Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Surveillance Outcome Program (GnSOP) Bloodstream Infection Annual Report 2022.

The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2022 survey was the tenth year to focus on blood stream infections caused by Enterobacterales, and the eighth year where Pseudomonas aeruginosa and Acinetobacter species were included. Fifty-five hospitals Australia-wide participated in 2022. The 2022 survey tested 9,739 isolates, comprising Enterobacterales (8,773; 90.1%), P. aeruginosa (840; 8.6%) and Acinetobacter species (126; 1.3%), using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2023). Key resistances included resistance to the third-generation cephalosporin ceftriaxone in 12.7%/12.7% (CLSI/EUCAST criteria) of Escherichia coli and in 6.6%/6.6% of Klebsiella pneumoniae complex. Resistance rates to ciprofloxacin were 13.7%/13.7% for E. coli; 7.8%/7.8% for K. pneumoniae complex; 5.3%/5.3% for Enterobacter cloacae complex; and 4.3%/10.0% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 2.8%/5.9%; 2.9%/8.7%; 18.3%/27.2%; and 6.1%/14.7% for the same four species, respectively. Twenty-nine Enterobacterales isolates from 28 patients were shown to harbour a carbapenemase gene: 18 blaIMP-4; four blaNDM-5; three blaNDM-1; one blaOXA-181; one blaOXA-244; one blaNDM-1 + blaOXA-181; and one blaNDM-5 + blaOXA-181. Transmissible carbapenemase genes were also detected among two Acinetobacter baumannii complex isolates (blaOXA-23) and one P. aeruginosa (blaNDM-1) in the 2022 survey.

The evolution and international spread of extensively drug resistant Shigella sonnei.

Shigella sonnei causes shigellosis, a severe gastrointestinal illness that is sexually transmissible among men who have sex with men (MSM). Multidrug resistance in S. sonnei is common including against World Health Organisation recommended treatment options, azithromycin, and ciprofloxacin. Recently, an MSM-associated outbreak of extended-spectrum ß-lactamase producing, extensively drug resistant S. sonnei was reported in the United Kingdom. Here, we aimed to identify the genetic basis, evolutionary history, and international dissemination of the outbreak strain. Our genomic epidemiological analyses of 3,304 isolates from the United Kingdom, Australia, Belgium, France, and the United States of America revealed an internationally connected outbreak with a most recent common ancestor in 2018 carrying a low-fitness cost resistance plasmid, previously observed in travel associated sublineages of S. flexneri. Our results highlight the persistent threat of horizontally transmitted antimicrobial resistance and the value of continuing to work towards early and open international sharing of genomic surveillance data.

Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Surveillance Outcome Program (GnSOP).

Abstract: The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2021 survey was the ninth year to focus on bloodstream infections caused by Enterobacterales, and the seventh year where Pseudomonas aeruginosa and Acinetobacter species were included. The 2021 survey tested 8,947 isolates, comprising Enterobacterales (8,104; 90.6%), P. aeruginosa (745; 8.3%) and Acinetobacter species (98; 1.1%), using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2022). Of the key resistances, resistance to the third-generation cephalosporin ceftriaxone was found in 12.5%/12.5% (CLSI/EUCAST criteria) of Escherichia coli and in 6.1%/6.1% of Klebsiella pneumoniae. Resistance rates to ciprofloxacin were 12.3%/12.3% for E. coli; 7.2%/7.2% for K. pneumoniae; 5.4%/5.4% for Enterobacter cloacae complex; and 3.7%/8.0% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 2.8%/6.5%; 2.9%/9.9%; 18.4%/28.1%; and 6.9%/12.8% for the same four species, respectively. Seventeen Enterobacterales isolates from 17 patients were shown to harbour a carbapenemase gene: 12 blaIMP-4; two blaNDM-7; one blaNDM-1; one blaOXA-181; and one blaKPC-2. No transmissible carbapenemase genes were detected among P. aeruginosa or Acinetobacter isolates in the 2021 survey.

Co-Occurrence of Multidrug Resistant Klebsiella pneumoniae Pathogenic Clones of Human Relevance in an Equine Pneumonia Case.

The global epidemiology of multidrug resistant Klebsiella pneumoniae, a serious threat to both animal and human health, is dominated by the spread of pathogenic clones, each separately evolving via acquisition of transferable antibiotic resistance or niche-specific virulence determinants. In horses, K. pneumoniae infection can lead to severe respiratory illness. Here, we characterized multiple isolates recovered from bronchial aspirates of a mare with pneumonia refractory to antibiotics. First, we used a combination of standard microbiology, bacteriophage cross-susceptibility and antibiotic resistance testing to profile the infecting K. pneumoniae population. The genomes of isolates with distinct fingerprints (pulsed-field gel electrophoresis) and unique combined bacteriophage/antibiotic profiles were then further analyzed using whole-genome sequencing. Adhesion to human epithelial cells and biofilm production were also measured as virulence indicators. Although it is commonly expected for one clone to dominate an infection episode, we identified five coexisting multidrug resistant K. pneumoniae sharing the same niche. One was a novel sequence type (ST4656), while the other four were all members of emerging human pathogenic clonal groups (ST307, ST628, ST893 and ST392). These isolates did not display significant differences from one another in terms of virulence or resistance and differed only in plasmid content from isolates implicated in severe human infections, with equal potential to prolong duration and severity of infection when sharing the same niche. This study highlights the importance of more precise surveillance and detection measures to uncover bacterial heterogeneity, reminding us that the "single clone" concept is not an absolute in invasive bacterial infections. IMPORTANCE Multidrug resistant Klebsiella pneumoniae are agents of life-threatening infections in animals and humans, with several multidrug resistant clones causing outbreaks of disease worldwide. It is generally accepted that only one clone will be dominant in an infection episode. In this study, we investigated K. pneumoniae isolates from a horse with severe pneumonia and demonstrated co-occurrence of multiple sequence types previously identified as emerging human pathogens. The equine isolates are not significantly different from one another in terms of virulence or resistance, with equal potential to prolong duration and severity of infection, and are indistinguishable from isolates recovered from humans, except for plasmid content. Our study highlights how the "one dominant clone" concept is not an absolute in severe infection, illustrating the need for improved diagnostics to track heterogeneity of infection, and reinforces the importance of cross-monitoring of environmental and human reservoirs of multidrug resistant pathogens.

Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Sepsis Outcome Programme (GnSOP) Annual Report 2020.

Abstract: The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2020 survey was the eighth year to focus on bloodstream infections caused by Enterobacterales, and the sixth year in which Pseudomonas aeruginosa and Acinetobacter species were included. Eight thousand seven hundred and fifty-two isolates, comprising Enterobacterales (7,871, 89.9%), P. aeruginosa (771, 8.8%) and Acinetobacter species (110, 1.3%), were tested using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2021). Of the key resistances, resistance to the third-generation cephalosporin ceftriaxone was found in 13.5%/13.5% (CLSI/EUCAST criteria) of Escherichia coli and 8.7%/8.7% of Klebsiella pneumoniae. Resistance rates to ciprofloxacin were 16.1%/16.1% for E. coli; 9.9%/9.9% for K. pneumoniae; 5.8%/5.8% for Enterobacter cloacae complex; and 4.5%/8.1% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 2.5%/6.6%; 3.9%/12.5%; 16.9%/26.3%; and 5.5%/14.4% for the same four species respectively. Thirty-two isolates from 32 patients were shown to harbour at least one carbapenemase gene: 19 blaIMP-4, three blaGES-5, two blaNDM-1, two blaNDM-5, two blaOXA-48, two blaOXA-181, one blaIMI-1, and one blaOXA-23+NDM-1.

Third generation cephalosporins and piperacillin/tazobactam have distinct impacts on the microbiota of critically ill patients.

Effective implementation of antibiotic stewardship, especially in critical care, is limited by a lack of direct comparative investigations on how different antibiotics impact the microbiota and antibiotic resistance rates. We investigated the impact of two commonly used antibiotics, third-generation cephalosporins (3GC) and piperacillin/tazobactam (TZP) on the endotracheal, perineal and faecal microbiota of intensive care patients in Australia. Patients exposed to either 3GC, TZP, or no ß-lactams (control group) were sampled over time and 16S rRNA amplicon sequencing was performed to examine microbiota diversity and composition. While neither treatment significantly affected diversity, numerous changes to microbiota composition were associated with each treatment. The shifts in microbiota composition associated with 3GC exposure differed from those observed with TZP, consistent with previous reports in animal models. This included a significant increase in Enterobacteriaceae and Enterococcaceae abundance in endotracheal and perineal microbiota for those administered 3GC compared to the control group. Culture-based analyses did not identify any significant changes in the prevalence of specific pathogenic or antibiotic-resistant bacteria. Exposure to clinical antibiotics has previously been linked to reduced microbiota diversity and increased antimicrobial resistance, but our results indicate that these effects may not be immediately apparent after short-term real-world exposures.

Comment on "Conserved phylogenetic distribution and limited antibiotic resistance of class 1 integrons revealed by assessing the bacterial genome and plasmid collection" by A.N. Zhang et al.

An article published in Microbiome in July 2018 uses incorrect definitions of integron integrase IntI1 and of class 1 integrons that affect the interpretation of the data.

Emergence of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump Conferring Resistance to Multiple Drugs, Including Tigecycline, in Klebsiella pneumoniae.

Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K.pneumoniae, Escherichia coli, and Salmonella TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniaetmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning.IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings-especially carbapenem-resistant K.pneumoniae Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a "One Health" perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

Plasmid DNA Isolation and Visualization: Isolation and Characterization of Plasmids from Clinical Samples.

Plasmids are important in carrying antibiotic resistance and other genes between bacterial cells, and a number of methods can be employed to characterize plasmids from clinical isolates. Single colonies typically obtained as part of hospital workflow can undergo S1 nuclease treatment to linearize plasmids followed by pulsed-field gel electrophoresis to enable determination of the number and sizes of plasmids present. Hybridization of S1/PFGE gels can be used to associate replicon types and passenger genes, such as those conferring antibiotic resistance, with a particular plasmid band. Individual plasmids, obtained by conjugation or transformation, can be compared by gel electrophoresis following restriction digestion of plasmid DNA prepared by alkaline lysis methods, including using specialized kits.

Diversity of P1 phage-like elements in multidrug resistant Escherichia coli.

The spread of multidrug resistance via mobile genetic elements is a major clinical and veterinary concern. Pathogenic Escherichia coli harbour antibiotic resistance and virulence genes mainly on plasmids, but also bacteriophages and hybrid phage-like plasmids. In this study, the genomes of three E. coli phage-like plasmids, pJIE250-3 from a human E. coli clinical isolate, pSvP1 from a porcine ETEC O157 isolate, and pTZ20_1P from a porcine commensal E. coli, were sequenced (PacBio RSII), annotated and compared. All three elements are coliphage P1 variants, each with unique adaptations. pJIE250-3 is a P1-derivative that has lost lytic functions and contains no accessory genes. In pTZ20_1P and pSvP1, a core P1-like genome is associated with insertion sequence-mediated acquisition of plasmid modules encoding multidrug resistance and virulence, respectively. The transfer ability of pTZ20_1P, carrying antibiotic resistance markers, was also tested and, although this element was not able to transfer by conjugation, it was able to lysogenize a commensal E. coli strain with consequent transfer of resistance. The incidence of P1-like plasmids (~7%) in our E. coli collections correlated well with that in public databases. This study highlights the need to investigate the contribution of phage-like plasmids to the successful spread of antibiotic resistant pathotypes.

Combining sequencing approaches to fully resolve a carbapenemase-encoding megaplasmid in a Pseudomonas shirazica clinical strain.

Horizontal transfer of plasmids plays a pivotal role in dissemination of antibiotic resistance genes and emergence of multidrug-resistant bacteria. Plasmid sequencing is thus paramount for accurate epidemiological tracking in hospitals and routine surveillance. Combining Nanopore and Illumina sequencing allowed full assembly of a carbapenemase-encoding megaplasmid carried by multidrug-resistant clinical isolate FFUP_PS_41. Average nucleotide identity analyses revealed that FFUP_PS_41 belongs to the recently proposed new species Pseudomonas shirazica, related to the P. putida phylogenetic group. FFUP_PS_41 harbours a 498,516-bp megaplasmid (pJBCL41) with limited similarity to publicly-available plasmids. pJBCL41 contains genes predicted to encode replication, conjugation, partitioning and maintenance functions and heavy metal resistance. The |aacA7|blaVIM-2|aacA4| cassette array (resistance to carbapenems and aminoglycosides) is located within a class 1 integron that is a defective Tn402 derivative. This transposon lies within a 50,273-bp region bound by Tn3-family 38-bp inverted repeats and flanked by 5-bp direct repeats (DR) that composes additional transposon fragments, five insertion sequences and a Tn3-Derived Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach allowed full resolution of a carbapenemase-encoding megaplasmid from P. shirazica. Identification of novel megaplasmids sheds new light on the evolutionary effects of gene transfer and the selective forces driving antibiotic resistance.

Mobile Genetic Elements Associated with Antimicrobial Resistance.

Strains of bacteria resistant to antibiotics, particularly those that are multiresistant, are an increasing major health care problem around the world. It is now abundantly clear that both Gram-negative and Gram-positive bacteria are able to meet the evolutionary challenge of combating antimicrobial chemotherapy, often by acquiring preexisting resistance determinants from the bacterial gene pool. This is achieved through the concerted activities of mobile genetic elements able to move within or between DNA molecules, which include insertion sequences, transposons, and gene cassettes/integrons, and those that are able to transfer between bacterial cells, such as plasmids and integrative conjugative elements. Together these elements play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes. This review aims to outline the characteristics of the major types of mobile genetic elements involved in acquisition and spread of antibiotic resistance in both Gram-negative and Gram-positive bacteria, focusing on the so-called ESKAPEE group of organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli), which have become the most problematic hospital pathogens.

Proposal for assignment of allele numbers for mobile colistin resistance (mcr) genes.

The initial report of the mcr-1 (mobile colistin resistance) gene has led to many reports of mcr-1 variants and other mcr genes from different bacterial species originating from human, animal and environmental samples in different geographical locations. Resistance gene nomenclature is complex and unfortunately problems such as different names being used for the same gene/protein or the same name being used for different genes/proteins are not uncommon. Registries exist for some families, such as bla (ß-lactamase) genes, but there is as yet no agreed nomenclature scheme for mcr genes. The National Center for Biotechnology Information (NCBI) recently took over assigning bla allele numbers from the longstanding Lahey ß-lactamase website and has agreed to do the same for mcr genes. Here, we propose a nomenclature scheme that we hope will be acceptable to researchers in this area and that will reduce future confusion.

Ecological effects of cefepime use during antibiotic cycling on the Gram-negative enteric flora of ICU patients.

This study examines the impact of cefepime and APP-ß (antipseudomonal penicillin/ ß-lactamase inhibitor combinations) on Gram-negative bacterial colonization and resistance in two Australian ICUs. While resistance did not cumulatively increase, cefepime (but not APP-ß treatment) was associated with acquisition of antibiotic resistant Enterobacteriaceae, consistent with an ecological effect. Analysis of the resident gut E. coli population in a subset of patients showed an increase in markers of horizontal gene transfer after cefepime exposure that helps explain the increase in APP-ß resistance and reminds us that unmeasured impacts on the microbiome are key outcome determinants that need to be fully explored.

Automated annotation of mobile antibiotic resistance in Gram-negative bacteria: the Multiple Antibiotic Resistance Annotator (MARA) and database.

Background: Multiresistance in Gram-negative bacteria is often due to acquisition of several different antibiotic resistance genes, each associated with a different mobile genetic element, that tend to cluster together in complex conglomerations. Accurate, consistent annotation of resistance genes, the boundaries and fragments of mobile elements, and signatures of insertion, such as DR, facilitates comparative analysis of complex multiresistance regions and plasmids to better understand their evolution and how resistance genes spread. Objectives: To extend the Repository of Antibiotic resistance Cassettes (RAC) web site, which includes a database of 'features', and the Attacca automatic DNA annotation system, to encompass additional resistance genes and all types of associated mobile elements. Methods: Antibiotic resistance genes and mobile elements were added to RAC, from existing registries where possible. Attacca grammars were extended to accommodate the expanded database, to allow overlapping features to be annotated and to identify and annotate features such as composite transposons and DR. Results: The Multiple Antibiotic Resistance Annotator (MARA) database includes antibiotic resistance genes and selected mobile elements from Gram-negative bacteria, distinguishing important variants. Sequences can be submitted to the MARA web site for annotation. A list of positions and orientations of annotated features, indicating those that are truncated, DR and potential composite transposons is provided for each sequence, as well as a diagram showing annotated features approximately to scale. Conclusions: The MARA web site (http://mara.spokade.com) provides a comprehensive database for mobile antibiotic resistance in Gram-negative bacteria and accurately annotates resistance genes and associated mobile elements in submitted sequences to facilitate comparative analysis.

Locally Acquired mcr-1 in Escherichia coli, Australia, 2011 and 2013.

We identified discrete importation events of the mcr-1 gene on incompatibility group IncI2 plasmids in Escherichia coli isolated from patients in New South Wales, Australia, in 2011 and 2013. mcr-1 is present in a small minority of colistin-resistant Enterobacteriaceae and appears not to be established locally.

Plasmid interference for curing antibiotic resistance plasmids in vivo.

Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.