Genomic Insights Into the Mechanism of Carbapenem Resistance Dissemination in Enterobacterales From a Tertiary Public Heath Setting in South Asia.

SUMMARY: 10.6% patients were CRE positive. Only 27% patients were prescribed at least 1 antibiotic to which infecting pathogen was susceptible. Burn and ICU admission and antibiotics exposures facilitate CRE acquisition. Escherichia coli ST167 was the dominant CRE clone. BACKGROUND: Given the high prevalence of multidrug resistance (MDR) across South Asian (SA) hospitals, we documented the epidemiology of carbapenem-resistant Enterobacterales (CRE) infections at Dhaka Medical College Hospital between October 2016 and September 2017. METHODS: We enrolled patients and collected epidemiology and outcome data. All Enterobacterales were characterized phenotypically and by whole-genome sequencing. Risk assessment for the patients with CRE was performed compared with patients with carbapenem-susceptible Enterobacterales (CSE). RESULTS: 10.6% of all 1831 patients with a clinical specimen collected had CRE. In-hospital 30-day mortality was significantly higher with CRE [50/180 (27.8%)] than CSE [42/312 (13.5%)] (P = .001); however, for bloodstream infections, this was nonsignificant. Of 643 Enterobacterales isolated, 210 were CRE; blaNDM was present in 180 isolates, blaOXA-232 in 26, blaOXA-181 in 24, and blaKPC-2 in 5. Despite this, ceftriaxone was the most commonly prescribed empirical antibiotic and only 27% of patients were prescribed at least 1 antibiotic to which their infecting pathogen was susceptible. Significant risk factors for CRE isolation included burns unit and intensive care unit admission, and prior exposure to levofloxacin, amikacin, clindamycin, and meropenem. Escherichia coli ST167 was the dominant CRE clone. Clustering suggested clonal transmission of Klebsiella pneumoniae ST15 and the MDR hypervirulent clone, ST23. The major trajectories involved in horizontal gene transfer were IncFII and IncX3, IS26, and Tn3. CONCLUSIONS: This is the largest study from an SA public hospital combining outcome, microbiology, and genomics. The findings indicate the urgent implementation of targeted diagnostics, appropriate antibiotic use, and infection-control interventions in SA public institutions.

Characterisation of Staphylococci species from neonatal blood cultures in low- and middle-income countries.

BACKGROUND: In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB). METHODS: We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015-2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age. RESULTS: From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017. CONCLUSIONS: In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome.

Tetracycline Resistance Mediated by tet(M) Has Variable Integrative Conjugative Element Composition in Mycoplasma hominis Strains Isolated in the United Kingdom from 2005 to 2015.

A minimal genome and absent bacterial cell wall render Mycoplasma hominis inherently resistant to most antimicrobials except lincosamides, tetracyclines, and fluoroquinolones. Often dismissed as a commensal (except where linked to preterm birth), it causes septic arthritis in immunodeficient patients and is increasingly associated with transplant failure (particularly lung) accompanying immunosuppression. We examined antimicrobial susceptibility (AST) on strains archived from 2005 to 2015 submitted to the Public Health England reference laboratory and determined the underlying mechanism of resistance by whole-genome sequencing (WGS). Archived M. hominis strains included 32/115 from invasive infection (sepsis, cerebrospinal [CSF], peritoneal, and pleural fluid) over the 10-year period (6.4% of all samples submitted from 2010 to 2015 were positive). No clindamycin resistance was detected, while two strains were resistant to moxifloxacin and levofloxacin (resistance mutations S83L or E87G in gyrA and S81I or E84V in parC). One of these strains and 11 additional strains were tetracycline resistant, mediated by tet(M) carried within an integrative conjugative element (ICE) consistently integrated at the somatic rumA gene; however, the ICEs varied widely in 5 to 19 associated accessory genes. WGS analysis showed that tet(M)-carrying strains were not clonal, refuting previous speculation that the ICE was broken and immobile. We found tet(M)-positive and -negative strains (including the multiresistant 2015 strain) to be equally susceptible to tigecycline and josamycin; however, the British National Formulary does not include guidance for these. Continued M. hominis investigation and AST surveillance (especially immunocompromised patients) is warranted, and the limited number of therapeutics needs to be expanded in the United Kingdom.

Legionella antimicrobial sensitivity testing: comparison of microbroth dilution with BCYE and LASARUS solid media.

OBJECTIVES: There is a lack of international unification for AST methodology for Legionella pneumophila. Current literature contains multiple possible methods and this study compares each of them to determine methodological concordance. METHODS: Antibiotic susceptibility of 50 L. pneumophila strains was determined using broth microdilution (BMD), serial antimicrobial dilution in traditional buffered charcoal yeast extract (BCYE) agar (as well as comparison with gradient strip overlay on BCYE) and in a novel charcoal-free agar (LASARUS) for rifampicin, azithromycin, levofloxacin and doxycycline. RESULTS: The deviation of tested media relative to BMD highlighted the overall similarity of BMD and LASARUS across all antimicrobials tested (within one serial dilution). BCYE agar dilution showed an increased MIC of up to five serial dilutions relative to BMD, while MICs by gradient strip overlay on BCYE were elevated by two to three serial dilutions, with the exception of doxycycline, which was decreased by three serial dilutions relative to MIC values determined by BMD. The MIC range for azithromycin was wider than for other antimicrobials tested and found to be caused by the presence or absence of the lpeAB gene. CONCLUSIONS: BMD-based antimicrobial susceptibility testing (AST) methodology should be the internationally agreed gold standard for Legionella spp. AST, as is common for other bacterial species. Traditional BCYE gave significantly elevated MIC results and its use should be discontinued for Legionella spp., while MIC determination using LASARUS solid medium gave results concordant (within one serial dilution) with BMD for all antimicrobials tested. To the best of our knowledge, this study is the first to identify the lpeAB gene in UK isolates.

Evaluation of the MYCOPLASMA IST3 urogenital mycoplasma assay in an international multicentre trial.

OBJECTIVES: To evaluate the accuracy, susceptibility and specificity of MYCOPLASMA IST3, the next generation of the most popular culture-based in vitro diagnostic device designed to detect, identify and test the susceptibility of urogenital mycoplasma infections. METHODS: MYCOPLASMA IST3 was evaluated against culture- and molecular-based gold standard methodologies to detect, identify, enumerate and determine antimicrobial resistance for Mycoplasma hominis and Ureaplasma species in 516 clinical samples collected across France, Serbia and the UK. Sample types included vulvovaginal/endocervical or urethral swabs (dry swab or eSwab®), semen and urine samples, which included blinded analysis following addition of a panel of 80 characterized control strains. RESULTS: Overall species identification was excellent for both Ureaplasma spp. (98.4% sensitivity, 99.7% specificity) and M. hominis (95.7% sensitivity, 100% specificity) relative to combined colony morphology on agar and quantitative PCR standards. Non-dilution-based bacterial load estimation by the assay was accurate between 83.7% (M. hominis) and 86.3% (Ureaplasma spp.) of the time (increased to 94.2% and 100%, respectively, if ±10-fold variance was allowed) relative to colonies counted on agar. Resistance accuracy for Ureaplasma spp. varied from gold standards for only 11/605 of individual tests (major error rate = 1.8%) and for 14/917 individual tests for M. hominis (major error rate = 1.5%). CONCLUSIONS: The redesigned MYCOPLASMA IST3 assay eliminated previous shortcomings by providing independent accurate resistance screening of M. hominis and Ureaplasma species, even in mixed infections, with CLSI-compliant thresholds. Specificity, sensitivity and enumeration estimates correlated closely with the confirmatory methods.

Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?

Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire's disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.

An Emerging Clone, Klebsiellapneumoniae Carbapenemase 2-Producing K. pneumoniae Sequence Type 16, Associated With High Mortality Rates in a CC258-Endemic Setting.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae has become a global priority, not least in low- and middle-income countries. Here, we report the emergence and clinical impact of a novel Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) sequence type (ST) 16 clone in a clonal complex (CC) 258-endemic setting. METHODS: In a teaching Brazilian hospital, a retrospective cohort of adult KPC-KP bloodstream infection (BSI) cases (January 2014 to December 2016) was established to study the molecular epidemiology and its impact on outcome (30-day all-cause mortality). KPC-KP isolates underwent multilocus sequence typing. Survival analysis between ST/CC groups and risk factors for fatal outcome (logistic regression) were evaluated. Representative isolates underwent whole-genome sequencing and had their virulence tested in a Galleria larvae model. RESULTS: One hundred sixty-five unique KPC-KP BSI cases were identified. CC258 was predominant (66%), followed by ST16 (12%). The overall 30-day mortality rate was 60%; in contrast, 95% of ST16 cases were fatal. Patients' severity scores were high and baseline clinical variables were not statistically different across STs. In multivariate analysis, ST16 (odds ratio [OR], 21.4; 95% confidence interval [CI], 2.3-202.8; P = .008) and septic shock (OR, 11.9; 95% CI, 4.2-34.1; P < .001) were independent risk factors for fatal outcome. The ST16 clone carried up to 14 resistance genes, including blaKPC-2 in an IncFIBpQIL plasmid, KL51 capsule, and yersiniabactin virulence determinants. The ST16 clone was highly pathogenic in the larvae model. CONCLUSIONS: Mortality rates were high in this KPC-KP BSI cohort, where CC258 is endemic. An emerging ST16 clone was associated with high mortality. Our results suggest that even in endemic settings, highly virulent clones can rapidly emerge demanding constant monitoring.

MYCO WELL D-ONE detection of Ureaplasma spp. and Mycoplasma hominis in sexual health patients in Wales.

The genital mycoplasmas are a unique group of inherently antibiotic-resistant sexually transmitted bacteria, often associated with non-gonococcal urethritis and bacterial vaginosis. The MYCO WELL D-ONE is a culture-based assay that aims to detect these organisms whilst concurrently screening them for antibiotic resistance. Urine and/or swabs from 856 informed and consented participants attending Welsh sexual health clinics were subjected to MYCO WELL D-ONE analysis, alongside qPCR and culture titration methodologies to determine sensitivity, specificity, PPV, NPV and accuracy. Resistance was confirmed by CLSI-compliant susceptibility testing and genetic mechanisms determined. The MYCO WELL D-ONE displayed a sensitivity and specificity of 91.98% and 96.44% for the detection of Ureaplasma spp., with sensitivity and specificity values of 78.23% and 98.84% for Mycoplasma hominis, compared with qPCR. Swabs harboured significantly greater bacterial loads than urine samples for both Ureaplasma spp. and M. hominis. Levofloxacin resistance rates, mediated by Ser83Leu mutation in ParC, for Ureaplasma spp. were 0.54%. Tetracycline resistance rates, mediated by tet(M), were 0.54% and 2% for Ureaplasma spp. and M. hominis, respectively; sequence analysis of tet(M)-positive Ureaplasma spp. and M. hominis strains isolated from a single individual confirmed separate resistance gene origins. The MYCO WELL D-ONE is a sensitive and specific assay for the detection of Ureaplasma spp. and M. hominis in genitourinary medicine samples, facilitating the accurate detection of these organisms within low-technology environments. While good for antibiotic resistance screening, accurate confirmation by MIC determination or molecular methods are required, and more optimally performed on urine samples.

Outbreak of Hypervirulent Multidrug-resistant Klebsiella variicola Causing High Mortality in Neonates in Bangladesh.

We report a clonal outbreak of multidrug-resistant (MDR) Klebsiella variicola (sequence type [ST] 771) in a Bangladeshi neonatal unit from October 2016 to January 2017, associated with high mortality (54.5%). During the outbreak, K. variicola ST771 acquired an MDR plasmid harboring blaNDM-1, linked to high exposure to ceftriaxone and amikacin.

Colonisation of hospital surfaces from low- and middle-income countries by extended spectrum ß-lactamase- and carbapenemase-producing bacteria.

Hospital surfaces can harbour bacterial pathogens, which may disseminate and cause nosocomial infections, contributing towards mortality in low- and middle-income countries (LMICs). During the BARNARDS study, hospital surfaces from neonatal wards were sampled to assess the degree of environmental surface and patient care equipment colonisation by Gram-negative bacteria (GNB) carrying antibiotic resistance genes (ARGs). Here, we perform PCR screening for extended-spectrum ß-lactamases (blaCTX-M-15) and carbapenemases (blaNDM, blaOXA-48-like and blaKPC), MALDI-TOF MS identification of GNB carrying ARGs, and further analysis by whole genome sequencing of bacterial isolates. We determine presence of consistently dominant clones and their relatedness to strains causing neonatal sepsis. Higher prevalence of carbapenemases is observed in Pakistan, Bangladesh, and Ethiopia, compared to other countries, and are mostly found in surfaces near the sink drain. Klebsiella pneumoniae, Enterobacter hormaechei, Acinetobacter baumannii, Serratia marcescens and Leclercia adecarboxylata are dominant; ST15 K. pneumoniae is identified from the same ward on multiple occasions suggesting clonal persistence within the same environment, and is found to be identical to isolates causing neonatal sepsis in Pakistan over similar time periods. Our data suggests persistence of dominant clones across multiple time points, highlighting the need for assessment of Infection Prevention and Control guidelines.

Genomic Analysis Reveals New Integrative Conjugal Elements and Transposons in GBS Conferring Antimicrobial Resistance.

Streptococcus agalactiae or group B streptococcus (GBS) is a leading cause of neonatal sepsis and increasingly found as an invasive pathogen in older patient populations. Beta-lactam antibiotics remain the most effective therapeutic with resistance rarely reported, while the majority of GBS isolates carry the tetracycline resistance gene tet(M) in fixed genomic positions amongst five predominant clonal clades. In the UK, GBS resistance to clindamycin and erythromycin has increased from 3% in 1991 to 11.9% (clindamycin) and 20.2% (erythromycin), as reported in this study. Here, a systematic investigation of antimicrobial resistance genomic content sought to fully characterise the associated mobile genetic elements within phenotypically resistant GBS isolates from 193 invasive and non-invasive infections of UK adult patients collected during 2014 and 2015. Resistance to erythromycin and clindamycin was mediated by erm(A) (16/193, 8.2%), erm(B) (16/193, 8.2%), mef(A)/msr(D) (10/193, 5.1%), lsa(C) (3/193, 1.5%), lnu(C) (1/193, 0.5%), and erm(T) (1/193, 0.5%) genes. The integrative conjugative elements (ICEs) carrying these genes were occasionally found in combination with high gentamicin resistance mediating genes aac(6')-aph(2″), aminoglycoside resistance genes (ant(6-Ia), aph(3'-III), and/or aad(E)), alternative tetracycline resistance genes (tet(O) and tet(S)), and/or chloramphenicol resistance gene cat(Q), mediating resistance to multiple classes of antibiotics. This study provides evidence of the retention of previously reported ICESag37 (n = 4), ICESag236 (n = 2), and ICESpy009 (n = 3), as well as the definition of sixteen novel ICEs and three novel transposons within the GBS lineage, with no evidence of horizontal transfer.

Comparing Long-Read Assemblers to Explore the Potential of a Sustainable Low-Cost, Low-Infrastructure Approach to Sequence Antimicrobial Resistant Bacteria With Oxford Nanopore Sequencing.

Long-read sequencing (LRS) can resolve repetitive regions, a limitation of short read (SR) data. Reduced cost and instrument size has led to a steady increase in LRS across diagnostics and research. Here, we re-basecalled FAST5 data sequenced between 2018 and 2021 and analyzed the data in relation to gDNA across a large dataset (n = 200) spanning a wide GC content (25-67%). We examined whether re-basecalled data would improve the hybrid assembly, and, for a smaller cohort, compared long read (LR) assemblies in the context of antimicrobial resistance (AMR) genes and mobile genetic elements. We included a cost analysis when comparing SR and LR instruments. We compared the R9 and R10 chemistries and reported not only a larger yield but increased read quality with R9 flow cells. There were often discrepancies with ARG presence/absence and/or variant detection in LR assemblies. Flye-based assemblies were generally efficient at detecting the presence of ARG on both the chromosome and plasmids. Raven performed more quickly but inconsistently recovered small plasmids, notably a ∼15-kb Col-like plasmid harboring bla KPC . Canu assemblies were the most fragmented, with genome sizes larger than expected. LR assemblies failed to consistently determine multiple copies of the same ARG as identified by the Unicycler reference. Even with improvements to ONT chemistry and basecalling, long-read assemblies can lead to misinterpretation of data. If LR data are currently being relied upon, it is necessary to perform multiple assemblies, although this is resource (computing) intensive and not yet readily available/useable.

Environmental surveillance of ESBL and carbapenemase-producing gram-negative bacteria in a Ghanaian Tertiary Hospital.

BACKGROUND: The burden of antibiotic resistant infection is mainly felt in low-to-middle income countries, where the rate of antimicrobial resistance is largely under-surveyed and under huge pressure from unregulated, disparate and often self-guided access to antimicrobials. Nosocomial infections from hospital environments have been shown to be a particularly prevalent source of multi-drug resistant strains, yet surveillance of hospital environmental contamination is often not investigated. METHODS: The study was prospective, observational and cross-sectional, sampling 231 high and low touch surfaces from 15th March to 13th April 2021, from five wards in the Cape Coast Teaching Hospital, Ghana. Microbial growth in the presence of vancomycin and either meropenem or cefotaxime was examined and bacterial species were identified by MALDI-TOF. The presence of common extended-spectrum ß-lactamases (ESBL) and carbapenemase antimicrobial resistance genes (ARG) were identified through PCR screening, which were confirmed by phenotypic antimicrobial susceptibility determination. Isolates positive for carbapenem resistance genes were sequenced using a multi-platform approach. RESULTS: We recovered microbial growth from 99% of swabs (n = 229/231) plated on agar in the absence of antimicrobials. Multiple sites were found to be colonised with resistant bacteria throughout the hospital setting. Bacteria with multi-drug resistance and ARG of concern were isolated from high and low touch points with evidence of strain dissemination throughout the environment. A total of 21 differing species of bacteria carrying ARG were isolated. The high prevalence of Acinetobacter baumannii carrying blaNDM-1 observed was further characterised by whole genome sequencing and phylogenetic analysis to determine the relationship between resistant strains found in different wards. CONCLUSION: Evidence of multiple clonal incursions of MDR bacteria of high sepsis risk were found in two separate wards for a regional hospital in Ghana. The prevalence of multiple blaNDM carrying species in combination with combinations of ESBLs was particularly concerning and unexpected in Africa. We also identify strains carrying tet(X3), blaVIM-5 or blaDIM-1 showing a high diversity of carbapenamases present as a reservoir in a hospital setting. Findings of multi-drug resistant bacteria from multiple environmental sites throughout the hospital will inform future IPC practices and aid research prioritisation for AMR in Ghana.

Early-Onset Neonatal Sepsis in Low- and Middle-Income Countries: Current Challenges and Future Opportunities.

Neonatal sepsis is defined as a systemic infection within the first 28 days of life, with early-onset sepsis (EOS) occurring within the first 72h, although the definition of EOS varies in literature. Whilst the global incidence has dramatically reduced over the last decade, neonatal sepsis remains an important cause of neonatal mortality, highest in low- and middle-income countries (LMICs). Symptoms at the onset of neonatal sepsis can be subtle, and therefore EOS is often difficult to diagnose from clinical presentation and laboratory testing and blood cultures are not always conclusive or accessible, especially in resource limited countries. Although the World Health Organisation (WHO) currently advocates a ß-lactam, and gentamicin for first line treatment, availability and cost influence the empirical antibiotic therapy administered. Antibiotic treatment of neonatal sepsis in LMICs is highly variable, partially caused by factors such as cost of antibiotics (and who pays for them) and access to certain antibiotics. Antimicrobial resistance (AMR) has increased considerably over the past decade and this review discusses current microbiology data available in the context of the diagnosis, and treatment for EOS. Importantly, this review highlights a large variability in data availability, methodology, availability of diagnostics, and aetiology of sepsis pathogens.

Diversity of lytic bacteriophages against XDR Klebsiella pneumoniae sequence type 16 recovered from sewage samples in different parts of the world.

Bacteriophages (phages) are viruses considered to be natural bacterial predators and widely detected in aquatic environments. Sewage samples are an important source of phage isolation since high density and diversity of bacterial cells are present, due to human, animal and household fluids. This study aims to investigate and characterise phages against an extremely drug-resistant (XDR) lineage, Klebsiella pneumoniae ST16, using sewage samples from different parts of the World. Sewage samples from Brazil, Bangladesh, Saudi Arabia, Thailand and the United Kingdom were collected and used to investigate phages against ten K. pneumoniae ST16 (hosts) recovered from infection sites. The phages were microbiological and genetically characterised by double-agar overlay (DLA), transmission electron microscopy and Illumina WGS. The host range against K. pneumoniae belonging to different sequence types was evaluated at different temperatures by spot test. Further phage characterisation, such as efficiency of plating, optimal phage temperature, and pH/temperature susceptibility, were conducted. Fourteen lytic phages were isolated, belonging to Autographiviridae, Ackermannviridae, Demerecviridae, Drexlerviridae, and Myoviridae families, from Brazil, Bangladesh, Saudi Arabia and Thailand and demonstrated a great genetic diversity. The viruses had good activity against our collection of clinical K. pneumoniae ST16 at room temperature and 37 °C, but also against other important Klebsiella clones such as ST11, ST15, and ST258. Temperature assays showed lytic activity in different temperatures, except for PWKp18 which only had activity at room temperature. Phages were stable between pH 5 and 10 with minor changes in phage activity, and 70 °C was the temperature able to kill all phages in this study. Using sewage from different parts of the World allowed us to have a set of highly efficient phages against an K. pneumoniae ST16 that can be used in the future to develop new tools to combat infections in humans or animals caused by this pathogen.

Effective phage cocktail to combat the rising incidence of extensively drug-resistant Klebsiella pneumoniae sequence type 16.

Bacteriophages are the most abundant organisms on Earth. As there are few effective treatment options against some pathogens, the interest in the bacteriophage control of multi-drug-resistant bacterial pathogens is escalating, especially for Klebsiella pneumoniae. This study aimed to develop a phage-based solution to the rising incidence of extensively drug-resistant clinical Klebsiella pneumoniae sequence type (ST16) infections starting from a set of phages recently characterized against this lineage. A phage-cocktail (Katrice-16) composed of eight lytic phages was characterized for potential use in humans. In vitro and in vivo broth inhibition and Galleria mellonella rescue assays were used to demonstrate the efficacy of this approach using a collection of 56 strains of K. pneumoniae ST16, with distinct genetic backgrounds that were collected from clinical infections from four disparate nations. Additionally, Katrice-16 anti-biofilm activity, synergism with meropenem, and activity in human body fluids were also assessed. Katrice-16 was highly active in vitro against our K. pneumoniae ST16 collection (AUC% median = 86.48%; Q1 = 83.8%; Q2 = 96.85%; Q3 = 98.85%). It additionally demonstrated excellent in vivo activity in G. mellonella rescue assays, even with larvae infected by isolates that exhibited moderate in vitro inhibition. We measured significant anti-biofilm activity over 12 h (p = .0113) and synergic activity with meropenem. In addition, we also demonstrate that Katrice-16 maintained high activity in human body fluids. Our results indicate that our cocktail will likely be an effective solution for human infections with this increasingly prevalent and often highly resistant bacterial clone.

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study.

BACKGROUND: Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. METHODS: The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. FINDINGS: Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69-234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04-74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37-2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. INTERPRETATION: Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. FUNDING: Bill & Melinda Gates Foundation.

Determination of In Vitro Antimicrobial Susceptibility for Lefamulin (Pleuromutilin) for Ureaplasma Spp. and Mycoplasma hominis.

Lefamulin is the first of the pleuromutilin class of antimicrobials to be available for therapeutic use in humans. Minimum inhibitory concentrations of lefamulin were determined by microbroth dilution for 90 characterised clinical isolates (25 Ureaplasma parvum, 25 Ureaplasma urealyticum, and 40 Mycoplasma hominis). All Mycoplasma hominis isolates possessed lefamulin MICs of ≤0.25 mg/L after 48 h (MIC50/90 of 0.06/0.12 mg/L), despite an inherent resistance to macrolides; while Ureaplasma isolates had MICs of ≤2 mg/L after 24 h (MIC50/90 of 0.25/1 mg/L), despite inherent resistance to clindamycin. Two U. urealyticum isolates with additional A2058G mutations of 23S rRNA, and one U. parvum isolate with a R66Q67 deletion (all of which had a combined resistance to macrolides and clindamycin) only showed a 2-fold increase in lefamulin MIC (1-2 mg/L) relative to macrolide-susceptible strains. Lefamulin could be an effective alternative antimicrobial for treating Ureaplasma spp. and Mycoplasma hominis infections irrespective of intrinsic or acquired resistance to macrolides, lincosamides, and ketolides. Based on this potent in vitro activity and the known good, rapid, and homogenous tissue penetration of female and male urogenital tissues and glands, further exploration of clinical efficacy of lefamulin for the treatment of Mycoplasma and Ureaplasma urogenital infections is warranted.

Defining Fluoroquinolone Resistance-Mediating Mutations from Non-Resistance Polymorphisms in Mycoplasma hominis Topoisomerases.

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24-48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics' surveillance.