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ABSTRACT The objective of this study was to examine the nasal microbiota in relation to otitis media (OM) status and nose health in Indigenous Australian children. Children 2 to 7 years of age were recruited from two northern Australian (Queensland) communities. Clinical histories were obtained through parent interviews and reviews of the medical records. Nasal cavity swab samples were obtained, and the children’s ears, nose, and throat were examined. DNA was extracted and analyzed by 16S rRNA amplicon next-generation sequencing of the V3/V4 region, in combination with previously generated culture data. A total of 103 children were recruited (mean age, 4.7 years); 17 (16.8%) were healthy, i.e., normal examination results and no history of OM. The nasal microbiota differed significantly in relation to OM status and nose health. Children with historical OM had greater relative abundance of Moraxella, compared to healthy children, despite both having healthy ears at the time of swabbing. Children with healthy noses had greater relative abundance of Staphylococcus aureus, compared to those with rhinorrhea. Dolosigranulum was correlated with Corynebacterium in healthy children. Haemophilus and Streptococcus were correlated across phenotypes. Ornithobacterium was absent or was present with low relative abundance in healthy children and clustered around otopathogens. It correlated with Helcococcus and Dichelobacter. Dolosigranulum and Corynebacterium form a synergism that promotes upper respiratory tract (URT)/ear health in Indigenous Australian children. Ornithobacterium likely represents “Candidatus Ornithobacterium hominis” and in this population is correlated with a novel bacterium that appears to be related to poor URT/ear health. IMPORTANCE Recurring and chronic infections of the ear (OM) are disproportionately prevalent in disadvantaged communities across the globe and, in particular, within Indigenous communities. Despite numerous intervention strategies, OM persists as a major health issue and is the leading cause of preventable hearing loss. In disadvantaged communities, this hearing loss is associated with negative educational and social development outcomes, and consequently, poorer employment prospects and increased contact with the justice system in adulthood. Thus, a better understanding of the microbial ecology is needed in order to identify new targets to treat, as well as to prevent the infections. This study used a powerful combination of 16S rRNA gene sequencing and extended culturomics to show that Dolosigranulum pigrum, a bacterium previously identified as a candidate protective species, may require cocolonization with Corynebacterium pseudodiphtheriticum in order to prevent OM. Additionally, emerging and potentially novel pathogens and bacteria were identified.

ABSTRACT Extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales continue to pose a major threat to human health worldwide. Given the limited therapeutic options available to treat infections caused by these pathogens, identifying additional effective antimicrobials or revisiting existing drugs is important. Ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae containing CTX-M-type ESBLs or AmpC, in addition to narrow-spectrum OXA and SHV enzymes, were selected from blood culture isolates obtained from the MERINO trial. Isolates had previously undergone whole-genome sequencing (WGS) to identify antimicrobial resistance genes. Cefotetan MICs were determined by broth microdilution (BMD) testing with a concentration range of 0.125 to 64 mg/liter; CLSI breakpoints were used for susceptibility interpretation. BMD was performed using an automated digital antibiotic dispensing platform (Tecan D300e). One hundred ten E. coli and 40 K. pneumoniae isolates were used. CTX-M-15 and CTX-M-27 were the most common beta-lactamases present; only 7 isolates had coexistent ampC genes. Overall, 98.7% of isolates were susceptible, with MIC50s and MIC90s of 0.25 mg/liter and 2 mg/liter (range, ≤0.125 to 64 mg/liter), respectively. MICs appeared higher among isolates with ampC genes present, with an MIC50 of 16 mg/liter, than among those containing CTX-M-15, which had an MIC50 of only 0.5 mg/liter. Isolates with an ampC gene exhibited an overall susceptibility of 85%. Presence of a narrow-spectrum OXA beta-lactamase did not appear to alter the cefotetan MIC distribution. Cefotetan demonstrated favorable in vitro efficacy against ESBL-producing E. coli and K. pneumoniae bloodstream isolates. IMPORTANCE Carbapenem antibiotics remain the treatment of choice for severe infection due to ESBL- and AmpC-producing Enterobacterales. The use of carbapenems is a major driver of the emergence of carbapenem-resistant Gram-negative bacilli, which are often resistant to most available antimicrobials. Cefotetan is a cephamycin antibiotic developed in the 1980s that demonstrates enhanced resistance to beta-lactamases and has a broad spectrum of activity against Gram-negative bacteria. Cefotetan holds potential to be a carbapenem-sparing treatment option. Data on the in vitro activity of cefotetan against ESBL-producing Enterobacterales remain scarce. Our study assessed the in vitro activity of cefotetan against ceftriaxone-nonsusceptible blood culture isolates obtained from patients enrolled in the MERINO trial.

Abstract Background Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. Methods DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. Results The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. Conclusions The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay’s suitability for direct screening of clinical specimens.

ABSTRACT Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of an mcr-1-positive multidrug-resistant Escherichia coli strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum β-lactamase blaCTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with E. coli bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent E. coli lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult. IMPORTANCE Escherichia coli ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an E. coli ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne mcr-1 gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage.