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Background/Objectives: Infections caused by Legionella species are primarily associated with Legionella pneumophila, but non-pneumophila species are increasingly implicated in human disease. Despite this, antimicrobial susceptibility testing (AST) data for non-pneumophila species remain scarce, and standardised testing protocols or resistance thresholds have not been established. This study aimed to address this gap by evaluating and comparing AST performance for non-pneumophila Legionella species relative to L. pneumophila using three methodologies. Methods: AST was conducted on 89 Legionella isolates using LASARUS agar dilution, buffered yeast extract broth microdilution (BYE-BMD), and BCYE-α agar dilution, against ampicillin, azithromycin, chloramphenicol, doxycycline, levofloxacin, and rifampicin. Growth performance and minimum inhibitory concentrations (MICs) were assessed after a 96 h incubation. Results: MIC profiles were obtained using LASARUS and BYE-BMD for 53.9% and 93.3% of isolates, respectively. While L. pneumophila reached sufficient turbidity in BYE-BMD after a 48 h incubation, non-pneumophila species required an extended incubation (72–96 h). Non-pneumophila species displayed broader MIC ranges against azithromycin (0.016–1 mg/L) and levofloxacin (0.016–0.25 mg/L), but a narrower rifampicin range (≤0.0005–0.032 mg/L) relative to L. pneumophila. L. longbeachae exhibited a higher MIC50 for rifampicin despite overlapping susceptibility ranges across all species (0.001–0.016 mg/L). Conclusions: This study demonstrates species-specific differences in Legionella susceptibility and highlights the limitations in extrapolating L. pneumophila-based AST data. Azithromycin MICs in non-pneumophila species exceeded those of L. pneumophila, raising clinical concern. While BYE-BMD was the most effective method for MIC determination, three species required BCYE-α due to poor growth. These findings support developing standardised, species-specific AST protocols and thresholds amid rising macrolide resistance and the increasing detection of non-pneumophila infections.

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.

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 β-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 bla NDM-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 bla NDM carrying species in combination with combinations of ESBLs was particularly concerning and unexpected in Africa. We also identify strains carrying tet (X3), bla VIM-5 or bla DIM-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.

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 β-lactamases ( bla CTX-M-15 ) and carbapenemases ( bla NDM , bla OXA-48 -like and bla KPC ), 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. In hospitals, surfaces present as a reservoir for bacteria pathogens, potentially leading to nosocomial infections. In this work, authors aim to profile extended-spectrum β lactamase- and carbapenemase-carrying bacterial species colonising neonatal hospital wards and causing neonatal sepsis.

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.

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.

Hospitalisation and routine antibiotic treatment are recommended for children with complicated severe acute malnutrition (SAM) but this may exacerbate antimicrobial resistance. Here, we investigate carriage of Gram-negative bacteria in children under five years of age receiving treatment for SAM in Niger, comparing the frequency of colonisation with bacteria carrying resistance genes at admission, during hospital stay and at discharge. E. coli isolates carrying a bla NDM-5 gene were selected for whole-genome sequencing. Rectal colonisation with bacteria carrying ß-lactamase genes is high, with 76% (n = 1042/1371) of children harbouring bacteria carrying a bla CTXM-1 -group gene and 25% (n = 338/1371) carrying a bla NDM-5 gene. Over two-thirds of children who did not carry bacteria with a carbapenemase gene at admission are colonised with bacteria carrying a carbapenemase gene at discharge (n = 503/729, 69%). E. coli ST167 carrying bla NDM-5 gene is recovered from 11% (n = 144/1371) of children. Here we highlight infection control and bacterial AMR transmission concerns amongst a vulnerable population in need of medical treatment. Hospital treatment for children with severe malnutrition may facilitate antibiotic resistance. Here, using rectal swabs from 1,371 children receiving treatment for severe acute malnutrition in Niger, the authors identify high rates of bacteria carrying carbapenemase genes, highlighting the urgent need to prioritize infection control.

Background: Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of neonatal sepsis in high-income countries. While intrapartum antibiotic screening reduces this risk, increasing resistance to macrolides and lincosamides in Europe since the 1990s has limited therapeutic options for penicillin-allergic patients. Reports of reduced beta-lactam susceptibility in GBS further emphasise the need for robust antimicrobial resistance (AMR) surveillance. However, broth microdilution (BMD) methods are unsuitable for large-scale antimicrobial susceptibility testing (AST). Objective: To demonstrate that agar-dilution AST provides equivalent results to broth dilution methods, with superior capacity for high-throughput screening. Methods: Agar-dilution and microdilution AST methods were compared using a panel of 24 characterised susceptible and resistant GBS strains for benzylpenicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, levofloxacin, tetracycline, and vancomycin. Minimum inhibitory concentration (MIC) agreements were evaluated, and resistance profile correlations were assessed using Cohen’s kappa values. Results: Agar-dilution demonstrated >90% agreement with BMD MIC for most antimicrobials, except vancomycin (87.5%), erythromycin (83.33%), and tetracycline (52.78%). Cohen’s kappa values indicated strong agreement (0.88–1.00) for resistance determination. Agar-dilution avoided “trailing growth” issues associated with BMD and facilitated easier detection of non-GBS contaminants. Conclusions: Agar-dilution is a valid method for high-throughput AMR surveillance of retrospective cohorts (96 isolates per plate) and is critical for identifying emerging GBS resistance trends and informing therapeutic guidelines. However, due to the large number of plates required per antimicrobial, it is impractical for routine clinical diagnostics.

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.

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.