Azithromycin therapy for prevention of chronic lung disease of prematurity (AZTEC): a multicentre, double-blind, randomised, placebo-controlled trial.

BACKGROUND: Systematic reviews have reported conflicting evidence on whether macrolide antibiotics reduce rates of chronic lung disease of prematurity (CLD) in at-risk preterm infants born at less than 30 weeks' gestation, including in those colonised with pulmonary Ureaplasma spp. Since an adequately powered trial has been lacking, we aimed to assess if the macrolide azithromycin improved survival without the development of physiologically defined moderate or severe CLD in preterm infants. METHODS: AZTEC was a multicentre, double-blind, randomised, placebo-controlled trial conducted in 28 tertiary neonatal intensive care units in the UK. Infants were eligible if they were born at less than 30 weeks' gestation and had received at least 2 h of either non-invasive (continuous positive airway pressure or humidified high flow nasal cannula therapy) or invasive respiratory support (via endotracheal tube) within 72 h of birth. Eligible infants were randomly allocated in a 1:1 ratio using random permuted blocks of four to receive either intravenous azithromycin at 20 mg/kg per day for 3 days followed by 10 mg/kg for 7 days, or to placebo. Allocation was stratified by centre and gestational age at birth (<28 weeks vs ≥28 weeks). Azithromycin and placebo vials were encased in tamper-evident custom cardboard cartons to ensure masking for clinicians, parents, and the research team. The primary outcome was survival without development of physiologically defined moderate or severe CLD at 36 weeks' postmenstrual age. Outcomes and safety were analysed on an intention-to-treat basis (all randomly allocated infants, regardless of any post-randomisation events). The study was registered with ISRCRN (11650227) and is closed. FINDINGS: Infants were recruited between Oct 9, 2019, and March 22, 2022. 799 (53·1%) of 1505 eligible infants underwent random allocation; three infants were withdrawn, including consent to use their data, leaving 796 infants for analysis. Survival without moderate or severe CLD occurred in 166 (42%) of 394 infants in the intervention group and 179 (45%) of 402 in the placebo group (three-level adjusted OR [aOR] 0·84, 95% CI 0·55-1·29, p=0·43). Pulmonary Ureaplasma spp colonisation did not influence treatment effect. Overall, seven serious adverse events were reported for the azithromycin group (five graded as severe, two as moderate), and six serious adverse events were reported in the placebo group (two severe, two moderate, and two mild), as assessed by the local principal investigators. INTERPRETATION: Since prophylactic use of azithromycin did not improve survival without development of physiologically-defined CLD, regardless of Ureaplasma spp colonisation, it cannot be recommended in clinical practice. FUNDING: UK National Institute for Health and Care Research.

Baseline azithromycin resistance in the gut microbiota of preterm born infants.

BACKGROUND: Macrolides, including azithromycin, are increasingly used in preterm-born infants to treat Ureaplasma infections. The baseline carriage of macrolide resistance genes in the preterm stool microbiota is unknown. OBJECTIVES: Identify carriage of azithromycin resistant bacteria and the incidence of macrolide resistant genes. METHODS: Azithromycin resistant bacteria were isolated from serial stool samples obtained from preterm infants (≤32 weeks' gestation) by culturing aerobically/anaerobically, in the presence/absence of azithromycin. Using quantitative PCR, we targeted 6 common macrolide resistance genes (erm(A), erm(B), erm(C), erm(F), mef(A/E), msr(A)) in DNA extracted from selected bacteria resistant to azithromycin. RESULTS: From 89 stool samples from 37 preterm-born infants, 93.3% showed bacterial growth in aerobic or anaerobic conditions. From the 280 azithromycin resistant isolates that were identified, Staphylococcus (75%) and Enterococcus (15%) species dominated. Macrolide resistance genes were identified in 91% of resistant isolates: commonest were erm(C) (46% of isolates) and msr(A) (40%). Multiple macrolide resistance genes were identified in 18% of isolates. CONCLUSION: Macrolide resistance is common in the gut microbiota of preterm-born infants early in life, most likely acquired from exposure to the maternal microbiota. It will be important to assess modulation of macrolide resistance, if macrolide treatment becomes routine in the management of preterm infants. IMPACT STATEMENT: Azithromycin resistance is present in the stool microbiota in the first month of life in preterm infants 91% of azithromycin resistant bacteria carried at least one of 6 common macrolide resistant genes Increasing use of macrolides in the preterm population makes this an important area of study.

Association of early and current life factors with telomere length in preterm-born children.

BACKGROUND: Telomeres shorten after each cell division. Since preterm-born babies are delivered early and often suffer from inflammatory conditions such as bronchopulmonary dysplasia (BPD), their telomere length may be altered. OBJECTIVES: We assessed associations of early and current life factors with telomere length in saliva samples obtained from 7-12-year-old children born at ≤34 weeks' gestation and term-born controls. STUDY DESIGN: Relative telomere length was measured by qPCR on extracted DNA. Groups were compared using independent t-tests or ANOVA with post-hoc correction. Linear regression analysis was also used. RESULTS: 534 children had satisfactory telomere data including 383 who were preterm-born (mean (SD) birthweight 1732g (558g), gestation 31.1 (2.6) weeks) and 151 term-born (3464g (510g); 39.8 (1.3) weeks). Telomere length was longer in children who had intrauterine growth restriction (IUGR) at birth: mean (SD): 464.6 (166.3) vs. 418.6 (110.7) in the no-IUGR group; in females: 440.2 (130.1) vs. 405.7 (101.5) in males; and in the least deprived group (397.8 (95.0) vs. 437.6 (121.9) most vs least deprivation quintile). Differences were most notable in females with IUGR. However, telomere length was not different between the preterm and term groups; the BPD and no BPD groups nor was it related to lung function or cardiovascular measurements. In multivariable regression analyses, telomere length was associated with sex, IUGR and deprivation with the greatest difference observed in females with IUGR. CONCLUSIONS: Telomere length was associated with sex, IUGR and deprivation, especially in females with IUGR, but not with prematurity, BPD, lung function or cardiovascular measurements.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

In vitro efficacy of imipenem-relebactam and cefepime-AAI101 against a global collection of ESBL-positive and carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: To evaluate the potential clinical in vitro efficacy of novel ß-lactam/ß-lactamase-inhibitor combinations - including imipenem-relebactam (IPM-REL) and cefepime-AAI101 (enmetazobactam) (FEP-AAI) - against contemporary multidrug-resistant (MDR) Enterobacteriaceae. METHODS: Agar-based MIC screening against MDR Enterobacteriaceae (n = 264) was used to evaluate the in vitro efficacy of IPM-REL and FEP-AAI, to compare the results with established combinations, and to investigate alternative ß-lactam partners for relebactam (REL) and enmetazobactam (AAI). The inhibition activities of REL, AAI and the comparators avibactam (AVI) and tazobactam, against isolated recombinant ß-lactamases covering representatives from all four Ambler classes of ß-lactamases, were tested using a fluorescence-based assay. RESULTS: Using recombinant proteins, all four inhibitors were highly active against the tested class A serine ß-lactamases (SBLs). REL and AVI showed moderate activity against the Class C AmpC from Pseudomonas aeruginosa and the Class D OXA-10/-48 SBLs, but outperformed tazobactam and AAI. All tested inhibitors lacked activity against Class B metallo-ß-lactamases (MBLs). In the presence of REL and IPM, but not AAI, susceptibility increased against Klebsiella pnuemoniae carbapenemase (KPC)-positive and OXA-48-positive isolates. Both aztreonam-AVI and ceftolozane-tazobactam were more effective than IPM-REL. In all the tested combinations, AAI was a more effective inhibitor of class A ß-lactamases (ESBLs) than the established inhibitors. CONCLUSION: The results lead to the proposal of alternative combination therapies involving REL and AAI to potentiate the use of ß-lactams against clinical Gram-negative isolates expressing a variety of lactamases. They highlight the potential of novel combinations for combating strains not covered by existing therapies.

Dissemination of genetically diverse NDM-1, -5, -7 producing-Gram-negative pathogens isolated from pediatric patients in Pakistan.

Aim: To determine the prevalence of New Delhi metallo-ß-lactamase (NDM)-producing Gram-negative pathogens isolated from children's samples. Materials & methods: Carbapenem-resistant clinical isolates (n = 117) were confirmed by VITEK® 2 compact system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing. MIC (µg/ml) of various antibiotics was determined by VITEK 2 compact system. Molecular characterization of the isolates was performed by PCR, DNA sequencing, PFGE and DNA hybridization. Results: Out of 117 carbapenemase producers, 37 (31.6%) and 29 (24.7%) were Klebsiella pneumoniae and Acinetobacter baumannii, respectively. 72 (61.5%) isolates were NDM positive and among these 60, 9 and 3 were NDM-1, -5 and -7, respectively. Majority of the NDM-producing K. pneumoniae belonged to ST11 and ST273 while most of the Escherichia coli belonged to ST405 and ST101. blaNDM were mainly located on 150kb plasmids. MIC displayed high resistance against ß-lactams drugs including carbapenems, and the most sensitive drugs were tigecycline and colistin. Conclusion: Dissemination of blaNDM-producing pathogens, particularly in children clinical settings, is a matter of great public health concern.

In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii.

OBJECTIVES: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants. METHODS: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity. RESULTS: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin. CONCLUSIONS: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

The polymyxin derivative NAB739 is synergistic with several antibiotics against polymyxin-resistant strains of Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii.

The antibiotic crisis has reinstated polymyxins, once abandoned because of their toxicity. Now, preclinical studies have revealed better tolerated and more effective derivatives of polymyxins such as NAB739. Simultaneously, polymyxin-resistant (PMR) strains such as the mcr-1 strains have received lots of justified publicity, even though they are still very rare. Here we show that NAB739 sensitizes the PMR strains to rifampin, a classic "anti-Gram-positive" antibiotic excluded by the intact outer membrane (OM) permeability barrier, as well as to retapamulin, the surrogate of lefamulin, an antibiotic under development against Gram-positive bacteria. Polymyxin B was used as a comparator. The combination of NAB739 and rifampin was synergistic against ten out of eleven PMR strains of Escherichia coli (Fractional Synergy Indices, FICs, 0.14-0.19) and that of NAB739 and retapamulin against all the tested eleven strains (FICs 0.19-0.25). Against PMR Klebsiella pneumoniae (n = 7), the FICs were 0.13-0.27 for NAB739 + rifampin and 0.14-0.28 for NAB739+retapamulin. Against Acinetobacter baumannii (n = 2), the combination of NAB739 and rifampin had the FIC of 0.09-0.19. Furthermore, NAB739 and meropenem were synergistic (FICs 0.25-0.50) against four out of five PMR strains that were simultaneously resistant to meropenem.

Clinical Validation of SensiTest Colistin, a Broth Microdilution-Based Method To Evaluate Colistin MICs.

The global spread of multidrug-resistant Gram-negative bacteria has led to the return of colistin for treating severe infections. Recently, different plasmid-mediated genes conferring resistance to this drug were described and reported worldwide. International committees (EUCAST/CLSI) reevaluated inconsistencies surrounding colistin antimicrobial susceptibility testing (AST), concluding that broth microdilution (BMD) should serve as the reference method for AST. The development of an accurate, reproducible commercial test based on BMD is therefore highly desirable. SensiTest Colistin (STC), a BMD-based compact 4-test panel containing the lyophilized antibiotic in 7 2-fold dilutions (0.25 to 16 µg/ml) was here compared with the EUCAST-CLSI standard reference method (BMD) and, for some isolates, with the automated Phoenix 100 system (PHX). A total of 353 bacterial strains were evaluated by two different laboratories; 137 isolates were resistant to colistin (19 were intrinsically resistant, 83 harbored the mcr-1 gene). Essential agreement (EA) between STC and BMD was obtained for 339 out of the 353 strains tested (96.0%). Overall categorical agreement was obtained for 349 out of the 353 strains analyzed (98.9%). Two major errors (MEs; 0.93%) and two very major errors (VMEs; 1.46%) were documented. STC appeared to be a simple but highly reliable test with good reproducibility even with panels stored at room temperature or at 35°C. Moreover, STC showed a good performance with strains carrying the mcr-1 gene, with a 98.8% EA. As the secondary endpoint of our study, VMEs for PHX were documented for 6 isolates (10%).

Isolation of Separate Ureaplasma Species From Endotracheal Secretions of Twin Patients.

Isolation of Ureaplasma spp. from preterm neonates and the association with development of bronchopulmonary dysplasia has been previously investigated. However, few studies have contrasted the nature of infection in twins. In this article, we report that dizygotic twins (1 girl, 1 boy) born at 24 weeks gestation both yielded culturable Ureaplasma from endotracheal secretions. The samples were part of a serial blind collection cohort of ventilated premature neonates, and analysis of repeat cultures showed stable, separate infections over a period of 17 and 21 days, respectively. Immunoblot and probe-specific quantitative polymerase chain reaction analysis determined that Twin 1 was solely infected with Ureaplasma parvum (specifically, serovar 6 by gene sequencing), whereas Twin 2 was solely infected with Ureaplasma urealyticum (specifically, genotype A- serovars 2, 5, and 8 by gene sequencing). Immunoblot analysis found that the major surface antigen (multiple-banded antigen) altered relative mass for both strains during the course of infection. Quantitative polymerase chain reaction analysis of extracted endotracheal aspirates confirmed no evidence of mixed infection for either twin. Failure of sentinel ventilated preterm infants on the same ward to acquire Ureaplasma infection after the first week of birth suggests no cot-to-cot transfer of Ureaplasma infection occurred. This study demonstrated not only a contrasting clinical outcome for a set of twins infected with 2 separate species of Ureaplasma, but also the first real-time demonstration of multiple-banded antigen alteration and evolution of Ureaplasma over the course of a clinical infection.

Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies.

Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections.

Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models.

Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to "sense" pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.

Correction: Synergic Activation of Toll-Like Receptor (TLR) 2/6 and 9 in Response to Ureaplasma parvum & urealyticum in Human Amniotic Epithelial Cells.

[This corrects the article DOI: 10.1371/journal.pone.0061199.].