KLPN outbreak and a comparison of colonizing and invasive isolates revealed a long term colonization of patients with the same strain.

High diversity of invasive Haemophilus influenzae isolates in France and the emergence of resistance to third generation cephalosporins by alteration of ftsI gene

BackgroundInvasive infections due to Haemophilus influenzae are infrequent following the implementation of vaccination against H. influenzae of serotype b. However, their changing epidemiology may not be clear due to a lack of appropriate genotyping methods combined with antibiotic susceptibility analyses which do not discriminate invasive and non-invasive isolates. We aimed to describe recent epidemiological trends of invasive H. influenzae infections in France and explore the microbiological characteristics of invasive versus non-invasive isolates. MethodsAll culture- and PCR-confirmed cases due to H. influenzae isolated from a sterile site, that were received at the French national reference centre for H. influenzae during the year 2017 (n = 138) were characterized by whole genome sequencing (WGS), serotyping and antibiotic susceptibility testing. We also included 100 isolates that were received from non-invasive infections. FindingsMost of the non-invasive isolates were non-typeable (99%) and this proportion was significantly less among invasive isolates 75%, p < 0.0001). Serotype f was the most frequently observed but serotypes b and a were also present among invasive isolates. WGS analysis suggested a serotype b to a capsule switching event. Non-typeable isolates showed extensive heterogeneity. Antibiotic susceptibility testing indicated that 24% of the invasive isolates were resistant to ampicillin but this percentage was significantly higher (51%, p < 0.001) among the non-invasive isolates. Moreover, the proportion of beta-lactamase negative ampicillin resistant isolates (BLNAR) was significantly higher among non-invasive isolates compared to that of invasive isolates (24% versus 7%, p < 0.001). BLNAR isolates were linked to modification in the ftsI gene encoding the penicillin binding protein 3 (PBP3). In particular, ftsI alleles that harboured the mutations D350N, S357N, M377I and S385T were resistant to ampicillin and third generation cephalosporins. These isolates were more frequent among non-invasive isolates. InterpretationOur data suggest that invasive H. influenzae isolates differed phenotypically and genotypically from non-invasive isolates. The high proportion of ampicillin resistance by mutation in ftsI among non-invasive isolates may suggest a biological cost of these mutations on the function of PBP3 that can lead to lower bacterial invasiveness. WGS should be used routinely for the characterization of H. influenzae isolates in order to reliably follow the emergence, spread and mechanism of antibiotic resistance.

Remarkable Genome Stability among emm1 Group A Streptococcus in Belgium over 19 Years.

During the last two decades, there has been a public health concern of severe invasive infections caused by Group A Streptococcus (GAS) of the emm1 genotype. This study investigated the dynamics of emm1 GAS during 1994–2013 in Belgium. emm1 GAS isolated from blood, tissue, and wounds of patients with invasive infections (n = 23, S1–S23), and from patients with uncomplicated pharyngitis (n = 15, NS1–NS15) were subjected to whole-genome mapping (WGM; kpn) (Opgen). Whole-genome sequencing was performed on 25 strains (WGS; S1–S23 and NS6–NS7) (Illumina Inc.). Belgian GAS belonged to the M1T1 clone typified by the 36-kb chromosomal region encoding extracellular toxins, NAD+-glycohydrolase and streptolysin O. Strains from 1994–1999 clustered together with published strains (MGAS5005 and M1476). From 2001 onward, invasive GAS showed higher genomic divergence in the accessory genome and harbored on average 7% prophage content. Low evolutionary rate (2.49E-008; P > 0.05) was observed in this study, indicating a highly stable genome. The studied invasive and pharyngitis isolates were no genetically distinct populations based on the WGM and core genome phylogeny analyses. Two copies of the speJ superantigen were present in the 1999 and 2010 study strains (n = 3), one being chromosomal and one being truncated and associated with phage remnants.This study showed that emm1 GAS in Belgium, compared with Canada and UK M1 strains, were highly conserved by harboring a remarkable genome stability over a 19-year period with variations observed in the accessory genome.

The potential of fosfomycin for multi-drug resistant sepsis: an analysis of in vitro activity against invasive paediatric Gram-negative bacteria.

Antimicrobial resistance (AMR) is of increasing global concern, threatening to undermine recent progress in reducing child and neonatal mortality. Repurposing older antimicrobials is a prominent strategy to combat multidrug-resistant sepsis. A potential agent is fosfomycin, however, there is scarce data regarding its in vitro activity and pharmacokinetics in the paediatric population. We analysed a contemporary, systematically collected archive of community-acquired (CA) and hospital-acquired (HA) paediatric Gram-negative bacteraemia isolates for their susceptibility to fosfomcyin. MICs were determined using agar serial dilution methods and validated by disk diffusion testing where breakpoints are available. Disk diffusion antimicrobial susceptibility testing was also conducted for current empirical therapies (ampicillin, gentamicin, ceftriaxone) and amikacin (proposed in the literature as a new combination empirical therapeutic option). Fosfomycin was highly active against invasive Gram-negative isolates, including 90 % (202/224) of Enterobacteriaceae and 96 % (22/23) of Pseudomonas spp. Fosfomycin showed high sensitivity against both CA isolates (94 %, 142/151) and HA isolates (81 %, 78/96; P =0.0015). CA isolates were significantly more likely to be susceptible to fosfomycin than the current first-line empirical therapy (96 % vs 59 %, P <0.0001). Extended spectrum β-lactamases (ESBL) production was detected in 34 % (85/247) of isolates with no significant difference in fosfomycin susceptibility between ESBL-positive or -negative isolates [73/85 (86 %) vs 147/162 (91 %) respectively, P =0.245]. All isolates were susceptible to a fosfomycin-amikacin combination. Gram-negative paediatric bacteraemia isolates are highly susceptible to fosfomycin, which could be combined with aminoglycosides as a new, carbapenem-sparing regimen to achieve excellent coverage to treat antimicrobial-resistant neonatal and paediatric sepsis.

Molecular Epidemiology of Invasive Staphylococcus aureus Infections and Concordance with Colonization Isolates

To characterize Staphylococcus aureus isolates recovered from hospitalized children and to determine the concordance between colonizing and invasive isolates. Children with culture-confirmed, community-onset, invasive S aureus infections were enrolled in this prospective case series from a large children's hospital over a 5-year period. Colonization isolates were obtained from the anterior nares, oropharynx, and inguinal folds and were compared with invasive isolates via repetitive-element, sequence-based polymerase chain reaction testing. Isolates with a ≥96% genetic match were characterized as concordant. A total of 86 S aureus isolates (44 invasive, 42 colonization) were collected from 44 children with invasive infections. Clinical isolates were genetically diverse, 64% of invasive isolates were methicillin-susceptible S aureus (MSSA), and 59% of cases had a colonizing S aureus isolate at the time of hospitalization. Of those who were colonized, at least 1 of their colonization isolates was indistinguishable from the infecting isolate in 88% of cases. Patients with invasive MSSA were significantly more likely to have a concordant MSSA colonization isolate present compared with patients with invasive methicillin-resistant S aureus (MRSA) (61% vs 38%, P<.05). Invasive MSSA infection was more common than MRSA infection in this pediatric cohort, and patients with MSSA infection were significantly more likely than those with MRSA infection to have concordant colonizing isolates across multiple anatomic sites. These findings warrant larger scale validation and may have important infection control and epidemiologic implications, as unlike MRSA, transmissibility of MSSA largely is ignored in healthcare settings.

Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016.

BackgroundThe emergence of antifungal resistance threatens effective treatment of invasive fungal infection (IFI). Invasive candidiasis is the most common health care–associated IFI. We evaluated the activity of fluconazole (FLU) against 20 788 invasive isolates of Candida (37 species) collected from 135 medical centers in 39 countries (1997–2016). The activity of anidulafungin, caspofungin, and micafungin (MCF) was evaluated against 15 308 isolates worldwide (2006–2016).MethodsSpecies identification was accomplished using phenotypic (1997–2001), genotypic, and proteomic methods (2006–2016). All isolates were tested using reference methods and clinical breakpoints published in the Clinical and Laboratory Standards Institute documents.ResultsA decrease in the isolation of Candida albicans and an increase in the isolation of Candida glabrata and Candida parapsilosis were observed over time. Candida glabrata was the most common non–C. albicans species detected in all geographic regions except for Latin America, where C. parapsilosis and Candida tropicalis were more common. Six Candida auris isolates were detected: 1 each in 2009, 2013, 2014, and 2015 and 2 in 2016; all were from nosocomial bloodstream infections and were FLU-resistant (R). The highest rates of FLU-R isolates were seen in C. glabrata from North America (NA; 10.6%) and in C. tropicalis from the Asia-Pacific region (9.2%). A steady increase in isolation of C. glabrata and resistance to FLU was detected over 20 years in the United States. Echinocandin-R (EC-R) ranged from 3.5% for C. glabrata to 0.1% for C. albicans and C. parapsilosis. Resistance to MCF was highest among C. glabrata (2.8%) and C. tropicalis (1.3%) from NA. Mutations on FKS hot spot (HS) regions were detected among 70 EC-R isolates (51/70 were C. glabrata). Most isolates harboring FKS HS mutations were resistant to 2 or more ECs.ConclusionsEC-R and FLU-R remain uncommon among contemporary Candida isolates; however, a slow and steady emergence of resistance to both antifungal classes was observed in C. glabrata and C. tropicalis isolates.

Metabolic analyses reveal common adaptations in two invasive Haemophilus influenzae strains.

Non-typeable Haemophilus influenzae (NTHi) is a major pathogen in upper and lower respiratory tract infections in humans, and is increasingly also associated with invasive disease. We have examined two unrelated NTHi invasive disease isolates, R2866 and C188, in order to identify metabolic and physiological properties that distinguish them from respiratory tract disease isolates such as Hi2019. While the general use of the Hi metabolic network was similar across all three strains, the two invasive isolates secreted increased amounts of succinate, which can have anti-inflammatory properties. In addition, they showed a common shift in their carbon source utilization patterns, with strongly enhanced metabolism of nucleoside substrates, glucose and sialic acid. The latter two are major compounds present in blood and cerebrospinal fluid (CSF). Interestingly, C188 and R2866 also shared a reduced ability to invade or survive intracellularly in 16HBE14 bronchial epithelial cells relative to Hi2019 (4-fold (4 h), 25-fold (24 h) reduction). Altered metabolic properties, such as the ones observed here, could arise from genomic adaptations that NTHi undergo during infection. Together these data indicate that shifts in substrate preferences in otherwise conserved metabolic pathways may underlie strain niche specificity and thus have the potential to alter the outcomes of host-NTHi interactions.

Phylogenetic relationships and regional spread of meningococcal strains in the meningitis belt, 2011-2016.

BackgroundHistorically, the major cause of meningococcal epidemics in the meningitis belt of sub-Saharan Africa has been Neisseria meningitidis serogroup A (NmA), but the incidence has been substantially reduced since the introduction of a serogroup A conjugate vaccine starting in 2010. We performed whole-genome sequencing on isolates collected post-2010 to assess their phylogenetic relationships and inter-country transmission.MethodsA total of 716 invasive meningococcal isolates collected between 2011 and 2016 from 11 meningitis belt countries were whole-genome sequenced for molecular characterization by the three WHO Collaborating Centers for Meningitis.FindingsWe identified three previously-reported clonal complexes (CC): CC11 (n = 434), CC181 (n = 62) and CC5 (n = 90) primarily associated with NmW, NmX, and NmA, respectively, and an emerging CC10217 (n = 126) associated with NmC. CC11 expanded throughout the meningitis belt independent of the 2000 Hajj outbreak strain, with isolates from Central African countries forming a distinct sub-lineage within this expansion. Two major sub-lineages were identified for CC181 isolates, one mainly expanding in West African countries and the other found in Chad. CC10217 isolates from the large outbreaks in Nigeria and Niger were more closely related than those from the few cases in Mali and Burkina Faso.InterpretationsWhole-genome based phylogenies revealed geographically distinct strain circulation as well as inter-country transmission events. Our results stress the importance of continued meningococcal molecular surveillance in the region, as well as the development of an affordable vaccine targeting these strains.FundMeningitis Research Foundation; CDC's Office of Advanced Molecular Detection; GAVI, the Vaccine Alliance.

Phase variation of Opa proteins in hypervirulent serogroup W meningococcal isolates

Serotype W, sequence type 11 Neisseria meningitidis are an increasing cause of morbidity and mortality. The mechanisms underpinning the success of this clone remain unclear. Meningococci express up to four Opa proteins, which mediate adhesion to/invasion of host cells. Opa expression undergoes phase variation – a high frequency ON/OFF switch in gene expression due to insertion/deletions of pentameric repeats (5′-CTCTT-3′) in the coding region. NadA functions as an adhesion, and is phase-variable through tetrameric repeats in a regulatory. WGS and PCR-based fragment size analysis of 121 invasive and 51 carriage MenW:ST11 strains was conducted to determine gene expression patterns between invasive and carriage. Inactivating mutations were constructed in opa, pilE and nadA genes of two representative MenW isolates. These mutants were tested in in vitro infection assays for adhesion and invasion of A549 cells. We observe that four opa loci are present in all MenW:cc11 isolates and that OpaB and OpaD share 100 % sequence similarity. Repeat number variability was detected between the ‘original UK’ strain and the novel ‘2013-strain’ of the hypervirulent MenW:cc11 South American sublineage. No significant differences in the patterns of Opa expression were observed between invasive and carriage isolates. In vitro assays with pilE and nadA deletion suggest that NadA has an effect on invasion, while the double mutant shows a reduction in both adhesion and invasion. Our findings indicate that the Opa proteins do not contribute to the invasiveness of MenW:cc11 strains, but may give a niche specific advantage by enhancing phase variation-mediated immune evasion.

Genomic analysis of Klebsiella pneumoniae isolates from Malawi reveals acquisition of multiple ESBL determinants across diverse lineages.

ObjectivesESBL-producing Klebsiella pneumoniae (KPN) pose a major threat to human health globally. We carried out a WGS study to understand the genetic background of ESBL-producing KPN in Malawi and place them in the context of other global isolates.MethodsWe sequenced genomes of 72 invasive and carriage KPN isolates collected from patients admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi. We performed phylogenetic and population structure analyses on these and previously published genomes from Kenya (n = 66) and from outside sub-Saharan Africa (n = 67). We screened for presence of antimicrobial resistance (AMR) genetic determinants and carried out association analyses by genomic sequence cluster, AMR phenotype and time.ResultsMalawian isolates fit within the global population structure of KPN, clustering into the major lineages of KpI, KpII and KpIII. KpI isolates from Malawi were more related to those from Kenya, with both collections exhibiting more clonality than isolates from the rest of the world. We identified multiple ESBL genes, including blaCTX-M-15, several blaSHV, blaTEM-63 and blaOXA-10, and other AMR genes, across diverse lineages of the KPN isolates from Malawi. No carbapenem resistance genes were detected; however, we detected IncFII and IncFIB plasmids that were similar to the carbapenem resistance-associated plasmid pNDM-mar.ConclusionsThere are multiple ESBL genes across diverse KPN lineages in Malawi and plasmids in circulation that are capable of carrying carbapenem resistance. Unless appropriate interventions are rapidly put in place, these may lead to a high burden of locally untreatable infection in vulnerable populations.

Genomic correlates of extraintestinal infection are linked with changes in cell morphology in Campylobacter jejuni.

Campylobacter jejuni is the most common cause of bacterial diarrheal disease in the world. Clinical outcomes of infection can range from asymptomatic infection to life-threatening extraintestinal infections. This variability in outcomes for infected patients has raised questions as to whether genetic differences between C. jejuni isolates contribute to their likelihood of causing severe disease. In this study, we compare the genomes of ten C. jejuni isolates that were implicated in extraintestinal infections with reference gastrointestinal isolates, in order to identify unusual patterns of sequence variation associated with infection outcome. We identified a collection of genes that display a higher burden of uncommon mutations in invasive isolates compared with gastrointestinal close relatives, including some that have been previously linked to virulence and invasiveness in C. jejuni. Among the top genes identified were mreB and pgp1, which are both involved in determining cell shape. Electron microscopy confirmed morphological differences in isolates carrying unusual sequence variants of these genes, indicating a possible relationship between extraintestinal infection and changes in cell morphology.